Methods for diagnosing and treating muscular dystrophy diseases

Abstract

A method for diagnosing facioscapulohumeral muscular dystrophy (FSHD) using one or more biomarkers is provided. More particularly, provided is a method for diagnosing FSHD in a subject, the method comprising detecting the presence of a biomarker or change in level of a biomarker in a biological sample from the subject, wherein the biomarker is wherein the biomarker is mannose binding lectin 2 (MBL2), junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), complement component 7 (C7), complement component 9 (C9), tenascin C (TNG), lumican (LUM), phosphatidylinositol-glycan-specific phospholipase D (GPLD1 ), complement component 4 binding protein, beta chain (C4BPB), carnosine dipeptidase (CNDP1), or immunoglobulin kappa variable 2-30 (IGKV2-30), or a combination of any two or more of MBL2, JUP, DSP, CLEC3B, C7, C9, TNG, LUM, GPLD1, C4BPB, CNDP1, or IGKV2-30. In some aspects, when the change in level of the biomarker is an increase in the level of MBL2, JUP, DSP, CLEC3B, C7, C9, TNG, and / or LUM, or a combination of any thereof, the subject is diagnosed as having FSHD. In some aspects, when the change in level of the biomarker is a decrease in the level of GPLD1, C4BPB, CNDP1, and / or IGKV2-30, or a combination of any thereof, the subject is diagnosed as having FSHD. Also provided is a method for determining efficacy of a therapeutic treatment for FSHD in a subject, the method comprising measuring the level of a biomarker in a biological sample from the subject before and after the treatment, and determining that the treatment is effective in treating FSHD if the level of the biomarker is decreased or increased relative to a reference level or to the level of the biomarker in the sample from the subject before treatment, wherein when the biomarker is mannose binding lectin 2 (MBL2), junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), complement component 7 (C7), complement component 9 (C9), tenascin C (TNG), or lumican (LUM), or a combination of any two or more of MBL2, JUP, DSP, CLEC3B, C7, C9, TNG, or LUM, the treatment is effective when the level of the biomarker decreases, or wherein when the biomarker is phosphatidylinositol-glycan-specific phospholipase D (GPLD1), complement component 4 binding protein, beta chain (C4BPB), carnosine dipeptidase (CNDP1), or immunoglobulin kappa variable 2-30 (IGKV2-30), or a combination of any two or more of GPLD1, C4BPB, CNDP1, or IGKV2-30, the treatment is effective when the level of the biomarker increases. In various aspects, the method further comprises treating the subject diagnosed with FSHD.

Description

METHODS FOR DIAGNOSING AND TREATING MUSCULAR DYSTROPHY DISEASESFIELD

[0001] This disclosure relates to the field of diagnosing a subject having a muscular dystrophy disease including, but not limited to, facioscapulohumeral muscular dystrophy (FSHD). In some aspects, the disclosure provides methods of using one or more biomarkers, or a combination of biomarkers, to detect the muscular dystrophy. In some aspects, the disclosure provides methods for diagnosing and treating a subject after the muscular dystrophy is diagnosed. Additionally, the disclosure provides methods for determining efficacy of a therapeutic treatment for FSHD in a subject by measuring levels of one or more biomarkers before and / or after treatment.BACKGROUND

[0002] Muscular dystrophies (MDs) are a group of genetic diseases. The group is characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some forms of MD develop in infancy or childhood, while others may not appear until middle age or later. The disorders differ in terms of the distribution and extent of muscle weakness (some forms of MD also affect cardiac muscle), the age of onset, the rate of progression, and the pattern of inheritance.

[0003] Facioscapulohumeral dystrophy (FSHD) is among the most commonly inherited muscular dystrophies, estimated to affect as many as 870,000 individuals. Classical descriptions of FSHD presentation include progressive muscle weakness in the face, shoulder-girdle and arms, but disease can manifest more broadly, including in muscles of the trunk and lower extremities. Variability is also commonly seen within individuals, as asymmetrical weakness is common. Age-at-onset can range from early childhood to adulthood, and is usually related to disease severity, where earlier onset is often associated with more severe muscle weakness. Although most patients with FSHD have a normal life span, respiratory insufficiency can occur, and the disease can be debilitating, as approximately 25% of affected individuals may become wheelchair dependent by their fifties, and even earlier in more severe forms of the disease, while others maintain lifelong ambulation.

[0004] FSHD is caused by aberrant expression of the double homeobox 4 gene (DUX4), which produces a transcription factor that is toxic to skeletal muscle. DUX4 is normally functional during the four-cell stage of human development but repressed thereafter in essentially all other tissues, except perhaps the testes, thymus, and skin. In skeletal muscles of people with FSHD, specific genetic and epigenetic factors conspire to permit DUX4 de-repression, where it then initiates several aberrant gene expression cascades, including those involved in differentiation abnormalities, oxidative stress, inflammatory infiltration, cell death and muscle atrophy.

[0005] Effective FSHD-targeted therapies would dramatically improve patient quality of life, but currently there are no approved treatments that slow FSHD progression or improve muscle weakness. FSHD has no cure or specific drug treatments, but there are supportive treatments that can help manage symptoms, including non-steroidal anti-inflammatory drugs and physical therapy. Further, some therapies, such as ARO-DUX4 (is an RNA interference (RNAi) conjugate designed to target DUX4 transcript; ClinicalTrials.gov ID: NCT06131983), RO7204239 (is a humanized monoclonal antibody that binds to human latent myostatin; ClinicalTrials.gov ID: NCT05548556), and AOC 1020 (is an Antibody Oligonucleotide Conjugate that targets DUX4 transcript; ClinicalTrials.gov ID: NCT06547216), are in clinical trials. Other therapies are being developed to silence genes, such as DUX4, to inhibit DUX4 expression in FSHD.

[0006] FSHD is often diagnosed through a combination of clinical examination, genetic testing, and other tests, but there remains a need in the art for new methods for diagnosing, predicting disease progression (prognosis) and treating FSHD.SUMMARY

[0007] The disclosure provides methods of diagnosing facioscapulohumeral dystrophy (FSHD) in a subject, the method comprising detecting the presence or change in level of a biomarker in a biological sample from the subject. In some aspects, the sample of the subject comprises extracellular vesicles (EVs) isolated from a bodily fluid of the subject.

[0008] The disclosure provides a method of diagnosing facioscapulohumeral dystrophy (FSHD) in a subject, the method comprising detecting the presence of a biomarker or change in level of a biomarker in a biological sample from the subject, wherein the biomarker is mannose binding lectin 2 (MBL2), junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), complement component 7 (C7), complement component 9 (C9), tenascin C (TNC), lumican (LUM), phosphatidylinositol-glycan-specific phospholipase D (GPLD1 ), complement component 4 binding protein, beta chain (C4BPB), carnosine dipeptidase (CNDP1 ), or immunoglobulin kappa variable 2-30 (IGKV2-30), or a combination of any two or more of MBL2, JUP, DSP, CLEC3B, 09, TNC, LUM, GPLD1 , C4BPB, CNDP1 , or IGKV2-30.

[0009] In some aspects, when the change in level of the biomarker is an increase in level of MBL2, JUP, DSP, CLEC3B, C7, C9, TNC, or LUM, or a combination of any two or more ofMBL2, JLIP, DSP, CLEC3B, C7, C9, TNC, or LUM, the subject is diagnosed as having FSHD.

[0010] In some aspects, when the change in level of the biomarker is a decrease in level of GPLD1 , C4BPB, CNDP1 , or IGKV2-30, or a combination of any two or more of GPLD1 , C4BPB, CNDP1 , or IGKV2-30, the subject is diagnosed as having FSHD.

[0011] In some aspects, the change in the level of the biomarker is compared to a reference level or to a control level. In some aspects, the change in the level of the biomarker is measured as being statistically significant compared to the reference level or to the control level.

[0012] In some aspects, the sample is tissue or fluid isolated from the subject. In some aspects, the fluid is plasma or serum. In some aspects, the fluid is plasma. In some aspects, the sample is an extracellular vesicle (EV) isolated from plasma of the subject. In some aspects, the biomarker is a protein. In some aspects, the biomarker is a nucleic acid. In some aspects, the method of diagnosing further comprises treating the subject after diagnosing the subject with FSHD. In some aspects, treating the subject comprises administering to the subject an effective amount of any of a non-steroidal anti-inflammatory drug, ARO-DUX4, RO7204239, and / or AGO 1020, or a combination of any thereof, and / or treating with physical therapy. In some aspects, treating the subject comprises administering an effective amount of a gene therapy to the subject.

[0013] In some aspects, MBL2 and GPLD1 are biomarkers indicative of FSHD in female subjects. In some aspects, JUP, CLEC3B, DSP, and C4BPB are biomarkers indicative of FSHD in male FSHD subjects. In some aspects, MBL2 level is increased in female FSHD subjects. In some aspects, MBL2 level is increased at least or about 2-fold, about 3-fold, about 4-fold, or about 5-fold in female FSHD subjects when compared to female control subjects. In some aspects, GPLD1 level is decreased in female FSHD subjects. In some aspects, GPLD1 level is decreased at least or about 1 .5-fold, about 2-fold, or about 2.3 fold in female FSHD subjects when compared to female control subjects.

[0014] In some aspects, JUP, CLEC3B, DSP, and C4BPB are biomarkers indicative of FSHD in male subjects. In some aspects, JUP, CLEC3B and DSP are exclusively expressed in male FSHD subjects and not present in male control subjects. In some aspects, JUP, CLEC3B, and DSP are increased in male FSHD subjects compared to male control subjects.

[0015] The disclosure provides a method for determining efficacy of a therapeutic treatment for FSHD in a subject, the method comprising measuring the level of a biomarker in a biological sample from the subject before and after the treatment, and determining thatthe treatment is effective in treating FSHD if the level of the biomarker is decreased or increased relative to a reference level or to the level of the biomarker in the sample from the subject before treatment, wherein when the biomarker is mannose binding lectin 2 (MBL2), junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), complement component 7 (C7), complement component 9 (C9), tenascin C (TNC), or lumican (LUM), or a combination of any two or more of MBL2, JUP, DSP, CLEC3B, C7, C9, TNC, or LUM, the treatment is effective when the level of the biomarker decreases, or wherein when the biomarker is phosphatidylinositol-glycan-specific phospholipase D (GPLD1 ), complement component 4 binding protein, beta chain (C4BPB), carnosine dipeptidase (CNDP1), or immunoglobulin kappa variable 2-30 (IGKV2-30), or a combination of any two or more of GPLD1 , C4BPB, CNDP1 , or IGKV2-30, the treatment is effective when the level of the biomarker increases.

[0016] In some aspects, the sample is tissue or fluid isolated from the subject. In some aspects, the fluid is plasma or serum. In some aspects, the fluid is plasma. In some aspects, the sample is an extracellular vesicle (EV) isolated from plasma of the subject. In some aspects, the biomarker is a protein. In some aspects, the biomarker is a nucleic acid. In some aspects, the method of diagnosing further comprises treating the subject after diagnosing the subject with FSHD. In some aspects, treating the subject comprises administering to the subject an effective amount of any of a non-steroidal anti-inflammatory drug, ARO-DUX4, RO7204239, and / or AGO 1020, or a combination of any thereof, and / or treating with physical therapy.

[0017] The disclosure provides a method of diagnosing facioscapulohumeral dystrophy (FSHD) in a female subject, the method comprising detecting a change in level of a biomarker in a biological sample from the subject, wherein the biomarker is mannose binding lectin 2 (MBL2) or phosphatidylinositol-glycan-specific phospholipase D (GPLD1 ), or a combination of MBL2 and GPLD1 , wherein when the level of MBL2 is increased in the subject relative to a reference level or a control level, and / or wherein when the level of GPLD1 is decreased in the subject relative to a reference level or a control level, the subject is diagnosed as having FSHD.

[0018] The disclosure provides a method of diagnosing facioscapulohumeral dystrophy (FSHD) in a male subject, the method comprising detecting the presence or a change in level of a biomarker in a biological sample from the subject, wherein the biomarker is junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), or complement component 4 binding protein, beta chain (C4BPB), or a combination of any two or more of JUP, DSP, CLEC3B, or C4BPB, the subject is diagnosed as having FSHD. In some aspects, the subject is diagnosed as having FSHD when JUP, CLEC3B, or DSP, or a combination ofany thereof, is present in the biological sample. In some aspects, the subject is diagnosed as having FSHD wherein the level of any of JUP, CLEC3B, or DSP, or a combination thereof, is increased in the subject compared to the level of the level of any of JUP, CLEC3B, or DSP in a control.

[0019] In some aspects, the sample is tissue or fluid isolated from the subject. In some aspects, the fluid is plasma or serum. In some aspects, the fluid is plasma. In some aspects, the sample is an extracellular vesicle (EV) isolated from plasma of the subject. In some aspects, the biomarker is a protein. In some aspects, the biomarker is a nucleic acid. In some aspects, the method of diagnosing further comprises treating the subject after diagnosing the subject with FSHD. In some aspects, treating the subject comprises administering to the subject an effective amount of any of a non-steroidal anti-inflammatory drug, ARO-DUX4, RO7204239, and / or AOC 1020, or a combination of any thereof, and / or treating with physical therapy.

[0020] Further aspects and advantages of the disclosure will be apparent to those of ordinary skill in the art from a review of the following detailed description, taken in conjunction with the drawings. It should be understood, however, that the detailed description (including the drawings and the specific examples), while indicating embodiments of the disclosed subject matter, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Fig. 1 A-E shows a characterization of plasma EVs isolated by Size Exclusion (SEC). Fig. 1 A is a transmission electron microscopy (TEM) image showing an EV and its lipid bilayer membrane. Fig. 1 B-C shows size and concentration of EVs determined by Nanoparticle Tracking Analysis (NTA). Fig. 1 D shows a Venn diagram of overlapping proteins between this study and the Exocarta Database for plasma EV proteins. Fig. 1 E shows enrichment analysis of proteins identified in this study. Enrichment analysis was conducted with DAVID software for Gene Ontology (GO) Terms with Cellular Compartment sub analysis. Top 5 significantly (Bonferroni corrected pval<0.05) enriched terms are displayed. * pval < 0.05, ** pval < 0.01 by two-tailed unpaired t test.

[0022] Fig. 2A-B shows principal component analysis using the proteins identified by mass spectrometry. Fig. 2A shows a clustering of proteins grouped by condition and gender. Fig. 2B shows a clustering of proteins identified in female cohorts grouped by disease severity as defined by clinical severity score.

[0023] Fig. 3A-E shows differential protein levels in plasma EVs of FSHD patients andhealthy controls. Proteins with >3 spectral counts detected on the mass spectrometer were included in the analysis. Volcano plots of the female cohort (Fig. 3A) and the male cohort (Fig. 3D) are shown. FDR corrected pval<0.1 and log2FC >1 (dashed lines) were used as a cutoff for significance. Labeled are proteins that met the cutoff criteria. Fig. 3B and E are heatmaps of relative levels (i.e., z-scores) displaying all proteins that are significantly changing using nominal pval<0.05, and disease severity based on clinical severity score. Fig. 3C shows confirmation of the mass spectrometry results by western blot for MBL2.

[0024] Fig. 4A-B shows the confirmation of EV localization of potential biomarkers using western blot analysis. Western blots were carried out on EVs treated with 20 pg / mL Proteinase K in the presence of 20 pM PMSF or 1% Triton X-100. Proteinase K-resistant proteins (lane 3) are considered EV proteins. Fig. 4B shows repetition of Proteinase K treatment of three more EV samples showing MBL2 is retained in EVs.

[0025] Fig. 5 shows differential protein biomarker levels (expressed in mass spectrometry spectra counts) in plasma EVs of FSHD patients and healthy controls. MBL2 and GPLD1 were identified as protein biomarkers in female FSHD patients and JUP, CLEC3B and DSP were identified as protein biomarkers in male FSHD patients. MBL2 increased in female FSHD patients by 5±3 fold (N=8-11 , p-value=0.0011 , two-tailed unpaired t-test) when compared to female healthy control participants. GPLD1 decreased in female FSHD patients by 2.3±0.5 fold (N=8-11 , p-value=0.0005, two-tailed unpaired t-test) when compared to female healthy control participants. JUP, CLEC3B and DSP were exclusively expressed in male FSHD patients and absent in male healthy control participants. These preliminary results indicate possible differences in the presence of biomarkers between the sexes, but studies are being repeated in a new validation cohort and on a larger population size to confirm differential expression depending on sex.

[0026] Fig. 6A-E shows differential protein levels in plasma EVs of FSHD patients and healthy controls. For this analysis, the minimal spectral count cutoff was increased from 3 to 5 to restrict the analysis on more abundant proteins. Volcano plots of the female cohort (Fig. 6A) and male cohort (Fig. 6D) are shown. FDR corrected pval<0.1 and log2FC >1 (dashed lines) were used as a cutoff for significance. Labeled are proteins that met the cutoff criteria. Fig. 6B and 6E provide heatmaps of relative levels (i.e., z-scores) displaying all proteins that are significantly changing using nominal pval<0.05, and disease severity based on clinical severity score. Fig. 6C shows confirmation of the mass spectrometry results by western blot for MBL2.

[0027] Fig. 7A-C shows correlations of EV-protein levels with confounding factors. Fig.7A-B shows Pearson correlations of MBL2 with age. Fig 7C shows Pearson correlations ofC4BPB with FSHD-COM score. The Pearson correlation coefficient (R) and statistical significance p is computed for each EV protein. Confidence intervals (95%) for each correlation are indicated by gray shade. * pval < 0.05 by two-tailed unpaired t test or oneway ANOVA.

[0028] Fig. 8A-E shows the identification of additional EV protein biomarkers in FSHD patients by using an additional study cohort (cohort 2). Patients were not sub-grouped by age or sex and comparisons were done with all healthy controls vs FSHD patients. Fig. 8A shows age distribution by disease status and sex in cohort 1 , made of 36 participants (11 healthy controls and 25 FSHD patients) and cohort 2, made of 38 participants (17 healthy controls and 21 FSHD patients). Fig. 8B-C shows volcano plots showing differentially expressed proteins based on nominal and FDR adjusted p value in both cohorts (FSHD vs healthy controls (cohort 1 ; Fig 8B) and cohort 2; Fig. 8C). Fig. 8D shows that combining cohorts increased the statistical power and revealed additional EV biomarkers (CNDP1 , IGKV2-30, C7 and LUM at False Discovery Rate [FDR]<0.1 ). Fig. 8E shows the levels of four proteins (C7, CNDP1 , IGKV2-30, and LUM) meeting the FDR adjusted p-value cut off in the combined data displayed across cohorts. * Nominal p value < 0.05, ** p value < 0.01 , *** p value < 0.001 by Wald test.

[0029] Fig. 9A-B shows the correlation of potential new FSHD biomarkers with disease confounding factors. Fig. 9A shows the correlation of CNDP1 with age, showing decreased trends of CNDP1 with age in FSHD patients (R: - 0.27) and an increase in healthy controls (R: 0.28) (AR=0.55). Fig. 9B shows the correlation of LUM with FSHD-COM score (an 18- item performance-based functional composite outcome measure assessing the legs, shoulders and arms, trunk, hands, and balance / mobility). Correlation of LUM with FSHD- COM score was adjusted for sex using ‘ppcor’ partial correlation assay. Pearson R correlation and p value for the correlation is displayed. 95% confidence intervals are indicated by curve shadows.

[0030] Fig. 10A-D shows EV protein biomarkers in FSHD patients in two cohorts after adjusting for sex as a variable. Fig. 10A-C are volcano plots for cohort 1 (Fig. 10A), cohort 2 (Fig. 10B) and combined cohorts 1 and 2 (Fig. 10C), after adjusting for sex as a variable, showing differentially expressed proteins based on nominal and FDR adjusted p value in both cohorts. Following this analysis, TNC in cohort 2 and LUM, C7 and C9 in the combined cohort were up-regulated and had a log2Fold-Change (FC)>0.5 with a False Discovery Rate [FDR]<0.1). Fig. 10D shows the levels (normalized counts) of C7, C9 and LUM proteins, after adjusting for sex as a variable, meeting the log2FC and FDR adjusted p-value cut offs in the combined data displayed across cohorts. * Nominal p value < 0.05, ** p value < 0.01 ,*** p value < 0.001 by Wald test.DETAILED DESCRIPTION

[0031] The disclosure provides a novel method for testing a subject for a muscular dystrophy. More specifically, the disclosure provides a novel method for diagnosing a subject with facioscapulohumeral dystrophy (FSHD). In some aspects, the method comprises detecting the presence or change in level of a biomarker in a biological sample from the subject. In some aspects, the biomarker is mannose binding lectin 2 (MBL2), junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), complement component 7 (C7), complement component 9 (C9), tenascin C (TNC), lumican (LUM), phosphatidylinositol-glycan-specific phospholipase D (GPLD1 ), complement component 4 binding protein, beta chain (C4BPB), carnosine dipeptidase (CNDP1), or immunoglobulin kappa variable 2-30 (IGKV2-30), or a combination of any two or more of MBL2, JUP, DSP, CLEC3B, C7, C9, TNC, LUM, GPLD1 , C4BPB, CNDP1 , or IGKV2-30. In some aspects, the change in level of the biomarker is an increase in level of MBL2, JUP, DSP, CLEC3B, C7, C9, TNC, or LUM, or a combination of any two or more of MBL2, JUP, DSP, CLEC3B, C7, C9, TNC, or LUM, the subject is diagnosed as having FSHD. In some aspects, the change in level of the biomarker is a decrease in level of GPLD1 , C4BPB, CNDP1 , or IGKV2-30, or a combination of any two or more of GPLD1 , C4BPB, CNDP1 , or IGKV2-30, the subject is diagnosed as having FSHD. Thus, the disclosure relates to the identification of various biomarkers, e.g., proteins or nucleic acids, alone and in combination, as biomarkers for muscular dystrophy, and some specific aspects, FSHD. More specifically, the disclosure provides fast and robust methods of determining if a subject has FSHD, is at risk of FSHD, or is responding to a treatment for FSHD, by measuring a level of at least one biomarker, e.g., a level of protein or nucleic acid, in a biological sample from a subject.

[0032] The disclosure also provides a method of diagnosing and treating a subject for FSHD, wherein diagnosing comprises detecting the presence or the change in level of a biomarker in a biological sample from the subject, wherein the biomarker is MBL2, JUP, DSP, CLEC3B, 07, 09, TNC, LUM, GPLD1 , C4BPB, CNDP1 , or IGKV2-30, or a combination of at least two of the biomarkers, and wherein treating comprises administering to the subject any therapy or therapeutic used in the treatment of FSHD. In some aspects, treating includes administering an effective amount of a non-steroidal anti-inflammatory drug, and / or ARO-DUX4, and / or RO7204239, and / or AGO 1020, and / or providing physical therapy. In some aspects, treating includes administering an effective amount of a gene therapy for FSHD.

[0033] The disclosure further provides a method for determining efficacy of a therapeutic treatment for FSHD in a subject, the method comprising measuring the level of a biomarker in a biological sample from the subject before and after the treatment, and determining thatthe treatment is effective in treating FSHD if the level of the biomarker is changed, i.e., decreased or increased, relative to a reference level or to the level of the biomarker in the sample from the subject before treatment. In some aspects, the change in level of the biomarker is a decrease in level of MBL2, JLIP, DSP, CLEC3B, C7, C9, TNC, and / or LUM if the treatment is effective. In some aspects, the change in level of the biomarker is an increase in level of GPLD1 , C4BPB, CNDP1 , and / or IGKV2-30 if the treatment is effective.

[0034] In some aspects, the sample is tissue or fluid isolated from the subject. In some aspects, the fluid is plasma or serum. In some more specific aspects, the fluid is plasma. In some aspects, the sample is an extracellular vesicle (EV) isolated from plasma of the subject. In some aspects, the biomarker is a protein or a fragment thereof. In some aspects, the biomarker is a mRNA or a fragment thereof. In some aspects, a combination of biomarkers, including protein and / or nucleic acid fragments of the biomarkers, are present in the sample of the subject and are used in diagnosing the subject as having FSHD and / or determining whether the subject is responding to a therapeutic treatment for FSHD.

[0035] Before any embodiments of the subject matter of the disclosure are explained in detail, it is to be understood that the disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the figures and examples. Accordingly, the disclosure embraces other embodiments and is practiced or carried out in various ways.

[0036] It is noted here that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. The terms "including," "comprising," "containing," or "having" and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

[0037] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues linked via peptide bonds. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides. The term "protein", as used herein, includes a fragment of a protein or any portion of the protein smaller than the full-length protein or protein expression product. Fragments are deletion analogs of the full-length protein wherein one or more amino acid residues have been removed from the amino terminus (protein) and / or the carboxy terminus of the full-length protein.

[0038] The term "nucleic acid" or "nucleic acid sequence" or "nucleic acid molecule" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or doublestranded form. The term nucleic acid is used interchangeably with gene, deoxyribonucleicacid, complementary DNA (cDNA), ribonucleic acid, messenger RNA (mRNA), oligonucleotide, and polynucleotide. The term "nucleic acid", as used herein, includes a fragment of a nucleic acid or any portion of the nucleic acid smaller than the full-length nucleic acid. Fragments include deletion analogs of the full-length nucleic acid wherein one or more nucleotides have been removed from the 5' end and / or the 3' end of the full-length nucleic acid.

[0039] A "biomarker" in the context of the disclosure encompasses, without limitation, proteins, nucleic acids, and metabolites, together with their polymorphisms, mutations, variants, modifications, subunits, fragments, protein-ligand complexes, and degradation products, protein-ligand complexes, elements, related metabolites, and other analytes or sample-derived measures. In some aspects, therefore, a biomarker includes a protein or a fragment thereof or a nucleic acid or a fragment thereof. In additional aspects, one or more biomarkers are measured together to provide an array for the prediction that the subject will positively respond to a treatment regimen. In exemplary aspects of the disclosure, a biomarker is a protein.

[0040] In some aspects, the methods, kits, and uses of the disclosure comprise a biomarker or two or more biomarkers in combination. In some aspects, a kit of the disclosure comprises reagents, such as antibodies or nucleic acids, for detecting the presence of the biomarker or the combination of any two or more biomarkers.

[0041] A biomarker, e.g., protein or nucleic acid, of the disclosure is any one or more of the following: mannose binding lectin 2 (MBL2), junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), complement component 7 (C7), complement component 9 (C9), tenascin C (TNC), lumican (LUM), phosphatidylinositol-glycan-specific phospholipase D (GPLD1), complement component 4 binding protein, beta chain (C4BPB), carnosine dipeptidase (CNDP1 ), or immunoglobulin kappa variable 2-30 (IGKV2-30). In some aspects, a biomarker may be a combination of any two or more of the biomarkers of the disclosure. In some aspects, therefore, the combination may be a combination of three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or 12 or more. In aspects of the disclosure, the term “MBL2” as used herein refers to an MBL2 protein or nucleic acid. In aspects of the disclosure, the term "JUP" as used herein refers to a JUP protein or nucleic acid. In aspects of the disclosure, the term "DSP" as used herein refers to a DSP protein or nucleic acid. In aspects of the disclosure, the term "CLEC3B" as used herein refers to a CLEC3B / tetranectin protein or nucleic acid. In aspects of the disclosure, the term "C7" as used herein refers to a C7 protein or nucleic acid. In aspects of the disclosure, the term "C9" as used herein refers to a C9 protein or nucleic acid. In aspects of the disclosure, the term "TNC" as used herein refers to a TNC protein ornucleic acid. In aspects of the disclosure, the term "LUM" as used herein refers to a LUM protein or nucleic acid. In aspects of the disclosure, the term "GPLD1" as used herein refers to a GPLD1 / PHLD protein or nucleic acid. In aspects of the disclosure, the term “C4BPB" as used herein refers to a C4BPB protein or nucleic acid. In aspects of the disclosure, the term "CNDP1" as used herein refers to a CNDP1 protein or nucleic acid. In aspects of the disclosure, the term " IGKV2-30" as used herein refers to a IGKV2-30 protein or nucleic acid.

[0042] MBL2 (Uniprot P11226) is also referred to as mannose-binding protein C and is a calcium-dependent lectin involved in innate immune defense (PubMed:35102342). MBL2 binds mannose, fucose and N-acetylglucosamine on different microorganisms and activates the lectin complement pathway. MBL2 binds to late apoptotic cells, as well as to apoptotic blebs and to necrotic cells, but not to early apoptotic cells, facilitating their uptake by macrophages. MBL2 may bind DNA. Upon SARS coronavirus-2 / SARS-CoV-2 infection, MBL2 activates the complement lectin pathway which leads to the inhibition SARS-CoV-2 infection and a reduction of the induced inflammatory response.

[0043] JUP (Uniprot G3R3M9) is common junctional plaque protein. The membrane- associated plaques are architectural elements in an important strategic position to influence the arrangement and function of both the cytoskeleton and the cells within the tissue. The presence of plakoglobin in both the desmosomes and in the intermediate junctions suggests that it plays a central role in the structure and function of submembranous plaques. JUP acts as a substrate for VE-PTP and is required by it to stimulate VE-cadherin function in endothelial cells. JUP replace beta-catenin in E-cadherin / catenin adhesion complexes which are proposed to couple cadherins to the actin cytoskeleton.

[0044] DSP (Uniprot P15924) is a major high molecular weight protein of desmosomes. DSP regulates profibrotic gene expression in cardiomyocytes via activation of the MAPK14 / p38 MAPK signaling cascade and increase in TGFB1 protein abundance.

[0045] Tetranectin (CLEC3B) or TETN or CLEC3B gene (Uniprot P05452) binds to plasminogen and to isolated kringle 4 (PubMed:35331648). CLEC3B is apparently involved in the packaging of molecules destined for exocytosis, and plays a role in retinal function.

[0046] Complement component 7 (C7) (Uniprot P10643) is a constituent of the membrane attack complex (MAC) that plays a key role in the innate and adaptive immune response by forming pores in the plasma membrane of target cells. C7 serves as a membrane anchor. C7 is a protein crucial for the body's defense against pathogens, acting as a key component of the membrane attack complex (MAC). C7's primary function is to bind to the C5bC6 complex, forming the C5b7 complex, which is essential for MAC assembly and targetmembrane binding. The C7 gene (HGNC:1346; ENSG00000112936) encodes a serum glycoprotein that forms a membrane attack complex together with complement components C5b, C6, C8, and C9 as part of the terminal complement pathway of the innate immune system. The C7 protein encoded by this gene contains a cholesterol-dependent cytolysin / membrane attack complex / perforin-like (CDC / MACPF) domain and belongs to a large family of structurally related molecules that form pores involved in host immunity and bacterial pathogenesis.

[0047] Complement component 9 (C9) (Uniprot P02748) is a constituent of the membrane attack complex (MAC) that plays a key role in the innate and adaptive immune response by forming pores in the plasma membrane of target cells (PubMed:26841934, PubMed:9212048, PubMed:9634479). C9 is the pore-forming subunit of the MAC (PubMed:26841934, PubMed:30111885, PubMed:4055801 ). In contrast to homologous proteins, such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerization, C9 assembles directly onto the nascent MAC from solution. The C9 gene (HGNC: 1358, ENSG00000113600) encodes the final component of the complement system.

[0048] Tenascin C (TNC) (Uniprot P24821) is an extracellular matrix protein implicated in guidance of migrating neurons as well as axons during development, synaptic plasticity as well as neuronal regeneration. TNC promotes neurite outgrowth from cortical neurons grown on a monolayer of astrocytes. TNC is a ligand for integrins alpha-8 / beta-1 , alpha- 9 / beta-1 , alpha-V / beta-3 and alpha-V / beta-6. In tumors, stimulates angiogenesis by elongation, migration and sprouting of endothelial cells (PubMed: 19884327). The TNC gene (HGNC:5318; ENSG00000041982) encodes an extracellular matrix protein with a spatially and temporally restricted tissue distribution. The TNC protein is homohexameric with disulfide-linked subunits, and contains multiple EGF-like and fibronectin type-ll I domains.

[0049] Lumican (LUM) (Uniprot P51884) is an extracellular matrix proteoglycan that plays a role in regulating collagen fibril structure and assembly. LUM is part of the small leucine- rich repeat proteoglycans (SLRPs) family and is involved in various cellular processes, including cell adhesion, migration, and proliferation. LUM is a structure regulatory proteoglycan of collagen-rich tissues, with cell instructive properties through interactions with a number of cell surface receptors in tissue repair, thereby regulating cell proliferation, differentiation, inflammation and the innate and humoral immune systems to combat infection. The LUM gene (HGNC: 6724, ENS00000139329) is alternatively called keratan sulfate proteoglycan lumican (KSPG lumican).

[0050] Phosphatidylinositol-glycan-specific phospholipase D (GPLD1 ) or GPLD1 gene(Uniprot P80108) or PHLD human hydrolyzes the inositol phosphate linkage in proteins anchored by phosphatidylinositol glycans (GPI-anchor) thus releasing these proteins from the membrane. The GPLD1 gene (HGNC: 4459; NCBI Gene: 2822; Ensembl: ENSG00000112293; OMIM®: 602515; UniProtKB / Swiss-Prot: P80108) encodes GPLD1 in humans. The GPLD1 enzyme is involved in cleaving the glycosylphosphatidylinositol (GPI) anchor from proteins, releasing them from the plasma membrane. GPLD1 also has roles in various processes like regulating high-density lipoprotein clearance and insulin secretion.

[0051] Complement component 4 binding protein, beta chain (C4BPB) (Uniprot P20851) is a protein that controls the classical pathway of complement activation. C4BPB binds as a cofactor to C3b / C4b inactivator (C3blNA), which then hydrolyzes the complement fragment C4b. It also accelerates the degradation of the C4bC2a complex (C3 convertase) by dissociating the complement fragment C2a. C4BPB also interacts with anticoagulant protein S and with serum amyloid P component, and the beta chain binds protein S. The C4BPB gene (HGNC: 1328; NCBI Gene: 725; Ensembl: ENSG00000123843; OMIM®: 120831 ; UniProtKB / Swiss-Prot: P20851) encodes C4BPB has a regulatory role in the coagulation system also, mediated through the beta-chain binding of protein S, a vitamin K-dependent protein that serves as a cofactor of activated protein C.

[0052] Carnosine dipeptidase (CNDP1 ) (Uniprot J3KRP0), a member of the M20 metalloprotease family, is an enzyme that breaks down the dipeptide carnosine, a naturally occurring compound in the brain and muscles. CNDP1 is also known as carnosinase 1 , glutamate carboxypeptidase-like protein 2 (CPGL-2), or X-His dipeptidase. CNDP1 is a metalloprotease, meaning it requires a metal ion for its activity. The CNDP1 gene (HGNC:20675; Ensembl: ENSG00000150656; Gene ID84735) encodes CNDP1 , a homodimeric dipeptidase identified as human carnosinase. This gene contains trinucleotide (CTG) repeat length polymorphism in the coding region.

[0053] Immunoglobulin kappa variable 2-30 (IGKV2-30) (Uniprot P06310) is the V region of the variable domain of immunoglobulin light chains that participates in the antigen recognition. IGKV2-30 is predicted to enable antigen binding activity and be involved in the immune response. IGKV2-30 is located in blood microparticles and extracellular exosomes. The IGKV2-30 gene (HGNC: 5785; NCBI Gene: 28919; Ensembl: ENSG00000243238; UniProtKB / Swiss-Prot: P06310) encodes the IGKV2-30 protein.

[0054] The disclosure includes the use of any biomarker or combination of biomarkers described herein in any of the disclosed methods, kits, uses and the like. In some aspects, the combination comprises two or more biomarkers, or three or more biomarkers, or four or more biomarkers, or five or more biomarkers, or six or more biomarkers, or seven or morebiomarkers, or eight or more biomarkers, or nine or more biomarkers, or 10 or more biomarkers, or 11 or more biomarkers, or 12 or more biomarkers, or more.

[0055] In some aspects, an increase in the level of the biomarker compared to the level of the biomarker at an initial measurement is indicative of progression of the disease. In some aspects, a decrease in the level of the biomarker compared to the level of the biomarker at an initial measurement is indicative of regression of the disease, or possible response to a therapeutic treatment regimen.

[0056] Thus, in some aspects, an increase in the level of MBL2, JLIP, DSP, CLEC3B, C7, C9, TNC, or LUM, or any combination thereof, compared to an initial level or a reference level are indicative of progression of FSHD; and in some aspects, a decrease in the level of GPLD1 , C4BPB, CNDP1 , or IGKV2-30, or any combination thereof, compared to an initial level or a reference level is indicative of progression of FSHD.

[0057] In some aspects of the disclosure, the level of a biomarker or the combination of biomarkers is measured in a sample from a subject and compared to a reference level. A “reference level”, as used herein, provides a known comparator for measuring the level or amount of biomarker, e.g., protein or nucleic acid, present in a biological sample from a subject.

[0058] In some aspects of the disclosure, the reference level is a mean level of the biomarker in a biological sample from a population of subjects determined to be healthy or not to have FSHD. In some aspects, the population of subjects is, optionally, matched to the subject in other parameters, such as one or more of the following: age, sex, body mass index (BMI) and the like. In various aspects, the level of the biomarker is a relative level. In some aspects, the level of the biomarker is an absolute level. In some aspects, the level of the biomarker is within the range of the mean level of the biomarker in a population of healthy subjects or subjects determined not to have FSHD.

[0059] In some aspects of the disclosure, a control or control level is used. A control or control level, in various aspects, is a level of the biomarker in a biological sample from a subject, or the mean level from a population of subjects, determined to be healthy or not to have a dystrophinopathy or FSHD.

[0060] In some aspects of the disclosure, the level of a biomarker or the levels of a combination of biomarkers is measured in a sample from a subject before and after a therapeutic treatment. Thus, a first level and a second level is measured. In such aspects, the first level is the level of the biomarker or the levels of each of the biomarkers in the sample from the subject before the therapeutic treatment. In such aspects, the second level is the level of the biomarker or the levels of each of the biomarkers in the sample from thesubject after the therapeutic treatment. In some aspects, the level of a biomarker or the levels of multiple biomarkers is measured at multiple times throughout the course of a therapeutic treatment. In such aspects, the reference level is the level of the biomarker in the subject before the therapeutic treatment or agent is administered (i.e., baseline).

[0061] In various aspects, an increased level of biomarker is a level greater than the reference level. In various aspects, an increase in the level of the biomarker in a subject is at least or about 1% greater, at least or about 2% greater, at least or about 3% greater, at least or about 4% greater, at least or about 5% greater, at least or about 6% greater, at least or about 7% greater, at least or about 8% greater, at least or about 9% greater, at least or about 10% greater, at least or about 11% greater, at least or about 12% greater, at least or about 13% greater, at least or about 14% greater, at least or about 15% greater, at least or about 16% greater, at least or about 17% greater, at least or about 18% greater, at least or about 19% greater, at least or about 20% greater, at least or about 21% greater, at least or about 22% greater, at least or about 23% greater, at least or about 24% greater, at least or about 25% greater, at least or about 26% greater, at least or about 27% greater, at least or about 28% greater, at least or about 29% greater, at least or about 30% greater, at least or about 35% greater, at least or about 40% greater, at least or about 45% greater, at least or about 50% greater, at least or about 55% greater, at least or about 60% greater, at least or about 65% greater, at least or about 70% greater, at least or about 75% greater, at least or about 80% greater, at least or about 85% greater, at least or about 90% greater, at least or about 95% greater, at least or about 100% greater, at least or about greater than 100% greater than the reference level, or the level of the biomarker before treatment.

[0062] In additional aspects, an increase in the level of the biomarker in a subject is at least or about 1 / 10 greater, at least or about 1 / 9 greater at least or about 1 / 8 greater, at least or about 1 / 7 greater, at least or about 1 / 6 greater, at least or about 1 / 5 greater, at least or about 1 / 4 greater, at least or about 1 / 3 greater, at least or about 1 / 2 greater, at least or about 1 times greater, at least or about 1 .5 times greater, at least or about 2.0 times greater, at least or about 2.5 times greater, at least or about 3.0 times greater, at least or about 3.5 times greater, at least or about 4.0 times greater, at least or about 4.5 times greater, at least or about 5 times greater, at least or about 10 times greater, at least or about 15 times greater, at least or about 20 times greater, at least or about 25 times greater, at least or about 30 times greater, at least or about 35 times greater, at least or about 40 times greater, at least or about 45 times greater, at least or about 50 times greater, at least or about 55 times greater, at least or about 60 times greater, at least or about 65 times greater, at least or about 70 times greater, at least or about 75 times greater, at least or about 80 times greater, at least or about 85 times greater, at least or about 90 times greater, at least orabout 100 times greater, or at least or about greater than about 100 times than the reference level, or the level of the biomarker before treatment.

[0063] In some aspects, this increase in the level of the biomarker is referred to as “fold greater”, or at least or about 1 / 10 fold greater, at least or about 1 / 9 fold greater, at least or about 1 / 8 fold greater, at least or about 1 / 7 fold greater, at least or about 1 / 6 fold greater, at least or about 1 / 5 fold greater, at least or about 1 / 4 fold greater, at least or about 1 / 3 fold greater, at least or about 1 / 2 fold greater, at least or about 1 fold greater, at least or about 1 .5 fold greater, at least or about 2.0 fold greater, at least or about 2.5 fold greater, at least or about 3.0 fold greater, at least or about 3.5 fold greater, at least or about 4.0 fold greater, at least or about 4.5 fold greater, at least or about 5 fold greater, at least or about 10 fold greater, at least or about 15 fold greater, at least or about 20 fold greater, at least or about 25 fold greater, at least or about 30 fold greater, at least or about 35 fold greater, at least or about 40 fold greater, at least or about 45 fold greater, at least or about 50 fold greater, at least or about 55 fold greater, at least or about 60 fold greater, at least or about 65 fold greater, at least or about 70 fold greater, at least or about 75 fold greater, at least or about 80 fold greater, at least or about 85 fold greater, at least or about 90 fold greater, at least or about 100 fold greater, or at least or about greater than about 100 fold than the reference level, or the level of the biomarker before treatment.

[0064] In various aspects, a decreased level of a biomarker is a level lesser than the reference level. In various aspects, a decrease in the level of the biomarker is at least or about 1% lesser, at least or about 2% lesser, at least or about 3% lesser, at least or about 4% lesser, at least or about 5% lesser, at least or about 6% lesser, at least or about 7% lesser, at least or about 8% lesser, at least or about 9% lesser, at least or about 10% lesser, at least or about 11% lesser, at least or about 12% lesser, at least or about 13% lesser, at least or about 14% lesser, at least or about 15% lesser, at least or about 16% lesser, at least or about 17% lesser, at least or about 18% lesser, at least or about 19% lesser, at least or about 20% lesser, at least or about 21% lesser, at least or about 22% lesser, at least or about 23% lesser, at least or about 24% lesser, at least or about 25% lesser, at least or about 26% lesser, at least or about 27% lesser, at least or about 28% lesser, at least or about 29% lesser, at least or about 30% lesser, at least or about 35% lesser, at least or about 40% lesser, at least or about 45% lesser, at least or about 50% lesser, at least or about 55% lesser, at least or about 60% lesser, at least or about 65% lesser, at least or about 70% lesser, at least or about 75% lesser, at least or about 80% lesser, at least or about 85% lesser, at least or about 90% lesser, at least or about 95% lesser, at least or about 100% lesser, or about lesser than 100% lesser than the reference level, or the level of the biomarker before treatment.

[0065] In additional aspects, a decrease in the level of the biomarker in a subject is at least or about 1 / 10 lesser, at least or about 1 / 9 lesser at least or about 1 / 8 lesser, at least or about 1 / 7 lesser, at least or about 1 / 6 lesser, at least or about 1 / 5 lesser, at least or about 1 / 4 lesser, at least or about 1 / 3 lesser, at least or about 1 / 2 lesser, at least or about 1 times lesser, at least or about 1 .5 times lesser, at least or about 2.0 times lesser, at least or about 2.5 times lesser, at least or about 3.0 times lesser, at least or about 3.5 times lesser, at least or about 4.0 times lesser, at least or about 4.5 times lesser, or at least or about 5 times lesser, at least or about 10 times lesser, at least or about 15 times lesser, at least or about 20 times lesser, at least or about 25 times lesser, at least or about 30 times lesser, at least or about 35 times lesser, at least or about 40 times lesser, at least or about 45 times lesser, at least or about 50 times lesser, at least or about 55 times lesser, at least or about 60 times lesser, at least or about 65 times lesser, at least or about 70 times lesser, at least or about 75 times lesser, at least or about 80 times lesser, at least or about 85 times lesser, at least or about 90 times lesser, at least or about 100 times lesser, or at least or about lesser than about 100 times lesser than the reference level, or the level of the biomarker before treatment.

[0066] In additional aspects, a decrease in the level of the biomarker in a subject is at least or about 1 / 10 fold lesser, at least or about 1 / 9 fold lesser at least or about 1 / 8 fold lesser, at least or about 1 / 7 fold lesser, at least or about 1 / 6 fold lesser, at least or about 1 / 5 fold lesser, at least or about 1 / 4 fold lesser, at least or about 1 / 3 fold lesser, at least or about 1 / 2 fold lesser, at least or about 1 fold lesser, at least or about 1 .5 fold lesser, at least or about 2.0 fold lesser, at least or about 2.5 fold lesser, at least or about 3.0 fold lesser, at least or about 3.5 fold lesser, at least or about 4.0 fold lesser, at least or about 4.5 fold lesser, or at least or about 5 fold lesser, at least or about 10 fold lesser, at least or about 15 fold lesser, at least or about 20 fold lesser, at least or about 25 fold lesser, at least or about 30 fold lesser, at least or about 35 fold lesser, at least or about 40 fold lesser, at least or about 45 fold lesser, at least or about 50 fold lesser, at least or about 55 fold lesser, at least or about 60 fold lesser, at least or about 65 fold lesser, at least or about 70 fold lesser, at least or about 75 fold lesser, at least or about 80 fold lesser, at least or about 85 fold lesser, at least or about 90 fold lesser, at least or about 100 fold lesser, or at least or about lesser than about 100 fold lesser than the reference level, or the level of the biomarker before treatment.

[0067] In some aspects, the biomarker is indicative of FSHD in subject based upon the sex of the subject. Thus, in some aspects, the biomarker is indicative of FSHD in a female subject. Likewise, in some aspects, the biomarker is indicative of FSHD in a male subject.

[0068] In some aspects, MBL2 and GPLD1 are biomarkers indicative of FSHD in female subjects. In some aspects, JUP, CLEC3B and DSP are biomarkers indicative of FSHD in male FSHD subjects. In some aspects, MBL2 level is increased in female FSHD subjects. In some aspects, MBL2 level is increased at least or about 2-fold in female FSHD subjects when compared to female control subjects. In some aspects, GPLD1 level is decreased in female FSHD subjects. In some aspects GPLD1 level is decreased at least or about 1 .5-fold in female FSHD subjects compared to female control subjects.

[0069] In some aspects, JUP, CLEC3B and DSP are biomarkers indicative of FSHD in male subjects. In some aspects, JUP, CLEC3B and DSP are exclusively expressed in male FSHD subjects and not present in male control subjects. In some aspects, JUP, CLEC3B, and DSP are increased in male FSHD subjects compared to male control subjects.

[0070] In some aspects, the level is a spectral count level. In some aspects, spectral counting simply counts the number of spectra identified for a given peptide in different biological samples and then integrates the results for all measured peptides of the protein(s) that are quantified.

[0071] In various aspects of the disclosure, level of the protein biomarker is detected or quantitatively measured in a biological sample by any suitable means known in the art for quantifying protein including, but not limited to, an immunoassay (e.g., ELISA, RIA), immunoturbidimetry, rapid immunodiffusion, laser nephelometry, visual agglutination, quantitative Western blot analysis, multiple reaction monitoring-mass spectrometry (MRM Proteomics), Lowry assay, Bradford assay, BCA assay, UV spectroscopic assays, such as a UV spectroscopic assay, surface plasmon resonance, or SOMAscan® assay. In various aspects, therefore, the level measured is an absolute level or is a relative level.

[0072] In some aspects, the protein biomarker is simply measured for the presence or absence of the biomarker.

[0073] In various aspects of the disclosure, the level of the nucleic acid biomarker is detected or quantitatively measured in a biological sample by any suitable means known in the art for quantifying nucleic acid including, but not limited to, RNA sequencing (RNA-seq), high-throughput sequencing (HT-seq), PCR, quantitative PCR, qT-PCR, RT-qPCR, digital PCR, real-time PCR, direct digital quantification, serial analysis of gene expression (SAGE), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), branched DNA (bDNA) assays, and / or Northern or Southern blotting. In various aspects, therefore, the level measured is an absolute level or is a relative level.

[0074] In some aspects, the nucleic acid biomarker is simply measured for the presence or absence of the biomarker.

[0075] In some aspects, the level of the biomarker is a relative level, for example, a Z- score. In some aspects, such relative levels are assessed with consideration of a false discovery rate (FDR), which is the metric for global confidence assessment of a large-scale proteomics dataset (PMID: 26519173; Aggarwal et al., Methods Mol Biol . 2016:1362:119- 28. doi: 10.1007 / 978-1 -4939-3106-4_7).

[0076] In some aspects, the level of the biomarker is measured in male versus female subjects. Thus, in some exemplary aspects, the biomarker or the level of the biomarker is relevant for one sex versus the other sex. Thus, in some aspects, the biomarker and the change in the level of the biomarker is indicative of FSHD in females, and in other aspects, the biomarker and the change in the level of the biomarker is indicative of FSHD in males.

[0077] In various aspects, any of these methods is performed on protein or nucleic acid isolated from or present in a biological sample obtained from a human subject. In some aspects, the biological sample is blood. In some aspects, the sample is serum. In exemplary aspects, the biological sample is plasma. In even more exemplary aspects, the biological sample is an extracellular vesicle (EV) isolated from plasma, or in some other aspects an EV isolated from some other tissue or bodily fluid.

[0078] Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are naturally released from almost all types of cells but, unlike a cell, cannot replicate. EVs are cell- derived membrane-surrounded vesicles that carry bioactive molecules and are released by a cell to deliver such bioactive molecules into the extracellular environment to recipient cells. Thus, EVs primarily act in cell-to-cell communication, delivering cargo from donor to recipient cells and modulating their physiological condition. Classical EVs are exosomes, microvesicles, and apoptotic bodies. EVs are different sizes (ranging from 50 to 500 nm) and have different contents based on the type and state of their producer cells. EVs carry tissuespecific molecules (i.e. lipids, RNA, DNA, peptides and proteins), and thus are exploited herein for the identification of biomarkers specific for the detection of FSHD. Additionally, such biomarkers specific for FSHD could be used to diagnose and / or predict disease progression, stratify patients for clinical trials, and assess efficiency or efficacy of potential FSHD therapies.

[0079] In some aspects, the sample or biological sample is taken prior to treatment to measure the level of the biomarker before treatment. In some aspects, the sample is taken before and after treatment with a therapeutic agent or therapeutic regimen to measure the level of the biomarker before or after treatment. In some aspects, the sample is taken over multiple time points to monitor the effect of a therapeutic treatment or therapeutic regimen over time. Thus, in some aspects, a first value of the biomarker, e.g., protein level, iscompared to a second value or is compared to multiple values taken over a period of time. In some aspects, the sample is taken before, during, and after treatment with a therapeutic agent or therapeutic regimen to measure the level of the biomarker before, during, or after treatment to determine the effect of a biomarker during treatment. In some aspects, patients have a full clinical assessment at baseline (i.e., time 0) or prior to a first treatment with a therapeutic agent.

[0080] “Measuring” or “measurement” means assessing the presence, quantity or level of a biomarker, e.g. a protein or a nucleic acid, within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substance, or otherwise evaluating the values or categorization of a subject’s clinical parameters.

[0081] The terms “level” and “amount” are used herein interchangeably to mean the concentration of biomarker, e.g., protein or nucleic acid, present in a biological sample. In some aspects, protein and / or nucleic acid is prepared from the sample and the “level” and / or “amount” is the level or amount of a particular protein and / or nucleic acid of interest. In some aspects, it is the level or amount of the protein or nucleic acid biomarker. In some aspects, the level or amount is relative to a reference level or an initial level in the subject. In some aspects, the change in level is a change in level which is determined to be a statistically significant change in level relative to the initial level or to a previous level, or to a reference level.

[0082] In some aspects, the methods include measuring the level of at least one or more of the biomarkers. In some aspects, the methods include measuring the level of a combination of biomarkers. In some aspects, the level is a protein level in a biological sample. In some aspects, the level is a nucleic acid level in a biological sample. In some aspects, the level is measured in the EV, or in the EV after the EV is isolated from the sample. In some aspects, plasma EVs are isolated by SEC and then lysed to extract protein or mRNA. In some aspects, EVs are lysed with a detergent or a protein denaturant. In some aspects, such detergent is sodium dodecyl sulfate or other organic sodium salt. In some aspects, such detergent is 5% SDS. In some aspects, after the EVs are lysed, the EVs are sonicated to extract proteins or mRNA. In some aspects, the methods of the disclosure simply detect the presence or the absence of the biomarker as an indicator of disease. In some aspects, the methods of the disclosure detect a change in the level of the biomarker as an indicator of disease.

[0083] In some aspects, the methods of the disclosure comprise treating a subject. As used herein, the terms "treat", “treating”, and "treatment" refer to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slowdown (lessen) an undesired physiological change associated with a disease or disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or disorder prior to other treatments, such as surgery, stabilization of a disease or disorder (i.e., where the disease or disorder does not worsen), delay or slowing of the progression of a disease or disorder, amelioration or palliation of the disease or disorder, and remission (whether partial or total) of the disease or disorder, whether detectable or undetectable, and prevention of disease or disorder recurrence. "Treatment", in some aspects, also means prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or disorder, those prone to or at risk of having the disease or disorder, or those in which the disease or disorder is to be prevented.

[0084] In some aspects, the disclosure includes treating a muscular dystrophy (MD). "Treating" includes ameliorating or inhibiting one or more symptoms of a muscular dystrophy including, but not limited to, muscle wasting, muscle weakness, myotonia, skeletal muscle problems, abnormalities of the retina, hip weakness, facial weakness, abdominal muscle weakness, joint and spinal abnormalities, lower leg weakness, shoulder weakness, hearing loss, muscle inflammation, and nonsymmetrical weakness. In various aspects, such MD is FSHD.

[0085] FSHD is among the most commonly inherited muscular dystrophies, estimated to affect as many as 870,000 individuals. Classical descriptions of FSHD presentation include progressive muscle weakness in the face, shoulder-girdle and arms, but disease can manifest more broadly, including in muscles of the trunk and lower extremities. Variability is also commonly seen within individuals, as asymmetrical weakness is common. Age-at-onset can range from early childhood to adulthood, and is usually related to disease severity, where earlier onset is often associated with more severe muscle weakness. Although most patients with FSHD have a normal life span, respiratory insufficiency can occur, and the disease can be debilitating, as approximately 25% of affected individuals may become wheelchair dependent by their fifties, and even earlier in more severe forms of the disease, while others maintain lifelong ambulation.

[0086] FSHD is caused by aberrant expression of the double homeobox 4 gene (DUX4), which produces a transcription factor that is toxic to skeletal muscle. DUX4 is normally functional during the two-cell stage of human development but repressed thereafter in essentially all other tissues, except perhaps the testes. In skeletal muscles of people with FSHD, specific genetic and epigenetic factors conspire to permit DUX4 de-repression, where it then initiates several aberrant gene expression cascades, including those involved in differentiation abnormalities, oxidative stress, inflammatory infiltration, cell death and muscleatrophy. This disease presents as a slowly progressive asymmetric muscle weakness that involves the facial, scapular, and upper arm muscles mainly.

[0087] A patient with FSHD experiences the consequences of this muscle damage, such as pain, chronic fatigue, sleeping difficulties, physical inactivity, and reduced social participation. We have treatments to address these consequences. Treating FSHD, in some aspects, includes, but is not limited to administering to the subject a non-steroidal antiinflammatory drug, ARO-DUX4, RO7204239, AOC 1020, and / or physical therapy, or a combination of any thereof. In some aspects, therefore, the treatment is a gene therapy for FSHD. In some aspects, treatment includes the administration of a therapeutic which reduces DLIX4 expression. DLIX4 is overexpressed in a person suffering from FSHD, and when DUX4 is overexpressed, it is highly toxic, causing caspase-3-mediated apoptosis, and causing a negative effect on myogenesis. In addition, the activation of the DUX4 gene involves proteins that cause atrophy and protein degradation. DUX4 also activates protein of the innate immune system which could explain why there are inflammatory infiltrates mainly in the perivascular region. Thus, treatment, although not clinically available today, include means for inhibiting DLIX4 expression or overexpression. Such treatments include but are not limited to RNA interference (RNAi) therapies, such as siRNA, shRNA, and miRNAs targeting DUX4.

[0088] In various aspects, the methods of the disclosure are carried out after diagnosis and, therefore, are methods of treating or ameliorating disease. In some aspects, the efficacy of treatment is monitored by observing outcome measures. Such outcome measures include, but are not limited to, measuring to determine if there is improved muscle strength, improved muscle function, improved mobility, improved stamina, or a combination of two or more thereof in the subject after treatment. Such outcome measures are important in determining muscular dystrophy progression in the subject and are measured by various tests known in the art.

[0089] Combination therapies are also contemplated by the disclosure. Combination as used herein includes simultaneous treatment or sequential treatments. Combinations of methods of the disclosure with standard medical treatments (e.g., corticosteroids and / or immunosuppressive drugs) or with other inhibitory RNA constructs are specifically contemplated, as are combinations with other therapies such as those disclosed in International Publication No. WO 2013 / 016352, which is incorporated by reference herein in its entirety.

[0090] A combination therapy, as used herein, includes both simultaneous treatment(s) and sequential treatment(s). Combinations of methods described herein with standardmedical treatments and supportive care are specifically contemplated, as are combinations with therapies, such as glucocorticoids. All types of glucocorticoids are included for use in the combination therapies disclosed herein. Such glucocorticoids include, but are not limited to, prednisone, prednisolone, dexamethasone, deflazacort, beclomethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, and triamcinolone.

[0091] The disclosure also provides a kit comprising reagents to determine the presence of the biomarker in the sample from the subject. Thus, in some aspects, the kit comprises antibodies or nucleic acids that could bind to and detect the presence of the biomarker in the sample of the subject. Thus, in some aspects, such kit comprises an antibody or a combination of antibodies to any one or more of the biomarker(s) or a nucleic acid or a combination of nucleic acids that bind to any one or more of the biomarker(s), or other reagents to detect the presence or change in level of the biomarker protein or nucleic acid.

[0092] In the context of the disclosure, the term "kit" means two or more components, one of which corresponds to a reagent to detect the presence of the biomarker in a sample, and the other which corresponds to a container, recipient, instructions, or otherwise. A kit, therefore, in various aspects, is a set of products that are sufficient to achieve a certain goal, which can be marketed as a single unit.

[0093] The kit may additionally contain directions or instructions for use (e.g. in the form of a leaflet or instruction manual), means for detecting the presence or the level of the biomarker protein or nucleic acid in the sample, and / or means for diluting the protein and / or nucleic acid. In some aspects, the kit comprises a label and / or instructions that describes use of the reagents provided in the kit.

[0094] This entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. The disclosure also includes, for instance, all embodiments of the disclosure narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the disclosure described as a genus, all individual species are considered separate aspects of the disclosure. With respect to aspects of the disclosure described or claimed with "a" or "an," it should be understood that these terms mean "one or more" unless context unambiguously requires a more restricted meaning.

[0095] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to thespecific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the disclosure.

[0096] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term."

[0097] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having."

[0098] When used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0099] In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

[0100] It should be understood that this disclosure is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the subject matter of the disclosure, which is defined solely by the claims.

[0101] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0102] A better understanding of the disclosure and of its advantages will be obtained from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.EXAMPLES

[0103] Additional aspects and details of the disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting.Example 1 Materials and Methods

[0104] Patient selection:

[0105] The study cohort (ReSolve FSHD) consisted of 25 FSHD patients (11 females, 14 males) and 11 healthy controls (8 females, 3 males). All participants were adults, and the age distribution was as follows: In the FSHD group: Ages 20-40: 5, 40-6: 13, 60+: 7, in the control group: Ages 20-40: 5, 40-60: 3, 60+: 3.

[0106] Extracellular vesicle (EV) isolation:

[0107] For proteomic analyses, plasma EVs were isolated by size-exclusion chromatography (SEC). This method allows for efficient EV separation from soluble components and most lipoproteins. While there are various other ways to isolate EVs (e.g., filtration, differential centrifugation, density gradient and immunoprecipitation), most of these methods are exclusively targeted for either EV yield or specificity. In contrast, SEC is efficient in both parameters, and therefore ideal for biomarker discovery. 500uL of each precleared plasma sample was loaded onto Izon qEV35 chromatography columns, and using an automated fraction collector (Izon), the first five fractions were collected and combined. EV size and concentration were monitored by Nanoparticle Tracking Analysis (NTA, Nanosight). The EV structure was characterized by transmission electron microscopy (TEM). EV quality was assessed by western blot to evaluate the presence of serum EV markers (e.g., CD9), serum proteins (e.g., ApoB, C3). To eliminate bias in subsequent analyses, EV isolation was performed blinded. Samples were then batched to ensure FSHD and healthy controls were analyzed together.

[0108] Protein isolation and Mass spectrometry:

[0109] Plasma EVs isolated by SEC were lysed with 5% SDS and sonicated to extract proteins. A typical sample preparation yielded at least 200 pg of protein from 500 pL of plasma. A total of 100 pg of protein was used for mass spectrometry (MS), while the rest was stored for Western Blot analysis. For MS, the proteins were digested via Strap or Rapigest depending on the sample quantity and following the Core’s protocol. Liquid Chromatography was then carried out with tandem MS (LC-MS / MS). Data were searched using Mascot Daemon by Matrix Science version 2.7.0 (Boston, MA) via Proteome Discoverer (version 2.4 Thermo Scientific) and searched the database against the mostrecent Uniprot databases. Label free quantitation was performed using the spectral count approach, in which the relative protein quantitation was measured by comparing the number of MS / MS spectra identified from the same protein in each of the multiple LC-MS / MS datasets.

[0110] Statistical Analysis'.

[0111] The differential expression of both EV-associated protein levels was assessed using the R package DESeq2, which is designed for RNA-seq count data but can also be used for protein spectral count data. To control for testing of many transcripts and proteins, false discovery rates (FDR) were computed from raw p values, with FDR<0.1 and log2FC >1 used as a cutoff for significance.

[0112] Immunoblotting:

[0113] Potential biomarkers were identified by mass spectrometry with western blot analysis. The location of the biomarkers (either inside or outside EVs) was then determined by treating the EVs with Proteinase K (20 ug / ml) which would only retain the molecules packed within the EVs because the protein would be protected in the EV by their lipid bilayer membrane. PMSF (20uM) was added to the EVs one hour post treatment to inhibit Proteinase K and ensure it would not be accessible to EV proteins prior to western blot. As an additional control, EV membranes were treated simultaneously with Proteinase K and 1% Triton X-100, which ruptures EV membranes, allowing Proteinase K to digest intraluminal EV proteins.Example 2 EV isolation from FSHD plasma and mass spectrometry analysis

[0114] Extracellular vesicles (EVs) were isolated from patient plasma using size exclusion chromatography (SEC). The isolated EVs were microscopically characterized (Fig. 1A). Next, using nanoparticle tracking analysis, it was determined that both size and concentration of EVs were reduced in FSHD patients (Fig. 1 B-C). The content of the EVs was next profiled using mass spectrometry (MS). MS analysis identified 338 proteins with at least 2 spectral counts in both cohorts. 52 proteins were detected out of 90 proteins indexed in the plasma EV database of Exocarta (Fig. 1 D). Enrichment analysis (DAVID, Go Terms “Cellular Compartments”) verified the enrichment of EV proteins in the dataset (Fig. 1 E). Next, a PCA analysis was carried out to see whether there is an overall clustering of the samples based on condition and gender (Fig. 2A). The analysis illustrated that gender was the main determinant of the overall protein profiles, regardless of the condition. The analysis also further illustrated that there was not a further separation based on disease severity (Fig. 2B). Thus, further analysis of biomarkers in EV proteins was conducted separately for malesand females.Example 3 FSHD specific changes in plasma EVs

[0115] To identify potential FSHD biomarkers, statistical analysis was carried out between FSHD and healthy controls in both genders. In the female cohort, MBL2 was identified as a potential FSHD biomarker, as its level was significantly increased in EVs collected from FSHD patients (false discovery rate (FDR) adjusted p.val<0.1 and log2FC >1). FDR is the metric for global confidence assessment of a large-scale proteomics dataset (PMID: 26519173; Aggarwal et al., Methods Mol Biol . 2016:1362:119-28. doi: 10.1007 / 978- 1 -4939-3106-4 7). On the other hand, the level of GPLD1 was significantly reduced (FDR adjusted p.val<0.1 and log2FC<-1 ) (Fig. 3A). Using the same criteria, three biomarkers were identified in the male cohort based upon significantly increased expression. These were JUP, DSP, and CLEC3B (Fig. 3D). The relative levels (Z-scores) of all changing proteins was visualized (nominal p.val<0.05) to display any potential correlation with disease severity (Figs. 3B and 3E). In particular, MBL2 levels were consistently higher in female patients with moderate to severe FSHD (Fig. 3B). Furthermore, increased MBL2 expression in FSHD females was verified using western blot (Fig. 3C).Example 4Specific packaging of MBL2 within EVs

[0116] While SEC effectively separates EVs from plasma proteins, isolated EVs may naturally be coated with proteins acquired from plasma. To determine the localization of these biomarkers, we treated them with Proteinase K that selectively degrades surface proteins and potential contaminants. This treatment effectively removed plasma apoliprotein B (ApoB) and complement protein C3 which are known to coat the EV ‘corona’, while not affecting the levels of EV tetraspanin CD9. Interestingly, the experiments showed that MBL2 and JUP were largely protected by proteinase K treatment, indicating that these two proteins are entrapped within EVs (Fig. 4A). The retainment of MBL2 in EVs was further verified after Proteinase K treatment using three more EV samples (Fig. 4B).Example 5Additional FSHD specific changes in plasma EVs

[0117] Additional plasma samples for the FSHD cohort were obtained from Dr. Rabi Tawil (University of Rochester, NY, USA) as part of the ReSolve FSHD study (Table 1). For all samples, plasma was collected using anti-coagulant (EDTA) treated collection tubes, and each sample was assigned a blinding code so that all batches of samples were tested in a blinded fashion.

[0118] Table 1. Patient clinical data of two cohorts.CSS: Clinical Severity Score. FSHD-COM: FSHD composite score.

[0119] To identify any additional potential FSHD biomarkers, statistical analysis between FSHD and healthy controls in both genders was carried out. In the female cohort, MBL2 was identified as a potential FSHD biomarker, as its level was significantly increased in EVs collected from FSHD patients (FDR adjusted p.val<0.1 and log2FC >1). On the other hand, the level of GPLD1 was significantly reduced (FDR adjusted p.val<0.1 and log2FC<-1 ) (Fig. 6A). Using the same criteria, four potential biomarkers significantly changing in the male FSHD cohort, i.e., JUP, DSP, C9, and C4BP, were identified (Fig. 6D). The relative levels (Z- scores) of all changing proteins (nominal p.val<0.05) across patients and healthy controls (Figs. 6B and 6E) also were visualized. Considering the dynamic and variable nature of FSHD, the differentially expressed protein levels were correlated with confounding factors. The assessed confounding factors included age, FSHD composite score (FSHD-COM), clinical severity score (CSS), and D4Z4 contraction size. FSHD-COM is a comprehensive, 18 item performance-based functional composite outcome measure assessing the legs, shoulders and arms, trunk, hands, and balance / mobility. A trend of positive correlation of MBL2 levels with age in FSHD patients (Fig. 7A) was observed. When sex is analyzed separately, we observed that this stemmed from the male cohort in which the MBL2 levels significantly increased with age (Fig. 7B). It is noteworthy that, in the differential protein expression analyses (Fig. 6A-E), MBL2 was identified as a potential biomarker only in the female cohort. The correlation analyses suggest that while female patients maintain an overall elevated levels of MBL2 in all ages, MBL2 levels reach equivalent levels with age in male patients. Moreover, a significant negative correlation was observed in C4BPB levels inmale FSHD patients with FSHD-COM score (Fig. 7C). Accordingly, study results indicate that C4BPB is progressively decreased in patients with unfavorable FSHD-COM scores (i.e., higher scores). No significant correlation was observed for the other tested potential biomarkers and confounding factors.Example 6Additional specific packing of MBL2 within EVs

[0120] While SEC effectively separates EVs from plasma proteins, isolated EVs may naturally be coated with proteins acquired from plasma. To determine the localization of these biomarker proteins, the proteins were treated with Proteinase K that selectively degrades surface proteins and potential contaminants. This treatment effectively removed plasma apoliprotein ApoB and complement protein C3 which are known to coat the EV ‘corona’, while not affecting the levels of EV tetraspanin CD9. Interestingly, it was shown that MBL2 and JUP were largely protected by proteinase K treatment, suggesting that they are entrapped within EVs (Fig. 4A). The study also further verified the retainment of MBL2 in EVs after Proteinase K treatment using three more EV samples (Fig. 4B).Example 7Identification of additional EV protein biomarkers in FSHD patients

[0121] Additional EV protein biomarkers were identified in FSHD patients by using an additional study cohort (cohort 2). Patients were not sub-grouped by age or sex and comparisons were done with all healthy controls vs FSHD patients. The age distribution by disease status and sex in cohort 1 , made of 36 participants (11 healthy controls and 25 FSHD patients) and cohort 2, made of 38 participants (17 healthy controls and 21 FSHD patients) are shown in Fig. 8A.

[0122] Volcano plots showing differentially expressed proteins based on nominal and false discovery rate (FDR) adjusted p values in both cohorts are provided in Fig. 8B and Fig. 8C. Combining cohorts increased the statistical power and revealed additional EV biomarkers (carnosine dipeptidase (CNDP1), immunoglobulin kappa variable 2-30 (IGKV2- 30), complement component 7 (C7), and lumican (LUM) at False Discovery Rate [FDR]<0.1 ; Fig. 8D).

[0123] In cohort 2: Tenascin C (TNC) was upregulated in FSHD patients. In combined cohort: Carnosine dipeptidase (CNDP1) and Immunoglobulin Kappa Variable 2-30 (IGKV2- 30) were significantly downregulated in FSHD patients, and Lumican (LUM) was significantly upregulated in FSHD patients. LUM and CNDP1 were further correlated with disease confounding factors.

[0124] The levels of four proteins meeting the FDR adjusted p-value cut off in the combined data are displayed across cohorts (Fig. 8E). * Nominal p value < 0.05, ** p value < 0.01 , *** p value < 0.001 by Wald test.

[0125] Thus, these experiments showed that additional biomarkers, i.e., C7, TNC, LUM, CNDP1 , and IGKV2-30, are relevant in the diagnosis, monitoring, and treatment of FSHD. More specifically, these experiments showed that C7, TNC, and LUM are upregulated in FSHD patients, and CNDP1 and IGKV2-30 are downregulated in FSHD patients.Example 8Identification of additional EV protein biomarkers in FSHD patients

[0126] The identification of additional EV protein biomarkers in FSHD patients in two cohorts was carried out after adjusting for sex as a variable. Fig. 10A-D shows EV protein biomarkers in FSHD patients in two cohorts after adjusting for sex as a variable. Fig. 10A-C are volcano plots for cohort 1 (Fig. 10A), cohort 2 (Fig. 10B) and combined cohorts 1 and 2 (Fig. 10C), after adjusting for sex as a variable, showing differentially expressed proteins based on nominal and FDR adjusted p value in both cohorts. Following this analysis, TNC in cohort 2 and LUM, C7 and C9 in the combined cohort were up-regulated and had a log2Fold-Change (FC)>0.5 with a False Discovery Rate [FDR]<0.1). Fig. 10D shows the levels (normalized counts) of C7, C9 and LUM proteins, after adjusting for sex as a variable, meeting the log2FC and FDR adjusted p-value cut offs in the combined data displayed across cohorts. * Nominal p value < 0.05, ** p value < 0.01 , *** p value < 0.001 by Wald test.

[0127] These experiments showed that C7, C9, and LUM are relevant biomarkers in the diagnosis, monitoring, and treatment of FSHD. More specifically, these experiments showed that C7, C9, and LUM are upregulated in FSHD patients.

[0128] The foregoing description is given for clearness of understanding only, and no unnecessary limitations should be understood therefrom, as modifications within the scope of the invention may be apparent to those having ordinary skill in the art.

[0129] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0130] Throughout the specification, where compositions are described as including components or materials, it is contemplated that the compositions can also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise. Likewise, where methods are described as including particular steps, itis contemplated that the methods can also consist essentially of, or consist of, any combination of the recited steps, unless described otherwise. The invention illustratively disclosed herein suitably may be practiced in the absence of any element or step which is not specifically disclosed herein.

[0131] The practice of a method disclosed herein, and individual steps thereof, can be performed manually and / or with the aid of automation provided by electronic equipment. Although processes have been described with reference to particular embodiments, a person of ordinary skill in the art will readily appreciate that other ways of performing the acts associated with the methods may be used. For example, the order of various of the steps may be changed without departing from the scope or spirit of the method, unless described otherwise. In addition, some of the individual steps can be combined, omitted, or further subdivided into additional steps.All patents, publications and references cited herein are hereby fully incorporated by reference. In case of conflict between the present disclosure and incorporated patents, publications and references, the present disclosure should control.

Claims

CLAIMS\Ne claim:1 . A method of diagnosing facioscapulohumeral dystrophy (FSHD) in a subject, the method comprising detecting the presence of a biomarker or change in level of a biomarker in a biological sample from the subject, wherein the biomarker is mannose binding lectin 2 (MBL2), junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), complement component 7 (C7), complement component 9 (C9), tenascin C (TNC), lumican (LUM), phosphatidylinositol-glycan-specific phospholipase D (GPLD1 ), complement component 4 binding protein, beta chain (C4BPB), carnosine dipeptidase (CNDP1), or immunoglobulin kappa variable 2-30 (IGKV2-30), or a combination of any two or more of MBL2, JUP, DSP, CLEC3B, 07, 09, TNC, LUM, GPLD1 , C4BPB, CNDP1 , or IGKV2-30.

2. The method of claim 1 , wherein when the change in level of the biomarker is an increase in level of MBL2, JUP, DSP, CLEC3B, 07, 09, TNC, or LUM, or a combination of any two or more of MBL2, JUP, DSP, CLEC3B, 07, 09, TNC, or LUM, the subject is diagnosed as having FSHD.

3. The method of claim 1 , wherein when the change in level of the biomarker is a decrease in level of GPLD1 , C4BPB, CNDP1 , or IGKV2-30, or a combination of any two or more of GPLD1 , C4BPB, CNDP1 , or IGKV2-30, the subject is diagnosed as having FSHD.

4. The method of any one of claims 1-3, wherein the change in level of the biomarker is an increase or decrease compared to a reference level or to a control level.

5. The method of claim 4, wherein the change in level of the biomarker is measured as being statistically significant compared to the reference level or to the control level.

6. The method of any one of claims 1-5, wherein the sample is tissue or fluid isolated from the subject.

7. The method of claim 6, wherein the fluid is plasma or serum.

8. The method of claim 6 or 7, wherein the fluid is plasma.

9. The method of any one of claims 1-8, wherein the sample is an extracellular vesicle(EV) isolated from plasma of the subject.

10. The method of any one of claims 1-8, wherein the biomarker is a protein.11 . The method of any one of claims 1-8, wherein the biomarker is a nucleic acid.

12. The method of any one of claims 1-11 further comprising treating the subject after diagnosing the subject with FSHD, wherein treating comprises administering to the subject an effective amount of any of a) a non-steroidal anti-inflammatory drug, b) ARO-DUX4, c) RO7204239, d) AOC 1020, and / or e) physical therapy, or a combination of any thereof.

13. A method for determining efficacy of a therapeutic treatment for facioscapulohumeral dystrophy (FSHD) in a subject, the method comprising measuring the level of a biomarker in a biological sample from the subject before and after the treatment, and determining that the treatment is effective in treating FSHD if the level of the biomarker is decreased or increased relative to a reference level or to the level of the biomarker in the sample from the subject before treatment, wherein when the biomarker is mannose binding lectin 2 (MBL2), junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), complement component 7 (C7), complement component 9 (C9), tenascin C (TNC), or lumican (LUM), or a combination of any two or more of MBL2, JUP, DSP, CLEC3B, C7, C9, TNC, or LUM, the treatment is effective when the level of the biomarker decreases, or wherein when the biomarker is phosphatidylinositol-glycan-specific phospholipase D (GPLD1 ), complement component 4 binding protein, beta chain (C4BPB), carnosine dipeptidase (CNDP1 ), or immunoglobulin kappa variable 2-30 (IGKV2-30), or a combination of any two or more of GPLD1 , C4BPB, CNDP1 , or IGKV2-30, the treatment is effective when the level of the biomarker increases.

14. The method of claim 13, wherein the sample is tissue or fluid isolated from the subject.

15. The method of claim 14, wherein the fluid is plasma or serum.

16. The method of claim 14 or 15, wherein the fluid is plasma.

17. The method of any one of claims 13-16, wherein the sample is an extracellular vesicle (EV) isolated from plasma of the subject.

18. The method of any one of claims 13-17, wherein the biomarker is a protein.

19. The method of any one of claims 13-17, wherein the biomarker is a nucleic acid.

20. The method of any one of claims 13-19, wherein the treatment comprises administering to the subject an effective amount of any of a) a non-steroidal anti-inflammatory drug, b) ARO-DUX4, c) RO7204239, d) AOC 1020, and / or e) physical therapy, or a combination of any thereof.21 . A method of diagnosing facioscapulohumeral dystrophy (FSHD) in a female subject, the method comprising detecting a change in level of a biomarker in a biological sample from the subject, wherein the biomarker is mannose binding lectin 2 (MBL2) or phosphatidylinositol-glycan-specific phospholipase D (GPLD1 ), or a combination of MBL2 and GPLD1 , wherein when the level of MBL2 is increased in the subject relative to a reference level or a control level, and / or wherein when the level of GPLD1 is decreased in the subject relative to a reference level or a control level, the subject is diagnosed as having FSHD.

22. A method of diagnosing facioscapulohumeral dystrophy (FSHD) in a male subject, the method comprising detecting the presence or a change in level of a biomarker in a biological sample from the subject, wherein the biomarker is junction plakoglobin (JUP), desmoplakin (DSP), tetranectin (CLEC3B), or complement component 4 binding protein, beta chain (C4BPB), or a combination of any two or more of JUP, DSP, CLEC3B, or C4BPB, the subject is diagnosed as having FSHD.

23. The method of claim 22, wherein the subject is diagnosed as having FSHD when JUP, CLEC3B, or DSP, or a combination thereof, is present in the biological sample.

24. The method of claim 22, wherein the subject is diagnosed as having FSHD wherein the level of any of JUP, CLEC3B, or DSP is increased in the subject compared to the level of the level of any of JUP, CLEC3B, or DSP in a control.

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