Methods, kits and systems for determining multiple sclerosis status and methods for treating multiple sclerosis based on same

Abstract

The present disclosure includes, among other things, methods, kits, and systems for determining the status of MS in a subject. In various embodiments, the present disclosure relates to the use of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation that are characteristic of the status of MS. In some embodiments, differential modifications and / or differential accessibility are detected and quantified at one or more genomic loci of a biological sample, e.g., in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from a subject with MS. In various embodiments a determined status is useful, e.g., in selecting treatment for and / or treating MS.

Description

Attorney Docket: 2014191-0043 METHODS, KITS AND SYSTEMS FOR DETERMINING MULTIPLE SCLEROSIS STATUS AND METHODS FOR TREATING MULTIPLE SCLEROSIS BASED ON SAME CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U. S. Provisional Patent Application 63 / 690,278 filed on September 3, 2024, the entire contents of which is hereby incorporated by reference. BACKGROUND

[0002] Multiple sclerosis (MS) is an immune-driven disease characterized by demyelination and axonal damage in the central nervous system (CNS). It is twice as common in females and most often diagnosed between 20–40 years of age. The MS prevalence in the United States has been steadily increasing over the past several decades, with approximately 58 cases per 100,000 persons in 1975 increasing to 309.2 cases per 100,000 persons (450.1 per 100,000 for females) during 2010–2019. This roughly translates to 1 in 300 people in the United States living with MS, with the incidence being the highest for Black people (10.2 per 100,000 person- years versus 6.2 in white people).

[0003] MRI is the current gold standard imaging procedure for diagnosing and monitoring MS. MRIs are expensive, however. There are also some important limitations on the ability of MRI to detect MS. For example, several diseases and syndromes are routinely mistaken for MS in clinical settings. These include migraine (22% of cases), fibromyalgia (15% of cases), nonspecific or nonlocalizing neurological symptoms with abnormal MRI presentation (12% of cases), conversion disorder / functional neurological disorder (11% of cases), and neuromyelitis optica (NMO) (6% of cases). Other diseases that may be considered in a differential diagnosis of MS include myelin oligodendrocyte glycoprotein antibody disease (MOGAD), CNS vasculitis, neurosarcoidosis, CNS manifestations of autoimmune diseases such as Sjögren’s syndrome, systemic lupus erythematosus (SLE), and antiphospholipid antibody syndrome, chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids, and12953047v1 Page 1 of 224Attorney Docket: 2014191-0043 Beçhet’s syndrome. MRI also does not provide insight into the biological mechanisms underlying MS in a subject, and therefore is limited in its ability to determine, e.g., which subjects are likely to respond well to treatment with a certain therapeutic, which subjects are more likely to exhibit worsening disease state, and which subjects are likely to be prone to relapse. SUMMARY

[0004] The present disclosure is based, at least in part, on the demonstration that MS in a subject can be detected and characterized by detecting and quantifying the presence of histone modifications and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample obtained or derived from the subject. The present disclosure also encompasses methods where chromatin accessibility and / or binding of one or more transcription factors are detected at the one or more genomic loci instead of (or in addition to) histone modifications and / or DNA methylation.

[0005] Liquid biopsies are now widely utilized in clinical oncology to detect cancer recurrence and inform therapeutic decisions. However, most commercially available cfDNA assays only detect tumor genomic alterations and not all disease states have a characteristic genomic alteration that can be used for detection. For example, MS is associated with a number of environmental factors, and while certain alleles have been associated with an increased risk of MS, it is currently not possible (and may be impossible) to detect the presence of MS on the basis of genome sequencing alone, let alone characterizing disease states. Among other things, the present disclosure provides tools to analyze multiple epigenomic features from patient plasma, including DNA methylation, chromatin accessibility, and histone modifications. Among other things, the present disclosure demonstrates that epigenomic cfDNA profiling can be used to detect MS in patients as well as characterize disease severity. Diagnosing and monitoring of MS by cfDNA profiling would be immediately clinically actionable, as guidelines recommend that MS be treated with different drugs depending on disease severity.

[0006] The present disclosure includes, among other things, technologies for the determination of MS status and for the detection, monitoring, and / or treatment of MS based on MS status. In various embodiments, the present disclosure relates to the measurement of histone modifications in a sample obtained or derived from a subject to detect and / or treat MS based on12953047v1 Page 2 of 224Attorney Docket: 2014191-0043 MS status. The present disclosure includes, among other things, histone modification measurements in cell-free DNA (cfDNA) that are characteristic of MS, and which in various embodiments are useful, e.g., for detecting, monitoring, selecting treatment for, and / or treating MS. The present disclosure includes, among other things, histone modification measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating MS. In some embodiments, histone modification measurements in cfDNA can be used to detect or determine resistance of MS in a subject to a therapy. In some embodiments, histone modification measurements in cfDNA can be used to monitor progression of MS (e.g., progression in a subject currently being administered a therapy). In some embodiments, histone modification measurements in cfDNA can be used to inform therapeutic selection for a subject with MS (e.g., determine an initial therapy, predict patients that are likely to respond to a given therapy, and / or determine when therapy should be changed for a subject). In some embodiments, histone modification measurements in cfDNA can be used as a complement to other methods (e.g., imaging methods and / or symptom-based methods) for monitoring MS (e.g., performed concurrently with other methods and / or performed subsequent to other methods, e.g., to monitor disease progression).

[0007] In various embodiments, the present disclosure includes exemplary genomic loci that are differentially modified in MS vs. healthy subjects. In various embodiments, genomic loci differentially modified in cfDNA are or include one or more enhancers. In various embodiments, genomic loci differentially modified in cfDNA are or include one or more promoters.

[0008] In various embodiments, a genomic locus is differentially modified if it is characterized by increased or decreased histone modification as compared to a reference (e.g., a sample from a healthy subject). Increased or decreased histone modification can be or include, e.g., increased or decreased histone methylation (hypermethylation or hypomethylation, respectively) of one or more particular methylation marks, or a combination thereof; increased or decreased pan-methylation; increased or decreased histone acetylation (hyperacetylation or hypoacetylation, respectively) of one or more particular acetylation marks, or a combination thereof; and / or increased or decreased pan-acetylation (e.g., pan-H3 acetylation). In various embodiments, histone methylation can be or include histone methylation marks selected from H3K4me1, H3K4me2, H3K4me3, or a combination thereof. In various embodiments, histone12953047v1 Page 3 of 224Attorney Docket: 2014191-0043 methylation can be or include H3K4me3. In various embodiments, histone acetylation can be or include histone acetylation marks selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, or a combination thereof. In various embodiments, histone acetylation can be or include H3K27ac.

[0009] In various embodiments, the present disclosure relates to the measurement of DNA methylation in a sample obtained or derived from a subject to detect and / or treat MS. The present disclosure includes, among other things, DNA methylation measurements in cell-free DNA (cfDNA) that are characteristic of MS, and which in various embodiments are useful, e.g., for detecting, monitoring, selecting treatment for, and / or treating MS. The present disclosure includes, among other things, DNA methylation measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating MS. The present disclosure includes, among other things, DNA methylation measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating MS. In some embodiments, DNA methylation measurements in cfDNA can be used to detect or determine resistance of MS to a therapy. In various embodiments, the present disclosure includes exemplary genomic loci that are differentially DNA methylated in MS. In various embodiments, a genomic locus is differentially modified if it is characterized by increased or decreased DNA methylation as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, genomic loci differentially modified in cfDNA are or include one or more enhancers. In various embodiments, genomic loci differentially modified in cfDNA are or include one or more promoters.

[0010] The present disclosure further relates, in various embodiments, to the measurement of chromatin accessibility in cell-free DNA (cfDNA) to determine MS status. The present disclosure includes, among other things, chromatin accessibility measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating MS. The present disclosure includes, among other things, chromatin accessibility measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating MS. In some embodiments, chromatin accessibility measurements in cfDNA can12953047v1 Page 4 of 224Attorney Docket: 2014191-0043 be used to detect or determine resistance of MS to a therapy. In various embodiments, the present disclosure includes genomic loci that are differentially accessible in MS. In various embodiments, genomic loci differentially accessible in cfDNA are or include one or more enhancers. In various embodiments, genomic loci differentially accessible in cfDNA are or include one or more promoters.

[0011] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with chromatin accessibility.

[0012] In various embodiments, a genomic locus is differentially accessible if it is characterized by increased or decreased chromatin accessibility as compared to a reference (e.g., a sample from a healthy subject). Increased or decreased histone modification can be or include, e.g., increased or decreased accessibility as determined by various chromatin accessibility assays known in the art.

[0013] The present disclosure further relates, in various embodiments, to the measurement of transcription factor binding in cell-free DNA (cfDNA) to determine MS status. The present disclosure includes, among other things, transcription factor binding measurements in cfDNA that are characteristic of MS, which in various embodiments are useful, e.g., in detecting, monitoring, selecting treatment for, and / or treating MS. In some embodiments, transcription factor binding measurements in cfDNA can be used to detect or determine resistance of MS in a subject to a therapy. In various embodiments, the present disclosure includes genomic loci that are differentially bound by transcription factors in MS. In various embodiments, genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more enhancers. In various embodiments, genomic loci that are differentially bound by transcription factors in cfDNA are or include one or more promoters.

[0014] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific12953047v1 Page 5 of 224Attorney Docket: 2014191-0043 theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with transcription factor binding.

[0015] In various embodiments, a genomic locus is differentially bound by transcription factors if it is characterized by increased or decreased transcription factor binding as compared to a reference (e.g., a sample from a healthy subject). Increased or decreased transcription factor binding can be or include, e.g., increased or decreased transcription factor binding as determined by various transcription factor binding assays known in the art.

[0016] In one aspect, the present disclosure provides a method of determining MS status in a subject, the method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) binding of one or more transcription factors, and / or (iv) DNA methylation.

[0017] In some embodiments, the one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, and pan-acetylation. In some embodiments, the histone modification assay detects H3K4me3 modifications. In some embodiments, the histone modification assay detects H3K27ac modifications. In some embodiments, the histone modification assay is selected from ChIP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.

[0018] In some embodiments, chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and a DNase hypersensitivity assay.

[0019] In some embodiments, binding of one or more transcription factors is quantified using a transcription factor binding assay. In some embodiments, the transcription factor binding assay is selected from ChIP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN12953047v1 Page 6 of 224Attorney Docket: 2014191-0043 (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.

[0020] In some embodiments, DNA methylation is quantified using Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq).

[0021] In some embodiments, a method comprises quantifying two or more of the following, each at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) transcription factor binding, and / or (iv) DNA methylation. In some embodiments, the method comprises quantifying two or more histone modifications, e.g., quantifying H3K4me3 and H3K27ac modifications. In some embodiments, a method comprises quantifying one or more histone modifications and DNA methylation, e.g., quantifying H3K4me3 and / or H3K27ac modifications and DNA methylation. In some embodiments, a method comprises quantifying H3K4me3 modifications, H3K27ac modifications and DNA methylation.

[0022] In some embodiment, a biological sample is a liquid biopsy sample, e.g., a plasma sample, serum sample, or urine sample.

[0023] In some embodiments, quantification of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the one or more genomic loci as compared to a reference indicates that the subject has MS. In some embodiments, the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and / or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a cohort of healthy subjects.

[0024] In some embodiments, a method comprises quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in Tables 1-6. In some embodiments, a method comprises quantifying H3K4me3 modifications for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 1. In some embodiments, a method comprises quantifying H3K27ac modifications for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2. In some embodiments, a method comprises12953047v1 Page 7 of 224Attorney Docket: 2014191-0043 quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 3. In some embodiments, a method comprises quantifying H3K4me3 modifications for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 4. In some embodiments, a method comprises quantifying H3K27ac modifications for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 5. In some embodiments, a method comprises quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 6.

[0025] In some embodiments, the activity of one or more transcription factors is determined by measuring transcription factor binding. In some embodiments, the activity of the one or more transcription factors is assessed using a method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) binding of one or more transcription factors, and / or (iv) DNA methylation. In some embodiments, the one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, and pan-acetylation. In some embodiments, the histone modification assay detects H3K4me3 modifications. In some embodiments, the histone modification assay detects H3K27ac modifications.

[0026] In some embodiments, a sample is obtained from a subject having MS wherein imaging of MS is not possible and / or feasible and / or when it is not possible to detect / diagnose MS using imaging or symptoms alone.

[0027] Among other things, the present disclosure provides a method of detecting multiple sclerosis (MS) in a subject, the method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) binding of one or more transcription factors, and / or12953047v1 Page 8 of 224Attorney Docket: 2014191-0043 (iv) DNA methylation.

[0028] In some embodiments, one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, and pan-acetylation.

[0029] In some embodiments, a histone modification assay detects H3K4me3 modifications.

[0030] In some embodiments, a histone modification assay detects H3K27ac modifications.

[0031] In some embodiments, a histone modification assay is selected from ChIP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.

[0032] In some embodiments, chromatin accessibility can be quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and a DNase hypersensitivity assay.

[0033] In some embodiments, binding of one or more transcription factors can be quantified using a transcription factor binding assay.

[0034] In some embodiments, a transcription factor binding assay is selected from ChIP- seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.

[0035] In some embodiments, DNA methylation can be quantified using Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq).12953047v1 Page 9 of 224Attorney Docket: 2014191-0043

[0036] In some embodiments, methods described herein comprise quantifying two or more of the following, each at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) transcription factor binding, and / or (iv) DNA methylation.

[0037] In some embodiments, methods described herein comprise quantifying two or more histone modifications.

[0038] In some embodiments, methods described herein comprise quantifying H3K4me3 and H3K27ac modifications.

[0039] In some embodiments, methods described herein comprise quantifying one or more histone modifications and DNA methylation.

[0040] In some embodiments, methods described herein comprise quantifying H3K4me3 and / or H3K27ac modifications and DNA methylation.

[0041] In some embodiments, methods described herein comprise quantifying H3K4me3 modifications, H3K27ac modifications and DNA methylation.

[0042] In some embodiments, a liquid biopsy sample is a plasma sample, serum sample, or urine sample.

[0043] In some embodiments, methods described herein comprise quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in Tables 1-6.

[0044] In some embodiments, an increase or decrease of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci (e.g., one or more loci described herein (e.g., one or more loci listed in Tables 1-6) as compared to a reference indicates that the subject has MS.

[0045] In some embodiments, methods described herein comprise quantifying:12953047v1 Page 10 of 224Attorney Docket: 2014191-0043 (a) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, or 700 genomic loci in Table 1; (b) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 genomic loci in Table 2; (c) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500, genomic loci in Table 3; (d) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 genomic loci in Table 4; (e) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, or 600 genomic loci in Table 5; (f) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, or 150 genomic loci in Table 6; or (g) or any combination of (a)-(f).

[0046] In some embodiments, one or more genomic loci comprise one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell identity genes, or one or more regulatory regions thereof, and an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell identity genes, or one or more regulatory regions thereof as compared to a reference indicates that the subject has MS.

[0047] In some embodiments, one or more genomic loci comprise one or more enhancer regions associated with one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell, and an increase in enhancer signal at the one or more enhancer regions associated with the one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell identity gene indicates that the subject has MS.

[0048] In some embodiments, the one or more cell identity genes of an oligodendrocyte progenitor cell comprise NSUN5, DAAM2, CNTN2, or any combination thereof. In some12953047v1 Page 11 of 224Attorney Docket: 2014191-0043 embodiments, one or more enhancer regions associated with NSUN5 include chr7:72713576- 72716059. In some embodiments, one or more enhancer regions of DAAM2 include chr6:39788740-39793623.

[0049] In some embodiments, one or more genomic loci comprise one or more enhancer regions of NSUN5, DAAM2, CNTN2, or any combination thereof; wherein an increase in enhancer signal at the one or more enhancer regions of NSUN5, DAAM2, CNTN2, or any combination thereof indicates that a subject has MS.

[0050] In some embodiments, one or more genomic loci comprise one or more gene markers of synaptic plasticity or one or more regulatory regions thereof, and an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the one or more gene markers of synaptic plasticity or one or more regulatory regions thereof as compared to a reference indicates that a subject has MS.

[0051] In some embodiments, one or more genomic loci comprise one or more promoter regions associated with one or more marker genes of synaptic plasticity, wherein an increase in promoter signal at one or more promoter regions associated with one or more marker genes of synaptic plasticity indicates that a subject has MS. In some embodiments, one or more marker genes of synaptic plasticity include SQSTM1 and / or LILRB2.

[0052] In some embodiments, one or more genomic loci comprise one or more promoter regions of SQSTM1 and / or LILRB2, and an increase in promoter signal as compared to a reference indicates that the subject has MS. In some embodiments, one or more promoter regions of LILRB2 include chr19:54,782,258-54,785,464. In some embodiments, one or more promoter regions for SQSTM1 include chr5:179232387-179234388.

[0053] In some embodiments, one or more genomic loci comprise one or more MS-risk alleles or one or more regulatory regions thereof, and an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the MS-risk alleles or one or more regulatory regions thereof as compared to a reference indicates that a subject has MS.

[0054] In some embodiments, one or more genomic loci comprise one or more promoter regions associated with one or more MS-risk alleles, wherein an increase in promoter signal at12953047v1 Page 12 of 224Attorney Docket: 2014191-0043 the one or more promoter regions associated with the one or more MS-risk alleles indicates that the subject has MS.

[0055] In some embodiments, one or more genomic loci comprise TNFRSF14 or one or more regulatory regions thereof, and wherein an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at TNFRSF14 or one or more regulatory regions thereof as compared to a reference indicates that a subject has MS.

[0056] In some embodiments, one or more genomic loci comprise a TNFRSF14 promoter region, wherein an increase in promoter signal at the TNFRSF14 promoter region as compared to a reference indicates a subject has MS.

[0057] In some embodiments, a reference is a predetermined threshold, a measurement from a liquid biopsy sample, and / or a normalized value. In some embodiments, a reference is a measurement from a liquid biopsy sample obtained from a subject or a cohort of subjects that have not been diagnosed with MS.

[0058] In some embodiments, MS is progressive MS or relapsing remitting MS.

[0059] Among other things, the present disclosure describes a method for determining MS disease severity in a subject, comprising: performing a method described herein for determining MS status in a subject, and comparing a value obtained from performing said method to a reference.

[0060] In some embodiments, one or more genomic loci comprise one or more microglial cell identity genes or one or more regulatory regions thereof, and an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more microglial cell identity genes or one or more regulatory regions thereof as compared to a reference indicates that a subject has severe MS.

[0061] In some embodiments, one or more genomic loci comprise one or more enhancer regions of one or more microglial cell identity genes, and an increase in enhancer signal (e.g., H3K27ac) in one or more enhancer regions of one or more microglial cell identity genes indicates a subject has severe MS.12953047v1 Page 13 of 224Attorney Docket: 2014191-0043

[0062] In some embodiments, one or more microglial cell identity genes include MAP4K4. In some embodiments, one or more enhancer regions of MAP4K4 include chr2:102513113-102515404.

[0063] In some embodiments, a reference is a predetermined threshold, a measurement from a liquid biopsy sample, and / or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a subject or a cohort of subjects that have diagnosed with moderate or severe MS (e.g., subjects having moderate or high TSPO PET signal, respectively).

[0064] Among other things, the present disclosure describes a method of determining MS disease progression in a subject, the method comprising determining, at a first time point and a second time point, MS severity in the subject, wherein MS severity is determined at each point in time using a method described herein.

[0065] In some embodiments, a method described herein can be used to predict disease flare and / or progression in a subject.

[0066] In some embodiments, one or more genomic loci comprise one or more microglial cell identity genes or one or more regulatory regions thereof and / or one or more neutrophil cell identity genes or one or more regulatory regions thereof, and an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more microglial cell identity genes or one or more regulatory regions thereof and / or one or more neutrophil cell identity genes or one or more regulatory regions thereof as compared to a reference indicates that the subject has an increased likelihood of experiencing disease flare and / or progression as compared to a reference, wherein the reference is optionally a plasma sample obtained for a subject or cohort of subjects having MS and not exhibiting an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more microglial cell identity genes or one or more regulatory regions thereof and / or one or more neutrophil cell identity genes or one or more regulatory regions thereof.

[0067] In some embodiments, one or more genomic loci comprise one or more enhancer regions of one or more microglial cell identity genes.12953047v1 Page 14 of 224Attorney Docket: 2014191-0043

[0068] In some embodiments, a subject has previously been determined to have MS (e.g., using MRI and / or PET imaging).

[0069] Among other things, the present disclosure describes method of treating a subject, comprising determining the MS status of the subject using a method described herein, and: (a) if the subject is determined to have MS, administering an MS therapeutic; and (b) if the subject is not determined to have MS, not administering an MS therapeutic.

[0070] In some embodiments, the present disclosure describes a method of treating a subject having MS, comprising determining MS severity in the subject using a method described herein, wherein: (a) if the subject is determined to have severe MS, administering a high or moderate efficacy MS therapeutic; and (b) if the subject is determined to have moderate disease severity, administering a low efficacy MS therapeutic.

[0071] Among other things, the present disclosure describes a method of treating MS in a subject, comprising determining MS disease progression in the subject using a method described herein, wherein the subject is being administered a first MS therapeutic at the first time point and the second time point, and wherein: (a) if MS disease severity is determined to increase between the first and the second time points, administering a second MS therapeutic, wherein the second therapeutic has a higher efficacy than the first MS therapeutic; and (b) if MS disease severity is determined to stay the same and / or decrease between the first and the second time points, continuing treatment with the first MS therapeutic. In some embodiments, a first MS therapeutic is a low efficacy MS therapeutic and a second MS therapeutic is a moderate or high efficacy MS therapeutic. In some embodiments, a first MS therapeutic is a moderate efficacy MS therapeutic the second MS therapeutic is a high efficacy MS therapeutic.

[0072] In some embodiments, a low efficacy MS therapeutic is an interferon (e.g., beta- or beta- ), glatiramer acetate, or teriflunomide). In some embodiments, a moderate efficacy12953047v1 Page 15 of 224Attorney Docket: 2014191-0043 MS therapeutic is cladribine, an S1p inhibitor (e.g., fingolimod, siponimod, ozanimod, or ponesimod), or a fumarate (e.g., dimethyl, diroximel, or monomethyl fumarate). In some embodiments, a high efficacy MS therapeutic is Ocrelizumab, Ofatumumab, Natalizumab, or Alemtuzumab.

[0073] Among other things, the present disclosure describes a kit comprising reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Tables 1-6.

[0074] In some embodiments, a kit comprises reagents for quantifying: (a) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, or 700 genomic loci in Table 1; (b) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 genomic loci in Table 2; (c) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500, genomic loci in Table 3; (d) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 genomic loci in Table 4; (e) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, or 600 genomic loci in Table 5; (f) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, or 150 genomic loci in Table 6; or (g) or any combination of (a)-(f).

[0075] In some embodiments, a kit comprises one or more antibodies for use in ChIP- seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac- modified histones.12953047v1 Page 16 of 224Attorney Docket: 2014191-0043

[0076] In some embodiments, a kit comprises one or more methyl-binding domains for use in MBD-seq. In some embodiments, a kit comprises one or more antibodies that bind methylated DNA for use in MeDIP.

[0077] In some embodiments, a kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample.

[0078] In some embodiments, a kit comprises reagents for library preparation for sequencing.

[0079] In some embodiments, a kit comprises reagents for sequencing.

[0080] In some embodiments, a kit comprises instructions for determining if a subject has MS.

[0081] Among other things, the present disclosure describes a non-transitory computer readable storage medium encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method described herein.

[0082] Among other things, the present disclosure describes a system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform a method described herein.

[0083] Among other things, the present disclosure describes a system comprising a sequencer configured to generate a sequencing data set from a sample; and a non-transitory computer readable storage medium described herein and / or a computer system described herein. In some embodiments, a sequencer is configured to generate a Whole Genome Sequencing (WGS) data set from the sample.

[0084] In some embodiments, a system comprises a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample. In some embodiments, a sample preparation device comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.12953047v1 Page 17 of 224Attorney Docket: 2014191-0043

[0085] In some embodiments, one or more genomic loci are selected from Tables 1-6.

[0086] In some embodiments of a system described herein, a device comprises reagents for quantifying: (a) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, or 700 genomic loci in Table 1; (b) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 genomic loci in Table 2; (c) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500, genomic loci in Table 3; (d) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 genomic loci in Table 4; (e) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, or 600 genomic loci in Table 5; (f) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, or 150 genomic loci in Table 6; or (g) or any combination of (a)-(f).

[0087] In some embodiments, a system comprises one or more antibodies for use in ChIP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.

[0088] In some embodiments, a system comprises reagents that comprise one or more methyl-binding domains for use in MBD-seq.

[0089] In some embodiments, a system described herein comprises a device comprising reagents for isolation of cell-free DNA (cfDNA) from a biological sample, optionally a liquid biopsy sample.

[0090] In some embodiments, a system described herein comprises a device comprising reagents for library preparation for sequencing.12953047v1 Page 18 of 224Attorney Docket: 2014191-0043

[0091] In some embodiments, a system described herein comprises a sequencer comprising reagents for sequencing. BRIEF DESCRIPTION OF THE DRAWING

[0092] Fig.1 provides an outline of a comprehensive epigenomic platform described herein, that can offer dynamic resolution into target and pathway biology from 1 mL of plasma. Cell free DNA exists in circulation as chromatin fragments that maintain epigenetic modifications on histones and DNA. Binding agents that detect markers of active enhancers (e.g., H3K27ac), active promoters (e.g., H3K4me3), and DNA methylation can be used to enrich for associated DNA fragments from a small volume of sample (e.g., 1 mL of plasma) and sequenced to define genome-wide epigenomic maps that capture the underlying transcriptional state of cells in a subject. Shown, for illustrative purposes, is an example of H3K27ac, H3K4me3, and DNAme signal obtained at the TROP2 locus and the loci of enhancers associated with TROP2.

[0093] Fig.2: Identification of MS-associated Signals Across Genomic and Epigenomic Analytes. (A) Plasma samples from Multiple Sclerosis (MS) patients display elevated abundance of cell free mitochondrial DNA, quantified by the percent of de-duplicated shallow whole genome reads mapping to the mitochondrial genome. (B)-(D) Promoter, enhancer, and DNA regions genome-wide were assessed for elevated / depleted signal in MS patients compared to healthy subjects, revealing hundreds of regions with MS-associated alterations in signal strength.

[0094] Fig.3: MS-Associated Promoter Signal Increases at Synaptic Pathways and Risk Genes. (A)-(C) Each gene in the genome was tested for enrichment / depletion of activate promoter signal within + / - 1kb of its TSS, which identified genes like TNFRSF14 (visualized in (A), quantified in (B)) that were found to have increased signal in MS patient plasma compared to healthy. Housekeeping genes ACTB and GAPDH are also shown as internal controls. TNFRSF14 and its ligand TNFSF14 were previously identified as candidate target genes of MS- associated risk alleles (previous MS-associated alleles shown in (C), adapted from The International Multiple Sclerosis Genetics Consortium & The Wellcome Trust Case Control Consortium 2. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple12953047v1 Page 19 of 224Attorney Docket: 2014191-0043 sclerosis. Nature 476, 214–219 (2011)). TNFRSF14 displayed the highest promoter signal of any gene in the genome in 7 / 18 tested MS samples. (D) Samples from rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients were tested to compare promoter and enhancer signals across autoimmune diseases. Enriched TNFRSF14 promoter signal was detected only in the MS patient population. (E) Genes were rank ordered by their MS vs. healthy differential promoter signal (normalized enrichment score, x-axis), and gene set enrichment analysis (GSEA) was performed. Terms from the Gene Ontology Consortium Biological Process (GO:BP) set with normalized enrichment scores above 1.3 and FDR-adjusted p-values below 0.05 are highlighted in purple. (F) Promoter signal quantification of two genes from the synaptic plasticity gene ontology terms (SWSTM1 and LILRB2) highlighted in (E).

[0095] Fig.4: MS-Associated Enhancers Are Enriched For Neural Cell Type Genes. (A) Differentially modified enhancer regions were assigned to their nearest gene (within 50 kb), after which genes were rank ordered by their most differential (MS vs healthy) enhancer signal and gene set enrichment analysis (GSEA) was performed utilizing the MSigDB cell type signature gene sets (C8). Terms with normalized enrichment scores greater than 1.3 and FDR- adjusted p-values below 0.01 are highlighted in purple and select neural cell types are labeled (with simplified term names). (B-C) Enhancer signal at oligodendrocyte progenitor cell identity genes (NSUN5, DAAM2, and CNTN2) and housekeeping genes (ACTB, GAPDH) are visualized in (B) and quantified in (C). (D) Multiple immune cell types (as determined using GSEA) were surprisingly under-enriched for MS-associated enhancer signals (underenriched immune cell types highlighted in purple). (E) None of the gene ontology terms highlighted in (A) or (D) were found to be significantly enriched when the same test was performed for enhancer regions associated with control autoimmune samples from RA / SLE patients. Without wishing to be bound by theory, it is thought that the cell identity type genes indicate cell types that are dying. MS enriched biological processes include (oligodendrocyte differentiation, neurogenesis, spinal cord development, neuron projection development, glial cell differentiation, and certain non-neural pathways.

[0096] Fig.5: Elevated Microglia and Neutrophil Enhancers Detected in High TSPO-PET Patients. (A) An MS patient cohort was sub-stratified based on an observed bimodal distribution in TSPO-PET (PK11195 DVR, NAWM) signal (dashed line indicates12953047v1 Page 20 of 224Attorney Docket: 2014191-0043 TSPO PET Signal (HRRT NAWM PK11195 DVR) threshold used to distinguish subjects with “moderate TSPO-PET Signal” from those with “highest TSPO-PET signal”). Enhancer regions were tested for differential signal between the two subgroups. (B) Microglia- and neutrophil- associated cell identity genes displayed preferential MS-elevated enhancer signal, although no individual loci (e.g., the microglia-associated gene MAP4K4) was found to show a statistically significant difference in signal after multiple testing correction. (C) Compares enhancer signal for one exemplary microglial cell identity gene (MAP4K4) to TSPO PET signal. As shown, a positive correlation was observed between MAP4K4 enhancer signal and TSPO PET signal, indicating that methods provided herein can be used to measure disease severity. DETAILED DESCRIPTION

[0097] The present disclosure is based, at least in part, on the demonstration that MS in a subject can be detected and characterized by detecting and quantifying the presence of histone modifications and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample, e.g., a plasma sample obtained or derived from the subject. The present disclosure also encompasses methods where chromatin accessibility and / or binding of one or more transcription factors are detected at the one or more genomic loci instead of (or in addition to) histone modifications and / or DNA methylation. MS status

[0098] Multiple sclerosis (MS) is an immune-driven disease characterized by demyelination and axonal damage in the central nervous system (CNS). It is twice as common in females and most often diagnosed between 20–40 years of age. The MS prevalence in the United States has been steadily increasing over the past several decades, with approximately 58 cases per 100,000 persons in 1975 increasing to 309.2 cases per 100,000 persons (450.1 per 100,000 for females) during 2010–2019. This roughly translates to 1 in 300 people in the United States living with MS, with the incidence being the highest for Black people (10.2 per 100,000 person- years versus 6.2 in white people).12953047v1 Page 21 of 224Attorney Docket: 2014191-0043

[0099] The neuronal damage that is characteristic of MS disrupts the ability of parts of the nervous system to transmit signals, resulting in a range of signs and symptoms, including physical, mental, and sometimes psychiatric problems. Symptoms can include double vision, vision loss, eye pain, muscle weakness, and loss of sensation or coordination. MS can take several forms, with new symptoms either occurring in isolated attacks (relapsing forms) or building up over time (progressive forms). In relapsing forms of MS, between attacks, symptoms may disappear completely, although some permanent neurological problems often remain, especially as the disease advances. In progressive forms of MS, bodily function slowly deteriorates once symptoms manifest and will steadily worsen if left untreated.

[0100] The cause of MS is unclear, but the underlying mechanism is thought to be either cellular destruction caused by the immune system or failure of myelin-producing cells. Proposed causes include immune dysregulation, genetics, and environmental factors, such as viral infections. MS is usually diagnosed based on presenting signs and symptoms and the results of supporting medical tests.

[0101] Criteria for MS diagnosis rely in part on the identification and characterization of CNS lesions. These criteria have undergone numerous changes over time. The original Schumacher criteria published in 1965 defined MS as spatial and temporal dissemination of focal neurological deficits. The current 2017 McDonald criteria still support the use of surrogate markers to fulfill criteria for dissemination in space and time. For example, having simultaneous enhancing and nonenhancing lesions in the first attack fulfills the dissemination-in-time criteria without requiring a second clinical attack. Oligoclonal bands, a marker of inflammatory reaction chronicity, can also fulfill the dissemination-in-time criteria. The revised criteria aim to increase sensitivity in detecting MS to expedite treatment and prevent disability. For the current dissemination-in space criteria, MS lesions must be seen in at least two of the following four locations: cortical / juxtacortical, periventricular, infratentorial, and spinal cord.

[0102] Typical clinical syndromes for MS include optic neuritis, internuclear ophthalmoplegia, facial sensory loss or trigeminal neuralgia, ataxia, and partial transverse myelitis. Optic neuritis is characterized by reduced visual acuity, afferent pupillary defects, and impaired color vision, and is typically unilateral in MS. Visual acuity in optic neuritis is often12953047v1 Page 22 of 224Attorney Docket: 2014191-0043 better than no light perception. Pain during eye movement is common. Visual deficits regularly nadir at 2 weeks and recover within 4 weeks.

[0103] Weakness or numbness in MS typically presents over hours to days, unlike strokes that present within minutes. MS symptoms often last for days to months, with some symptoms becoming permanent. MS relapse typically lasts for at least 24 hours. When a fluctuation of prior MS symptoms occurs for less than 24 hours, it is considered a “pseudorelapse,” which is common after the acute inflammatory period. Risk factors for a pseudorelapse include infection, stress, and heat.

[0104] MRI is the current gold standard imaging procedure for diagnosing and monitoring MS. MRIs are expensive, however. There are also some important limitations on the ability of MRI to detect MS. For example, several diseases and syndromes are routinely mistaken for MS in clinical settings. These include migraine (22% of cases), fibromyalgia (15% of cases), nonspecific or nonlocalizing neurological symptoms with abnormal MRI presentation (12% of cases), conversion disorder / functional neurological disorder (11% of cases), and neuromyelitis optica (NMO) (6% of cases). Other diseases that may be considered in a differential diagnosis of MS include myelin oligodendrocyte glycoprotein antibody disease (MOGAD), CNS vasculitis, neurosarcoidosis, CNS manifestations of autoimmune diseases such as Sjögren’s syndrome, systemic lupus erythematosus (SLE), and antiphospholipid antibody syndrome, chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids, and Beçhet’s syndrome. MRI also does not provide insight into the biological mechanisms underlying MS in a subject, and therefore is limited in its ability to determine, e.g., which subjects are likely to respond well to treatment with a certain therapeutic, which subjects are more likely to exhibit worsening disease state, and which subjects are likely to be prone to relapse.

[0105] Cerebrospinal fluid (CSF) analysis can also be used in the diagnosis and characterization of MS and typically consists of the cell count, protein and glucose levels, oligoclonal bands, and the IgG index and synthesis rate. Oligoclonal bands are observed in 80% of first MS attacks and in more than 90% of patients with MS at some point. CSF tests that are considered less sensitive or specific for diagnosing MS mimics include angiotensin-converting enzyme and myelin basic protein (MBP); however, these tests can be considered in certain cases12953047v1 Page 23 of 224Attorney Docket: 2014191-0043 where necessary. A meningitis panel or bacterial or fungal cultures may be necessary to rule out infection. Atypical features in MS include absence of oligoclonal bands, cell count >50 WBC / mm3, and a protein concentration of >100 mg / dL. Oligoclonal band testing paired with (or replaced by) measurement of kappa free light chains (kFLC) in the CSF increases diagnostic yield. Comparative studies have found CSF kFLC concentrations to be highly sensitive and specific in all forms of MS without the need for a corresponding serum sample.

[0106] Positron emission tomography (PET)-imaging and radioligands binding to the 18 kDa translocator protein (TSPO) (Banati et al. “The peripheral benzodiazepine binding site in the brain in multiple sclerosis: quantitative in vivo imaging of microglia as a measure of disease activity.” Brain 123.11 (2000): 2321-2337; Giannetti et al. “Increased PK11195-PET binding in normal-appearing white matter in clinically isolated syndrome.” Brain 138.1 (2015): 110-119; Rissanen et al. “In vivo detection of diffuse inflammation in secondary progressive multiple sclerosis using PET imaging and the radioligand 11C-PK11195.” Journal of Nuclear Medicine 55.6 (2014): 939-944) can be used to monitor innate immune cell activation in the central nervous system (CNS), which is a prominent pathological feature of multiple sclerosis (MS). MS-patients display increased TSPO-PET-measurable microglial activation in the normal appearing white matter (NAWM) and in thalamus. This phenomenon is most pronounced in advanced MS, and increased TSPO-binding also predicts later disease progression. Microglia and macrophage activation at the edge of chronic lesions and in the NAWM associate with MS disease type, disease severity and future progression, but factors promoting and maintaining the increased innate immune cell activation in MS brain are largely unknown. MS therapies

[0107] No cure for multiple sclerosis is known. Current treatments are aimed at mitigating inflammation and resulting symptoms from acute flares and prevention of further attacks with disease-modifying medications. Physical therapy and occupational therapy, along with patient-centered symptom management, can help with patient’s ability to function. Long- term outcomes are difficult to predict; better outcomes are more often seen in women, those who12953047v1 Page 24 of 224Attorney Docket: 2014191-0043 develop the disease early in life, those with a relapsing course, and those who initially experienced few attacks. Acute MS relapse management

[0108] Acute flares in a patient with MS are associated with new symptoms or old ones that have worsened. Most patients with MS flares will not require emergency care unless they present with strength, gait, or vision impairment. The use of intravenous (IV) corticosteroids (e.g., 1 g of IV methylprednisolone daily for 3–5 days) may shorten flare duration, but does not impact the overall disability outcome. One exception is optic neuritis, for which the Optic Neuritis Treatment Trial (Optic Neuritis Study Group. “Multiple sclerosis risk after optic neuritis: final optic neuritis treatment trial follow-up.” Arch Neurol 65 (2008): 727-732) found that patients treated with high dose steroids had better visual outcomes at 6 months.

[0109] Oral prednisone at 1.25 g daily can be a substitute for 1 g of IV methylprednisolone. Plasma exchange can be considered for patients with symptoms that are refractory to IV steroids. IV immunoglobin monotherapy can be considered in cases of acute MS relapse in patients with contraindications to plasmapheresis and IV steroid therapy, although there is less evidence supporting this. Therapy for long-term MS management

[0110] As of March 2023, 24 different DMTs had been approved for MS treatment, including injectable, oral, and infused medications.

[0111] One area of practice in MS that has changed over the years is the early initiation of high-efficacy treatment. Recent research supported high-efficacy therapy that commences within 2 years of disease onset, since it is associated with lessened disability after 6–10 years compared with delaying such therapies. In one comparison study of patients who underwent high-efficacy therapy versus those who underwent moderate-efficacy therapy, 68% and 52% of patients achieved no evidence of disease activity (NEDA) at 1 year, respectively, and 52% and 19% achieved NEDA at 2 years.12953047v1 Page 25 of 224Attorney Docket: 2014191-0043

[0112] However, not all patients require high-efficacy therapy; clinically stable patients on mild- or moderate-efficacy therapy do not need to change their medication regimen. Similarly, patients with relatively few lesions or older patients with MS do not require such strong medications. Mild-efficacy therapies Interferons

[0113] Interferons (IFNs) including beta- -1990s. They work by binding to cell surface receptors and initiating signaling pathways that lead to increased antiviral, antiproliferative, and immunomodulatory gene products that ultimately inhibit proinflammatory cytokines and T-cell activation. Initially considered a platform therapy, tolerability restricts their use due to common flu-like side effects from injections. Glatiramer acetate

[0114] Glatiramer acetate (GA) functions via two mechanisms: 1) reducing interleukin-17 and IFN- thereby inhibiting proinflammatorycytokines, and 2) mimicking myelin basic protein (MBP) regions, thereby functioning as a decoy receptor via molecular mimicry to be targeted by the immune system of the body.

[0115] GA is the only MS drug that does not require laboratory monitoring. The main side effect is skin hardening at the injection site. Teriflunomide

[0116] Teriflunomide is an oral therapy that inhibits dihydroorotate dehydrogenase, which in turn reduces the levels of activated B and T lymphocytes. Teriflunomide is often administered to patients who have been treated with mild-efficacy injectables and who experience injection fatigue. There may also be a use for teriflunomide in older patients who wish to de-escalate treatment. Moderate-to-high efficacy therapies12953047v1 Page 26 of 224Attorney Docket: 2014191-0043 Cladribine

[0117] Cladribine (2-chlorodeoxyadenosine) works by incorporating into DNA and inhibiting DNA polymerase and ribonucleotide reductase (enzymes involved in DNA synthesis and transcription), thereby creating DNA strand breaks. Cladribine consists of two oral treatment courses administered approximately 1 year apart and has been found to deplete the total T- and B-cell counts by 40%–50% and 80%, respectively.

[0118] Cladribine may be considered a high-efficacy therapy after both courses. Side effects include lymphopenia (in 21.6% of cases) and herpetic infections. There is a black-box warning for malignancies, although more than 10 years of safety data had not indicated increased malignancy risk. However, additional cladribine treatment during the 2 years after the second treatment course is associated with increased malignancy incidence (0.91 events per 100 patient- years). s1p inhibitors

[0119] s1p inhibitors such as fingolimod, siponimod, ozanimod, and ponesimod block the egress of lymphocytes from lymph nodes. They are effective at preventing MS relapse, but also have the side effect of increased infection risk, including that of progressive multifocal leukoencephalopathy (PML) and cryptococcal meningitis, as well as the risk of rebound relapse after drug discontinuation; there have been documented cases of severe MS rebound after fingolimod withdrawal. Fumarates

[0120] Fumarates (dimethyl, diroximel, and monomethyl fumarate) are oral medications that work via various pathways to both suppress proinflammatory cytokines (e.g., NF-kB) and activate the nuclear factor E2 pathway that leads to increased antioxidant enzyme synthesis. Taking fumarates twice daily has been found to approximately halve the frequency of MS relapses.

[0121] Dimethyl fumarate was approved for relapsing-remitting MS (RRMS) in 2013. Up to 30% of patients treated with dimethyl fumarate can experience nausea or flushes.12953047v1 Page 27 of 224Attorney Docket: 2014191-0043

[0122] Nausea rates are lower for diroximel fumarate. This drug class is considered to have a lower infection risk than high-efficacy therapy. High-efficacy therapies Ocrelizumab

[0123] Ocrelizumab was approved for both relapsing-remitting multiple sclerosis (RRMS) and primary progressive MS (PPMS) in 2017. It works by depleting CD20 B cells and is administered via infusion every 6 months.

[0124] Although MS is historically considered a T-cell disease, blockade of B-cell autoreactivity inhibits neuroinflammation through several pathways, including 1) preventing B cells from acting as antigen-presenting cells that activate autoreactive T cells, 2) preventing B cells from releasing proinflammatory cytokines, 3) preventing B cells from transforming into plasma cells that may produce myelin-directed autoantibodies, and 4) preventing B cells from forming meningeal lymphoid follicles. An interim analysis of an open-label phase IIIb study found that patients with MS treated using ocrelizumab demonstrated a 75% increase in NEDA -Furthermore, a post-hoc analysis in the phase IIIORATORIO study on PPMS found that ocrelizumab slowed the accumulation of T2-weighted lesions. The side effects of ocrelizumab include decreased responsiveness to vaccines and increased risks of mucosal and herpetic infections. Ofatumumab

[0125] Another anti-CD20 B-cell therapy, ofatumumab, was approved for RRMS in 2020 and is administered via monthly self-injection. Its side-effects profile is similar to that of ocrelizumab. Given the lack of a need for premedication, patients can inject ofatumumab at home. Natalizumab

[0126] Natalizumab is a monoclonal antibody that was approved for MS in 2004 and is administered via infusion every 4 weeks. The advantage of natalizumab over conventional DMTs is decreased peripheral immunosuppression due to a more-targeted effect. Natalizumab12953047v1 Page 28 of 224Attorney Docket: 2014191-0043 -mediated leukocyte transmigration across the blood–brain barrier. It should be noted that screening for JC virus (JCV) is necessary in patients taking natalizumab, since JCV can cause PML.

[0127] Natalizumab was initially withdrawn from the market due to PML cases in 2005 but returned in 2006 with a Risk Evaluation and Mitigation Strategy (REMS) program based on JCV serology. The current PML rate is <1:10,000 in JCV-negative patients. Monitoring laboratory tests include CBC with differential, CMP, and JC virus every 6 months. Alemtuzumab

[0128] Alemtuzumab is an anti-CD52 monoclonal antibody with high efficacy that is administered via injection. It is often utilized for RRMS and in MS cases that do not respond to multiple medications due to its side-effects profile that includes significant risks of secondary autoimmune thyroid disease, TB, HSV, and infusion reactions. Subjects and Samples

[0129] A sample analyzed using methods, kits and systems provided herein can be any biological sample including any processed sample that includes cell free DNA (cfDNA) derived from a biological sample. In various embodiments, a sample analyzed using methods, kits and systems provided herein can be a sample obtained from a mammalian subject. In various embodiments, a sample analyzed using methods, kits and systems provided herein can be a sample obtained from a human subject.

[0130] In various instances, a human subject is a subject diagnosed or seeking diagnosis as having, diagnosed as, or seeking diagnosis as at risk of having, and / or diagnosed as or seeking diagnosis as at immediate risk of having MS. In various instances, a human subject is a subject identified as needing MS status screening. In certain instances, a human subject is a subject identified as needing MS status screening by a medical practitioner.

[0131] The subject may not have undergone previous treatments for MS, such as the treatments recited in this disclosure. In other embodiments, the subject has undergone previous treatments for MS, such as the treatments recited in this disclosure.12953047v1 Page 29 of 224Attorney Docket: 2014191-0043

[0132] In various embodiments a subject has one or more biomarkers and / or risk factors for MS. In certain embodiments, a human subject is identified as in need of MS status screening based on an initial MS diagnosis. In various instances, a human subject is a subject not yet diagnosed as having, not at risk of having, not at immediate risk of having, not diagnosed as having, and / or not seeking diagnosis for MS.

[0133] In various embodiments, a sample from a subject, e.g., a human can be obtained from a liquid biopsy. In certain embodiments, a sample and / or reference is obtained from serum, plasma, or urine. In certain embodiments, the sample is serum. In certain embodiments, a sample comprises cell free DNA (cfDNA). In certain embodiments, a sample is derived from about 1 mL of blood obtained from the subject. In certain embodiments, a sample is derived from about 0.5-2 mL of blood obtained from the subject, e.g., about 0.5 to 1.75 mL, about 0.5 to 1.5 mL, about 0.75 to 1.25 mL or about 0.9 to 1.1 mL of blood.

[0134] In various embodiments, a sample is a sample of cell-free DNA (cfDNA). cfDNA is typically found in human biofluids (e.g., plasma, serum, or urine) in short, double-stranded fragments. The concentration of cfDNA is typically low, but can significantly increase under particular conditions, including without limitation pregnancy, autoimmune disorders, myocardial infarction, and cancer.

[0135] cfDNA can provide a real-time or nearly real time metric of status of a source tissue. cfDNA demonstrates a half-life in blood of about 2 hours, such that a sample taken at a given time provides a relatively timely reflection of the status of a source tissue.

[0136] Various methods of isolating nucleic acids from a sample (e.g., of isolating cfDNA from blood or plasma) are known in the art. Nucleic acids can be isolated using, without limitation, standard DNA purification techniques, by direct gene capture (e.g., by clarification of a sample to remove assay-inhibiting agents and capturing a target nucleic acid, if present, from the clarified sample with a capture agent to produce a capture complex and isolating the capture complex to recover the target nucleic acid).

[0137] Reagents and protocols for obtaining and analyzing cfDNA, such as circulating in blood or other tissue, are commercially available as described in the Examples and well-known in the art.12953047v1 Page 30 of 224Attorney Docket: 2014191-0043

[0138] In various embodiments, samples can be collected from individuals repeatedly over a period of time (e.g., once daily, weekly, monthly, annually, biannually, etc.). In various embodiments, such samples can be used to verify results from earlier detections and / or to identify an alteration in biological pattern because of, for example, disease progression, resistance to therapy, treatment, remission, and the like. For example, subject samples can be taken and monitored every month, every two months, or combinations of one, two, or three- month intervals according to the present disclosure. In various embodiments, samples can be collected for monitoring over time beginning at or at certain clinically determined stages, such as at resistance to a therapy, before radiographic progression, after radiographic progression, and / or at tissue biopsy. In addition, the MS status obtained at different points in time can be conveniently compared with each other, as well as with those of normal controls during the monitoring period, thereby providing the subject’s own values, as an internal, or personal, control for long-term monitoring.

[0139] Samples include materials prepared by processes including, without limitation, steps such as concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives, addition of calibrants, addition of protease inhibitors, addition of denaturants, desalting, concentration and / or extraction of sample nucleic acids, and / or amplification of sample nucleic acids (e.g., by PCR or other nucleic acid amplification techniques). Samples also include materials prepared by techniques that isolate, e.g., nucleosomes or transcription factors and / or nucleic acids associated with nucleosomes or transcription factors.

[0140] Removal from a sample of proteins that are not desirable for a relevant purpose or context (e.g., high abundance, uninformative, or undetectable proteins) can be achieved using high affinity reagents, high molecular weight filters, ultracentrifugation and / or electrodialysis. High affinity reagents include antibodies or other reagents (e.g., aptamers) that selectively bind to high abundance proteins. Sample preparation can also include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques. Molecular weight filters include membranes that separate molecules based on size and molecular weight. Such filters may further employ reverse osmosis, nanofiltration, ultrafiltration and12953047v1 Page 31 of 224Attorney Docket: 2014191-0043 microfiltration. Ultracentrifugation is the centrifugation of a sample at about 15,000-60,000 rpm while monitoring with an optical system the sedimentation (or lack thereof) of particles. Electrodialysis is a procedure which uses an electromembrane or semipermeable membrane in a process in which ions are transported through semi-permeable membranes from one solution to another under the influence of a potential gradient. Since the membranes used in electrodialysis may have the ability to selectively transport ions having positive or negative charge, reject ions of the opposite charge, or to allow species to migrate through a semipermeable membrane based on size and charge, it renders electrodialysis useful for concentration, removal, or separation of electrolytes.

[0141] Separation and purification in the present disclosure may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography (e.g., in capillary, column or on a chip). Electrophoresis is a method that can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip. Examples of gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof. A gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity electrophoresis) or substrates (zymography) and incorporation of a pH gradient. Examples of capillaries used for electrophoresis include capillaries that interface with an electrospray.

[0142] Capillary electrophoresis (CE) is preferred for separating complex hydrophilic molecules and highly charged solutes. CE technology can also be implemented on microfluidic chips. Depending on the types of capillary and buffers used, CE can be further segmented into separation techniques such as capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP) and capillary electrochromatography (CEC). An embodiment to couple CE techniques to electrospray ionization involves the use of volatile solutions, for example, aqueous mixtures containing a volatile acid and / or base and an organic such as an alcohol or acetonitrile.

[0143] Capillary isotachophoresis (CITP) is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities. Capillary zone electrophoresis (CZE), also known as free-solution CE (FSCE), is12953047v1 Page 32 of 224Attorney Docket: 2014191-0043 based on differences in the electrophoretic mobility of the analytes, determined by the charge on the analytes, and the frictional resistance the analytes encounter during migration, which is often directly proportional to the size of the analytes. Capillary isoelectric focusing (CIEF) allows weakly-ionizable amphoteric molecules, to be separated by electrophoresis in a pH gradient. CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE.

[0144] Separation and purification techniques used in the present disclosure can include any chromatography procedures known in the art. Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), etc.

[0145] In some embodiments, whole blood is collected from a subject, and a plasma layer is separated by centrifugation. cfDNA may be then extracted from the plasma using methods known in the art. Histone Modifications, Chromatin Accessibility and Transcription Factor Binding

[0146] Histone methylation is understood to increase or decrease expression of associated coding sequences, depending on which histone residue is methylated. Histone methylation is an essential modification that can cause monomethylation (me1), dimethylation (me2), and trimethylation (me3) of several amino acids, thus directly affecting heterochromatin formation, gene imprinting, X chromosome inactivation, and gene transcriptional regulation. Histone methyltransferases promote monomethylation, dimethylation, or trimethylation of histones while histone demethylases promote demethylation of histones. In general, lysine (Lys or K), arginine (Arg or R), and rarely histidine (His or H) are the most common histone methyl acceptors. Histone methylation only occurs at specific lysine and arginine sites of histone H3 and H4. In histone H3, lysine 4, 9, 26, 27, 36, 56, and 79 and arginine 2, 8, and 17 can be methylated. By comparison, histone H4 has fewer methylation sites, in which only lysine 5, 12, and 20 and arginine 3 can be methylated. Histone methylation is often associated with transcriptional activation or inhibition of downstream genes. The methylation of histone H3K4, R8, R17, K26,12953047v1 Page 33 of 224Attorney Docket: 2014191-0043 K36, K79, H4R3, and K12 can activate gene transcription. However, the methylation of histone H3K9, K27, K56, H4K5, and K20 can inhibit gene transcription. Thus, for example, H3K4 methylation generally activates gene expression, while H3K27 methylation generally represses gene expression.

[0147] Histone acetylation occurs predominantly at lysine residues and is generally understood to increase expression of associated coding sequences. Without wishing to be bound by any theory, acetylation of lysine residues is thought to neutralize lysine’s positive charge and thereby cause histones to drift away from DNA, which has a negative charge. The released structure facilitates access to transcriptional machinery such as transcription factors and RNA polymerase II. Histone acetylation and deacetylation are generally catalyzed by histone acetyltransferases (HATs) and HDACs, respectively. Acetyl-CoA can be a source and co-factor of acetylation. In regulatory regions, HATs can acetylate histones and recruit HAT-containing complexes to activate the transcriptional process. For instance, H3K9ac and H3K27ac levels can be associated with promoter and enhancer activities. Furthermore, H3K27ac enhances not only the kinetics of transcriptional activation, but also accelerates the transition of RNA polymerase II from the initiation state to the elongation state.

[0148] Differential modification of a genomic locus (e.g., differential histone methylation and / or differential histone acetylation) can refer to, or be determined by or detected as, a comparative difference or change in modification status of one or more genomic loci between a first sample, condition, disease, or state and a second or reference sample, condition, disease, or state. Those of skill in the art will appreciate that a reference is typically produced by measurement using a methodology identical, similar, or comparable to that by which a compared non-reference measurement was taken.

[0149] Chromatin accessibility can refer to the degree to which nuclear macromolecules are able to physically contact DNA and is determined in part by the occupancy and modification status of nucleosomes. Modified histones can regulate chromatin accessibility through a variety of mechanisms, such as altering transcription factor (TF) binding through steric hindrance and modulating nucleosome affinity for active chromatin remodelers. The topological organization of nucleosomes across the genome is non-uniform: while histones can be densely arranged within facultative and constitutive heterochromatin, histones can be depleted at regulatory loci,12953047v1 Page 34 of 224Attorney Docket: 2014191-0043 including within enhancers, insulators and transcribed gene bodies. Active regulatory elements of the genome are generally accessible.

[0150] Differential accessibility of a genomic locus can refer to, or be determined by or detected as, a comparative difference or change in modification status of one or more genomic loci between a first sample, condition, disease, or state and a second or reference sample, condition, disease, or state. Those of skill in the art will appreciate that a reference is typically produced by measurement using a methodology identical, similar, or comparable to that by which a compared non-reference measurement was taken.

[0151] A reference can be a value or set of values that are predetermined or derived from a sample or set of samples. A reference can be a sample or set of samples. A reference value can be a predetermined threshold value, a value that varies in accordance with circumstances (e.g., according to patient subpopulation, age, weight, or other variables), or a ratio. Reference ratios can be ratios relating to the modification and / or accessibility of multiple loci within individual samples and / or references, or across or between samples and / or references. In various embodiments, a reference can have or represent a normal, non-diseased state. In some embodiments, such as for staging of disease or for evaluating the efficacy of treatment, a reference can have or represent a diseased state, e.g., MS. In some embodiments, a reference can represent MS by being obtained from a subject diagnosed as having MS (e.g., based on imaging, symptoms, and / or CSF analysis).

[0152] In certain instances, a reference is a non-contemporaneous sample from the same source, e.g., a prior sample from the same source, e.g., from the same subject. In certain instances, a reference for the modification status of one or more genomic loci (e.g., one or more differentially modified genomic loci) can be the modification status of the one or more genomic loci (e.g., one or more differentially modified genomic loci) in a sample (e.g., a sample from a subject), or a plurality of samples, known to represent a particular state (e.g., MS). In certain instances, a reference for the accessibility status of one or more genomic loci (e.g., one or more differentially accessible genomic loci) can be the accessibility status of the one or more genomic loci (e.g., one or more differentially accessible genomic loci) in a sample (e.g., a sample from a subject), or a plurality of samples, known to represent a particular state (e.g., MS).12953047v1 Page 35 of 224Attorney Docket: 2014191-0043

[0153] In some illustrative but non-limiting embodiments of the present disclosure differential modification or differential accessibility can refer to a differential (e.g., between a sample and a reference) with an absolute log2(fold-change) that is greater than or equal to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or more, or any range in between, inclusive, e.g., as measured according to an assay provided herein.

[0154] Enhancers are genomic loci that can be differentially modified or differentially accessible in and / or between conditions, diseases, and other states. Enhancers are cis-acting DNA regulatory regions that are thought to bind trans-acting proteins that contribute to expression patterns of associated genes. Chromatin ImmunoPrecipitation sequencing (ChIP-seq) of histone modifications (e.g., acetylation) have identified millions of enhancers in mammalian genomes. The number of active enhancers in any given cell type is estimated to be in the tens of thousands. Certain transcription factors (TFs), sometimes referred to as “master” transcription factors, associate with active enhancers with important impacts on gene expression and cell function. Certain such transcription factors preferentially associate with enhancers that regulate genes required for establishing cell identity and function, including enhancer domains known as “super-enhancers”. Moreover, master TFs can participate in inter-connected auto-regulatory circuitries or “cliques” that are self-reinforcing, show marked cell selectivity, and function to maintain cell state and / or cell survival. Techniques for Detecting and Quantifying Histone Modifications and Transcription Factor Binding

[0155] Various techniques of molecular biology are well known in the art and / or disclosed in the present application for detecting and quantifying histone modifications and / or transcription factor binding. In some embodiments, the methods, kits and systems of present disclosure involve the detection and quantification of histone modifications and / or transcription factor binding in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA. Chromatin ImmunoPrecipitation (ChIP) is one technique of molecular biology useful in detecting and quantifying histone modifications and transcription12953047v1 Page 36 of 224Attorney Docket: 2014191-0043 factor binding in samples. CUT&RUN or CUT&Tag are other more recent techniques that can also be used to detect and quantify histone modifications and transcription factor binding sites.

[0156] ChIP can involve various steps including one or more of fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. ChIP has become a very widely used tissue-based technique for determining the in vivo location of binding sites of various transcription factors and histones. Because the proteins are captured at the sites of their binding with DNA, ChIP helps to detect DNA-protein interactions that take place in living cells. More importantly, ChIP can be coupled to many commonly used molecular biology techniques such as PCR and real-time PCR, PCR with single-stranded conformational polymorphism, Southern blot analysis, Western blot analysis, cloning, and microarray. The resulting versatility has increased the potential of this technique.

[0157] ChIP of tissue samples usually involves cross-linking of the chromatin-bound proteins by formaldehyde, followed by sonication or nuclease treatment to obtain small DNA fragments. Immunoprecipitation can be then carried out using specific antibodies to the DNA- binding protein of interest. The DNA can be then released from the proteins and analyzed using various methods. ChIP has also been used to study RNA-protein interactions. X-ChIP methods utilize fixed chromatin fragmented by sonication, while the N-ChIP methods utilize native chromatin, which can be unfixed and nuclease digested.

[0158] The first step of the technique can be the cross-linking of DNA and proteins. Formaldehyde is one of the most used cross-linking agents. One advantage of using formaldehyde can be the ease of reversibility of the cross-links and its ability to form bonds that span approximately 2 angstroms. This means that formaldehyde can bind molecules in close association with each other. Generally, formaldehyde can be added to the medium in the cell culture flask or plate. It enters the cells through the cell membrane and cross-links the proteins to the chromatin. Formaldehyde fixation of tumor tissues has also been done. Other cross-linking agents that have been used include chemicals such as methylene blue and acridine orange, cisplatin, dimethylarsinic acid, potassium chromate, and ultraviolet (UV) light and lasers.

[0159] Harvested chromatin can be sonicated in one or more sonication cycles. DNA can be typically broken into to 100–500 bp fragments to pinpoint the location of the DNA sequence of interest. An alternative to sonication can be nuclease digestion of the chromatin, e.g., in N-12953047v1 Page 37 of 224Attorney Docket: 2014191-0043 ChIP methods. Purification of chromatin can be achieved using a cesium chloride (CsCl) gradient centrifugation.

[0160] Chromatin can be immunoprecipitated using one or more antibodies that bind a target epitope. For example, an antibody used in ChIP can selectively bind a particular transcription factor or one or more particular histone modifications, such as one or more particular histone acetylation modifications or histone methylation modifications. In some embodiments, an antibody used to bind a target epitope can be a “pan” antibody (e.g., a pan- acetylation antibody, a pan-methylation antibody, an antibody that binds a group of histone modifications associated with increased transcription activation, and / or an antibody that binds a group of histone modifications associated with increased transcription repression). The antibody against the protein of interest is allowed to bind to the protein-DNA complex, and the complex can be then precipitated. Immunosorbants commonly used to separate the antigen-antibody complex from the lysate include salmon sperm DNA-protein A-Sepharose®, protein G, magnetic beads, and other engineered immunoprecipitation systems known to those of skill in the art.

[0161] Immunoprecipitated DNA can be eluted. Once the DNA of interest is isolated, many detection and quantification methods can be used to study the isolated gene fragments. Commonly utilized methods include PCR, real-time PCR, slot blot hybridization, microarray techniques, and deep or next-generation sequencing. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. ChIP-seq can be used to map DNA-binding proteins, e.g., transcription factor binding sites and histone modifications in a genome-wide manner.

[0162] Cell-free Chromatin ImmunoPrecipitation sequencing (cfChIP-seq) involves applying ChIP-seq to samples that include cell-free DNA, e.g., liquid biopsy samples including cfDNA such as plasma samples including cfDNA (e.g., see Sadeh et al., Nat Biotechnol (2021) 39: 586–598 and Jang et al., Life Sci Alliance (2023) 6(12):e202302003 the entire contents of each of which are incorporated herein by reference). In some embodiments, cfChIP-seq uses antibodies or antibody fragments that bind specific histone modifications (e.g., H3K4me3 and / or H3K27ac) and / or transcription factors that are coupled (covalently or non-covalently) to beads, e.g., magnetic beads such as Dynabeads® magnetic beads and incubated with a volume, e.g., about 1 mL of thawed plasma obtained from a subject. Without limitation, exemplary antibodies12953047v1 Page 38 of 224Attorney Docket: 2014191-0043 that bind H3K4me3 include PA5-27029 (available from Thermo Fisher Scientific in Waltham, MA) and C15410003 (available from Diagenode in Denville, NJ) and exemplary antibodies that bind H3K27ac include ab21623 or ab4729 (both available from Abcam in Cambridge, UK) and C15210016 (available from Diagenode in Denville, NJ).

[0163] In some embodiments, the antibodies or antibody fragments can be covalently coupled to beads, e.g., epoxy beads. In some embodiments, the antibodies or antibody fragments can be non-covalently coupled to beads, e.g., Protein A or Protein G beads such as Dynabeads® Protein A or Dynabeads® Protein G beads. After washing, a cfDNA library is then typically prepared from the captured cfDNA. Library preparation can be done on-bead or after releasing the captured cfDNA by digestion of bound histones, e.g., using proteinase K. The cfDNA library is then sequenced to generate reads of captured cfDNA sequences, e.g., by next-generation sequencing (NGS) as is known in the art. The reads are then analyzed, e.g., aligned and counted using standard bioinformatic techniques as is known in the art. A cfChIP-seq bioinformatic pipeline can include, e.g., alignment of sequence reads to a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference.

[0164] CUT&Tag involves antibody-based binding of a target protein, e.g., transcription factor or histone modification of interest, where antibody incubation is directly followed by the shearing of the chromatin and library preparation (see Kaya-Okur et al., Nat Comm (2019) 10:1930). CUT&Tag assays take advantage of a Tn5 transposase that is fused with Protein A to direct the enzyme to the antibody bound to its target on chromatin. Tn5 transposase is pre-loaded with sequencing adapters (generating the assembled pA-Tn5 adapter transposome) to carry out antibody-targeted tagmentation. In a typical CUT&Tag assay samples are incubated with an antibody immobilized on Concanavalin A-coated magnetic beads to facilitate subsequent washing steps. Cells can be incubated with a primary antibody specific for the target protein of interest followed by incubation with a secondary antibody. Samples can then be incubated with assembled transposomes, which consist of Protein A fused to the Tn5 transposase enzyme that is conjugated to NGS adapters. After incubation, unbound transposome can be washed away using stringent conditions. Tn5 is a Mg2+-dependent enzyme so Mg2+can be added to activate the reaction, which results in the chromatin being cut close to the protein binding site and12953047v1 Page 39 of 224Attorney Docket: 2014191-0043 simultaneous addition of the NGS adapter DNA sequences. Chromatin cleavage and library preparation can be achieved in one single step.

[0165] CUT&RUN is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing (see Skene and Henikoff, Elife (2017) 6:1-35, Skene et al., Nat Protoc (2018) 13:1006-1019). As only targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has low background levels. In an example CUT&RUN assay, a sample is incubated with an antibody or antibody fragment that binds the target protein, e.g., transcription factor or histone modification of interest. The sample is then incubated with Protein-A-MNase after which CaCl2can be added to initiate the calcium dependent nuclease activity of MNase to cleave the DNA around the target protein. The protein- A-MNase reaction can be quenched by adding chelating agents (EDTA and EGTA). Cleaved DNA fragments are then liberated, extracted, and used to construct a sequencing library. Techniques for Detecting and Quantifying Chromatin Accessibility

[0166] Various techniques of molecular biology are well known in the art and / or disclosed in the present application for detecting and quantifying chromatin accessibility. In some embodiments, the methods, kits and systems of the present disclosure involve the detection and quantification of chromatin accessibility in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA. ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and DNase hypersensitivity assays are exemplary techniques of molecular biology useful in detecting and quantifying chromatin accessibility in samples.

[0167] DNase hypersensitivity assays can use the non-specific DNA endonuclease Deoxyribonuclease I (DNase I), which selectively digests accessible DNA regions. DNase I hypersensitivity sites (DHS) identified by DNase-seq include open chromatin regulatory regions. A typical DNase hypersensitivity assay can include a first step in which nuclei are isolated from cells using lysis buffer, and nuclei are digested using DNase I. DNA fragment sizes are measured12953047v1 Page 40 of 224Attorney Docket: 2014191-0043 to identify optimal digestion using gel electrophoresis. Biotinylated linkers can be ligated to the ends of digested DNA after polishing to make blunt ends, and the DNA can then be isolated. DNA with biotinylated linker can be digested by restriction endonuclease MmeI and captured by streptavidin coated Dynabeads® to generate short tags to which a second sequencing adaptor can be ligated. A second linker can be ligated and amplified to generate a library for sequencing. A DNase-seq bioinformatic pipeline can include, e.g., alignment of sequence reads to a reference genome with BWA or Bowtie2. Aligned reads can be used to call and quantify peaks as compared to a reference.

[0168] MNase-seq determines chromatin accessibility with micrococcal nuclease (MNase) that preferentially digests nucleosome-free, protein-unbound DNA. A typical MNase- seq assay can include a first step in which nuclei are isolated from either native or crosslinked chromatin and digested using MNase with titration. In vivo formaldehyde crosslinking step that is designed to capture the interaction between proteins and DNA. This crosslinking allows bound proteins to shield their associated DNA from digestion by MNase. Following crosslinking, samples are digested with MNase, which can be specifically activated by addition of Ca2+ to the buffer. Digestion can be halted by chelating the reaction, at which point the samples are RNase treated, crosslinks are reversed, and proteins are digested away from the chromatin. DNA can then be isolated via a phenol-chloroform extraction. Uncut DNA is purified and mononucleosome bands are isolated and excised through gel electrophoresis. Isolated DNA can be amplified by adding adapters to generate a library, and sequenced. MNase-seq primarily sequences regions of DNA bound by histones or other proteins. Therefore, it indirectly determines which regions of DNA are accessible by directly determining which regions are bound to nucleosomes or proteins.

[0169] FAIRE-seq is a method in which nucleosome-depleted regions of DNA (NDRs) are isolated from chromatin. A typical FAIRE-seq assay can include a first step in which cells are fixed using formaldehyde so that histones are crosslinked to interacting DNA. Crosslinked chromatin can then be sheared by sonication that generates protein-free DNA and protein- crosslinked DNA fragments. Protein-free DNA can be isolated using a phenol–chloroform extraction: DNA crosslinked with protein stays in organic phase, while protein-free DNA stays in aqueous phase. Highly crosslinked DNA remains in the organic phase and the non-crosslinked12953047v1 Page 41 of 224Attorney Docket: 2014191-0043 DNA is pulled to the aqueous phase. Non-crosslinked DNA from the aqueous phase can then be amplified and sequenced. Reads enriched in the sequencing pool tend to have lower nucleosome and transcription factor binding and are therefore inferred to come from accessible regions.

[0170] NOMe-seq is a method to identify nucleosome-depleted regions of DNA (NDRs) with M.CviPI methyltransferase that methylates cytosine in GpC dinucleotides not protected by nucleosomes or other proteins. Unlike CmpG, GpCmin the human genome does not occur naturally in most cell types. GpCmlevels at open chromatin regions can be compared to background signals and used to detect and quantify NDRs. A typical NOMe-seq protocol can include a step in which samples are treated with M.CviPI and S-adenosylhomocysteine (SAM) to methylate accessible GpC sites. M.CviPI treated DNA can be sheared using a sonicator, so that DNA fragments can be sequenced. DNA is treated with bisulfite, which converts unmethylated cytosine to uracil using sodium bisulfite, while methylated cytosine is unaffected. A library is generated using adapters and sequenced. Accessible chromatin is expected to have high levels of GpCmbut low levels of CmpG. Therefore, NOMe-seq identifies NDRs using the two separate methylation analyses that serve as independent (but opposite) measures, providing matched chromatin designations for each regulatory element.

[0171] ATAC-seq uses hyperactive Tn5 transposase that preferentially cuts accessible chromatin regions and simultaneously inserts adapters to the fragmented region (Buenrostro et al., Nat Methods (2013) 10(12):1213-1218 the entirety of which is incorporated herein by reference). A typical ATAC-seq assay can include a first step in which samples are incubated with Tn5 transposase. DNA can then be isolated and purified. DNA fragmented and tagged by Tn5 transposase can be purified and then amplified to generate a library and sequenced for analysis. Techniques for Detecting and Quantifying DNA Methylation

[0172] Various techniques of molecular biology are well known in the art and / or disclosed in the present application for detecting and quantifying DNA methylation. In some embodiments, the methods, kits and systems of the present disclosure involve the detection and quantification of chromatin accessibility in samples, e.g., in liquid biopsy samples including cfDNA such as plasma samples including cfDNA. Bisulfite sequencing (BS-Seq), Whole12953047v1 Page 42 of 224Attorney Docket: 2014191-0043 Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq) are exemplary techniques of molecular biology useful in detecting and quantifying chromatin accessibility in samples.

[0173] DNA methylation typically refers to the methylation of the 5’ position of cytosine (mC) by DNA methyltransferases (DNMT). It is a major epigenetic modification in humans and many other species. In mammals, most DNA methylations occur within the context of CpG dinucleotides. DNA methylation is thought to be a repressive chromatin modification. Aberrant methylation can lead to many diseases including cancers (Robertson, Nat Rev Genet (2005) 6:597–610 and Bergman and Cedar, Nat Struct Mol Biol (2013) 20:274–281).

[0174] Bisulfite sequencing (BS-Seq) or Whole-Genome Bisulfite Sequencing (WGBS) is a well-established protocol to detect methylated cytosines in genomic DNA. In this method, genomic DNA is treated with sodium bisulfite and then sequenced, providing single-base resolution of methylated cytosines in the genome. Upon bisulfite treatment, unmethylated cytosines are deaminated to uracils which, upon sequencing, are converted to thymidines. Simultaneously, methylated cytosines resist deamination and are read as cytosines. The location of the methylated cytosines can then be determined by comparing treated and untreated sequences.

[0175] MeDIP-seq was first reported by Weber et al., Nat Genet (2005) 37:853–862. In a typical MeDIP-seq protocol, antibody or antibody-fragment that binds 5-methylcytidine (5mC) is used to enrich methylated DNA fragments, then these fragments are sequenced and analyzed. If using 5mC-specific antibodies or antibody fragments, methylated DNA is isolated from genomic DNA via immunoprecipitation. Anti-5mC antibodies are incubated with fragmented genomic DNA and precipitated, followed by DNA purification and sequencing.

[0176] Methyl-CpG-Binding Domain sequencing (MBD-seq) is similar to MeDIP-seq except that it uses methyl binding domain (MBD) proteins instead of antibodies or antibody fragments to bind methylated DNA. In a typical MBD-seq protocol, genomic DNA is first sonicated and incubated with tagged MBD proteins that can bind methylated cytosines. The protein-DNA complex is then precipitated with antibody-conjugated beads that are specific to the MBD protein tag, followed by DNA purification and sequencing.12953047v1 Page 43 of 224Attorney Docket: 2014191-0043 Classifiers

[0177] In some embodiments, the present disclosure provides methods for obtaining a classifier, e.g., a validated classifier that can be used to determine MS status. In some embodiments, a subject is determined to have a validated epigenetic profile indicative of MS based on analysis of a biological sample, optionally of cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject, wherein the presence of the validated epigenetic profile has been determined using a validated classifier.

[0178] For illustration purposes and without limitation, in an exemplary embodiment of the present disclosure, the validated classifier may be obtained by: (a) determining a genomic profile of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation in biological samples obtained from a first cohort of subjects who have previously been determined to have an MS; (b) determining a genomic profile of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation in biological samples obtained from a second cohort of healthy subjects or subjects who have previously been determined not to have MS; (c) comparing the genomic profile determined in step (a) and the genomic profile determined in step (b), to identify genomic loci that have statistically different histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels (“differential loci”); (d) training a classifier on histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels in the differential loci to distinguish between (i) samples from one or more biological samples obtained from the first cohort, and (ii) samples from one or more biological samples obtained from the second cohort, to identify samples having a profile of histone modification, chromatin accessibility, binding of transcription factor, and / or DNA methylation levels (“epigenetic profile”) that indicates that the samples are likely obtained from the first cohort; and (e) obtaining the validated classifier by validating the classifier from step (d) on a third cohort comprising an independent and blinded group of subjects with and without12953047v1 Page 44 of 224Attorney Docket: 2014191-0043 MS and selecting a threshold such that the validated classifier predicts MS, with an area under the receiver operating characteristic (AUROC) greater than 0.5 (e.g., greater than 0.55, greater than 0.6, greater than 0.65, greater than 0.7, greater than 0.75, greater than 0.8, greater than 0.85, greater than 0.9, or greater than 0.95), wherein subjects falling within the group of predicted MS display the validated epigenetic profile and subjects that do not fall within the group of MS lack the validated epigenetic profile.

[0179] A person of ordinary skill will appreciate that other methods can be used to obtain a classifier, e.g., a validated classifier that can be used to determine MS status and that the present disclosure is not limited to classifiers obtained in accordance with this method. Exemplary Genomic Loci

[0180] The present disclosure includes the identification of exemplary genomic loci that are differentially modified and / or differentially accessible in MS. See Tables 1-6 which show the chromosomal coordinates of each genomic locus, a measure of signal detected at each locus (BaseMean), fold-change observed in MS vs. healthy controls (log2FoldChange), statistical measures of significance (pvalue and padj) and the gene each loci is associated with (Symbol). The genomic loci are grouped by gene. Tables 1-3 list loci for which an increase in epigenetic modifications was observed in MS subjects as compared to healthy subjects. Tables 4-6 list loci for which a decrease in epigenetic modifications was observed in MS subjects as compared to healthy subjects.

[0181] The present disclosure is not limited to methods that use the exact same chromosomal coordinates that are recited in Tables 1-6. The present disclosure encompasses methods that use any of the genomic loci in Table 1-6 and also subregions thereof, i.e., references herein to methods that involve detecting and / or quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci of Table 1-6 encompasses methods that detect these marks anywhere within these genomic loci including within any subregions. For example, where Table 1 references chr1:10056254-10058255 as a genomic locus for detecting and / or quantifying H3K4me3 modification, this encompasses methods that detect and / or quantify H3K4me3 modification at any position or sub-region of chr1:10056254-10058255, e.g., methods that detect12953047v1 Page 45 of 224Attorney Docket: 2014191-0043 and / or quantify H3K4me3 modification within chr1:10056354-10058155, etc. In some embodiments, a subregion may span at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 or at least 3000 contiguous base pairs that are located between the lower and upper coordinates of a genomic locus recited in Tables 1-6. In some embodiments, a subregion may span less than 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500 or at least 3000 contiguous base pairs that are located between the lower and upper coordinates of a genomic locus recited in Tables 1-6. In some embodiments, a subregion may have the same central coordinate as a genomic locus recited in Tables 1-6. In some embodiments, a subregion may have a different central coordinate as a genomic locus recited in Tables 1-6. It is also to be understood that the lower / upper coordinates of the genomic loci in Tables 1-6 are approximate and that the present disclosure encompasses methods where any one or more of the genomic loci are expanded by increasing the size of the genomic locus by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or up to 50% in one or both directions.

[0182] In some embodiments a classifier is generated using a set of differentially modified and / or differentially accessible genomic loci that are correlated with MS. Sequence reads that fall into each selected genomic locus are analyzed and counted, e.g., as described herein including the Examples. In some embodiments, counts from genomic loci that are correlated with MS are aggregated. Other ways of using the genomic loci and related sequencing data to generate and apply a classifier to determine MS status are described herein and known in the art, e.g., without limitation, methods that use a learning statistical classifier system or a combination of learning statistical classifier systems.

[0183] In some embodiments, exemplary genomic loci from one or more of Tables 1-6 are used in a monomodal classifier, e.g., a classifier that uses a single histone modification (e.g., H3K4me3 or H3K27ac) or DNA methylation at one or more genomic loci for purposes of determining MS status. In some embodiments, exemplary genomic loci from any one of Table 1- 6, or any combination thereof, are used in combination in a multimodal classifier, e.g., a classifier that uses more than one histone modification (e.g., H3K4me3 and H3K27ac) or one or more histone modifications (e.g., H3K4me3 and / or H3K27ac) and DNA methylation at one or more genomic loci for purposes of determining MS status.12953047v1 Page 46 of 224Attorney Docket: 2014191-0043

[0184] In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and / or binding of a transcription factor at one or more loci provided in one or more of Tables 1-6. In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and / or binding of a transcription factor at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 600, 800, 1,000, 1,500, 2,000, 3,000, 4,000, or more loci listed in one or more of Tables 1-6 (e.g., 1-200, 5- 200, 10-200, 15-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 1-150, 5-150, 10-150, 15-150, 20-150, 30-150, 40-150, 50-150, 60-150, 70-150, 80-150, 90-150, 100-150, 1-100, 5-100, 10-100, 15-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80- 100, 90-100). In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and / or binding of a transcription factor at each of the loci provided in one or more of Tables 1-6. In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and / or binding of a transcription factor for at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in one or more of Tables 1-6. In some embodiments, a method described herein comprises quantifying one or more of a histone modification, DNA methylation, chromatic accessibility and / or binding of a transcription factor for at least a percent of loci identified in any one of Tables 1-6 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100%. Differential H3K4me3 modification

[0185] Exemplary genomic loci demonstrating differential H3K4 methylation (in particular H3K4 trimethylation, H3K4me3) in MS vs. subjects not having MS are provided in Tables 1 and 4 which shows the chromosomal coordinates of each genomic locus. Table 1 lists exemplary loci in which, in some embodiments, H3K4me3 modifications are increased in MS subjects vs. healthy subjects, and Table 4 lists loci in which, in some embodiments, H3K4me3 modifications are decreased in MS subjects vs. healthy subjects. The genomic loci are sorted by12953047v1 Page 47 of 224Attorney Docket: 2014191-0043 fold increase in Tables 1 and 4. “Symbol” corresponds to the gene each differentially modified loci was associated with. “lfcSE” and “stat” correspond to the standard error and test statistic values calculated by DEseq in a differential test. “Entrez” is the entrez gene ID.

[0186] A person of skill in the art will recognize that the methods disclosed herein do not require that every genomic locus listed in Tables 1 and 4 be assessed for H3K4me3 modification. Instead, a subset of loci may be assessed for H3K4me3 modification. Subsets of the genomic loci of Tables 1 and 4 can be selected (e.g., for use in determining MS status) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and / or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)). Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier. Those of skill in the art will appreciate that such subsets of loci of Tables 1 and 4, and loci included in such subsets, are together, individually, and / or in randomly selected subsets, at least as informative (e.g., as statistically significant and / or reliable) for uses disclosed herein, e.g., for determining MS status. The present disclosure particularly includes, among other things, subsets of the genomic loci of Tables 1 and 4, which have an absolute log2(fold-change) of 3.0 or higher, 2.9 or higher, 2.8 or higher, 2.7 or higher, 2.6 or higher, 2.5 or higher, 2.4 or higher, 2.3 or higher, 2.2 or higher, 2.1 or higher, 2.0 or higher, 1.9 or higher, 1.8 or higher, 1.7 or higher, 1.6 or higher, 1.5 or higher, 1.4 or higher, 1.3 or higher, 1.2 or higher, 1.1 or higher, 1.0 or higher, 0.9 or higher, 0.8 or higher, 0.7 or higher, 0.6 or higher, or 0.5 or higher. The present disclosure also includes subsets of the genomic loci of Tables 1 and 4, which have an absolute log2(fold-change) of 2.8 to less than 3.0, 2.6 to less than 2.8, 2.4 to less than 2.6, 2.2 to less than 2.4, 2.0 to less than 2.2, 1.8 to less than 2.0, 1.6 to less than 1.8, 1.4 to less than 1.6, 1.2 to less than 1.4, 1.0 to less than 1.2, 0.8 to less than 1.0, 0.6 to less than 0.8, 0.4 to less than 0.6, or 0.4 to less than 0.6.

[0187] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, or 700 loci identified in Tables 1 and 4 (or any subset thereof) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least a number of loci identified in a Tables 1 and12953047v1 Page 48 of 224Attorney Docket: 2014191-0043 4 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, or 700 is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Tables 1 and 4 (e.g., about 1 to about 700, about 5 to about 700, about 10 to about 700, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least a percent of loci identified in Tables 1 and 4 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).

[0188] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Tables 1 and 4). In some embodiments, a subject12953047v1 Page 49 of 224Attorney Docket: 2014191-0043 from which the sample is obtained or derived, is determined to have a particular MS status if at least one of the top 10 loci identified in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one of the top 50 loci identified in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 10 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference.

[0189] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one of the top 10 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified in Tables 1 and 4 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 1 and 4 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 2000, 2500,12953047v1 Page 50 of 224Attorney Docket: 2014191-0043 or 3000 loci identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one of the top 50 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) identified in Tables 1 and 4 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 1 and 4 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 1 and 4 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).

[0190] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one or more promoter regions associated with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 of the genes listed in Tables 1 and 4 (or any subset thereof) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a12953047v1 Page 51 of 224Attorney Docket: 2014191-0043 subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one or more promoter regions associated with one of more of the genes listed in Tables 1 and 4 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more promoter regions associated with at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 genes identified in Tables 1 and 4 (e.g., about 1 to about 1000, about 5 to about 1000, about 10 to about 1000, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more promoter regions of at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of the genes identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least promoter regions of a percent of genes identified in Tables 1 and 4 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).

[0191] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more promoter regions associated with at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the genes having the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,12953047v1 Page 52 of 224Attorney Docket: 2014191-0043 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Tables 1 and 4). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one or more promoter regions of one or more of the genes identified as having one of the top 10 loci in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more promoter regions of one or more of the genes having one of the top 25 loci identified in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at one or more promoter regions of one or more genes identified as having one of the top 50 loci identified in Tables 1 and 4 is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more promoter regions of at least five of the genes identified as having the top 10 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more promoter regions of at least five genes identified as having one of the top 25 loci identified in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more promoter regions of at least five of the genes identified as having one of top 50 loci in Tables 1 and 4 are differentially H3K4me3 modified as compared to a reference.

[0192] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more promoter regions of one or more genes (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,12953047v1 Page 53 of 224Attorney Docket: 2014191-0043 or 10) identified as having one of the top 10 loci identified in Tables 1 and 4 and one or more promoter regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 genes identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one or more promoter regions of one of more genes identified as having one of the top 25 loci in Tables 1 and 4 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and one or more promoter regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 genes identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more promoter regions of one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) genes identified as having one of the top 50 loci identified in Tables 1 and 4 and one or promoter regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 genes identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more promoter regions of five or more genes identified as having one of the top 25 loci in Tables 1 and 4 and one or more promoter regions associated with at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 genes identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a12953047v1 Page 54 of 224Attorney Docket: 2014191-0043 sample or subject from which the sample is derived, is determined to have a particular MS status if one or more promoter regions of at least five the genes identified in Tables 1 and 4 as having one of the top 50 loci and one or more promoter regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, or 2200 genes identified in Tables 1 and 4 (or any subset thereof) in total are differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).

[0193] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more promoter regions of one or more marker genes of synaptic plasticity (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10; e.g., one or more marker genes of synaptic plasticity listed in Table 1) are differentially H3K4me3 modified (e.g., has increased H3K4me3 modifications) as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more promoter regions of SQSTM1 (e.g., chr5:179232387- 179234388), LILRB2 (e.g., chr19:54783399-54785400; chr19:54783483-54785484; chr19:54783738-54785739; or chr19:54784033-54786034), or a combination thereof is differentially H3K4me3 modified (e.g., has increased H3K4me3 modifications) as compared to a reference (e.g., a sample from a healthy subject).

[0194] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more promoter regions of one or more MS- risk alleles (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10; e.g., one or more MS-risk alleles listed in Table 1) are differentially H3K4me3 modified (e.g., have increased H3K4me3 modifications) as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more promoter regions of TNFRSF14 (e.g., chr1:2486803-2488804) is differentially H3K4me3 modified as compared to a reference (e.g., a sample from a healthy subject).

[0195] In various embodiments, differentially H3K4me3 modified refers to a methylation status characterized by an increase or decrease in a value measuring methylation (e.g., of read12953047v1 Page 55 of 224Attorney Docket: 2014191-0043 counts and / or normalized read counts for a given genomic locus), and / or a mean, median and / or mode thereof, and / or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to 16-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16- fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16- fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e- 5, 1e-5, 5e-6, or 1e-6. In various embodiments, an increase or decrease in a value measuring methylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1-fold to 10- fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0- fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold, 1.2-fold to 4.0-fold, 1.4-fold to 4.0-fold, 1.6-fold to 4.0-fold, 1.8-fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6- fold to 4.0-fold, 2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold, optionally where the statistical significance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e-5, 1e-5, 5e-6, or 1e-6.

[0196] In various embodiments, a promoter region refers to a region a certain number of nucleotides upstream of a gene (e.g., 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 nucleotides upstream of a gene). In some embodiments, a promoter region refers to a region identified in Tables 1 and 4.

[0197] In some embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, or 700 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject).12953047v1 Page 56 of 224Attorney Docket: 2014191-0043

[0198] In some embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject).

[0199] In some embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if (a) H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, or 700 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and (b) H3K4me3 modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject). In some embodiments, a sample or subject from which the sample is obtained or derived is determined to have a particular status if: H3K4me3 modifications for at least 1 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 1 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject). H3K4me3 modifications for at least 5 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 5 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject). H3K4me3 modifications for at least 10 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 10 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject). H3K4me3 modifications for at least 25 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for12953047v1 Page 57 of 224Attorney Docket: 2014191-0043 at least 25 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject). H3K4me3 modifications for at least 50 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 50 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject). H3K4me3 modifications for at least 75 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 75 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject). H3K4me3 modifications for at least 100 loci identified in Table 1 are found to be increased relative to a reference (e.g., a healthy subject), and H3K4me3 modifications for at least 100 loci identified in Table 4 are found to be decreased relative to a reference (e.g., a healthy subject). Differential H3K27ac modification

[0200] Exemplary genomic loci demonstrating differential H3K27ac modification in MS vs. subjects not having MS are provided in Tables 2 and 5 which shows the chromosomal coordinates of each genomic locus. Table 2 lists exemplary loci in which, in some embodiments, H3K27ac modifications are increased in MS subjects vs. healthy subjects, and Table 5 lists exemplary loci in which, in some embodiments, H3K27ac modifications are decreased in MS subjects vs. healthy subjects. The genomic loci are sorted by fold increase in Tables 2 and 5. “Symbol” corresponds to the gene each differentially modified loci was associated with. “lfcSE” and “stat” correspond to the standard error and test statistic values calculated by DEseq in a differential test. “Entrez” is the entrez gene ID.

[0201] A person of skill in the art will recognize that the methods disclosed herein do not require that every genomic locus listed in Tables 2 and 5 be assessed for H3K27ac modification. Instead, a subset of loci may be assessed for H3K27ac modification. Subsets of the genomic loci of Tables 2 and 5 can be selected (e.g., for use in determining MS status) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and / or a particular threshold of differential between12953047v1 Page 58 of 224Attorney Docket: 2014191-0043 relevant states (e.g., a measured log2(fold-change)). Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier. Those of skill in the art will appreciate that such subsets of loci of Tables 2 and 5, and loci included in such subsets, are together, individually, and / or in randomly selected subsets, at least as informative (e.g., as statistically significant and / or reliable) for uses disclosed herein, e.g., for determining MS status. The present disclosure particularly includes, among other things, subsets of the genomic loci of Tables 2 and 5, which have an absolute log2(fold-change) of 4.2 or higher, 4.1 or higher, 3.0 or higher, 2.9 or higher, 2.8 or higher, 2.7 or higher, 2.6 or higher, 2.5 or higher, 2.4 or higher, 2.3 or higher, 2.2 or higher, 2.1 or higher, 2.0 or higher, 1.9 or higher, 1.8 or higher, 1.7 or higher, 1.6 or higher, 1.5 or higher, 1.4 or higher, 1.3 or higher, 1.2 or higher, 1.1 or higher, 1.0 or higher, 0.9 or higher, 0.8 or higher, 0.7 or higher, 0.6 or higher, or 0.5 or higher. The present disclosure also includes subsets of the genomic loci of Tables 2 and 5, which have an absolute log2(fold-change) of 4.0 to less than 4.2, 3.8 to less than 4.0, 3.6 to less than 3.8, 3.4 to less than 3.6, 3.2 to less than 3.4, 3.0 to less than 3.2, 2.8 to less than 3.0, 2.6 to less than 2.8, 2.4 to less than 2.6, 2.2 to less than 2.4, 2.0 to less than 2.2, 1.8 to less than 2.0, 1.6 to less than 1.8, 1.4 to less than 1.6, 1.2 to less than 1.4, 1.0 to less than 1.2, 0.8 to less than 1.0, 0.6 to less than 0.8, or 0.4 to less than 0.6.

[0202] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 loci identified in Tables 2 and 5 (or any subset thereof) are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least a number of loci identified in a Tables 2 and 5 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to12953047v1 Page 59 of 224Attorney Docket: 2014191-0043 have a particular MS status if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Tables 2 and 5 (e.g., about 1 to about 1000, about 5 to about 1000, about 10 to about 1000, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least a percent of loci identified in Tables 2 and 5 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).

[0203] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Tables 2 and 5). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one of the top 10 loci identified in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy12953047v1 Page 60 of 224Attorney Docket: 2014191-0043 subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one of the top 50 loci identified in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 10 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).

[0204] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one of the top 10 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified in Tables 2 and 5 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 2 and 5 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000 loci identified in Tables 2 and 5 in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status12953047v1 Page 61 of 224Attorney Docket: 2014191-0043 if at least one of the top 50 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) identified in Tables 2 and 5 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000loci identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 2 and 5 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000 loci identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 2 and 5 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, or 1000loci identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).

[0205] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more enhancer regions associated with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 of the genes listed in Tables 2 and 5 (or any subset thereof) are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more enhancer regions associated with one of more of the genes listed in Tables 2 and 5 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to12953047v1 Page 62 of 224Attorney Docket: 2014191-0043 have a particular MS status if one or more enhancer regions associated with at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 genes identified in Tables 2 and 5 (e.g., about 1 to about 1000, about 5 to about 1000, about 10 to about 1000, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more enhancer regions of at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of the genes identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least enhancer regions of a percent of genes identified in Tables 2 and 5 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).

[0206] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more enhancer regions associated with at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the genes having the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Tables 2 and 5). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one or more enhancer regions of one or more of the genes identified as having one of the top 10 loci in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more enhancer regions12953047v1 Page 63 of 224Attorney Docket: 2014191-0043 of one or more of the genes having one of the top 25 loci identified in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at one or more enhancer regions of one or more genes identified as having one of the top 50 loci identified in Tables 2 and 5 is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more enhancer regions of at least five of the genes identified as having the top 10 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more enhancer regions of at least five genes identified as having one of the top 25 loci identified in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more enhancer regions of at least five of the genes identified as having one of top 50 loci in Tables 2 and 5 are differentially H3K27ac modified as compared to a reference.

[0207] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more enhancer regions of one or more genes (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified as having one of the top 10 loci identified in Tables 2 and 5 and one or more enhancer regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 genes identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one or more enhancer regions of one of more genes identified as having one of the top 25 loci in Tables 2 and 5 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and one or more enhancer regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450,12953047v1 Page 64 of 224Attorney Docket: 2014191-0043 500, 600, 700, 800, 900, 1000, or 1100 genes identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more enhancer regions of one or more (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) genes identified as having one of the top 50 loci identified in Tables 2 and 5 and one or enhancer regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 genes identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more enhancer regions of five or more genes identified as having one of the top 25 loci in Tables 2 and 5 and one or more enhancer regions associated with at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 genes identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more enhancer regions of at least five the genes identified in Tables 2 and 5 as having one of the top 50 loci and one or more enhancer regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or 1100 genes identified in Tables 2 and 5 (or any subset thereof) in total are differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).

[0208] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more enhancer regions of one or more marker genes of synaptic plasticity (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10; e.g., one or more marker genes of synaptic plasticity listed in Table 2) are differentially H3K27ac modified (e.g., to have increased H3K27ac modifications) as compared to a reference (e.g., a sample from a healthy subject). In some12953047v1 Page 65 of 224Attorney Docket: 2014191-0043 embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more enhancer regions of SQSTM1 (e.g., chr5:179232387- 179234388), LILRB2 (e.g., chr19:54783399-54785400; chr19:54783483-54785484; chr19:54783738-54785739; or chr19:54784033-54786034), or a combination thereof is differentially H3K27ac modified (e.g., to have increased H3K27ac modifications) as compared to a reference (e.g., a sample from a healthy subject).

[0209] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more enhancer regions of one or more MS- risk alleles (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10; e.g., one or more MS-risk alleles listed in Table 2) are differentially H3K27ac modified (e.g., to have increased H3K27 modifications) as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more enhancer regions of TNFRSF14 (e.g., chr1:2486803-2488804) is differentially H3K27ac modified as compared to a reference (e.g., a sample from a healthy subject).

[0210] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more enhancer regions of one or more cell identity genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell are differentially H3K27ac modified (e.g., to have increased H3K27ac modifications) as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, the one or more one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell are selected from genes provided in Table 2. In some embodiments, the one or more one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell comprise NSUN5, DAAM2, CNTN2, or any combination thereof. In some embodiments, the one or more enhancer regions of NSUN5 include chr7:72713576-72716059. In some embodiments, the one or more enhancer regions of DAAM2 include chr6:39788740-39793623.

[0211] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more enhancer regions of one or more cell identity genes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) of a microglial cell is differentially12953047v1 Page 66 of 224Attorney Docket: 2014191-0043 H3K27ac modified (e.g., to have increased H3K27ac modifications) as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, the one or more one or more cell identity genes of a microglial cell comprise MAP4K4. In some embodiments, one or more enhancer regions of MAP4K4 include chr2:102513113-102515404.

[0212] In various embodiments, differentially H3K27ac modified refers to an acetylation status characterized by an increase or decrease in a value measuring acetylation (e.g., of read counts and / or normalized read counts for a given genomic locus), and / or a mean, median and / or mode thereof, and / or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to 16-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16- fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16- fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e- 5, 1e-5, 5e-6, or 1e-6. In various embodiments, an increase or decrease in a value measuring acetylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase or decrease of 0.1-fold to 10- fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0- fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold.1.2-fold to 4.0-fold.1.4-fold to 4.0-fold, 1.6-fold to 4.0-fold, 1.8-fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6- fold to 4.0-fold, 2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold, optionally where the statistical significance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e-5, 1e-5, 5e-6, or 1e-6.

[0213] In some embodiments, one or more enhancer regions of a recited gene are provided in Tables 2 and 5. In some embodiments, one or more enhancer regions of a recited gene corresponds to: (i) one or more loci with increased or decreased H3K27ac modifications as12953047v1 Page 67 of 224Attorney Docket: 2014191-0043 compared to a reference (e.g., a sample from a healthy subject) within a certain number of nucleotides (e.g., 50,000 nucleotides) of the recited gene; and / or (ii) one or more loci with increased or decreased H3K27ac modifications as compared to a reference (e.g., a sample from a healthy subject) that are closest to the recited gene in the genome.

[0214] In some embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, or 500 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject).

[0215] In some embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject).

[0216] In some embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if (a) H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, or 500 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and (b) H3K27ac modifications for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject). In some embodiments, a sample or subject from which the sample is obtained or derived is determined to have a particular status if: H3K27ac modifications for at least 1 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 1 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject). H3K27ac modifications for at least 5 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 512953047v1 Page 68 of 224Attorney Docket: 2014191-0043 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject). H3K27ac modifications for at least 10 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 10 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject). H3K27ac modifications for at least 25 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 25 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject). H3K27ac modifications for at least 50 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 50 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject). H3K27ac modifications for at least 75 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 75 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject). H3K27ac modifications for at least 100 loci identified in Table 2 are found to be increased relative to a reference (e.g., a healthy subject), and H3K27ac modifications for at least 100 loci identified in Table 5 are found to be decreased relative to a reference (e.g., a healthy subject). Differential DNA methylation

[0217] Exemplary genomic loci demonstrating differential DNA methylation in MS are provided in Tables 3 and 6 which show the chromosomal coordinates of each genomic locus. Table 3 lists exemplary loci in which, in some embodiments, increased DNA methylation is increased in MS subjects vs. healthy subjects, and Table 6 lists exemplary loci in which, in some embodiments, DNA methylation is decreased in MS subjects vs. healthy subjects. The genomic loci are sorted by fold increase in Tables 3 and 6. “Symbol” corresponds to the gene each12953047v1 Page 69 of 224Attorney Docket: 2014191-0043 differentially modified loci was associated with. “lfcSE” and “stat” correspond to the standard error and test statistic values calculated by DEseq in a differential test. “Entrez” is the entrez gene ID.

[0218] A person of skill in the art will recognize that the methods disclosed herein do not require that every genomic locus listed in Tables 3 and 6 be assessed for DNA methylation. Instead, a subset of loci may be assessed for DNA methylation. Subsets of the genomic loci of Tables 3 and 6 can be selected (e.g., for use in determining MS status) based on various performance criteria, e.g., to select genomic loci that demonstrate differential modification with a particular level of statistical significance and / or a particular threshold of differential between relevant states (e.g., a measured log2(fold-change)). Subsets of the genomic loci may also be selected based on an algorithm, e.g., during the process of obtaining a classifier. Those of skill in the art will appreciate that such subsets of loci of Tables 3 and 6, and loci included in such subsets, are together, individually, and / or in randomly selected subsets, at least as informative (e.g., as statistically significant and / or reliable) for uses disclosed herein, e.g., for determining MS status. See also the Examples of the present disclosure for experiments showing that informative classifiers can be generated using many different combinations of the loci. The present disclosure particularly includes, among other things, subsets of the genomic loci of Tables 3 and 6, which have an absolute log2(fold-change) of 10.0 or higher, 6.5 or higher, 6.0 or higher, 5.5 or higher, 5.0 or higher, 4.5 or higher, 4.0 or higher, 3.5 or higher, 3.0 or higher, 2.5 or higher, 2.0 or higher, 1.9 or higher, 1.8 or higher, 1.7 or higher, 1.6 or higher, 1.5 or higher, 1.4 or higher, 1.3 or higher, 1.2 or higher, 1.1 or higher, 1.0 or higher, 0.9 or higher, 0.8 or higher, 0.7 or higher, 0.6 or higher, or 0.5 or higher. The present disclosure also includes subsets of the genomic loci of Tables 3 and 6, which have an absolute log2(fold-change) of 10.0 or higher, 6.5 or higher, 6.0 or higher, 5.5 to less than 6.0, 5.0 to less than 5.5, 4.5 to less than 5.0, 4.0 to less than 4.5, 3.8 to less than 4.0, 3.6 to less than 3.8, 3.4 to less than 3.6, 3.2 to less than 3.4, 3.0 to less than 3.2, 2.8 to less than 3.0, 2.6 to less than 2.8, 2.4 to less than 2.6, 2.2 to less than 2.4, 2.0 to less than 2.2, 1.8 to less than 2.0, 1.6 to less than 1.8, 1.4 to less than 1.6, 1.2 to less than 1.4, 1.0 to less than 1.2, 0.8 to less than 1.0, or 0.6 to less than 0.8.

[0219] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) are differentially12953047v1 Page 70 of 224Attorney Docket: 2014191-0043 DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least a number of loci identified in a Tables 3 and 6 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 loci identified in Tables 3 and 6 (e.g., about 1 to about 650, about 5 to about 650, about 10 to about 650, about 25 to about 200, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 100, about 2 to about 200, about 5 to about 200, about 10 to about 200, about 20 to about 200, about 25 to about 200, about 50 to about 200, about 20 to about 150, about 50 to about 150, about 50 to about 100, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if at least a percent of loci identified in Tables 3 and 6 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).

[0220] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one (e.g., at least 2, at least 3, at least 4, at12953047v1 Page 71 of 224Attorney Docket: 2014191-0043 least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Tables 3 and 6). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one of the top 10 loci identified in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least one of the top 50 loci identified in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 10 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).

[0221] In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one of the top 10 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified in Tables 3 and 6 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a12953047v1 Page 72 of 224Attorney Docket: 2014191-0043 sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one of the top 25 loci identified in Tables 3 and 6 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least one of the top 50 loci (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) identified in Tables 3 and 6 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least five of the top 25 loci identified in Tables 3 and 6 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if at least five of the top 50 loci identified in Tables 3 and 6 and at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 loci identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).

[0222] In various embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 or more) of the genes listed in Tables 3 and 6 (or any subset thereof) are differentially DNA methylated as12953047v1 Page 73 of 224Attorney Docket: 2014191-0043 compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions in one or more of the genes listed in Tables 3 and 6 (or any subset thereof) having a lower bound selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, or 300 and an upper bound selected from 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In certain particular embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions associated with at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 genes identified in Tables 3 and 6 (e.g., about 1 to about 500, about 5 to about 500, about 10 to about 500, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 loci) are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In various embodiments a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions of at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of the genes identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In certain embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions of at a percent of genes identified in Tables 3 and 6 having a lower bound selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%, and an upper bound selected from 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% is found to be differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more regions of at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) of the genes having the top 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,12953047v1 Page 74 of 224Attorney Docket: 2014191-0043 300, 350, 400, 450, 500, 550, 600, or 650 identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject) (wherein, e.g., the “top” 10 loci refers to the loci with 10 highest absolute log2(fold-change) in Tables 3 and 6). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions of one or more of the genes identified as having one of the top 10 loci in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions of one or more of the genes having one of the top 25 loci identified in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions of one or more genes identified as having one of the top 50 loci identified in Tables 3 and 6 is differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions of at least five of the genes identified as having the top 10 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions of at least five genes identified as having one of the top 25 loci identified in Tables 3 and 6 are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In some embodiments, a subject from which the sample is obtained or derived, is determined to have a particular MS status if one or more regions of at least five of the genes identified as having one of top 50 loci in Tables 3 and 6 are differentially DNA methylated as compared to a reference. In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more regions of one or more genes (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or 10) identified as having one of the top 10 loci identified in Tables 3 and 6 and one or more enhancer regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 genes identified in Tables 3 and 6 (or any subset thereof) in total are12953047v1 Page 75 of 224Attorney Docket: 2014191-0043 differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more regions of at least one gene identified as having one of the top 25 loci in Tables 3 and 6 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or 25) and one or more regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 genes identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more regions of at least one (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, at least 15, at least 20, or at least 25, at least 30, at least 35, at least 40, at least 45, or 50) genes identified as having one of the top 50 loci identified in Tables 3 and 6 and one or more regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 genes identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more regions of five or more genes identified as having one of the top 25 loci in Tables 3 and 6 and one or more regions associated with at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 genes identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject). In various embodiments, a sample or subject from which the sample is derived, is determined to have a particular MS status if one or more regions of at least five the genes identified in Tables 3 and 6 as having one of the top 50 loci and one or more regions of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 genes identified in Tables 3 and 6 (or any subset thereof) in total are differentially DNA methylated as compared to a reference (e.g., a sample from a healthy subject).12953047v1 Page 76 of 224Attorney Docket: 2014191-0043

[0223] In various embodiments, differentially DNA methylated refers to a methylation status characterized by an increase or decrease in a value measuring methylation (e.g., of read counts and / or normalized read counts for a given genomic locus), and / or a mean, median and / or mode thereof, and / or a log thereof (e.g., log base 2 (log2)), of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40- fold, 45-fold, 50-fold, or greater, or any range in between, inclusive, such as 1% to 50%, 50% to 2-fold, 25% to 50-fold, 25% to 30-fold, 25% to 20-fold, 25% to 16-fold, 30% to 16-fold, 50% to 16-fold, 70% to 16-fold, 2-fold to 16-fold, 2.2-fold to 16-fold, 2.6-fold to 16-fold, 3-fold to 16- fold, 3.4-fold to 16-fold, 4-fold to 16-fold, 4.5-fold to 16-fold, 5.2-fold to 16-fold, 6-fold to 16- fold, 7-fold to 16-fold, or 8-fold to 16-fold, as compared to a reference, optionally where the statistical significance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e- 5, 1e-5, 5e-6, or 1e-6. In various embodiments, an increase or decrease in a value measuring methylation can be, or is expressed as, a log2(fold-change), e.g., a log2(fold-change) of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, or greater, or any range in between, inclusive, such as an increase of 0.1-fold to 10-fold, 0.2-fold to 5-fold, 0.2-fold to 4.0-fold, 0.4-4.0-fold, 0.4-fold to 4.0-fold, 0.6-fold to 4.0-fold, 0.8-fold to 4.0-fold, 1.0-fold to 4.0-fold.1.2-fold to 4.0-fold.1.4-fold to 4.0-fold, 1.6-fold to 4.0-fold, 1.8- fold to 4.0-fold, 2.0-fold to 4.0-fold, 2.2-fold to 4.0-fold, 2.4-fold to 4.0-fold, 2.6-fold to 4.0-fold, 2.8-fold to 4.0-fold, or 3.0-fold to 4.0-fold, optionally where the statistical significance of the increase or decrease is at least 5e-2, 1e-2, 5e-3, 1e-3, 5e-4, 1e-4, 5e-5, 1e-5, 5e-6, or 1e-6.

[0224] In some embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if DNA methylation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, or 500 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject).

[0225] In some embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if DNA methylation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,12953047v1 Page 77 of 224Attorney Docket: 2014191-0043 140, or 150 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject).

[0226] In some embodiments, a sample or subject from which the sample is obtained or derived, is determined to have a particular MS status if (a) DNA methylation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, or 500 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and (b) DNA methylation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject). In some embodiments, a sample or subject from which the sample is obtained or derived is determined to have a particular status if: DNA methylation for at least 1 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 1 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject). DNA methylation for at least 5 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 5 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject). DNA methylation for at least 10 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 10 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject). DNA methylation for at least 25 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 25 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject). DNA methylation for at least 50 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 50 loci12953047v1 Page 78 of 224Attorney Docket: 2014191-0043 identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject). DNA methylation for at least 75 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 75 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject). DNA methylation for at least 100 loci identified in Table 3 are found to be increased relative to a reference (e.g., a healthy subject), and DNA methylation for at least 100 loci identified in Table 6 are found to be decreased relative to a reference (e.g., a healthy subject). Differential chromatin accessibility or transcription factor binding

[0227] Genomic loci provided in Tables 1-6 can also demonstrate differential chromatin accessibility or transcription factor binding in MS.

[0228] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with chromatin accessibility. In various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with chromatin accessibility.

[0229] In some embodiments, without wishing to be limited to any particular scientific theory, chromatin accessibility corresponds and / or is correlated with H3K4me3 modifications. As a result, in some embodiments, MS status may be determined by detecting and quantifying chromatin accessibility at one or more genomic loci in Tables 1 and 3 in accordance with the section above discussing exemplary genomic loci with differential H3K4me3 modifications.

[0230] In some embodiments, without wishing to be limited to any particular scientific theory, chromatin accessibility corresponds and / or is correlated with H3K27ac modifications. As a result, in some embodiments, MS status may be determined by detecting and quantifying chromatin accessibility at one or more genomic loci in Tables 2 and 5 in accordance with the section above discussing exemplary genomic loci with differential H3K27ac modifications.12953047v1 Page 79 of 224Attorney Docket: 2014191-0043

[0231] In some embodiments, without wishing to be limited to any particular scientific theory, chromatin accessibility corresponds and / or is correlated with DNA methylation. As a result, in some embodiments, MS status may be determined by detecting and quantifying chromatin accessibility at one or more genomic loci in Tables 3 and 6 in accordance with the section above discussing exemplary genomic loci with differential DNA methylation.

[0232] In various embodiments, without wishing to be bound by any particular scientific theory, histone methylation (e.g., H3K4me3) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific theory, histone acetylation (e.g., H3K27ac) corresponds and / or is correlated with transcription factor binding. In various embodiments, without wishing to be bound by any particular scientific theory, DNA methylation corresponds and / or is correlated with transcription factor binding.

[0233] In some embodiments, without wishing to be limited to any particular scientific theory, binding of RNA pol II corresponds and / or is correlated with H3K4me3 modifications. As a result, in some embodiments, MS status may be determined by detecting and quantifying binding of RNA pol II at one or more genomic loci in Tables 1 and 4 in accordance with the section above discussing exemplary genomic loci with differential H3K4me3 modifications.

[0234] In some embodiments, without wishing to be limited to any particular scientific theory, binding of p300, mediator complex, cohesin complex or RNA pol II corresponds and / or is correlated with H3K27ac modifications. As a result, in some embodiments, MS status may be determined by detecting and quantifying binding of p300, mediator complex, cohesin complex or RNA pol II at one or more genomic loci in Tables 2 and 5 in accordance with the section above discussing exemplary genomic loci with differential H3K27ac modifications. Applications

[0235] Methods, kits and systems of the present disclosure include analysis of differentially modified and / or differentially accessible genomic loci to determine MS status. Methods, kits and systems of the present disclosure can be used in any of a variety of applications. For example, methods, kits and systems of the present disclosure can be used in detecting and / or treating MS. Methods, kits and systems of the present disclosure can also be used to detect or determine resistance of MS to a therapy or transformation of MS.12953047v1 Page 80 of 224Attorney Docket: 2014191-0043

[0236] In various embodiments, methods, kits and systems of the present disclosure can be applied to an asymptomatic human subject. As used herein, a subject can be referred to as “asymptomatic” if the subject does not report, and / or demonstrate by non-invasively observable indicia (e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or autoimmune screening), sufficient characteristics of MS to support a medically reasonable suspicion that the subject is likely suffering from MS. Detection of early- stage MS can be achieved using methods, kits and systems of the present disclosure, with attendant medical benefits including potential for early treatment and attendant improvement in therapeutic outcomes.

[0237] In various embodiments, methods, kits and systems of the present disclosure can be applied to a symptomatic human subject. As used herein, a subject can be referred to as “symptomatic” if the subject report, and / or demonstrates by non-invasively observable indicia (e.g., without one, several, or all of device-based probing, tissue sample analysis, bodily fluid analysis, surgery, or MS screening), sufficient characteristics of MS to support a medically reasonable suspicion that the subject is likely suffering from MS. In various embodiments a sample from a subject, where the subject is suspected of having MS, can be assayed according to one or more embodiments of the present disclosure to determine if the subject in fact has MS.

[0238] In some embodiments, methods, kits and systems of the present disclosure can be used to determine that a subject has MS that correlates with a prior determination of MS (e.g., based on imaging, symptoms, and / or CSF analysis).

[0239] In some embodiments, methods, kits and systems of the present disclosure can be used to validate or confirm a prior determination that a subject has MS.

[0240] Those of skill in the art will appreciate that regular, preventive, and / or prophylactic screening to determine MS status improves diagnosis of MS, including and / or particularly early-stage MS. Thus, the present disclosure provides, among other things, methods, kits and systems particularly useful for the diagnosis and treatment of early-stage MS. Generally, and particularly in embodiments in which MS detection in accordance with the present disclosure is carried out annually, and / or in which a subject is asymptomatic at time of detecting, methods, kits and systems of the present disclosure are especially likely to detect early-stage MS.12953047v1 Page 81 of 224Attorney Docket: 2014191-0043 In various embodiments, detecting in accordance with methods, kits and systems of the present disclosure reduces MS symptoms.

[0241] In various embodiments MS status determination in accordance with the present disclosure is performed once for a given subject or multiple times for a given subject. In various embodiments, MS status determination in accordance with the present disclosure is performed on a regular basis, e.g., every six months, annually, every two years, every three years, every four years, every five years, or every ten years.

[0242] In various embodiments, methods, kits and systems disclosed herein provide a determination of MS status. In other instances, methods, kits and systems disclosed herein will be indicative of MS status but not definitive for MS status. In various instances in which methods, kits and systems of the present disclosure are used to determine MS status, the same can be followed by a further confirmatory assay, which further assay can confirm, support, undermine, or reject a determination resulting from a prior determination, e.g., a determination in accordance with the present disclosure. As used herein, a confirmatory assay can be a test that is currently recognized by medical practitioners, e.g., based on symptoms, imaging, CSF analysis, or other testing.

[0243] In various embodiments, MS status determination according to one or more methods, kits and / or systems disclosed herein is followed by treatment of MS. In various embodiments, treatment of MS includes administration of a therapeutic regimen including one or more therapies provided herein, including without limitation one or more of a low, moderate, or high efficacy MS therapy. In various embodiments, treatment of MS includes administration of a therapeutic regimen including one or more treatments provided herein as available, appropriate, and / or preferred for a particular MS status.

[0244] In various embodiments, methods, kits and systems can be used to determine whether a particular subject is likely to be and / or is characterized as responsive to a MS therapeutic agent. In some such embodiments, methods, kits and systems can be followed by treatment of the subject with a MS therapeutic agent.

[0245] In various embodiments, methods, kits and systems can be used to determine whether a particular subject is likely to be and / or is characterized as resistant to, non-responsive to, or not recommended treatment with an MS therapeutic agent. In some such embodiments,12953047v1 Page 82 of 224Attorney Docket: 2014191-0043 methods, kits and systems can be followed by treatment with a higher efficacy MS therapeutic agent.

[0246] Responsiveness can refer to the ability or likelihood of a therapy to cause a reduction in the number and / or size of MS lesions, improved symptoms, slowing of disease progression, shortening of flare duration, lessened disability, reduced disease activity, and / or an increased likelihood of achieving no evidence of disease activity status. Responsiveness can refer to improvement in prognosis. Responsiveness can refer to achievement of a treatment benefit, including e.g., improvement in one or more symptoms of MS. Responsiveness can be measured quantitatively (e.g., as in the case of MS lesion size and / or numbers; as in the case of measurement of histone modification, chromatin accessibility, transcription factor binding, or DNA methylation at one or more genomic loci; or as in the calculation of clinical benefit (CBR)), or qualitatively (e.g., by measures such as “pathological complete response” (pCR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD), or other qualitative criteria). Resistance can refer to the inability or unlikelihood of a therapy to achieve a desired therapeutic effect (e.g., a reduction MS lesions, lessened disability, reduced symptoms, or decreased flare frequency), or other treatment benefit such as, e.g., improvement in one or more symptoms of MS) in a subject. Resistance includes natural resistance. In certain embodiments, resistance includes the extent to which one or more desired therapeutic benefits results from administration of a therapy to a subject is less than that expected and / or achieved in a reference (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of benefit achieved in a reference).

[0247] In various embodiments, methods, kits and systems can be used to detect the clinical efficacy of a course of therapy for MS. For example, methods and / or compositions of the present disclosure could be used to determine the presence, absence, or MS status of a subject over the course of treatment. Methods and / or compositions of the present disclosure could be used in conjunction with, or confirmed by, other means of determining the presence, absence, or MS status of a subject including, for example measurements of lesion size or character by techniques such as MRI, measurements of immune activity using PET imaging, or by various qualitative, quantitative, or semi quantitative scoring systems including without limitation based on) in a qualitative fashion like “pathological complete response” (pCR), “clinical complete12953047v1 Page 83 of 224Attorney Docket: 2014191-0043 remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), or “clinical progressive disease” (cPD).

[0248] In some embodiments, methods, kits and systems for MS status determination provided herein can inform treatment and / or payment (e.g., reimbursement for or reduction of cost of medical care, such as detecting or treatment) decisions and / or actions, e.g., by individuals, healthcare facilities, healthcare practitioners, health insurance providers, governmental bodies, or other parties interested in healthcare cost.

[0249] In some embodiments, methods, kits and systems for MS status determination provided herein can inform decision making relating to whether health insurance providers reimburse a healthcare cost payer or recipient (or not), e.g., for (1) MS status determination itself (e.g., reimbursement for detecting otherwise unavailable, available only for periodic / regular detecting, or available only for temporally- and / or incidentally- motivated detecting); and / or for (2) treatment, including initiating, maintaining, and / or altering therapy, e.g., based on the determined MS status. For example, in some embodiments, methods, kits and systems for MS status determination provided herein are used as the basis for, to contribute to, or support a determination as to whether a reimbursement or cost reduction will be provided to a healthcare cost payer or recipient. In some instances, a party seeking reimbursement or cost reduction can provide results of MS status determination conducted in accordance with the present disclosure together with a request for such reimbursement or reduction of a healthcare cost. In some instances, a party making a determination as to whether or not to provide a reimbursement or reduction of a healthcare cost will reach a determination based in whole or in part upon receipt and / or review of results of MS status determination conducted in accordance with the present disclosure.

[0250] In various embodiments, MS status determination using methods, kits and systems disclosed herein can be used in classifying subjects and / or samples (e.g., MS subjects and / or samples). In various embodiments, methods, kits and systems disclosed herein can be used to generate a set of subjects, and / or samples identified according to the present methods, kits and systems each classified as corresponding to a particular MS status, and optionally using two or more of such classified subjects, and / or samples to identify biomarkers that distinguish12953047v1 Page 84 of 224Attorney Docket: 2014191-0043 the classes (i.e., distinguish the subjects, and / or samples according to their class, e.g., according to their MS status).

[0251] For illustration purposes and without limitation, in an exemplary assay of the present disclosure, samples obtained from a subject (e.g., a liquid biopsy sample including cfDNA, e.g., a plasma sample including cfDNA) is analyzed by ChIP-seq for a histone modification (e.g., H3K4me3 and / or H3K27ac). ChIP-seq sequence reads are aligned to human genome build hg19, e.g., using the Burrows-Wheeler Aligner (BWA). Non-uniquely mapping and redundant reads are optionally discarded. To provide one example of peak calling, MACS v2.1.1.20140616 can be used for ChIP-seq peak calling with a q-value (FDR) threshold of 0.01. ChIP-seq data quality can optionally be evaluated by any of one or more of a variety of measures, including total peak number, FriP (fraction of reads in peak) score, number of high- confidence peaks (e.g., enriched > ten-fold over background), and percent of peak overlap with “blacklist” DHS peaks derived from the ENCODE project (Amemiya et al., Sci Rep (2019) 9(1):9354). If the ChIP-seq data quality is below a particular threshold the data may be discarded and the assay repeated. ChIP-seq peaks that overlap with selected genomic loci that are differentially modified as provided herein for the relevant histone modification (Tables 1-2) can then be used to determine MS status. The number of reads overlapping the selected genomic loci for the relevant histone modification are summed, e.g., in some embodiments all the genomicloci that are differentially modified with an absolute log2(fold- . Insome embodiments, the average number of reads in the local background of each ChIP-seq peak is subtracted to improve signal to noise. The data can then be log2-transformed and quantile normalized to match the distribution of the data used to train the classifier. The normalized data can then be used as input into a classifier that was trained using the same histone modification and selected genomic loci. The classifier can then use the inputted data to determine <S status of the subject. It will be appreciated that this or similar approaches can be applied to assays of the present disclosure that quantify chromatin accessibility, transcription factor binding and / or DNA methylation.

[0252] For the avoidance of any doubt, those of skill in the art will appreciate from the present disclosure that methods, kits and systems for MS status determination of the present12953047v1 Page 85 of 224Attorney Docket: 2014191-0043 disclosure are at least for in vitro use. Accordingly, all aspects and embodiments of the present disclosure can be performed and / or used at least in vitro.

[0253] Those of skill in the art will also appreciate that, in certain embodiments, methods of the present disclosure can be implemented on and / or in conjunction with a computer program and computer system. In some embodiments, methods of the present disclosure can be implemented on and / or in conjunction with a non-transitory computer readable storage medium encoded with the computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform the method. A computer system can also store and manipulate data generated by methods of the present disclosure that comprise a plurality of genomic locus modification status and / or accessibility status changes / profiles, which data can be used by a computer system in implementing methods disclosed herein. In certain embodiments, a computer system (i) receives modification status and / or accessibility status data; (ii) stores the data; and (iii) compares the data in any number of ways described herein (e.g., analysis relative to appropriate references), e.g., to determine MS status. In certain embodiments, a computer system (i) compares the genomic locus modification and / or accessibility status to a reference; and (ii) outputs an indication of whether the modification status and / or accessibility status of the genomic locus is significantly different from the reference and / or provides a determination regarding MS status.

[0254] Numerous types of computer systems can be used to implement methods of the present disclosure according to knowledge possessed by a skilled artisan in the bioinformatics and / or computer arts. Several software components can be loaded into memory during operation of such a computer system. The software components can comprise both software components that are standard in the art and components that are special to the present disclosure (e.g., dCHIP software described in Lin et al., Bioinformatics (2004) 20:1233-1240, incorporated herein by reference in its entirety; radial basis machine learning algorithms (RBM) known in the art). Methods of the present disclosure can also be programmed or modeled in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including specific algorithms to be used, thereby freeing a user of the need to procedurally program individual equations and algorithms. Such packages include, e.g., Matlab from Mathworks (Natick, MA), Mathematica from Wolfram Research (Champaign, IL), S-Plus from12953047v1 Page 86 of 224Attorney Docket: 2014191-0043 MathSoft (Seattle, WA), R from R Foundation for Statistical Computing (Vienna, Austria), Python from Python Software Foundation (Wilmington, DE), or Perl from Perl Foundation (Holland, MI). In certain embodiments, a computer system comprises a database for storage of genomic locus modification status and / or accessibility status data. Such stored profiles can be accessed and used to perform comparisons of interest at a later point in time. In addition to the exemplary program structures and computer systems described herein, other, alternative program structures and computer systems will be readily apparent to the skilled artisan.

[0255] As demonstrated in the Examples, various algorithms can be applied to the comparison, between samples and references, of the modification status and / or accessibility status of genomic loci that are differentially modified in MS. In various embodiments, an algorithm can be a single learning statistical classifier system. Other suitable statistical algorithms are well known to those of skill in the art. For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., a panel of genomic loci of interest) and making decisions based upon such data sets. In some embodiments, a single learning statistical classifier system such as a classification tree (e.g., random forest) is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, those described in the Examples and also those using inductive learning (e.g., decision / classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines (e.g., Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms, Gauss-Newton algorithms, mixtures of Gaussians,12953047v1 Page 87 of 224Attorney Docket: 2014191-0043 gradient descent algorithms, and learning vector quantization (LVQ). In certain embodiments, methods of the present disclosure can include sending classification results to a medical practitioner, e.g., an oncologist. Formulation and Administration of Therapeutic Agents

[0256] The present disclosure includes methods where a therapeutic agent or regimen is administered to a subject based on the MS status. In general, the therapeutic agent or regimen provided herein will be available, appropriate, and / or preferred for the determined MS status. Those of skill in the art will be aware of recommended and / or governmentally approved formulations and / or dosages for various therapeutic agents provided herein.

[0257] The present disclosure includes pharmaceutical compositions for delivery of one or more therapeutic agents to a subject. As disclosed herein, a pharmaceutical composition may be in any form known in the art, including formulations for administration according to any route known in the art. A suitable means of administration can be selected based on the age and condition of a subject.

[0258] Pharmaceutical composition forms of the present disclosure can include, e.g., liquid, semi-solid and solid dosage forms. Pharmaceutical composition forms of the present disclosure can include, e.g., liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, and liposomes. Selection or use of any particular form may depend, in part, on the intended mode of administration and therapeutic application. Accordingly, the compositions can be formulated for administration by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection) or a non-parenteral mode. As used herein, parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection or infusion.

[0259] In some embodiments, the compositions provided herein are present in unit dosage form, which unit dosage form can be suitable for self-administration. Such a unit dosage form may be provided within a container, e.g., a pill, vial, cartridge, prefilled syringe, or disposable pen.

[0260] A pharmaceutical composition of the present disclosure can be in an injectable or infusible form. For example, the present disclosure includes sterile formulations for injection or12953047v1 Page 88 of 224Attorney Docket: 2014191-0043 infusion, which can be formulated in accordance with conventional pharmaceutical practices. Sterile solutions can be prepared by incorporating a composition described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Solutions can be formulated, e.g., using distilled water, physiological saline, or an isotonic solution containing glucose and other supplements such as D- sorbitol, D-mannose, D-mannitol, or sodium chloride as an aqueous solution for injection, optionally in combination with a suitable solubilizing agent, for example, an alcohol such as ethanol and / or a polyalcohol such as propylene glycol or polyethylene glycol, and / or a nonionic surfactant such as polysorbate 80™ or HCO-50, and the like. In the case of sterile powders for the preparation of sterile injectable solutions, methods for preparation include vacuum drying and freeze-drying that yield a powder of a composition described herein plus any additional desired ingredient (see below) from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin. In particular instances, a pharmaceutical composition can be formulated, for example, as a buffered solution at a suitable concentration and suitable for storage, e.g., at 2-8°C (e.g., 4°C).

[0261] In various embodiments, a pharmaceutical composition of the present disclosure can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration. Generally, dispersions are prepared by incorporating a composition described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0262] In various instances, a pharmaceutical composition can be formulated to include a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, without limitation, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0263] In certain embodiments, compositions can be formulated with a carrier that will protect the therapeutic agent against rapid release, such as a controlled release formulation,12953047v1 Page 89 of 224Attorney Docket: 2014191-0043 including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known in the art. See, e.g., J. R. Robinson (1978) “Sustained and Controlled Release Drug Delivery Systems,” Marcel Dekker, Inc., New York.

[0264] Route of administration can be parenteral, for example, administration by injection. Administration by injection can be by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. Administration can be systemic or local. In certain embodiments, a composition described herein can be therapeutically delivered to a subject by way of local administration. As used herein, “local administration” or “local delivery,” can refer to delivery that does not rely upon transport of the composition or therapeutic agent to its intended target tissue or site via the vascular system. For example, the composition may be delivered by injection or implantation of the composition or therapeutic agent or by injection or implantation of a device containing the composition or therapeutic agent. In certain embodiments, following local administration in the vicinity of a target tissue or site, the composition or therapeutic agent, or one or more components thereof, may diffuse to an intended target tissue or site that is not the site of administration.

[0265] A pharmaceutical composition can be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, a pharmaceutical composition can be formulated by suitably combining the therapeutic molecule with pharmaceutically acceptable vehicles or media, such as sterile water and physiological saline, vegetable oil, emulsifier, suspension agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, binder, followed by mixing in a unit dose form required for generally accepted pharmaceutical practices. Examples of oily liquid include sesame oil and soybean oil, and it may be combined with benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are a buffer such as a phosphate buffer, or sodium acetate buffer, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The formulated injection can be packaged in a suitable ampule.12953047v1 Page 90 of 224Attorney Docket: 2014191-0043

[0266] In various embodiments, subcutaneous administration can be accomplished by means of a device, such as a syringe, a prefilled syringe, an auto-injector (e.g., disposable or reusable), a pen injector, a patch injector, a wearable injector, an ambulatory syringe infusion pump with subcutaneous infusion sets, or other device for combining with a therapeutic agent for subcutaneous injection.

[0267] An injection system of the present disclosure may employ a delivery pen as described in U.S. Pat. No.5,308,341. Pen devices, most commonly used for self-delivery of insulin to patients with diabetes, are well known in the art. Such devices can include at least one injection needle, are typically pre-filled with one or more therapeutic unit doses of a solution that includes the therapeutic agent and are useful for rapidly delivering solution to a subject with as little pain as possible. One medication delivery pen includes a vial holder into which a vial of a therapeutic or other medication may be received. The pen may be an entirely mechanical device or it may be combined with electronic circuitry to accurately set and / or indicate the dosage of medication that is injected into the user. See, e.g., U.S. Pat. No.6,192,891. In some embodiments, the needle of the pen device is disposable and the kits include one or more disposable replacement needles. Pen devices suitable for delivery of any one of the presently featured compositions are also described in, e.g., U.S. Pat. Nos.6,277,099; 6,200,296; and 6,146,361, the disclosures of each of which are incorporated herein by reference in their entirety. A microneedle-based pen device is described in, e.g., U.S. Pat. No.7,556,615, the disclosure of which is incorporated herein by reference in its entirety. See also the Precision Pen Injector (PPI) device, MOLLYTM, manufactured by Scandinavian Health Ltd.

[0268] In certain embodiments, administration of a therapeutic agent as described herein is achieved by administering to a subject a nucleic acid encoding a therapeutic agent described herein. Nucleic acids encoding a therapeutic agent described herein can be incorporated into a gene construct to be used as a part of a gene therapy protocol to deliver nucleic acids that can be used to express and produce therapeutic agent within cells. Expression constructs of such components may be administered in any therapeutically effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, lentivirus, and herpes simplex virus-1 (HSV-1), or12953047v1 Page 91 of 224Attorney Docket: 2014191-0043 recombinant bacterial or eukaryotic plasmids. Viral vectors can transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized, polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4precipitation. Examples of suitable retroviruses include adenovirus-derived vectors, adeno-associated virus (AAV), pLJ, pZIP, pWE, and pEM which are known to those skilled in the art.

[0269] In some embodiments, a composition can be formulated for storage at a temperature below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition can be formulated for storage for up to 2 years (e.g., one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, 10 months, 11 months, 1 year, or 2 years) at 2-8°C (e.g., 4°C). Thus, in some embodiments, the compositions described herein are stable in storage for at least 1 year at 2-8°C (e.g., 4°C).

[0270] A pharmaceutical composition can include a therapeutically effective amount of a therapeutic agent described herein. Such effective amounts can be readily determined by one of ordinary skill in the art. A therapeutically effective amount can be an amount at which any toxic or detrimental effects of the composition are outweighed by therapeutically beneficial effects. In some embodiments, a dose can also be chosen to reduce or avoid production of antibodies or other host immune responses against a therapeutic agent. Those of skill in the art will appreciate that data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. In various embodiments, the amount of active ingredient included in a pharmaceutical composition is such that a suitable dose within the designated range can be administered to subjects. The dose and method of administration can vary depending on weight, age, condition, and other characteristics of a patient, and can be suitably selected as needed by those skilled in the art.

[0271] Pharmaceutical compositions including certain therapeutic agents, e.g., therapeutic antibodies, can be administered as a fixed dose, or in a milligram per kilogram (mg / kg) dose. While in no way intended to be limiting, an exemplary single dose of certain pharmaceutical compositions described herein can include certain therapeutic agents as described herein in an amount equal to, e.g., 0.001 to 1000 mg / kg, 1-1000 mg / kg, 1-100 mg / kg, 0.5-50 mg / kg, 0.1-100 mg / kg, 0.5-25 mg / kg, 1-20 mg / kg, and 1-10 mg / kg body weight. Exemplary12953047v1 Page 92 of 224Attorney Docket: 2014191-0043 dosages of a composition described herein include, without limitation, 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 8 mg / kg, or 20 mg / kg. The present disclosure is not limited to such ranges or dosages.

[0272] The present disclosure further includes methods of preparing pharmaceutical compositions of the present disclosure and kits including pharmaceutical compositions of the present disclosure.

[0273] In various embodiments, therapeutic agents of the present disclosure can be administered to a subject in a course of treatment that further includes administration of one or more additional therapeutic agents or therapies that are not therapeutic agents (e.g., surgery or radiation). Combination therapies of the present disclosure can include simultaneous exposure of a subject to therapeutic agents of two or more therapeutic regimens.

[0274] In certain embodiments, a therapeutic agent as described herein can be administered together with (e.g., at the same time and / or in the same composition as) an additional agent or therapy. In certain embodiments, a therapeutic agent of the present disclosure can be administered separately from an additional therapeutic agent or therapy (e.g., at a different time and / or in a different composition than the additional therapeutic agent or therapy). Dosing regimens of a therapeutic agent and one or more additional therapeutic agents with which it is administered in combination can be coordinated or independently determined. In various embodiments, an additional therapeutic agent or therapy administered in combination with a therapeutic agent as described herein can be administered at the same time as therapeutic agent, on the same day as therapeutic agent, or in the same week as therapeutic agent. In various embodiments, an additional therapeutic agent or therapy administered in combination with a therapeutic agent as described herein can be administered such that administration of the therapeutic agent and the additional therapeutic agent or therapy are separated by one or more hours before or after, one or more days before or after, one or more weeks before or after, or one or more months before or after administration of the therapeutic agent. In various embodiments, the administration frequency and / or dosage of one or more additional therapeutic agents can be the same as, similar to, or different from the administration frequency of a therapeutic agent. In some embodiments, the two or more regimens can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all “doses” of a first12953047v1 Page 93 of 224Attorney Docket: 2014191-0043 regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such therapeutic agents are administered in overlapping dosing regimens.

[0275] In certain embodiments, administration of a therapeutic agent can be to a subject having previously received, scheduled to receive, or in the course of a treatment regimen including an additional MS therapy. Administration of a therapeutic agent can, in some instances, improve delivery or efficacy of another therapeutic agent or therapy with which it is administered in combination.

[0276] It is contemplated that therapeutic agent combination therapies can demonstrate synergy and / or greater-than-additive effects between a therapeutic agent and one or more additional therapeutic agents with which it is administered in combination. A therapeutic agent can be administered in any effective amount as determined independently or as determined by the joint action of therapeutic agent and any of one or more additional therapeutic agents or therapies administered. Administration of the therapeutic agent may, in some embodiments, reduce the therapeutically effective dosage, required dosage, or administered dosage of the additional therapeutic agent or therapy relative to a reference regimen for administration of additional therapeutic agent or therapy or therapy absent the therapeutic agent. In certain embodiment, a composition described herein can replace or augment other previously or currently administered therapy. For example, upon treating with therapeutic agent, administration of one or more additional therapeutic agents or therapies can cease or diminish, e.g., be administered at lower levels. Kits

[0277] The present disclosure includes kits for detecting modification and / or accessibility of one or more genomic loci. In some embodiments, the present disclosure provides kits for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci. Kits of the present disclosure can include, e.g., reagents such as buffers and / or antibodies useful in the detection and quantification of histone modifications. In certain embodiments, a kit of the present disclosure can include at least one antibody that selective binds a histone modification selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, or H3K4me3, or pan12953047v1 Page 94 of 224Attorney Docket: 2014191-0043 acetylation. In certain embodiments, a kit of the present disclosure can include at least one antibody that selective binds H3K4me3 modifications. In certain embodiments, a kit of the present disclosure can include at least one antibody that selective binds H3K27ac modifications. A kit of the present disclosure can include instructional materials disclosing or describing the use of the kit in a method of determining MS status and / or treatment disclosed herein. In various embodiments, a kit of the present disclosure can include one or more therapeutic agents useful in the treatment of MS, e.g., as disclosed herein, optionally in combination with instruction materials for treatment of MS.

[0278] In some embodiments, a kit of the present disclosure comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from those provided in Tables 1-6.

[0279] In some embodiments, the kit comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 1. In some embodiments, the kit comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 4. In some embodiments, the kit comprises reagents for quantifying H3K27ac for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2. In some embodiments, the kit comprises reagents for quantifying H3K27ac for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 5. In some embodiments, the kit comprises one or more antibodies for use in ChIP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.

[0280] In some embodiments, the kit comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 3. In some embodiments, the kit comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 6. In some embodiments, the kit comprises one or more methyl-binding domains (e.g., for use in MBD-seq). In some embodiments, the kit comprises one or more antibodies that can bind methylated DNA (e.g., for use in MeDIP).

[0281] In some embodiments, the kit comprises reagents for measuring chromatin accessibility via an ATAC-seq assay.

[0282] In some embodiments, the kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample. In some embodiments, the kit comprises reagents for12953047v1 Page 95 of 224Attorney Docket: 2014191-0043 library preparation for sequencing. In some embodiments, the kit comprises reagents for sequencing. In some embodiments, the kit comprises instructions for determining if a subject has MS. Systems

[0283] The present disclosure includes systems for detecting modification and / or accessibility of one or more genomic loci. In some embodiments, the present disclosure provides systems for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci. Systems of the present disclosure can include a sequencer configured to generate a sequencing data set from a sample; and a non-transitory computer readable storage medium and / or a computer system.

[0284] In some embodiments, the non-transitory computer readable storage medium is encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform a method of the present disclosure.

[0285] In some embodiments, the computer system comprises a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform a method of the present disclosure.

[0286] In some embodiments, the sequencer is configured to generate a Whole Genome Sequencing (WGS) data set from the sample. In some embodiments, the system also includes a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample. The sample preparation device may include reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.

[0287] Systems of the present disclosure can include, e.g., reagents such as buffers and / or antibodies useful in the detection and quantification of histone modifications. In certain12953047v1 Page 96 of 224Attorney Docket: 2014191-0043 embodiments, a system of the present disclosure can include at least one antibody that selective binds a histone modification selected from H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, or H3K4me3, or pan acetylation. In certain embodiments, a system of the present disclosure can include at least one antibody that selective binds H3K4me3 modifications. In certain embodiments, a system of the present disclosure can include at least one antibody that selective binds H3K27ac modifications. A system of the present disclosure can include instructional materials disclosing or describing the use of the system in a method of determining MS status and / or treatment disclosed herein.

[0288] In some embodiments, a system of the present disclosure comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Tables 1-6.

[0289] In some embodiments, the system comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 1. In some embodiments, the system comprises reagents for quantifying H3K4me3 for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2. In some embodiments, the system comprises reagents for quantifying H3K27ac for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 2. In some embodiments, the system comprises reagents for quantifying H3K27ac for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 5. In some embodiments, the system comprises one or more antibodies for use in ChIP- seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac- modified histones.

[0290] In some embodiments, the system comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 3. In some embodiments, the system comprises reagents for quantifying DNA methylation for at least 5, 10, 20, 30, 40, or 50 genomic loci in Table 6. In some embodiments, the system comprises one or more methyl- binding domains (e.g., for use in MBD-seq). In some embodiments, the system comprises one or more antibodies that can bind methylated DNA (e.g., for use in MeDIP).

[0291] In some embodiments, the system comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample. In some embodiments, the sequencer comprises reagents for library preparation for sequencing. In some embodiments, the sequencer comprises12953047v1 Page 97 of 224Attorney Docket: 2014191-0043 reagents for sequencing. In some embodiments, the system comprises instructions for determining if a subject has MS.

[0292] In some embodiments, the system comprises reagents for measuring chromatin accessibility via an ATAC-seq assay. Definitions

[0293] “A” or “An”: The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” refers to one element or more than one element.

[0294] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context, to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” can encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or within a fraction of a percent, of the referenced value.

[0295] “Accessibility Status” or “Chromatin Accessibility Status”: As used herein, “accessibility status” or “chromatin accessibility status” of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of accessible chromatin. Accessibility status can be determined by various assays known in the art, including without limitation ChIP-seq as one example. Where two samples are separately analyzed by the same assay or comparable assays for detection of accessible DNA sequences, differences in chromatin accessibility status of genomic loci can be detected. Accessibility status can be compared to a standard or reference. A sample that has an accessibility status that differs in accessibility status from a standard or reference can be referred to as differentially modified. Suitable assays for determining chromatin accessibility are known in the art. Exemplary assays include ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing), FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), Mnase-seq (Micrococcal Nuclease digestion with sequencing), and / or a Dnase hypersensitivity assay.12953047v1 Page 98 of 224Attorney Docket: 2014191-0043

[0296] Administration: As used herein, the term “administration” typically refers to the administration of a disease appropriate (e.g., MS appropriate) treatment. In some embodiments, the disease appropriate treatment may comprise administering a composition to a subject, for example to achieve delivery of an agent that is, is included in, or is otherwise delivered by, the composition. In some embodiments, the disease appropriate treatment may comprise administering an appropriate surgical procedure or radiological procedure, optionally in combination with administration of a composition.

[0297] Agent: As used herein, the term “agent” may refer to any chemical or physical entity, including without limitation any of one or more of an atom, e.g., a radioactive atom, molecule, compound, conjugate, polypeptide, polynucleotide, polysaccharide, lipid, cell, or combination or complex thereof.

[0298] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen (e.g., a heavy chain variable domain, a light chain variable domain, and / or one or more CDRs). Thus, the term antibody includes, without limitation, human antibodies, non-human antibodies, synthetic and / or engineered antibodies, fragments thereof, and agents including the same. Antibodies can be naturally occurring immunoglobulins (e.g., generated by an organism reacting to an antigen). Synthetic, non-naturally occurring, or engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those of skill in the art.

[0299] As is well known in the art, typical human immunoglobulins are approximately 150 kD tetrameric agents that include two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other to form a structure commonly referred to as a “Y-shaped” structure. Typically, each heavy chain includes a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain includes three CH domains: CH1, CH2 and CH3. A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the immunoglobulin. Each light chain includes a light chain variable domain (VL) and a light chain constant domain (CL), separated from one another by another “switch.” Each variable domain contains three hypervariable loops12953047v1 Page 99 of 224Attorney Docket: 2014191-0043 known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). In each VH and VL, the three CDRs and four FRs are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of a heavy and / or a light chain are typically understood to provide a binding moiety that can interact with an antigen. Constant domains can mediate binding of an antibody to various immune system cells (e.g., effector cells and / or cells that mediate cytotoxicity), receptors, and elements of the complement system. Heavy and light chains are linked to one another by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. When natural immunoglobulins fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three- dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure.

[0300] In some embodiments, an antibody is a polyclonal, monoclonal, monospecific, or multispecific antibody (e.g., a bispecific antibody). In some embodiments, an antibody includes at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at least one heavy chain-light chain dimer, or a tetramer that includes two heavy chain monomers and two light chain monomers. Moreover, the term “antibody” can include (unless otherwise stated or clear from context) any art-known constructs or formats utilizing antibody structural and / or functional features including without limitation intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), Small Modular ImmunoPharmaceuticals (SMIPs), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®,12953047v1 Page 100 of 224Attorney Docket: 2014191-0043 Centyrins®, KALBITOR®s, chimeric antigen receptors (CARs), engineered T-cell receptors (TCRs), and antigen-binding fragments of any of the above.

[0301] In various embodiments, an antibody includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR) or variable domain. In some embodiments, an antibody can be a covalently modified (“conjugated”) antibody (e.g., an antibody that includes a polypeptide including one or more canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked with one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule). In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art.

[0302] An antibody including a heavy chain constant domain can be, without limitation, an antibody of any known class, including but not limited to, IgA, secretory IgA, IgG, IgE and IgM, based on heavy chain constant domain amino acid sequence (e.g. include but are not limited to human IgG1, IgG2, IgG3 and IgG4. “Isotype” refers to the Ab class or subclass (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes. As used herein, a “light chain” can be of a distinct type, e.g. amino acid sequence of the light chain constant domain. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human immunoglobulins. Naturally produced immunoglobulins are glycosylated, typically on the CH2 domain. As is known in the art, affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, antibodies produced and / or utilized in accordance with the present disclosure include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation.

[0303] In some embodiments, an antibody can be specific for a particular histone modification (e.g., an antibody can bind one histone modification, e.g., H3K27ac with a higher affinity than other histone modifications, under conditions that are commonly used in ChIP-seq12953047v1 Page 101 of 224Attorney Docket: 2014191-0043 experiments). In some embodiments, an antibody is specific for an H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, or H3K4me3 modification. In some embodiments, an antibody is specific for an H3K27ac modification. In some embodiments, an antibody is specific for an H3K4me3 modification.

[0304] In some embodiments, an antibody is a “pan” antibody. As used herein, the term pan antibody refers to an antibody that can bind a group of histone modifications having one or more features that are similar. In some embodiments, a pan antibody is a pan-methylation antibody (e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one methylated lysine, wherein the at least one methylated lysine can be at any one of a plurality of amino acid positions, e.g., in some embodiments, a pan-methylation antibody can bind an H3 protein comprising a methylated lysine at any position). In some embodiments, a pan antibody is a pan-acetylation antibody (e.g., an antibody that can bind a histone, e.g., H3 that comprises at least one acetylated lysine, wherein the at least one acetylated lysine can be at any one of a plurality of amino acid positions, e.g., a pan-acetylation antibody can bind an H3 protein comprising an acetylated lysine at any position). In some embodiments, a pan antibody can bind one or more histone modifications that are associated with transcription activation. In some embodiments, a pan antibody can bind one or more histone modifications that are associated with transcription silencing.

[0305] Antibody fragment: As used herein, an “antibody fragment” refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigen-binding portion or variable region thereof. An antibody fragment can be produced by any means. For example, in some embodiments, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment can be recombinantly produced, i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment can be wholly or partially synthetically produced. In some embodiments, an antibody fragment (particularly an antigen-binding antibody fragment) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids.12953047v1 Page 102 of 224Attorney Docket: 2014191-0043

[0306] Associated with: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and / or form of one is correlated with that of the other. For example, a particular entity (e.g., an epigenetic profile comprising one or more histone modifications at a set of genomic loci, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non- covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, or a combination thereof.

[0307] “Between” or “From”: As used herein, the term “between” refers to content that falls between indicated upper and lower, or first and second, boundaries, inclusive of the boundaries. Similarly, the term “from”, when used in the context of a range of values, indicates that the range includes content that falls between indicated upper and lower, or first and second, boundaries, inclusive of the boundaries.

[0308] Biological Sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell) of interest, as described herein. In some embodiments, a biological source is or includes an organism, such as a human subject. In some embodiments, a biological sample is or includes a biological tissue or fluid. In some embodiments, a biological sample can be or include cells, tissue, or bodily fluid. “Bodily fluids” refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., blood, serum, plasma, Cowper’s fluid or pre- ejaculate fluid, chyle, chyme, stool, interstitial fluid, intracellular fluid, lymph, menses, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vitreous humor, vomit). In some embodiments, a biological sample can be or include blood, blood components, cell-free DNA (cfDNA), ascites, biopsy samples, surgical specimens, cell-containing body fluids, sputum, saliva, feces, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, gynecological12953047v1 Page 103 of 224Attorney Docket: 2014191-0043 fluids, secretions, excretions, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings or lavages such as a ductal lavages or bronchoalveolar lavages, aspirates, scrapings, or bone marrow. In some embodiments, a biological sample is a liquid biopsy sample obtained from a bodily fluid. In some embodiments, a biological sample is or includes DNA obtained from a single subject or from a plurality of subjects. A biological sample can be a “primary sample” obtained directly from a biological source or can be a “processed sample”, i.e., a sample that was derived from a primary sample, e.g., via dilution, purification, mixing with one or more reagents, or any other processing step(s) as described herein. A biological sample can also be referred to as a “sample.”

[0309] Blood component: As used herein, the term “blood component” refers to any component of whole blood, including red blood cells, white blood cells, plasma, platelets, endothelial cells, mesothelial cells, epithelial cells, and cell-free DNA (cfDNA). Blood components also include the components of plasma, including proteins, metabolites, lipids, nucleic acids, and carbohydrates, and any other cells that can be present in blood, e.g., due to pregnancy, organ transplant, infection, injury, or disease.

[0310] Combination therapy: As used herein, the term “combination therapy” refers to administration to a subject of two or more therapeutic agents or therapeutic regimens such that the two or more therapeutic agents or therapeutic regimens together treat a disease, condition, or disorder of the subject. In some embodiments, the two or more therapeutic agents or therapeutic regimens can be administered simultaneously, sequentially, or in overlapping dosing regimens. Those of skill in the art will appreciate that combination therapy includes but does not require that the two therapeutic agents or therapeutic regimens be administered together in a single composition, nor at the same time.

[0311] Corresponding to: As used herein, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of skill in the art appreciate that residues in a provided polypeptide or polynucleotide sequence are often designated (e.g., numbered or12953047v1 Page 104 of 224Attorney Docket: 2014191-0043 labeled) according to the scheme of a related reference sequence (even if, e.g., such designation does not reflect literal numbering of the provided sequence). By way of illustration, if a reference sequence includes a particular amino acid motif at positions 100-110, and a second related sequence includes the same motif at positions 110-120, the motif positions of the second related sequence can be said to “correspond to” positions 100-110 of the reference sequence. Those of skill in the art appreciate that corresponding positions can be readily identified, e.g., by alignment of sequences, and that such alignment is commonly accomplished by any of a variety of known tools, strategies, and / or algorithms, including without limitation software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, Hhpred / Hhsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE. Two sequences can be identified as corresponding if they are identical or if they share substantial identity, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, e.g., over a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more residues. In various embodiments, a nucleic acid sequence can correspond to a sequence that is identical or substantially identical (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the complement of the nucleic acid sequence, e.g., over a length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more nucleic acid residues.

[0312] “Diagnosing”, “Detecting”, “Determining” or “Screening for”: As used herein, “diagnosing”, “detecting”, “determining”, “screening for” the presence of a condition or disease (e.g., MS), or a related state (e.g., responsiveness of MS to one or more MS therapies) includes the act, process, and / or outcome of determining whether, and / or the qualitative of quantitative probability that, a subject has or will develop the condition, disease, or related state. In some instances, diagnosing can include a determination relating to prognosis and / or likely response to one or more general or particular therapeutic agents or regimens.

[0313] Differentially accessible: As used herein, the term “differentially accessible” describes a genomic locus for which chromatin accessibility status differs between a first condition or sample and a second condition or sample (e.g., a standard or reference). A differentially accessible genomic locus can include a greater or smaller measured accessibility12953047v1 Page 105 of 224Attorney Docket: 2014191-0043 under a selected condition of interest, such as MS, as compared to a reference state, such as a healthy subject.

[0314] Differentially modified: As used herein, the term “differentially modified” describes a genomic locus for which histone modification status and / or DNA methylation status differs between a first condition or sample and a second condition or sample (e.g., a standard or reference). A differentially modified genomic locus can include a greater or smaller number or frequency of histone modification and / or DNA methylations under a selected condition of interest, such as MS, as compared to a reference state, such as a healthy subject.

[0315] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules) and / or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided sequences are known in the art. The term “% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between protein and nucleic acid sequences as determined by the match between strings of such sequences. “Identity” (often referred to as “similarity”) can be readily calculated by known methods, including those described in: Computational Molecular Biology (Lesk, A. M. ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W. ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M. and Griffin, H. G. eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G. ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J. eds.) Oxford University Press, NY (1992), each of which are separately incorporated by reference in their entirety. Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. For example, calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences (or the complement of one or both sequences) for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non- identical sequences can be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied12953047v1 Page 106 of 224Attorney Docket: 2014191-0043 by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally accounting for the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a computational algorithm, such as BLAST (basic local alignment search tool). Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp, Comp Appl Biosci (1989) 5(2):151-153), incorporated by reference herein in its entirety, with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul et al., J Mol Biol (1990) 215:403-410); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput Methods Genome Res [Proc Int Symp] (1994), Meeting Date 1992, 111-120. Eds. Suhai, Sandor. Plenum, New York, NY (the contents of each of which is separately incorporated herein by reference in its entirety). Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. “Default values” will mean any set of values or parameters, which originally load with the software when first initialized.

[0316] “Improve,” “increase,” “inhibit,” or “reduce”: As used herein, the terms “improve”, “increase”, “inhibit”, and “reduce”, and grammatical equivalents thereof, indicate qualitative or quantitative difference from a reference.

[0317] Methylation Status: As used herein, “methylation status” of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of DNA methylated sequences and / or the density (e.g., the measured density) of DNA methylation corresponding to the genomic locus. Methylation status can be determined by various assays known in the art, including without limitation Bisulfite12953047v1 Page 107 of 224Attorney Docket: 2014191-0043 sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq). Where two samples are separately analyzed by the same assay or comparable assays for detection of DNA methylated sequences, differences in methylation status of genomic loci can be detected. Methylation status can be compared to a standard or reference. A sample that has a methylation status that differs from a standard or reference can be referred to as differentially modified.

[0318] “Modification Status” or “Histone Modification Status”: As used herein, “modification status” or “histone modification status” of a genomic locus refers to the frequency with which DNA sequences corresponding to the genomic locus are identified in an assay for detection of DNA sequences associated with histones bearing one or more histone modifications (e.g., one or more particular histone modifications) and / or the density (e.g., the measured density) of histone modifications (e.g., one or more particular histone modifications) corresponding to the genomic locus. Modification status can be determined by various assays known in the art, including without limitation ChIP-seq as one example. Other well-known assays include CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing and CUT&Tag (Cleavage Under Targets and Tagmentation). Where two samples are separately analyzed by the same assay or comparable assays for detection of DNA sequences associated with histones bearing one or more histone modifications (e.g., one or more particular histone modifications), differences in modification status of genomic loci can be detected. Modification status can be compared to a standard or reference. A sample that has a modification status that differs in modification status or histone modification status from a standard or reference can be referred to as differentially modified.

[0319] Regulatory sequence: As used herein in the context of expression of a nucleic acid coding sequence, a regulatory sequence is a nucleic acid sequence that controls expression of a coding sequence, e.g., a promoter sequence or an enhancer sequence. In some embodiments, a regulatory sequence can control or impact one or more aspects of gene expression (e.g., cell- type-specific expression, inducible expression, etc.).

[0320] Subject: As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a disease, disorder or12953047v1 Page 108 of 224Attorney Docket: 2014191-0043 condition (e.g., MS). In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject is not suffering from a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject has one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a subject that has been tested for a disease, disorder, or condition, and / or to whom therapy has been administered. In some instances, a human subject can be interchangeably referred to as a “patient” or “individual”.

[0321] Therapeutic agent: As used herein, the term “therapeutic agent” refers to any agent that elicits a desired pharmacological effect when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population can be a population of model organisms or a human population. In some embodiments, an appropriate population can be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used for treatment of a disease, disorder, or condition (e.g., MS). In some embodiments, a therapeutic agent is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a therapeutic agent is an agent for which a medical prescription is required for administration to humans.

[0322] Therapeutically effective amount: As used herein, “therapeutically effective amount” refers to an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition (e.g., MS) in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular12953047v1 Page 109 of 224Attorney Docket: 2014191-0043 individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0323] Treatment: As used herein, the term “treatment” (also “treat” or “treating”) refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, or condition, or is administered for the purpose of achieving any such result. In some embodiments, such treatment can be of a subject who does not exhibit signs of the relevant disease, disorder, or condition and / or of a subject who exhibits only early signs of the disease, disorder, or condition (e.g., MS). Alternatively, or additionally, such treatment can be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, or condition. A “prophylactic treatment” includes a treatment administered to a subject who does not display signs or symptoms of a condition to be treated or displays only early signs or symptoms of the condition to be treated such that treatment is administered for the purpose of diminishing, preventing, or decreasing the risk of developing the condition. Thus, a prophylactic treatment functions as a preventive treatment against a condition. A “therapeutic treatment” includes a treatment administered to a subject who displays symptoms or signs of a condition and is administered to the subject for the purpose of reducing the severity or progression of the condition.12953047v1 Page 110 of 224Attorney Docket: 2014191-0043EXAMPLES

[0324] The present Examples demonstrate the identification and use of differentially modified and / or differentially accessible genomic loci in cfDNA in plasma samples obtained from subjects with MS. Loci identified in the present example can be useful, e.g., for detecting MS, characterizing MS disease severity, monitoring MS, and informing patient treatment decisions. Example 1: Materials and Methods

[0325] The present Example describes exemplary materials and methods that can be used to generate the sequencing data characterized in Example 2 to detect and characterize MS. Materials Plasma samples

[0326] Plasma samples were prepared from whole blood collected in EDTA blood collection tubes or Streck cell-free DNA BCT within 4-6 hours of collection and plasma wasstored at - multiple sclerosis (MS) patients andhealthy patients under a protocol approved by an IRB. MS patients had previously been determined to have progressive MS (PMS) or relapsing remitting MS (RRMS). Clinical evaluation and magnetic resonance imaging had previously been collected for MS patients. Methods Chromatin immunoprecipitation (ChIP)

[0327] Chromatin immunoprecipitation (ChIP) for histone marks (H3K4me3 and H3K27ac) in plasma samples was performed using methods similar to those previously described in Sadeh et al., Nat Biotechnol (2021) 39: 586-598 and Jang et al., Life Sci Alliance (2023) 6(12):e202302003. Briefly, about 1 mL frozen plasma was thawed and then prepared for ChIP. The thawed plasma was incubated with antibodies that bind H3K4me3 modifications or H3K27ac modifications that were previously conjugated to magnetic epoxy beads (Invitrogen)12953047v1 Page 111 of 224Attorney Docket: 2014191-0043 with constant mild shaking overnight. The beads were then washed and rinsed. Sequencing libraries were generated from purified immunoprecipitated sample DNA and then sequenced. Methylated DNA enrichment

[0328] Enrichment of DNA methylation was performed on DNA extracted from human plasma samples using the EpiMark® Methylated DNA Enrichment Kit (E2600S, available from New England Biolabs) following the manufacturer’s protocol. Briefly, cfDNA libraries were prepared and adaptors ligated. Then, the EpiMark® capture reagent was applied to each library sample following the manufacturer’s protocol. Enriched DNA libraries were amplified and sequenced. ChIP-seq and DNA methylation data analysis

[0329] ChIP-sequencing reads and MeDIP-sequencing reads were aligned to the human genome build hg19 using the Burrows-Wheeler Aligner (BWA) version 0.7.15. Non-uniquely mapping and redundant reads were discarded. MACS v2.2.7.1 was used for peak calling with a q- value (FDR) threshold of 0.01. Data quality was evaluated by a variety of measures, including total peak number, FrIP (fraction of reads in peak) score, number of high-confidence peaks (enriched > ten-fold over background), and percent of peak overlap with “blacklist” DHS peaks derived from the ENCODE project (Amemiya et al., Sci Rep (2019) 9(1):9354). Peaks were assessed for overlap with gene features and CpG islands using annotatr. IGV was used to visualize normalized read counts at specific genomic loci. Overlap of peaks was assessed using BEDTools and the GenomicRanges package in Bioconductor. Peaks were considered overlapping if they shared one or more nucleotide. Sequence data processing

[0330] cfChIP-seq reads were aligned to the hg19 human genome build using the Burrows-Wheeler Aligner (BWA) version 0.7.1740. Non-uniquely mapping and redundant reads were discarded. MACS v2.1.1.2014061641 was used for ChIP-seq peak calling with a q-value (FDR) threshold of 0.01. Fragment locations were converted to bed files using bedtools (v2.29.2)12953047v1 Page 112 of 224Attorney Docket: 2014191-0043 bamtobed with the -bedpe flag set. For analyses involving overlap with genomic regions, fragments were imported as Granges objects and collapsed to 1bp at the center of the fragment location to ensure a fragment can map to only one site.

[0331] ChIP-seq data quality was evaluated by several measures, including the number of total unique fragments and total peaks. The distribution of fragment sizes was assessed to verify the expected bi-modal or tri-modal distribution characteristic of cfDNA.

[0332] To assess immunoprecipitation specificity, on-target to off-target enrichment ratio was calculated. The enrichment ratio reflects the density of fragments mapping to sites that are marked in most cell types (on-target sites) compared to sites that are not marked in any cell type (off-target sites). On-target sites were identified from the 18-state chromHMM maps generated by Epimap (website: egg2.wustl.edu / roadmap / web_portal / chr_state_learning.html#exp_18state; accessed 10 / 4 / 21). For H3K4me3 on-target sites, 200bp windows were selected with any of the following “active” chromatin states in > 50% of tissues in Epimap: 1_TssA, 2_TssFlnk, 3_TssFlnkU, 4_TssFlnkD, 8_EnhG2, and 14_TssBiv. Off-target sites were defined as 200bp windows that lacked the on-target annotations in all of 129 samples used to generate chromatin state maps in Epimap. On-target and off-target windows were merged and retained if the merged windows spanned 1,000bp or more. Off-target regions within 10,000bp of on-target regions were excluded.

[0333] Unless otherwise specified, samples were included in downstream analysis if the following criteria were met: MBD: on-target to off-target enrichment ratio was > 4 and the product of the unique fragment number and enrichment ratio was > 1 x 107. Enhancers: on-target to off-target enrichment ratio was > 200 and the product of the unique fragment number and enrichment ratio was > 2 x 106. Promoters: on-target to off-target enrichment ratio was > 500 and the product of the unique fragment number and enrichment ratio was > 2 x 106.12953047v1 Page 113 of 224Attorney Docket: 2014191-0043 Identification of MS correlated regulatory elements

[0334] Normalized cfChIP-seq read counts at specific genomic loci were visualized with IGV v2.8.243. The fGSEA and GREAT tool48 (V3.0) (Korotkevich, G., Sukhov, V., Budin, N., Shpak, B., Artyomov, M. N., & Sergushichev, A. (2016). Fast gene set enrichment analysis. bioRxiv) were used to assess for enrichment of Gene Ontology (GO) and MsigDB biological process / cell type signature annotations among genes near CREs. Assessment of gene promoter activity based on H3K4me3

[0335] To estimate gene promoter activity, H3K4me3 was quantified within 1 kb of transcription start sites. Each peak was corrected for local background signal and the distribution of promoter counts were quantile normalized across samples. Differential promoter signals were quantified using Deseq2. Assessment of gene enhancer activity based on H3K27ac

[0336] H3K27ac cfChIP-seq peak calls present in 3 or greater MS / healthy samples were merged into a single Granges object and reduced to non-overlapping intervals. Peaks in high- noise regions were removed. Each peak was corrected for local background signal and the distribution of corrected enhancer counts were quantile normalized across samples. Differential enhancer signals were quantified using Deseq2. Each enhancer region was then linked to its nearest gene, within 50kb. If genes were linked to multiple enhancers, only the enhancer with the lowest adjusted p-value in the healthy vs MS differential test was retained. Genes were then rank ordered by the log2(Fold Change) of enhancer signal in the MS vs healthy test and gene set enrichment analysis was performed on the ordered list. To control for biases in enhancer-gene linking, this process was repeated 1000 times with the MS / healthy labels shuffled and the number of times a shuffled test produced a more extreme fGSEA normalized enrichment score than the true test was used to calculate an empirical p-value. For each gene set the more conservative of the true test fGSEA p-value and the empirical p-value was utilized for plotting and calling significance.12953047v1 Page 114 of 224Attorney Docket: 2014191-0043 Example 2: MS Detection and Characterization Using cfDNA Detected in MS Subjects

[0337] The present example describes an experiment that was performed to characterize differences in cfDNA and the epigenome of subjects with multiple sclerosis (MS). The present study involved 26 subjects having progressive MS, 4 subjects having relapsing remitting MS (RRMS), and 23 healthy controls (HC). Clinical evaluation and MRI was collected for subjects with MS. Blood samples were collected from healthy subjects and subjects having MS at regular intervals. For subjects having MS, TSPO-PET imaging using [11C]PK11195- ligand was conducted concurrently with blood sampling.1 mL of plasma was used to capture genome-wide activating enhancers, promoter signals, and repressive DNA hypermethylation. Dynamic epigenomic regions were identified in a discovery cohort comprising 18 MS patients and the HC group. Plasma from rheumatoid arthritis and systemic lupus erythematosus patients was also analyzed (3 subjects for each). Provided below is a table summarizing characteristics of subjects characterized in the present Example. Total Samples (Samples with 3 High Qualit Anal tes)

[0338] Genomic loci likely to differentiate MS and healthy subjects based on H3K4me3 modification, H3K27ac modification, and / or DNA methylation were first identified. To12953047v1 Page 115 of 224Attorney Docket: 2014191-0043 accomplish this, for each analyte, a universal peak map was created using plasma samples obtained from healthy subjects and MS subjects. Reads in each peak region were quantified in a manner that accounts for different sequencing depths and ChIP QC quality (e.g., based on count reads and then quantile normalizing each sample to a common reference distribution). Genomic loci that had differential analyte signal between MS and healthy subjects were determined using DESeq2 (Love et al., Genome Biol (2014) 15(12):550). These differential loci are shown in Table 1 (loci for which H3K4me3 modifications were found to increase), Table 4 (loci for which H3K4me3 modifications were found to decrease), Table 2 (loci for which H3K27ac modifications were found to increase), Table 5 (loci for which H3K27ac modifications were found to decrease), Table 3 (loci for which DNA methylation was found to increase) and Table 6 (loci for which DNA methylation was found to decrease) and grouped in accordance with the status they correlated with, i.e., Genomic locus (MS) or Genomic locus (healthy).

[0339] This analysis resulted in the identification of 2276 genomic loci having differential H3K4me3 signal (Tables 1 and 4), 1184 genomic loci having differential H3K27ac signal (Tables 2 and 5) and 690 genomic loci having differential DNA methylation (Tables 3 and 6) in the MS state as compared to the healthy state.

[0340] Whole genome sequencing was performed on the cfDNA collected in plasma samples in healthy and MS subjects. As shown in Fig.2(A), elevated amounts of mitochondrial DNA was surprisingly detected in MS subjects as compared to healthy subjects. This effect was also observed in RA and SLE subjects, demonstrating that detection of mitochondrial DNA in cfDNA can be a useful method for detecting immune disorders (e.g., autoimmune disorders (e.g., MS, Rheumatoid Arthritis (RA), and systemic lupus erythematosus (SLE))).

[0341] Promoter (H3K4me3), enhancer signal (H3K27ac), and DNA methylation signal was also compared between MS subjects and healthy subjects, and differentially modified regions were identified. Fig.2(B)-2(D) show volcano plots for all loci sequenced for each of H3K4me3, H3K27ac, and DNAme. As shown in Fig.2(B)-2(D), a number of loci were identified that were differentially modified in MS subjects.

[0342] Differentially modified promoter loci were then analyzed to identify biological mechanisms underlying the MS disease state. Figs.3(A)-(F) provide results of this analysis. As shown in Figs.3(A)-(D), TNFRSF14 was among the genes found to be differentially modified.12953047v1 Page 116 of 224Attorney Docket: 2014191-0043 As shown in Fig.3(B), MS subjects were found to have a statistically significant increase in promoter signal at the TNFRSF14 locus as compared to healthy subjects. Also shown, as an internal control is promoter signal for two housekeeping genes (ACTB and GAPDH). TNFRSF14 and its ligand, TNFS14, have previously been identified as MS risk alleles (see, e.g., The International Multiple Sclerosis Genetics Consortium & The Wellcome Trust Case Control Consortium 2. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nature 476, 214–219 (2011)). Thus, the results provided in Fig.3 confirm that methods described herein capture biologically relevant insights regarding the MS disease state. The data provided in the present experiment also demonstrates that the loci / genes / pathways / cell identity genes described herein can be used to characterize MS disease state in a subject. The loci used in the present experiment are as follows: GAPDH (chr12:6,637,940-6,649,353), TNFRSF14 (chr1:2,484,387-2,491,864), and ACTB (chr7:5,561,396-5,578,851). As shown in Fig.3(D), increased TNFRSF14 promoter signal was only observed in MS and not in other autoimmune disorders.

[0343] Genes were then rank ordered by MS vs. healthy differential promoter signal, and gene set enrichment analysis (GSEA) was performed to characterize differentially modified genomic loci. Results are shown in Fig.3(E). Also shown in Fig.3(E) are terms from the Gene Ontology Consortium Biological Process (GOBP) for loci with normalized enrichment scores above 1.3 and FDR-adjusted p-values below 0.05 (see loci highlighted in purple). As shown in Fig.3(E), genomic loci associated with synaptic plasticity were among the differentially modified loci detected. Fig.3(F) provides promoter signal quantification for two genes associated with synaptic plasticity (SQSTM1 and LILRB2). As shown in Fig.3(F), increased promoter signal was observed for each of SQSTM1 and LILRB2 as compared to healthy subjects. This increased signal was not observed in all autoimmune disorders characterized (see LILRB2 data, showing no increase in SLE subjects). Thus, the data provided in Fig.3 indicates that upregulation of genes associated with synaptic plasticity (e.g., promoter signal associated with SQSTM1 and LILRB2) can be useful for detection and / or characterization of MS. The particular genomic loci used for LILRB was chr19:54,782,258-54,785,464 and the particular genomic loci used from SQSTM1 was chr5:179232387-179234388.12953047v1 Page 117 of 224Attorney Docket: 2014191-0043

[0344] Enhancer regions were assigned to their nearest gene within 50 kb. Genes were then rank ordered by MS vs. healthy differential enhancer signal and GSEA was performed using MSigDB cell type signature gene sets (C8) to characterize cell types with which the detected loci were associated. Provided in Fig.4 are the results of said analysis. As shown in Fig.4(A), oligodendrocyte progenitor cells, fetal muscle Schwann cells, fetal cerebrum astrocytes, fetal cerebellum astrocytes, and embryonic ctx excitatory neurons (Ex 4) were all found to be enriched. These results were consistent with certain MS biological processes, including oligodendrocyte differentiation, neurogenesis, spinal cord development, neuron projection development, and glial cell differentiation. The enriched cell types also likely indicate preferential cell damage / death of the detected cell types. Thus, Fig.4(A) again shows that technologies provided herein can detect biologically significant MS disease pathways and also provides several exemplary markers that can be used, e.g., to detect and / or characterize MS in a subject. Figs.4(B) and 4(C) provide quantification of three genes that were found to have differential enhancer signal: NSUN5, DAAM2, and CNTN2, each of which are markers of oligodendrocyte development. As shown in Fig.4(C), statistically significant increases in enhancer signal was observed for each of these genes. Thus, Fig.4(C) demonstrates that NSUN5, DAAM2, and CNTN2 activity (e.g., enhancer activity) can each be useful for characterizing MS disease state in a subject. Also provided in Fig.4(C) is quantification of NSUN5, DAAM2, and CTN2 enhancer signal for RA and SLE; the data provided in Fig.4(C) shows that increased NSUN5, DAAM2, and CNTN2 enhancer signal was specific to MS.

[0345] Surprisingly, enhancer signal was found to be under enriched for certain immune related signals in MS. See results provided in Fig.3(D), showing that enhancer signal decreased for genes associated with B lymphocyte Ovary CL18, Bone Marrow Naïve T cell, Fetal Lung Mature B cells, and Bone Marrow Folicular B cells. Similar results were observed for promoter signals. Interestingly, under enrichment of promoter signal was not observed for all immune cells. In particular, upregulation of certain T cell associated genes (e.g., TNFRSF14) was found to be increased in MS. As shown in Fig.3(E), the under enrichment in enhancer signal observed in MS was not observed in other autoimmune disorders (in par...

Claims

Attorney Docket: 2014191-0043 CLAIMS What is claimed is:

1. A method of detecting multiple sclerosis (MS) in a subject, the method comprising: quantifying, at one or more genomic loci in a biological sample, optionally in cell-free DNA (cfDNA) from a liquid biopsy sample, obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) binding of one or more transcription factors, and / or (iv) DNA methylation.

2. The method of claim 1, wherein the one or more histone modifications are quantified using a histone modification assay that measures one or more of H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, and pan-acetylation.

3. The method of claim 2, wherein the histone modification assay detects H3K4me3 modifications.

4. The method of claim 2, wherein the histone modification assay detects H3K27ac modifications.

5. The method of any one of claims 1-4, wherein the histone modification assay is selected from ChIP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.

6. The method of any one of claims 1-5, wherein chromatin accessibility is quantified using a chromatin accessibility assay selected from ATAC-seq (Assay of Transpose Accessible Chromatin sequencing), NOMe-seq (Nucleosome Occupancy and Methylome sequencing),12953047v1 Page 210 of 224Attorney Docket: 2014191-0043 FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements sequencing), MNase-seq (Micrococcal Nuclease digestion with sequencing), and a DNase hypersensitivity assay.

7. The method of any one of claims 1-6, wherein the binding of one or more transcription factors is quantified using a transcription factor binding assay.

8. The method of claim 7, wherein the transcription factor binding assay is selected from ChIP-seq (Chromatin ImmunoPrecipitation sequencing), CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing, and CUT&Tag (Cleavage Under Targets and Tagmentation) sequencing.

9. The method of any one of claims 1-8, wherein DNA methylation is quantified using Bisulfite sequencing (BS-Seq), Whole Genome Bisulfite Sequencing (WGBS), Methylated DNA ImmunoPrecipitation sequencing (MeDIP-seq), or Methyl-CpG-Binding Domain sequencing (MBD-seq).

10. The method of any one of claims 1-9, comprising quantifying two or more of the following, each at one or more genomic loci in cell-free DNA (cfDNA) from a liquid biopsy sample obtained or derived from the subject: (i) one or more histone modifications, (ii) chromatin accessibility, (iii) transcription factor binding, and / or (iv) DNA methylation.

11. The method of claim 10, comprising quantifying two or more histone modifications.

12. The method of claim 11, comprising quantifying H3K4me3 and H3K27ac modifications.

13. The method of claim 10, comprising quantifying one or more histone modifications and DNA methylation.12953047v1 Page 211 of 224Attorney Docket: 2014191-004314. The method of claim 13, comprising quantifying H3K4me3 and / or H3K27ac modifications and DNA methylation.

15. The method of claim 14, comprising quantifying H3K4me3 modifications, H3K27ac modifications and DNA methylation.

16. The method of any one of claims 1-15, wherein the liquid biopsy sample is a plasma sample, serum sample, or urine sample.

17. The method of any one of claims 1-16, wherein the method comprises quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in Tables 1-6.

18. The method of any one of claims 1-17, wherein an increase or decrease of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the one or more genomic loci as compared to a reference indicates that the subject has MS.

19. The method of any one of claims 1-18, wherein the method comprises quantifying: (a) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, or 700 genomic loci in Table 1; (b) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 genomic loci in Table 2; (c) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500, genomic loci in Table 3; (d) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 genomic loci in Table 4; (e) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, or 600 genomic loci in Table 5;12953047v1 Page 212 of 224Attorney Docket: 2014191-0043 (f) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, or 150 genomic loci in Table 6; or (g) or any combination of (a)-(f).

20. The method of any one of the previous claims, wherein the one or more genomic loci comprise one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell identity genes, or one or more regulatory regions thereof, and wherein an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell identity genes, or one or more regulatory regions thereof as compared to a reference indicates that the subject has MS.

21. The method of claim 20, wherein the one or more genomic loci comprise one or more enhancer regions associated with one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell, and wherein the method comprises quantifying enhancer signal (e.g., H3K27ac) at the one or more enhancer regions associated with the one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell identity gene, and wherein an increase in enhancer signal at the one or more enhancer regions associated with the one or more cell identity genes of an oligodendrocyte progenitor cell, an astrocyte, and / or an excitatory neuron cell identity gene indicates that the subject has MS.

22. The method of claim 21, wherein the one or more cell identity genes of an oligodendrocyte progenitor cell comprise NSUN5, DAAM2, CNTN2, or any combination thereof, optionally wherein: (a) the one or more enhancer regions associated with NSUN5 include chr7:72713576-72716059; and / or12953047v1 Page 213 of 224Attorney Docket: 2014191-0043 (b) the one or more enhancer regions of DAAM2 include chr6:39788740-39793623.

23. The method of claim 22, wherein the one or more genomic loci comprise one or more enhancer regions of NSUN5, DAAM2, CNTN2, or any combination thereof; wherein the method comprises quantifying enhancer signal (e.g., H3K27ac modifications) at the one or more enhancer regions of NSUN5, DAAM2, CNTN2, or any combination thereof; and wherein an increase in enhancer signal at the one or more enhancer regions of NSUN5, DAAM2, CNTN2, or any combination thereof indicates that the subject has MS.

24. The method of any one of the previous claims, wherein the one or more genomic loci comprise one or more gene markers of synaptic plasticity or one or more regulatory regions thereof, and wherein an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the one or more gene markers of synaptic plasticity or one or more regulatory regions thereof as compared to a reference indicates that the subject has MS.

25. The method of claim 24, wherein the one or more genomic loci comprise one or more promoter regions associated with one or more marker genes of synaptic plasticity, wherein the method comprises quantifying promoter signal (e.g., H3K4me3) at the one or more promoter regions associated with the one or more marker genes of synaptic plasticity; and wherein an increase in promoter signal at the one or more promoter regions associated with one or more marker genes of synaptic plasticity indicates that the subject has MS.

26. The method of claim 24 or 25, wherein the one or more marker genes of synaptic plasticity comprise SQSTM1 and / or LILRB2.12953047v1 Page 214 of 224Attorney Docket: 2014191-0043 27. The method of claim 26, wherein the one or more genomic loci comprise one or more promoter regions of SQSTM1 and / or LILRB2, wherein the method comprises quantifying promoter signal (e.g., H3K4me3) at the one or more promoter regions of SQSTM1 and / or LILRB2, and wherein an increase in promoter signal as compared to a reference indicates that the subject has MS.

28. The method of claim 27, wherein the promoter region of LILRB2 comprises chr19:54,782,258-54,785,464 and / or wherein the promoter region for SQSTM1 comprises chr5:179232387-179234388.

29. The method of any one of the previous claims, wherein the one or more genomic loci comprise one or more MS-risk alleles or one or more regulatory regions thereof, and wherein an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at the MS-risk alleles or one or more regulatory regions thereof as compared to a reference indicates that the subject has MS.

30. The method of claim 29, wherein the one or more genomic loci comprise one or more promoter regions associated with one or more MS-risk alleles, wherein the method comprises quantifying promoter signal (e.g., H3K4me3) at the one or more promoter regions associated with the one or more MS-risk alleles, and wherein an increase in promoter signal at the one or more promoter regions associated with the one or more MS-risk alleles indicates that the subject has MS.

31. The method of any one of the previous claims,12953047v1 Page 215 of 224Attorney Docket: 2014191-0043 wherein the one or more genomic loci comprise TNFRSF14 or one or more regulatory regions thereof, and wherein an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at TNFRSF14 or one or more regulatory regions thereof as compared to a reference indicates that the subject has MS.

32. The method of claim 31, wherein the one or more genomic loci comprise a TNFRSF14 promoter region, wherein the method comprises quantifying promoter signal (e.g., H3K4me3 modifications) at the TNFRSF14 promoter region, and wherein an increase in promoter signal as compared to a reference indicates the subject has MS.

33. The method of any one of claims 19 to 21, wherein the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and / or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a subject or a cohort of subjects that have not been diagnosed with MS.

34. The method of any one of the previous claims, wherein the subject has progressive MS or relapsing remitting MS.

35. A method for determining MS disease severity in a subject, comprising performing the method of any one of claims 1 to 34, and comparing to a reference sample.

36. The method of claim 35, wherein the one or more genomic loci comprise one or more microglial cell identity genes or one or more regulatory regions thereof, and wherein an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more microglial12953047v1 Page 216 of 224Attorney Docket: 2014191-0043 cell identity genes or one or more regulatory regions thereof as compared to a reference indicates that the subject has severe MS.

37. The method of claim 36, wherein the one or more genomic loci comprise one or more enhancer regions of one or more microglial cell identity genes, and wherein an increase in enhancer signal (e.g., H3K27ac) in one or more enhancer regions of one or more microglial cell identity genes indicates the subject has severe MS.

38. The method of claim 37, wherein the one or more genomic loci comprise one or more enhancer regions of MAP4K4 (e.g., chr2:102513113-102515404).

39. The method of any one of claims 35-38, wherein the reference is a predetermined threshold, a measurement from a liquid biopsy sample, and / or a normalized value, optionally wherein the reference is a measurement from a liquid biopsy sample obtained from a subject or a cohort of subjects that have diagnosed with moderate or severe MS (e.g., subjects having moderate or high TSPO PET signal, respectively).

40. A method of determining MS disease progression in a subject, the method comprising determining, at a first time point and a second time point, MS severity in the subject, wherein MS severity is determined at each point in time using the method of any one of claims 35-39.

41. A method of predicting disease flare and / or progression in a subject, the method comprising performing the method of any one of claims 1-40.

42. The method of claim 41,12953047v1 Page 217 of 224Attorney Docket: 2014191-0043 wherein the one or more genomic loci comprise one or more microglial cell identity genes or one or more regulatory regions thereof and / or one or more neutrophil cell identity genes or one or more regulatory regions thereof, and wherein an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more microglial cell identity genes or one or more regulatory regions thereof and / or one or more neutrophil cell identity genes or one or more regulatory regions thereof as compared to a reference indicates that the subject has an increased likelihood of experiencing disease flare and / or progression as compared to a reference, wherein the reference is optionally a plasma sample obtained for a subject or cohort of subjects having MS and not exhibiting an increase of one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more microglial cell identity genes or one or more regulatory regions thereof and / or one or more neutrophil cell identity genes or one or more regulatory regions thereof.

43. The method of claim 42, wherein the one or more genomic loci comprise one or more enhancer regions of one or more microglial cell identity genes, and wherein the method comprises quantifying enhancer signal (e.g., H3K27ac modifications) at the one or more enhancer loci of the one or more microglial cell identity genes.

44. The method of any one of claims 35-43, wherein the subject has previously been determined to have MS (e.g., using MRI and / or PET imaging).

45. A method of treating a subject, comprising determining the MS status of the subject using the method of any one of claims 1-34, and: (a) if the subject is determined to have MS, administering an MS therapeutic; and (b) if the subject is not determined to have MS, not administering an MS therapeutic.12953047v1 Page 218 of 224Attorney Docket: 2014191-0043 46. A method of treating a subject having MS, comprising determining MS severity in the subject using the method of any one of claims 35-38, wherein (a) if the subject is determined to have severe MS, administering a high or moderate efficacy MS therapeutic; and (b) if the subject is determined to have moderate disease severity, administering a low efficacy MS therapeutic.

47. A method of treating MS in a subject, comprising determining MS disease progression in the subject using the method of claim 40, wherein the subject is being administered a first MS therapeutic at the first time point and the second time point, and wherein: (a) if MS disease severity is determined to increase between the first and the second time points, administering a second MS therapeutic, wherein the second therapeutic has a higher efficacy than the first MS therapeutic; and (b) if MS disease severity is determined to stay the same and / or decrease between the first and the second time points, continuing treatment with the first MS therapeutic.

48. The method of claim 47, wherein: (a) the first MS therapeutic is a low efficacy MS therapeutic and the second MS therapeutic is a moderate or high efficacy MS therapeutic, or (b) the first MS therapeutic is a moderate efficacy MS therapeutic and the second MS therapeutic is a high efficacy MS therapeutic.

49. The method of claims 47 or 48, wherein: the low efficacy MS therapeutic is an interferon (e.g., beta- or beta- ), glatirameracetate, or teriflunomide; the moderate efficacy MS therapeutic is cladribine, an S1p inhibitor (e.g., fingolimod, siponimod, ozanimod, or ponesimod), or a fumarate (e.g., dimethyl, diroximel, or monomethyl fumarate); and the high efficacy MS therapeutic is Ocrelizumab, Ofatumumab, Natalizumab, or Alemtuzumab.12953047v1 Page 219 of 224Attorney Docket: 2014191-004350. A kit comprising reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci, wherein the one or more genomic loci are selected from Tables 1-6.

51. The kit of claim 50, wherein the kit comprises reagents for quantifying: (a) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, or 700 genomic loci in Table 1; (b) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 genomic loci in Table 2; (c) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500, genomic loci in Table 3; (d) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 genomic loci in Table 4; (e) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, or 600 genomic loci in Table 5; (f) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, or 150 genomic loci in Table 6; or (g) or any combination of (a)-(f).

52. The kit of claim 51, wherein the kit comprises one or more antibodies for use in ChIP- seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac- modified histones.

53. The kit of claim 51 or 52, wherein the kit comprises one or more methyl-binding domains for use in MBD-seq or wherein the kit comprises one or more antibodies that bind methylated DNA for use in MeDIP.

54. The kit of any one of claims 50-53, wherein the kit comprises reagents for isolation of cell-free DNA (cfDNA) from a liquid biopsy sample.12953047v1 Page 220 of 224Attorney Docket: 2014191-004355. The kit of any one of claims 50-54, wherein the kit comprises reagents for library preparation for sequencing.

56. The kit of any one of claims 50-55, wherein the kit comprises reagents for sequencing.

57. The kit of any one of claims 50-56, wherein the kit comprises instructions for determining if a subject has MS.

58. A non-transitory computer readable storage medium encoded with a computer program, wherein the program comprises instructions that when executed by one or more processors cause the one or more processors to perform operations to perform the method of any one of claims 1- 49.

59. A computer system comprising a memory and one or more processors coupled to the memory, wherein the one or more processors are configured to perform operations to perform the method of any one of claims 1-49.

60. A system for determining the MS status of a subject, the system comprising a sequencer configured to generate a sequencing data set from a sample; and a non-transitory computer readable storage medium of claim 58 and / or a computer system of claim 59.

61. The system of claim 60, wherein the sequencer is configured to generate a Whole Genome Sequencing (WGS) data set from the sample.

62. The system of claim 60 or 61 further comprising a sample preparation device configured to prepare the sample for sequencing from a biological sample, optionally a liquid biopsy sample.12953047v1 Page 221 of 224Attorney Docket: 2014191-0043 63. The system of claim 62, wherein the sample preparation device comprises reagents for quantifying one or more histone modifications, chromatin accessibility, binding of one or more transcription factors, and / or DNA methylation at one or more genomic loci in cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.

64. The system of claim 63, wherein the one or more genomic loci are selected from Tables 1-6.

65. The system of any one of claims 62-64, wherein the device comprises reagents for quantifying: (a) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, or 700 genomic loci in Table 1; (b) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500 genomic loci in Table 2; (c) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, or 500, genomic loci in Table 3; (d) H3K4me3 modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 genomic loci in Table 4; (e) H3K27ac modifications for at least 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, or 600 genomic loci in Table 5; (f) DNA methylation for at least 5, 10, 20, 30, 40, 50, 100, or 150 genomic loci in Table 6; or (g) or any combination of (a)-(f).

66. The system of any one of claims 63-65, wherein the reagents comprise one or more antibodies for use in ChIP-seq, optionally wherein the one or more antibodies specifically bind H3K4me3- or H3K27ac-modified histones.

67. The system of any one of claims 63-66, wherein the reagents comprise one or more methyl-binding domains for use in MBD-seq.12953047v1 Page 222 of 224Attorney Docket: 2014191-004368. The system of any one of claims 63-67, wherein the device comprises reagents for isolation of cell-free DNA (cfDNA) from the biological sample, optionally the liquid biopsy sample.

69. The system of any one of claims 63-68, wherein the device comprises reagents for library preparation for sequencing.

70. The system of any one of claims 63-69, wherein the sequencer comprises reagents for sequencing.12953047v1 Page 223 of 224

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