Sphingolipid biomarkers for sphingosine phosphate lyase insufficiency syndrome and related conditions

Abstract

Methods and compositions for identifying the presence and severity of sphingosine phosphate lyase insufficiency syndrome (SPLIS) in a subject are provided. The methods and compositions provided herein also find use in monitoring effectiveness of treatment of a SPLIS patient receiving a treatment for the SPLIS. Embodiments of the methods and compositions of the disclosure are also useful for informing SPLIS patient outcomes and facilitating interventional clinical trials and natural history studies related to SPLIS. In addition, the methods and compositions of the present disclosure find use in facilitating treatment decisions for a subject having SPLIS.

Description

[0001] SPHINGOLIPID BIOMARKERS FOR SPHINGOSINE PHOSPHATE LYASE INSUFFICIENCY SYNDROME AND RELATED CONDITIONS

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under grant no. R21TR004262, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

[0004] INTRODUCTION

[0005] SGPL1 gene encodes the vitamin B6-dependent enzyme sphingosine- 1 -phosphate lyase (SPL, also known as, S1PL), which catalyzes the irreversible degradation of the bioactive sphingolipid sphingosine- 1 -phosphate (SIP) in the final step of sphingolipid degradation (Expert Opin Ther Targets. 2009;13(8):1013-25; J Lipid Res. 2019;60(3):456-63). SIP serves as a ligand for a family of ubiquitously expressed G protein-coupled receptors (SIPRs). The SIPRs control actin cytoskeleton organization, cell migration, morphology and cell survival by signaling through downstream targets including MAPK, AKT and Rho GTPases (J Recept Signal Transduct Res. 2017;37(5):437-46). SIP is generated from sphingosine by sphingosine kinases (SphKl / 2) via a phosphorylation event that can be reversed by lipid- and SIP-specific phosphatases (Sgppl / 2) (Biochim Biophys Acta Mol Cell Biol Lipids. 2018; 1863(11): 1413-22; J Lipid Res. 2015;56(l l):2048-60). However, only SPL can irreversibly degrade SIP. Thus, SPL is a critical regulator of SIP levels in blood and tissues (Expert Opin Ther Targets. 2009; 13(8): 1013-25).

[0006] How a loss of SPL function might manifest in humans has remained unknown until very recently. NPHS14, a previously unrecognized inborn error of metabolism was identified in 2017 to be caused by recessive mutations (i.e., bi-allelic pathogenic variants) in SGPL1 (J Clin Invest. 2017;127(3):912-28). Over the last two years, other groups have reported patients with SGPL1 mutations and similar presentations (J Clin Invest. 2017;127(3):942-53; Hum Mutat. 2017;38(4):365-72; Neurology. 2017;88(6):533-42; Clinical Kidney Journal. 2017;l-6; Brain Dev. 2018;40(6):480-3; J Clin Endocrinol Metab.2019 May 1; 104(5): 1484- 1490; Pediatr Nephrol. 2019;34(l):77-9). Patients affected by NPHS14, which has been renamed SPL insufficiency syndrome (SPLIS) (Adv Biol Regul 2019 71:128-140) exhibit one or more major disease features including steroid-resistant nephrotic (protein spilling) syndrome with focal segmental glomerulosclerosis (FSGS) pathology, neurological defects, ichthyosis, lymphopenia, and primary adrenal insufficiency (Adv Biol Regul 2019;71 :128-40). A wide range of severity is observed, with some patients dying in utero, others in infancy, while still others have presented late in the first decade of life and are living into adulthood, albeit requiring dialysis or kidney transplantation. Further, SPLIS diagnosis remains challenging, given the existence of similarly presenting diseases (such as, e.g., other sphingolipid disorders) and the prevalence of SGPL1 mutations of unknown pathogenicity.

[0007] SUMMARY

[0008] Thus, there is a need for improved and useful methods and compositions for reliably differentiating sphingosine phosphate lyase insufficiency syndrome (SPLIS) from similar diseases and conditions. Additionally, there is also a need for improved and useful methods for comparing SPLIS severity across a wide variety of phenotypically and genotypically different disease manifestations. This invention provides such new and useful methods and compositions, addressing the limitations mentioned above. The present disclosure is based on the discovery that certain signatures of sphingolipids may be used as biomarkers, or surrogate indicators, of the degree of sphingosine phosphate lyase (SPL) enzyme inactivation in patients with SPLIS. Specifically, by measuring a range of plasma sphingolipid analytes, a signature of lipid metabolites unique to SPLIS patients was discovered. This unique signature was found to correlate with disease activity and, accordingly, finds use not only as a biomarker for diagnosis, but additionally as a quality endpoint for natural history studies and interventional clinical trials.

[0009] Methods and compositions for identifying the presence and severity of sphingosine phosphate lyase insufficiency syndrome (SPLIS) in a subject are provided. The methods and compositions provided herein also find use in monitoring effectiveness of treatment of a SPLIS patient receiving a treatment for the SPLIS. Embodiments of the methods and compositions of the disclosure are also useful for informing SPLIS patient outcomes and facilitating interventional clinical trials and natural history studies related to SPLIS. In addition, the methods and compositions of the present disclosure find use in facilitating treatment decisions for a subject having SPLIS.

[0010] In one aspect, methods for identifying a subject as having sphingosine phosphate lyase insufficiency syndrome (SPLIS) are provided. Aspects of the methods include: assaying in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP, wherein a ratio of the SI P level to the dihydro-SIP level greater than a threshold ratio indicates the subject has SPLIS. In some embodiments, the method further includes assaying in a body fluid sample of the subject a level of one or more of ceramides, ceramide- 1 -phosphates, and hexosylceramides. In these cases, a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1 -phosphates higher than a threshold level of ceramide-1- phosphates, and / or a level of hexosylceramides higher than a threshold level of hexosylceramides may indicate the subject has SPLIS. In some embodiments, the SGPL1 gene of the subject is sequenced and, e.g., the subject is found to have a mutation in the SGPL1 gene. In some embodiments, the effect of the mutation on sphingosine phosphate lyase (SPL) activity is unknown. In other instances, the mutation is a known SPLIS pathogenic variant.

[0011] In certain embodiments, the subject has one or more of lymphopenia, steroid resistant necrotic kidney disease, renal fibrosis, kidney failure, adrenal insufficiency, neurological defects, ichthyosis, and hypercholesterolemia. In some embodiments, the subject has steroid resistant necrotic kidney disease, renal fibrosis, and / or kidney failure. In these instances, a urine sample of the subject may be assayed for protein levels in order to assess SPLIS severity. In some embodiments, the subject has one or more neurological defects. In these instances, a nerve conduction study is performed on the subject in order to assess SPLIS severity.

[0012] In certain embodiments, the method further includes treating the subject having SPLIS. In some embodiments, the treatment includes pyridoxine therapy and / or gene therapy. In some embodiments, the body fluid sample includes blood, serum, or plasma. In some embodiments, the body fluid sample includes plasma. In some embodiments, assaying the level of SIP and dihydro-SIP includes performing mass spectrometry such as, e.g., tandem mass spectrometry. In some embodiments, assaying the level of SIP and dihydro-SIP includes contacting the sample with specific binding member of SIP and a specific binding member of dihydro-SIP.

[0013] In certain embodiments, the subject has previously been diagnosed with and / or is being treated for a sphingolipid disorder other than SPLIS and / or a kidney disease other than SPLIS. In some embodiments, the sphingolipid disorder is dihydroceramide desaturase deficiency or ceramidase deficiency. In some embodiments, the kidney disease is focal segmental glomerulosclerosis. In some embodiments, wherein the ratio of the SIP level to the dihydro-SIP level greater than the threshold ratio, the method includes altering the non-SPLIS treatment, wherein altering the treatment includes tapering the dose and / or dosage of the treatment or terminating the treatment.

[0014] In another aspect, a method of providing a health evaluation for a subject based on the discovered SPLIS biomarkers / surrogate indicators is provided. Aspects of the methods include: assaying in a body fluid sample of a subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP in order to obtain a ratio of the SIP level to the dihydro-SIP level; generating a sphingosine phosphate lyase insufficiency syndrome (SPLIS) assessment for the subject using the ratio of the SIP level to the dihydro-SIP level; and providing a health evaluation for the subject based on the SPLIS assessment. In some embodiments, the method further includes assaying in the body fluid sample of the subject a level of one or more of ceramides, ceramide- 1- phosphates, and hexosylceramides in order to generate the SPLIS assessment.

[0015] In certain embodiments, the health assessment includes a predicted clinical outcome based on the SPLIS assessment. In some embodiments, the SGPL1 gene of the subject is sequenced and, e.g., the subject has a mutation in the SGPL1 gene. In some embodiments, the health evaluation includes a natural history study. In some embodiments, the subject is being treated for SPLIS. In some embodiments, the SPLIS treatment includes pyridoxine therapy, adeno-associated virus (AAV) mediated gene therapy, and / or induced pluripotent stem cell (iPSC) gene therapy. In some embodiments, the health evaluation includes an interventional clinical trial and, e.g., the ratio of the SIP level to the dihydro-SIP level is an endpoint of the clinical trial.

[0016] In another aspect, methods for monitoring response to treatment for sphingosine phosphate lyase insufficiency syndrome (SPLIS) of a subject receiving the treatment are provided. Aspects of the methods include: assaying in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP, wherein a ratio of the SIP level to the dihydro-SIP level lower than a threshold ratio indicates that the subject is responding positively to the treatment. Aspects of the methods may additionally include: assaying in a body fluid sample of the subject a level of ceramides, ceramide- 1 -phosphate, and hexosylceramides, wherein a level of ceramides lower than a threshold level of ceramides, a level of ceramide- 1- phosphate lower than a threshold level of ceramide- 1 -phosphate, and a level of hexosylceramides lower than a threshold level of hexosylceramides, indicates that the subject is responding positively to the treatment. In certain embodiments, the method further includes assaying a protein level in a urine sample of the subject, wherein a level of protein lower than a threshold level of protein indicates that the subject is responding positively to the treatment. In some embodiments, the treatment includes pyridoxine therapy. In some embodiments, the treatment includes adeno-associated virus (AAV) mediated gene therapy. In some embodiments, the treatment includes induced pluripotent stem cell (iPSC) gene therapy.

[0017] In certain embodiments, the body fluid sample includes blood, serum, or plasma. In some embodiments, the body fluid sample includes plasma. In some embodiments, assaying the level of SIP and dihydro-SIP includes performing mass spectrometry. In these cases, the mass spectrometry may include tandem mass spectrometry. In some embodiments, assaying the level of SIP and dihydro-SIP includes contacting the sample with specific binding member of SIP and a specific binding member of dihydro-SIP. In these cases, one or both of the specific binding members may be antibodies.

[0018] In certain embodiments, wherein the ratio of the SIP level to the dihydro-SIP level is higher than the threshold ratio, the method includes altering the treatment. Altering the treatment may include changing the treatment regimen or changing the active agent administered to the subject. In some embodiments, changing the treatment regimen includes increasing dose and / or dosage of the active agent administered to the subject. In some embodiments, wherein the ratio of the SIP level to the dihydro-SIP level is higher than the threshold ratio, the method includes tapering the dose and / or dosage of the treatment or terminating the treatment. In some embodiments, the method further includes assaying, in a body fluid sample of the subject whose treatment has been terminated, a level of SIP and dihydro-SIP, wherein a ratio of the SIP level to the dihydro-SIP level higher than a threshold ratio indicates that the subject has a relapse of SPLIS symptoms.

[0019] In another aspect, methods for monitoring relapse of sphingosine phosphate lyase insufficiency syndrome (SPLIS) symptoms in a subject treated for the SPLIS are provided. Aspects of the methods include: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1-phosphate (SIP) and dihydro-SIP to obtain a first ratio of SIP to dihydro-SIP; measuring at a second time point in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a second ratio of SIP to dihydro-SIP, wherein the second time point is after the first time point; wherein an increased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-S IP indicates relapse of the SPLTS, and wherein a lack of change between first and second ratios of SIP to dihydro-S IP or a decreased second ratio of SIP to dihydro-S IP compared to the first ratio of SIP to dihydro-S IP is indicative of a stable disease state. In some embodiments, the method further includes measuring in a body fluid sample of the subject a level of SIP and dihydro-S IP to obtain a ratio of SIP to dihydro-S IP at multiple points of time over a period, wherein a trend of increased ratio of SIP to dihydro-S IP is indicative of relapse of the SPLIS symptoms.

[0020] In certain embodiments, the body fluid sample includes blood, serum, or plasma. In some embodiments, the body fluid sample includes plasma. In some embodiments, measuring the level of SIP and dihydro-S IP includes performing mass spectrometry such as, e.g., tandem mass spectrometry. In some embodiments, measuring the level of SIP and dihydro-S IP includes contacting the sample with specific binding member of SIP and a specific binding member of dihydro-S IP.

[0021] In certain embodiments, the method further includes treating the subject having a relapse of the SPLIS symptoms. In some embodiments, the treatment includes pyridoxine therapy. In some embodiments, the treatment includes adeno-associated virus (AAV) mediated gene therapy. In some embodiments, the treatment includes induced pluripotent stem cell (iPSC) gene therapy.

[0022] In another aspect, methods for monitoring progression of sphingosine phosphate lyase insufficiency syndrome (SPLIS) in a subject are provided. Aspects of the methods include: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1- phosphate (SIP) and dihydro-S IP to obtain a first ratio of SIP to dihydro-S IP; measuring at a second time point in a body fluid sample of the subject a level of SIP and dihydro-S IP to obtain a second ratio of SIP to dihydro-S IP, wherein the second time point is after the first time point; wherein an increased second ratio of SIP to dihydro-S IP compared to the first ratio of SIP to dihydro-S IP is indicative of increased severity of SPLIS, wherein a lack of change between first and second ratios of SIP to dihydro-S IP is indicative of lack of change in severity of SPLIS, and wherein a decreased second ratio of SIP to dihydro-S IP compared to the first ratio of SIP to dihydro-S IP is indicative of improvement in SPLIS. In some embodiments, the method includes measuring in a body fluid sample of the subject a level of SIP and dihydro-S IP to obtain a ratio of S IP to dihydro-SIP at multiple points of time over a period, wherein a trend of increased ratio of SIP to dihydro-SIP is indicative of increased severity of SPLIS.

[0023] In another aspect, methods for monitoring responsiveness of a subject to a treatment for sphingosine phosphate lyase insufficiency syndrome (SPLIS) are provided. Aspects of the methods include: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain a first ratio of SIP to dihydro-SIP; measuring at a second time point in a body fluid sample of the subject a level of S IP and dihydro-SIP to obtain a second ratio of SIP to dihydro-SIP, wherein the second time point is after the first time point; wherein a decreased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP indicates a positive response to the treatment, wherein an increased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of increased severity of SPLIS, and wherein a lack of change between first and second ratios of SIP to dihydro-SIP is indicative of lack of change in severity of SPLIS. In some embodiments, the method includes measuring in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a ratio of SIP to dihydro-SIP at multiple points of time over a period, wherein a trend of decreased ratios of SIP to dihydro-SIP is indicative of positive response to the treatment.

[0024] In certain embodiments, the method further includes: measuring at the first time point in a body fluid sample of the subject a level of ceramides, ceramide- 1 -phosphates, and / or hexosylceramides to obtain a first level of ceramides, ceramide- 1 -phosphates, and / or hexosylceramides; measuring at the second time point in a body fluid sample of the subject a level of ceramides, ceramide- 1 -phosphates, and / or hexosylceramides to obtain a second level of ceramides, ceramide- 1-phosphates, and / or hexosylceramides; wherein a decreased second level of ceramides compared to the first level of ceramides, a decreased second level of ceramide- 1- phosphates compared to the first level of ceramide- 1 -phosphates, and / or a decreased second level of hexosylceramides compared to the first level of hexosylceramides, indicates a positive response to the treatment.

[0025] In another aspect, methods for treating a subject diagnosed with sphingosine phosphate lyase insufficiency syndrome (SPLIS) are provided. Aspects of the methods include: administering to a subject an effective amount of: a vitamin B6 compound; and / or a recombinant adeno-associated viral (rAAV) virion including a nucleic acid encoding sphingosine- 1 -phosphate lyase (SPL), wherein the subject is identified as in need for treatment for SPLTS based on having, in a body fluid sample, a ratio of sphingosine- 1 -phosphate (SIP) to dihydro-SIP higher than a threshold ratio of SIP to dihydro-SIP. In some embodiments, the subject is identified as in need for treatment for SPLIS based on having, in a body fluid sample, a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1 -phosphates higher than a threshold level of ceramide- 1 -phosphates, and / or a level of hexosylceramides higher than a threshold level of hexo sy Iceramides .

[0026] In another aspect, methods for treating a subject diagnosed with sphingosine phosphate lyase insufficiency syndrome (SPLIS) include: administering a pyridoxine therapy and / or a genetic therapy to the subject; measuring a level of at least one biomarker selected from sphingosine- 1 -phosphate (SIP) to dihydro-SIP ratio, ceramides level, ceramide- 1 -phosphates level, and hexosylceramides level in a biological sample of the subject after the administering; tapering or terminating the pyridoxine therapy and / or the genetic therapy if the level of the measured biomarker is below a threshold level of the biomarker; or continuing the pyridoxine therapy and / or the genetic therapy if the level of the measured biomarker is at or above a threshold level of the biomarker. In some embodiments, continuing the pyridoxine therapy includes changing the treatment regimen or changing the active agent administered to the subject to a different vitamin B6 vitamer. In some embodiments, the measuring includes measuring a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain the ratio of SIP to dihydro- SIP. In some embodiments, the measuring includes measuring a level of ceramides, ceramide- 1- phosphates, and / or hexosylceramides. In some embodiments, the genetic therapy includes adeno- associated virus (AAV) mediated gene therapy.

[0027] In another aspect, methods for treating a subject diagnosed with sphingosine phosphate lyase insufficiency syndrome (SPLIS) include: administering a pyridoxine therapy to the subject; measuring a level of at least sphingosine- 1 -phosphate (SIP) and dihydro-SIP in a biological sample of the subject to obtain a ratio of SIP to dihydro-SIP after the administering; if the ratio of SIP to dihydro-SIP is at or above a threshold ratio of SIP to dihydro-SIP changing the treatment regimen; or continuing the administering if the ratio of SIP to dihydro-SIP is below a threshold ratio of SIP to dihydro-SIP. In some embodiments, wherein changing the treatment regimen includes increasing dose and / or dosage of the pyridoxine therapy or changing the active agent administered to the subject to a different vitamin B6 vitamer. In some embodiments, the biological sample is serum, plasma, or blood. In some embodiments, the biological sample is plasma.

[0028] In another aspect, methods for diagnosing a subject as having phosphate lyase insufficiency syndrome (SPLIS) are provided. Aspects of the methods include: measuring a level of at least sphingosine- 1 -phosphate (SIP) and dihydro-SIP in a biological sample of the subject to obtain a ratio of SIP to dihydro-SIP, wherein a ratio of the measured SIP level and the measured dihydro-SIP level that is higher than a threshold ratio of SIP to dihydro-SIP indicates that the subject has SPLIS. In some embodiments, the method further includes measuring in a body fluid sample of the subject a level of one or more of ceramides, ceramide- 1 -phosphates, and hexosylceramides, wherein a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1 -phosphates higher than a threshold level of ceramide- 1 -phosphates, and / or a level of hexosylceramides higher than a threshold level of hexosylceramides, indicates the subject has SPLIS. In some embodiments, the SGPL1 gene of the subject is sequenced. In some embodiments, the subject has a mutation in the SGPL1 gene. In some embodiments, the method further includes administering a therapy for treatment of SPLIS.

[0029] BRIEF DESCRIPTION OF THE FIGURES

[0030] FIG. 1 depicts the sphingosine phosphate lyase (SPL) open reading frame encoded by the SGPL1 gene including the vitamin B6 binding homology domain, exons 1-15, and a selection of known mutations.

[0031] FIG. 2 illustrates a portion of the sphingolipid metabolic pathway with linkage to triacylglycerol and phosphatidylethanolamine synthesis.

[0032] FIG. 3 illustrates a portion of the ceramide pathway with linkage to ceramide- 1- phosphate, ethanolamine- 1 -phosphate, sulfatide, glyco sphingolipid, and sphingomyelin synthesis.

[0033] FIGS. 4A to 4B depict liquid chromatography and mass spectrometry systems that may be used to assay a fluid sample for disease biomarkers according to an embodiment of the invention.

[0034] FIG. 5 provides a table of specific tandem mass spectrometry conditions for different analytes according to an embodiment of the invention. FIG. 6 provides an elution profile of internal standards spiked in plasma according to an embodiment of the invention.

[0035] FIG. 7 illustrates plasma sphingosine- 1 -phosphate (SIP) levels in a healthy pediatric cohort in comparison to children with two types of inborn errors of sphingolipid metabolism.

[0036] FIGS. 8A to 8B illustrates dihydroceramides (DHCer) and ceramides (Cer) in a healthy individual and in an individual with dihydroceramide desaturase (DES) deficiency.

[0037] DETAILED DESCRIPTION

[0038] Methods and compositions for identifying the presence and severity of sphingosine phosphate lyase insufficiency syndrome (SPLIS) in a subject are provided. The methods and compositions provided herein also find use in monitoring effectiveness of treatment of a SPLIS patient receiving a treatment for the SPLIS. Embodiments of the methods and compositions of the disclosure are also useful for informing SPLIS patient outcomes and facilitating interventional clinical trials and natural history studies related to SPLIS. In addition, the methods and compositions of the present disclosure find use in facilitating treatment decisions for a subject having SPLIS.

[0039] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0040] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the ait to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0042] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0043] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0044] As will be apparent to those of skill in the ail upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. § 112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

[0045] DEFINITIONS

[0046] The terms “individual,” “subject,” and “patient,” used interchangeably herein, refer to a human.

[0047] The terms “polypeptide,” “peptide” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N- terminal methionine residues; immunologically tagged proteins; and the like. “NH2” refers to the free amino group present at the amino terminus of a polypeptide and “COOH” refers to the free carboxyl group present at the carboxyl terminus of a polypeptide in keeping with standard polypeptide nomenclature, J. Biol. Chem., 243 (1969), 3552-59 is used.

[0048] In the context of a polypeptide present in a biological sample, “polypeptide” refers to a naturally-occurring polypeptide present in an individual from whom the sample is obtained.

[0049] “Conservative amino acid substitution” refers to a substitution of one amino acid residue for another sharing chemical and physical properties of the amino acid side chain (e.g., charge, size, hydrophobicity / hydrophilicity). “Conservative substitutions” are intended to include substitution within the following groups of amino acid residues: gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr. Guidance for such substitutions can be drawn from alignments of amino acid sequences of polypeptides.

[0050] A “biomarker” or “marker” as used herein generally refers to one or more organic biomolecules (e.g., a polypeptide or a lipid), or a relationship between a plurality of biomarkers (e.g., a ratio between two different polypeptides or lipids) which is differentially present in a sample taken from a subject of one phenotypic status (e.g., having a disease) as compared with another phenotypic status (e.g., not having the disease or having a different level or severity of the disease or having a different disease). A biomarker is differentially present between different phenotypic statuses if the mean or median level of the biomarker in a first phenotypic status relative to a second phenotypic status is calculated to represent statistically significant differences. Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio. Biomarkers, alone or in combination, provide measures of relative likelihood that a subject belongs to a phenotypic status of interest (e.g., having a disease, and / or severity of a disease, and / or remission of the disease, etc.). As such, biomarkers can find use as markers for, for example, disease (diagnostics), severity of disease, therapeutic effectiveness of a treatment, and the like. Biomarkers are thus analytes in assays that facilitate diagnosis, theranostics, monitoring efficacy of treatment, monitoring presence absence or severity of disease, and the like.

[0051] A “biological fluid sample” or “body fluid sample” encompasses a variety of fluid sample types obtained from an individual. The definition encompasses whole blood and blood fractions (e.g., serum, plasma); and other liquid samples of biological origin (e.g., saliva, urine, bile fluid). “Blood sample” refers to a biological sample, which is obtained from blood of a subject, and includes whole blood and blood fractions (e.g., plasma or serum) suitable for analysis in the present methods. In general, separation of cellular' components and non-cellular components in a blood sample (e.g., by centrifugation) without coagulation provides a blood plasma sample, while such separation of coagulated (clotted) blood provides a blood serum sample. Examples of biological samples of blood include peripheral blood or samples derived from peripheral blood. The definition also includes samples that have been manipulated after their procurement, such as by treatment with reagents, dilution, or enrichment for certain components, such as one or more analyte(s) to be assayed. For example, a biological sample (e.g., blood) can be enriched for a fraction containing an analyte(s) of interest.

[0052] By “purified” is meant a compound of interest (e.g., a polypeptide) has been separated from components that accompany it in nature. “Purified” can also be used to refer to a compound of interest separated from components that can accompany it during manufacture (e.g., in chemical synthesis). In some embodiments, a compound is substantially pure when it is at least 50% to 60%, by weight, free from organic molecules with which it is naturally associated or with which it is associated during manufacture. In some embodiments, the preparation is at least 75%, at least 90%, at least 95%, or at least 99%, by weight, of the compound of interest. A substantially pure compound can be obtained, for example, by extraction from a natural source (e.g., bacteria), by chemically synthesizing a compound, or by a combination of purification and chemical modification. A substantially pure compound can also be obtained by, for example, enriching a sample that contains the compound. A substantially pure compound can also be obtained by recombinant or chemical synthetic production. Purity can be measured by any appropriate method, e.g., chromatography, mass spectroscopy, high performance liquid chromatography analysis, etc.

[0053] As used herein, the terms “determining”, “assessing”, “assaying”, “measuring” and “detecting” refer to both quantitative and semi-quantitative determinations. Where a quantitative determination is intended, the phrase “determining an amount” of an analyte and the like is used. Where either a quantitative and semi-quantitative determination is intended, the phrase “determining a level” of an analyte or “detecting” an analyte is used.

[0054] “Quantitative” assays in general provide information on the amount of an analyte in a sample relative to a reference (control), and are usually reported numerically, where a “zero” value can be assigned where the analyte is below the limit of detection. “Semi-quantitative” assays typically involve presentation of a numeric representation of the amount of the analyte in the specimen that is relative to a reference (e.g., a threshold, e.g., normal threshold or an abnormal threshold), where a “zero” value can be assigned where the analyte is below the limit of detection. In general, semi-quantitative results are compared against an accompanying reference interval to provide a qualitative interpretation of the result.

[0055] “Sensitivity” refers to the fraction of people with a phenotype (e.g., presence or absence of disease, severity of disease, etc.) that a test correctly identifies as positive. “Specificity” refers to the fraction of people without the phenotype (e.g., presence or absence of disease, severity of disease, etc.) that the test correctly identifies as negative. The fractions with respect to sensitivity and / or specificity may be presented as a percentage. Where expressed as percentages, specificity can be calculated by subtracting the sensitivity value for incorrect diagnosis from 100. For example, if a test used an algorithm for diagnosis of SPLIS also incorrectly identified SPLIS in 8% of cases, the specificity for SPLIS would be 92%. “Antibody” as used herein refers to an antigen-binding protein having one or more polypeptides that can be genetically cncodablc by immunoglobulin genes, or fragments of immunoglobulin genes, and which bind an antigen of interest. “Antibody” as used herein encompasses whole antibodies as well antigen-binding fragments of whole antibodies. Antigenbinding antibody fragments include, for example, Fab’, (Fab’)2, and the like. “Fab”’ as used herein refers to a minimal antigen-binding portion of an antibody that lacks an Fc portion (e.g., a heterodimer of a VH / VL pair of a tetrameric antibody). “(Fab’)2” refers to Fab molecules that are covalently linked, usually covalently linked as found in nature, which lack an Fc portion. It should be noted that while various antibody fragments may be defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. The term “antibody” encompasses polyclonal and monoclonal antibodies, and further encompasses antibodies of any class (e.g., IgM, IgG, and subclasses thereof). “Antibody” also encompasses hybrid antibodies, bispecific antibodies, heteroantibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof, which retain antigen binding.

[0056] The terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0057] METHODS OF IDENTIFYING AND CHARACTERIZING SPLIS

[0058] In one aspect, methods for identifying a subject as having sphingosine phosphate lyase insufficiency syndrome (SPLIS) are provided. Aspects of the methods include: assaying in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP, wherein a ratio of the SIP level to the dihydro-SIP level greater than a threshold ratio indicates the subject has SPLIS. In some embodiments, the method further includes assaying in a body fluid sample of the subject a level of one or more of ceramides, ceramide- 1 -phosphates, and hexosylceramides. In these cases, a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1 -phosphates higher than a threshold level of ceramide-l-phosphates, and / or a level of hexosylceramides higher than a threshold level of hexosylceramides may indicate the subject has SPLIS. In some embodiments, a level of SIP higher than a threshold level of SIP may indicate the subject has SPLIS.

[0059] In some embodiments, the SGPL1 gene of the subject is sequenced and, e.g., the subject is found to have a mutation in the SGPL1 gene. In some embodiments, the effect of the mutation on sphingosine phosphate lyase (SPL) activity is unknown. In other words, the SGPL1 mutation is a variant of unknown significance. In other instances, the mutation is a known SPLIS pathogenic variant. In some instances, the sequencing of the subject is used to identify the subject as having SPLIS. For example, one or more mutations in the SGPL1 gene of the subject (i.e., determined by sequencing) may indicate the subject has SPLIS. In other cases, one or more mutations in the SGPL1 gene of the subject (i.e., determined by sequencing) may indicate that the subject is a candidate for having SPLIS or, i.e., it is possible the subject has SPLIS.

[0060] In certain embodiments, the subject has one or more of lymphopenia, steroid resistant necrotic kidney disease, renal fibrosis, kidney failure, adrenal insufficiency, neurological defects, ichthyosis, and hypercholesterolemia. In some embodiments, the subject has steroid resistant necrotic kidney disease, renal fibrosis, and / or kidney failure. In these instances, a urine sample of the subject may be assayed for protein levels in order to assess SPLIS severity. In some embodiments, the subject has one or more neurological defects. In these instances, a nerve conduction study is performed on the subject in order to assess SPLIS severity.

[0061] In certain embodiments, the method further includes treating a subject having SPLIS (e.g., wherein the subject is determined to have a ratio of SIP level to dihydro-SIP level greater than a threshold ratio as described above). In some embodiments, the treatment includes pyridoxine therapy and / or gene therapy. In some embodiments, the treatment includes pyridoxine therapy wherein a therapeutically effective dose of a vitamin B6 compound (e.g., pyridoxine) is administered to the subject. In some embodiments, the treatment includes adeno- associated viral (AAV) mediated gene therapy wherein a therapeutically effective dose of a recombinant AAV (rAAV) virion comprising a nucleic acid encoding SPL is administered to the subject. Tn some embodiments, induced pluripotent stem cells (iPSC) are used to deliver a therapeutically effective dose of a functional SPL enzyme to the subject.

[0062] In some embodiments, the body fluid sample includes blood, serum, or plasma. In some cases, the body fluid sample includes plasma. In some embodiments, assaying the level of SIP and dihydro-SIP includes performing mass spectrometry such as, e.g., tandem mass spectrometry. In some embodiments, assaying the level of SIP and dihydro-SIP includes contacting the sample with specific binding member of SIP and a specific binding member of dihydro-SIP. In some embodiments, the specific binding member may be a protein such as, e.g., an antibody.

[0063] In certain embodiments, the subject has previously been diagnosed with and / or is being treated for a sphingolipid disorder other than SPLIS and / or a kidney disease other than SPLIS. In some embodiments, the sphingolipid disorder is dihydroceramide desaturase deficiency or ceramidase deficiency. In some embodiments, the kidney disease is focal segmental glomerulosclerosis. In some embodiments, wherein the ratio of the SIP level to the dihydro-SIP level greater than the threshold ratio, the method includes altering the non-SPLIS treatment, wherein altering the treatment includes tapering the dose and / or dosage of the treatment or terminating the treatment.

[0064] In another aspect, a method of providing a health evaluation for a subject based on the discovered SPLIS biomarkers / surrogate indicators is provided. Aspects of the methods include: assaying in a body fluid sample of a subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP in order to obtain a ratio of the SIP level to the dihydro-SIP level; generating a sphingosine phosphate lyase insufficiency syndrome (SPLIS) assessment for the subject using the ratio of the SIP level to the dihydro-SIP level; and providing a health evaluation for the subject based on the SPLIS assessment. In some embodiments, the method further includes assaying in the body fluid sample of the subject a level of one or more of ceramides, ceramide- 1- phosphates, and hexosylceramides in order to generate the SPLIS assessment.

[0065] The health evaluation is a qualitative or quantitative determination regarding one or more health related matters pertaining to the subject. The health evaluation, generated in accordance with embodiments of the methods, may vary. In certain embodiments, the health assessment includes a predicted clinical outcome based on the SPLIS assessment. In some embodiments, the SGPL1 gene of the subject is sequenced and, e.g., the subject has a mutation in the SGPL1 gene. In some embodiments, the health evaluation includes a natural history study. In other words, the health evaluation may make a determination pertaining to the health of the subject (such as, e.g., the significance of a SGPL1 mutation variant the subject has) based on information generated by / pertaining to a plurality of subjects. In these instances the assayed biomarker (e.g., a ratio of the SIP level to the dihydro-S IP level in the plasma of a subject) may be an endpoint of the natural history study. In some embodiments, the subject is being treated for SPLIS. In some embodiments, the SPLIS treatment includes pyridoxine therapy, adeno-associated virus (AAV) mediated gene therapy, and / or induced pluripotent stem cell (iPSC) gene therapy. In some embodiments, the health evaluation includes an interventional clinical trial and, e.g., the ratio of the SIP level to the dihydro-S IP level is an endpoint of the clinical trial.

[0066] In another aspect, methods for diagnosing a subject as having phosphate lyase insufficiency syndrome (SPLIS) are provided. Aspects of the methods include: measuring a level of at least sphingosine- 1 -phosphate (SIP) and dihydro-S IP in a biological sample of the subject to obtain a ratio of SIP to dihydro-S IP, wherein a ratio of the measured SIP level and the measured dihydro-S IP level that is higher than a threshold ratio of SIP to dihydro-S IP indicates that the subject has SPLIS. In some embodiments, the method further includes measuring in a body fluid sample of the subject a level of one or more of ceramides, ceramide- 1 -phosphates, and hexosylceramides, wherein a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1 -phosphates higher than a threshold level of ceramide- 1 -phosphates, and / or a level of hexosylceramides higher than a threshold level of hexosylceramides, indicates the subject has SPLIS. In some embodiments, the SGPL1 gene of the subject is sequenced. In some embodiments, the subject has a mutation in the SGPL1 gene. In some embodiments, the method further includes administering a therapy for treatment of SPLIS.

[0067] “Threshold biomarker level”, which may also be referred to herein as a “cutoff value” or “threshold value”, refers to a biomarker level that can be used to distinguish between a first condition and a second (e.g., between individuals who do not have SPLIS and individuals who have SPLIS or a subject having a different severity of SPLIS than another individual having SPLIS) such that a biomarker level in a sample that is above a control level indicates an increased likelihood of the second condition in the individual. Thus, a “control biomarker level” or “biomarker threshold value” refers to an assay value (e.g., amount of a biomarker)), which is an approximate value that distinguishes the likelihood that a condition is present in the individual tested from the likelihood that a condition is not present in the individual tested, with a preselected specificity and / or sensitivity.

[0068] For example, a biomarker threshold value can represent an approximate level of a biomarker that detects affected subjects at a desired sensitivity (e.g., at least 55%, at least about 60%, at least 70%, or at least 80% or more). Thus, for example, an individual having a biomarker level that is greater than a threshold value has at least about 60% or greater likelihood of having that condition.

[0069] It will be appreciated that the precise number value for control or threshold values can vary with the type of assay and reagents used to detect the biomarkers as well as the sensitivity and specificity desired from the assay. For example, the assay values upon which the threshold values for are based on may be obtained using, e.g., NIST plasma and commercial sphingolipid standards as controls. However, regardless of the assay and reagents used, the correlations between a threshold or cut off value of a biomarker and likelihood of a disease state (e.g., SPLIS) will be present regardless of the assays and reagents used. Thus, so long as the test samples are assayed for the biomarker (e.g., SIP / dihydro-SIP, SIP, ceramides, ceramide- 1 -phosphates, and / or hexosylceramides) using an assay platform and reagents of the same general type and similar sensitivity as the assay platform and reagents used to determine the control / threshold values of the biomarker, the findings upon which the methods of the present disclosure are based will be preserved.

[0070] Assays using a ratio of SIP / dihydro-SIP levels according to the present disclosure may facilitate a diagnosis of SPLIS with a desired sensitivity (e.g., at least 20%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95% or greater), and with a desired specificity (e.g., at least 80%, at least 85%, at least 90%, at least 95% or greater).

[0071] METHODS OF MONITORING SPLIS

[0072] In another aspect, methods for monitoring response to treatment for sphingosine phosphate lyase insufficiency syndrome (SPLIS) of a subject receiving the treatment are provided. Aspects of the methods include: assaying in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP, wherein a ratio of the SIP level to the dihydro-SIP level lower than a threshold ratio indicates that the subject is responding positively to the treatment. Aspects of the methods may additionally include: assaying in a body fluid sample of the subject a level of ceramides, ceramide- 1 -phosphate, and hcxosylccramidcs, wherein a level of ceramides lower than a threshold level of ceramides, a level of ceramide- 1- phosphate lower than a threshold level of ceramide- 1 -phosphate, and a level of hexosylceramides lower than a threshold level of hexosylceramides, indicates that the subject is responding positively to the treatment.

[0073] In certain embodiments, the method further includes assaying a protein level in a urine sample of the subject, wherein a level of protein lower than a threshold level of protein indicates that the subject is responding positively to the treatment. In some embodiments, the treatment includes pyridoxine therapy. In some embodiments, the treatment includes adeno-associated vims (AAV) mediated gene therapy. In some embodiments, the treatment includes induced pluripotent stem cell (iPSC) gene therapy.

[0074] In certain embodiments, the body fluid sample includes blood, serum, or plasma. In some embodiments, the body fluid sample includes plasma. In some embodiments, assaying the level of SIP and dihydro-SIP includes performing mass spectrometry. In these cases, the mass spectrometry may include tandem mass spectrometry. In some embodiments, assaying the level of SIP and dihydro-SIP includes contacting the sample with specific binding member of SIP and a specific binding member of dihydro-SIP. In these cases, one or both of the specific binding members may be antibodies.

[0075] In certain embodiments, wherein the ratio of the SIP level to the dihydro-SIP level is higher than the threshold ratio, the method includes altering the treatment, wherein altering the treatment includes changing the treatment regimen or changing the active agent administered to the subject. In some embodiments, changing the treatment regimen includes increasing dose and / or dosage of the active agent administered to the subject. In some embodiments, wherein the ratio of the SIP level to the dihydro-SIP level is higher than the threshold ratio, the method includes tapering the dose and / or dosage of the treatment or terminating the treatment. In some embodiments, the method further includes assaying, in a body fluid sample of the subject whose treatment has been terminated, a level of SIP and dihydro-SIP, wherein a ratio of the SIP level to the dihydro-SIP level higher than a threshold ratio indicates that the subject has a relapse of SPLIS symptoms. In another aspect, methods for monitoring relapse of sphingosine phosphate lyase insufficiency syndrome (SPLIS) symptoms in a subject treated for the SPLIS arc provided. Aspects of the methods include: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain a first ratio of SIP to dihydro-SIP; measuring at a second time point in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a second ratio of SIP to dihydro-SIP, wherein the second time point is after the first time point; wherein an increased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP indicates relapse of the SPLIS, and wherein a lack of change between first and second ratios of SIP to dihydro-SIP or a decreased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of a stable disease state. In some embodiments, the method further includes measuring in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a ratio of SIP to dihydro-SIP at multiple points of time over a period, wherein a trend of increased ratio of SIP to dihydro-SIP is indicative of relapse of the SPLIS symptoms.

[0076] In certain embodiments, the body fluid sample includes blood, serum, or plasma. In some embodiments, the body fluid sample includes plasma. In some embodiments, measuring the level of SIP and dihydro-SIP includes performing mass spectrometry such as, e.g., tandem mass spectrometry. In some embodiments, measuring the level of SIP and dihydro-SIP includes contacting the sample with specific binding member of SIP and a specific binding member of dihydro-SIP.

[0077] In certain embodiments, the method further includes treating the subject having a relapse of the SPLIS symptoms. In some embodiments, the treatment includes pyridoxine therapy. In some embodiments, the treatment includes adeno-associated virus (AAV) mediated gene therapy. In some embodiments, the treatment includes induced pluripotent stem cell (iPSC) gene therapy.

[0078] In another aspect, methods for monitoring progression of sphingosine phosphate lyase insufficiency syndrome (SPLIS) in a subject are provided. Aspects of the methods include: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1- phosphate (SIP) and dihydro-SIP to obtain a first ratio of SIP to dihydro-SIP; measuring at a second time point in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a second ratio of SIP to dihydro-SIP, wherein the second time point is after the first time point; wherein an increased second ratio of S 1 P to dihydro-S 1 P compared to the first ratio of S 1 P to dihydro-SIP is indicative of increased severity of SPLIS, wherein a lack of change between first and second ratios of SIP to dihydro-SIP is indicative of lack of change in severity of SPLIS, and wherein a decreased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of improvement in SPLIS. In some embodiments, the method includes measuring in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a ratio of SIP to dihydro-SIP at multiple points of time over a period, wherein a trend of increased ratio of SIP to dihydro-SIP is indicative of increased severity of SPLIS.

[0079] In another aspect, methods for monitoring responsiveness of a subject to a treatment for sphingosine phosphate lyase insufficiency syndrome (SPLIS) are provided. Aspects of the methods include: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain a first ratio of SIP to dihydro-SIP; measuring at a second time point in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a second ratio of SIP to dihydro-SIP, wherein the second time point is after the first time point; wherein a decreased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP indicates a positive response to the treatment, wherein an increased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of increased severity of SPLIS, and wherein a lack of change between first and second ratios of SIP to dihydro-SIP is indicative of lack of change in severity of SPLIS. In some embodiments, the method includes measuring in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a ratio of SIP to dihydro-SIP at multiple points of time over a period, wherein a trend of decreased ratios of SIP to dihydro-SIP is indicative of positive response to the treatment.

[0080] In certain embodiments, the method further includes: measuring at the first time point in a body fluid sample of the subject a level of ceramides, ceramide- 1 -phosphates, and / or hexosylceramides to obtain a first level of ceramides, ceramide- 1 -phosphates, and / or hexosylceramides; measuring at the second time point in a body fluid sample of the subject a level of ceramides, ceramide- 1 -phosphates, and / or hexosylceramides to obtain a second level of ceramides, ceramide- 1-phosphates, and / or hexosylceramides; wherein a decreased second level of ceramides compared to the first level of ceramides, a decreased second level of ceramide- 1- phosphates compared to the first level of ceramide- 1-phosphates, and / or a decreased second level of hexosylceramides compared to the first level of hexosylceramides, indicates a positive response to the treatment.

[0081] METHODS OF TREATING SPLIS

[0082] In another aspect, methods for treating a subject diagnosed with sphingosine phosphate lyase insufficiency syndrome (SPLIS) are provided. Aspects of the methods include: administering to a subject an effective amount of: a vitamin B6 compound; and / or a recombinant adeno-associated viral (rAAV) virion including a nucleic acid encoding sphingosine- 1-phosphate lyase (SPL), wherein the subject is identified as in need for treatment for SPLIS based on having, in a body fluid sample, a ratio of sphingosine- 1-phosphate (SIP) to dihydro-SIP higher than a threshold ratio of SIP to dihydro-SIP. In some embodiments, the subject is identified as in need for treatment for SPLIS based on having, in a body fluid sample, a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1 -phosphates higher than a threshold level of ceramide- 1 -phosphates, and / or a level of hexosylceramides higher than a threshold level of hexo sy Iceramides .

[0083] In another aspect, methods for treating a subject diagnosed with sphingosine phosphate lyase insufficiency syndrome (SPLIS) include: administering a pyridoxine therapy and / or a genetic therapy to the subject; measuring a level of at least one biomarker selected from sphingosine- 1-phosphate (SIP) to dihydro-SIP ratio, ceramides level, ceramide- 1 -phosphates level, and hexosylceramides level in a biological sample of the subject after the administering; tapering or terminating the pyridoxine therapy and / or the genetic therapy if the level of the measured biomarker is below a threshold level of the biomarker; or continuing the pyridoxine therapy and / or the genetic therapy if the level of the measured biomarker is at or above a threshold level of the bio marker. In some embodiments, continuing the pyridoxine therapy includes changing the treatment regimen or changing the active agent administered to the subject to a different vitamin B6 vitamer. In some embodiments, the measuring includes measuring a level of sphingosine- 1-phosphate (SIP) and dihydro-SIP to obtain the ratio of SIP to dihydro- SIP. In some embodiments, the measuring includes measuring a level of ceramides, ceramide- 1- phosphates, and / or hexosylceramides. In some embodiments, the genetic therapy includes adeno- associated virus (AAV) mediated gene therapy. In another aspect, methods for treating a subject diagnosed with sphingosine phosphate lyase insufficiency syndrome (SPLIS) include: administering a pyridoxine therapy to the subject; measuring a level of at least sphingosine- 1 -phosphate (SIP) and dihydro-SIP in a biological sample of the subject to obtain a ratio of SIP to dihydro-SIP after the administering; if the ratio of SIP to dihydro-SIP is at or above a threshold ratio of SIP to dihydro-SIP changing the treatment regimen; or continuing the administering if the ratio of SIP to dihydro-SIP is below a threshold ratio of SIP to dihydro-SIP. In some embodiments, wherein changing the treatment regimen includes increasing dose and / or dosage of the pyridoxine therapy or changing the active agent administered to the subject to a different vitamin B6 vitamer. In some embodiments, the biological sample is serum, plasma, or blood. In some embodiments, the biological sample is plasma.

[0084] BIOMARKERS

[0085] The biomarkers of the present disclosure (e.g., SIP / dihydro-SIP, SIP, ceramides, ceramide- 1 -phosphates, and / or hexosylceramides) may act as a surrogate indicator of the degree of sphingosine phosphate lyase (SPL) enzyme inactivation in patients with SPLIS. For example, patients with SPLIS may exhibit high levels of the SPL substrate SIP. Additionally, it was surprisingly discovered that a pronounced increase in the ratio of plasma S IP to dihydro-SIP levels was uniquely indicative of SPLIS and, further, indicative of the degree of SPL enzyme inactivation. This may arise from the fact that a biochemical feedback loop in the metabolic pathway exists. In this feedback mechanism, ceramide-mediated interactions with ORM proteins inhibit serine palmitoyltransferase. The high sphingolipid levels in SPLIS patients feeds back to reduce de novo sphingolipid biosynthesis, causing dihydro sphingolipid species (e.g., dihydro- SIP) to rise less severely than sphingolipids harboring a double bond in the sphingoid base.

[0086] The SIP / dihydroSIP ratio was found to be extremely high in all SPLIS patients examined. Further, the ratio was found to correlate with disease severity. Within individual patients, the ratio of SIP to dihydro-SIP levels was reduced after therapeutic interventions that improved clinical status (such as, e.g., nerve conduction studies and urine protein levels). This indicates that the SIP / dihydro-SIP biomarker correlates with disease activity. In contrast, patients with other sphingolipid disorders such as dihydroceramide desaturase deficiency, or ceramidase deficiency (Farber’s disease) were not found to have high SIP / dihydroSIP ratios. Similarly, while patients with focal segmental glomerulosclerosis (a similar kidney disease as SPLIS) may have elevated SIP levels, the SIP / dihydroSIP ratios of patients were not found to be elevated.

[0087] Additionally, SPLIS patients were found to have elevation of ceramides, ceramide- 1- phosphates, and hexosylceramides. As such, a second signature of elevated sphingolipids in these categories is characteristic of SPLIS patients and, e.g., ceramides, ceramide- 1 -phosphates, and hexosylceramides may be used as biomarkers in the above-described methods. FIG. 2 illustrates a portion of a sphingolipid metabolic pathway with linkage to triacylglycerol and phosphatidylethanolamine synthesis.

[0088] SUBJECTS

[0089] The methods of the present disclosure can be used to facilitate a diagnosis and / or monitoring of severity of SPLIS in any suitable subject having or suspected of having SPLIS. In certain aspects, the subject has, is suspected of having, or is at risk of having SPLIS.

[0090] SGPL1 gene encodes the vitamin B6-dependent enzyme SPL, which catalyzes the irreversible degradation of the bioactive sphingolipid sphingosine- 1 -phosphate (SIP) in the final step of sphingolipid degradation. SPLIS patients harbor inactivating mutations in SGPL1 gene and exhibit one or more major disease features including steroid-resistant nephrotic (protein spilling) syndrome with focal segmental glomerulosclerosis (FSGS) pathology, neurological defects (developmental delay or regression, ataxia, cranial nerve defects, seizures, and peripheral neuropathy), ichthyosis, cranial nerve palsies, bony abnormalities, hypocalcemia, hypothyroidism, gonadal defects, immunodeficiency (e.g., lymphopenia) and primary adrenal insufficiency with a wide range of severity. SPLIS patients have reduced life span with some patients dying in utero, others in infancy. SPLIS patients can present late in the first decade of life, and may survive to adulthood, albeit requiring dialysis or kidney transplantation.

[0091] A subject who may be treated, monitored, assayed, or diagnosed with the methods disclosed herein may be a newborn, an infant, a toddler, a child, a teenager, or an adult. In certain aspects, the subject may be a fetus in utero. The subject may exhibit one or more symptoms of SPLIS and / or have a mutation associated with lack of sufficient SPL activity. In some aspects, a subject may be treated for SPLIS prior to onset of symptoms of SPLIS, based on, e.g., a genetic test and / or a blood test. An inactivating SPL mutation that results in significant lack of enzyme activity may be one or more of nonsense mutations, splicing defects, and missense mutations.

[0092] In certain aspects, a subject who may be treated, assayed, or diagnosed with the methods of the disclosure may be asymptomatic but may be identified by low to undetectable SPL abundance and / or activity, e.g., in skin fibroblasts obtained from the subject by, e. g., a skin biopsy. In certain aspects, cells from a buccal swab may be assayed to determine low SPL level or activity. In certain aspects, a subject may be diagnosed as having or at risk of developing SPLIS prior to the onset of symptoms by next generation DNA sequence analysis of whole genome or whole exome in infants with lymphopenia or in a sibling of a previously diagnosed SPLIS patient. In certain aspects, lymphopenia may be persistent idiopathic lymphopenia diagnosed at birth. In some embodiments, a subject may be diagnosed as having or at risk of developing SPLIS prior to or after the onset of symptoms by detecting specific markers of naive T cells through any number of modalities or methods, as well as combinations thereof, as are known in the ail. For example, the subject may be identified or diagnosed by measuring recent thymic emigrants (using, e.g., flow cytometry) and / or through T cell receptor excision circle testing. In some cases, disease progression or response to therapy is monitored by detecting specific markers of naive T cells (e.g., by measuring recent thymic emigrants using flow cytometry and / or by performing T cell receptor excision circle testing), e.g., in conjunction with the biomarker-based monitoring methods of the disclosure as described above. In some embodiments, a subject who may be treated, assayed, or diagnosed with the methods disclosed herein may have a mutation of SGPL1 of unknown significance.

[0093] FIG. 1 the SPL open reading frame encoded by SGPL1 is shown, with exons 1-15 indicated. The vitamin B6 (e.g., pyridoxal 5'-phosphate [PLP]) binding homology domain is shown by a box. Mutations found are shown in the schematic.

[0094] BIOLOGICAL SAMPLES

[0095] Suitable biological samples useful in the methods of the present disclosure include biological fluids (e.g., a blood sample, e.g., whole blood, blood fraction (e.g., serum, plasma)). Where the biological sample is a blood sample, the blood sample can be obtained from fresh blood or stored blood (e.g., in a blood bank). The biological sample can be a blood sample expressly obtained for an assay of the present disclosure or a blood sample obtained for another purpose which can be subsampled for an assay of the present disclosure. Cell free biological fluid samples include scrum and plasma.

[0096] Samples can be manipulated after or during procurement, such as, by treatment with reagents (e.g., anti-coagulants), dilution, and / or enrichment for certain components for an analyte (s) to be assayed. Samples can be pre-treated as necessary by dilution in an appropriate buffer solution, concentrated if desired, or fractionated by any number of methods including but not limited to ultracentrifugation, fractionation by fast performance liquid chromatography (FPLC), or precipitation. Any of a number of standard aqueous buffer solutions, employing one of a variety of buffers, such as phosphate, Tris, or the like, at physiological pH can be used.

[0097] ASSAY FORMATS AND DETECTION METHODS

[0098] Biomarkers for analysis in connection with the methods of the present disclosure (e.g., SIP / dihydro-SIP, SIP, ceramides, ceramide- 1 -phosphates, and / or hexosylceramides) can be detected using a variety of methods, with methods suitable for quantitative and semi-quantitative assays being of particular interest. Examples of detection methods include, but are not limited to, methods for detection of a biomarker by mass spectrometric methods, mass spectrophotometry, HPLC, gas chromatography, cytokine / chemokine arrays (e.g., using cytokine / chemokine binding partners), and the like), binding to a specific binding partner (e.g., antibody) (e.g., ELISA (e.g., non-multiplex, multiplex (e.g., LUMINEX®, MESO DISCOVERY®), flow cytometry and the like).

[0099] The assay can include a series of suitable standards, e.g., a reagent for detection of a biomarker that serves as an internal control (which internal control may be present in the biological sample as obtained from the subject or spiked to include a known amount of the control), a separately assayed sample containing a known concentration of one or more biomarkers, and the like. Controls can be positive controls or negative controls. Where desired, multiple samples and standards can be assayed so that mean values can be obtained for each.

[0100] As described above, the methods of the present disclosure can be accomplished by mass spectrometric assays, which can be adapted for detection of biomarker(s) in a biological sample. Mass spectrometry-based methods exploit the differences in mass of biomarkers to facilitate detection. Examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, tandem, electrostatic sector analyzer and hybrids of these. Data generated by mass spectrometry methods can be analyzed with the use of a programmable computer. The computer program executes a program to analyze the data to indicate the number of biomarkers detected, and the strength of the signal (indicative of the amount of the biomarker), and the determined molecular mass for each biomarker detected. Data analysis can include steps of determining signal strength of a biomarker and removing data deviating from a predetermined statistical distribution. For example, the observed peaks can be normalized, by calculating the height of each peak relative to some reference.

[0101] The computer can transform the resulting data into various formats for display. The standard spectrum can be displayed, but in one useful format only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling biomarkers with nearly identical molecular weights to be more easily seen. In another useful format, two or more spectra are compared, conveniently highlighting unique biomarkers and biomarkers that are up- or down-regulated between samples. Using any of these formats, one can readily determine whether a particular biomarker is present in a sample.

[0102] Analysis generally involves the identification of peaks in the spectrum that represent signal from an analyte. Peak selection can be done visually, but software is available, as part of Ciphergen's PROTEINCHIP® software package, that can automate the detection of peaks. In general, this software functions by identifying signals having a signal-to-noise ratio above a selected threshold and labeling the mass of the peak at the centroid of the peak signal. One version of this software clusters all peaks appearing in the various spectra within a defined mass range, and assigns a mass (M / Z) to all the peaks that are near the mid-point of the mass (M / Z) cluster.

[0103] Software used to analyze the data can include code that applies an algorithm to the analysis of the signal to determine whether the signal represents a peak in a signal that corresponds to a biomarker according to the present invention. The software also can subject the data regarding observed biomarker peaks to classification tree or ANN analysis, to determine whether a biomarker peak or combination of biomarker peaks is present that indicates the status of the particular clinical parameter under examination. Analysis of the data may be “keyed” to a variety of parameters that are obtained, either directly or indirectly, from the mass spectrometric analysis of the sample. These parameters include, but are not limited to, the presence or absence of one or more peaks, the shape of a peak or group of peaks, the height of one or more peaks, the log of the height of one or more peaks, and other arithmetic manipulations of peak height data. The present disclosure provides compositions that find use, e.g., in practicing the methods of the present disclosure. In certain aspects, the compositions include an agent for detecting a biomarker of interest (e.g., SIP / dihydro-SIP, SIP, ceramides, ceramide- 1 -phosphates, and / or hexosylceramides). The compositions of the present disclosure may include a biological sample from a subject (e.g., a subject suspected of having SPLIS, an apparently healthy subject undergoing routine medical screening, a subject with an unspecified morbidity under investigation, a control subject, or other subject) or a control sample (e.g., serum, buffer, or the like in which one or more biomarkers of interest are present (e.g., added) for purposes of providing a control (e.g., a benchmark control that includes serum from a healthy individual spiked with each of the biomarkers to be detected in the assay) for the assay).

[0104] COMPUTER-IMPLEMENTED METHODS, SYSTEMS AND DEVICES

[0105] The present disclosure provides computer program products that, when executed on a programmable computer such as that described above can carry out the methods of the present disclosure. The subject matter described herein may be embodied in systems, apparatus, methods, and / or articles depending on the desired configuration. These various implementations may include implementation in one or more computer programs that are executable and / or interpretable on a programmable system including at least one programmable processor, which may be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device (e.g. video camera, microphone, joystick, keyboard, and / or mouse), and at least one output device (e.g. display monitor, printer, etc.).

[0106] In some instances the systems further include one or more computers for complete automation or partial automation of the methods described herein. In some embodiments, systems include a computer having a computer readable storage medium with a computer program stored thereon.

[0107] In embodiments, the system includes an input module, a processing module and an output module. The subject systems may include both hardware and software components, where the hardware components may take the form of one or more platforms, e.g., in the form of servers, such that the functional elements, i.e., those elements of the system that cany out specific tasks (such as managing input and output of information, processing information, etc.) of the system may be carried out by the execution of software applications on and across the one or more computer platforms represented of the system.

[0108] The processing module includes a processor which has access to a memory having instructions stored thereon for performing the steps of the subject methods. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, memory storage devices, input-output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or it may be one of other processors that are or will become available. The processor executes the operating system and the operating system interfaces with firmware and hardware in a well-known manner, and facilitates the processor in coordinating and executing the functions of various computer programs that may be written in a variety of programming languages, such as Java, Perl, C++, Python, other high-level or low-level languages, as well as combinations thereof, as is known in the art. The operating system, typically in cooperation with the processor, coordinates and executes functions of the other components of the computer. The operating system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics which provide feedback control, such as for example negative feedback control.

[0109] The system memory may be any of a variety of known or future memory storage devices. Examples include any commonly available random-access memory (RAM), magnetic medium such as a resident hard disk or tape, an optical medium such as a read and write compact disc, flash memory devices, or other memory storage device. The memory storage device may be any of a variety of known or future devices, including a compact disk drive, a tape drive, a removable hard disk drive, or a diskette drive. Such types of memory storage devices typically read from, and / or write to, a program storage medium (not shown) such as, respectively, a compact disk, magnetic tape, removable hard disk, or floppy diskette. Any of these program storage media, or others now in use or that may later be developed, may be considered a computer program product. As will be appreciated, these program storage media typically store a computer software program and / or data. Computer software programs, also called computer control logic, typically are stored in system memory and / or the program storage device used in conjunction with the memory storage device.

[0110] In some embodiments, a computer program product is described including a computer usable medium having control logic (computer software program, including program code) stored therein. The control logic, when executed by the processor the computer, causes the processor to perform functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. Implementation of the hardware state machine so as to perform the functions described herein will be apparent to those skilled in the relevant arts.

[0111] Memory may be any suitable device in which the processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including magnetic or optical disks or tape or RAM, or any other suitable device, either fixed or portable). The processor may include a general-purpose digital microprocessor suitably programmed from a computer readable medium carrying necessary program code. Programming can be provided remotely to processor through a communication channel, or previously saved in a computer program product such as memory or some other portable or fixed computer readable storage medium using any of those devices in connection with memory. For example, a magnetic or optical disk may carry the programming, and can be read by a disk writer / reader. Systems of the invention also include programming, e.g., in the form of computer program products, algorithms for use in practicing the methods as described above. Programming according to the present invention can be recorded on computer readable media, e.g., any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; portable flash drive; and hybrids of these categories such as magnetic / optical storage media.

[0112] The processor may also have access to a communication channel to communicate with a user at a remote location. By remote location is meant the user is not directly in contact with the system and relays input information to an input manager from an external device, such as a computer connected to a Wide Area Network (“WAN”), telephone network, satellite network, or any other suitable communication channel, including a mobile telephone (i.e., smartphone). In some embodiments, systems according to the present disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface can be configured for wired or wireless communication, including, but not limited to, radio frequency (RF) communication (e.g., Radio-Frequency Identification (RFID), Zigbee communication protocols, WiFi, infrared, wireless Universal Serial Bus (USB), Ultra-Wide Band (UWB), Bluetooth® communication protocols, and cellular communication, such as code division multiple access (CDMA) or Global System for Mobile communications (GSM).

[0113] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., physical ports or interfaces such as a USB port, an RS-232 port, or any other suitable electrical connection port to allow data communication between the subject systems and other external devices such as a computer terminal (for example, at a physician’s office or in hospital environment) that is configured for similar complementary data communication.

[0114] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol to enable the subject systems to communicate with other devices such as computer terminals and / or networks, communication enabled mobile telephones, personal digital assistants, or any other communication devices which the user may use in conjunction.

[0115] In one embodiment, the communication interface is configured to provide a connection for data transfer utilizing Internet Protocol (IP) through a cell phone network, Short Message Service (SMS), wireless connection to a personal computer (PC) on a Local Area Network (LAN) which is connected to the internet, or WiFi connection to the internet at a WiFi hotspot.

[0116] In one embodiment, the subject systems are configured to wirelessly communicate with a server device via the communication interface, e.g., using a common standard such as 802.11 or Bluetooth® RF protocol, or an IrDA infrared protocol. The server device may be another portable device, such as a smart phone, Personal Digital Assistant (PDA) or notebook computer; or a larger device such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), as well as an input device, such as buttons, a keyboard, mouse or touchscreen. In some embodiments, the communication interface is configured to automatically or scmi-automatically communicate data stored in the subject systems, c.g., in an optional data storage unit, with a network or server device using one or more of the communication protocols and / or mechanisms described above.

[0117] Output controllers may include controllers for any of a variety of known display devices for presenting information to a user, whether a human or a machine, whether local or remote. If one of the display devices provides visual information, this information typically may be logically and / or physically organized as an array of picture elements. A graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing graphical input and output interfaces between the system and a user, and for processing user inputs. The functional elements of the computer may communicate with each other via system bus. Some of these communications may be accomplished in alternative embodiments using network or other types of remote communications. The output manager may also provide information generated by the processing module to a user at a remote location, e.g., over the Internet, phone or satellite network, in accordance with known techniques. The presentation of data by the output manager may be implemented in accordance with a variety of known techniques. As some examples, data may include SQL, HTML or XML documents, email or other files, or data in other forms. The data may include Internet URL addresses so that a user may retrieve additional SQL, HTML, XML, or other documents or data from remote sources. The one or more platforms present in the subject systems may be any type of known computer platform or a type to be developed in the future, although they typically will be of a class of computer commonly referred to as servers. However, they may also be a main-frame computer, a workstation, or other computer type. They may be connected via any known or future type of cabling or other communication system including wireless systems, either networked or otherwise. They may be co-located, or they may be physically separated. Various operating systems may be employed on any of the computer platforms, possibly depending on the type and / or make of computer platform chosen. Appropriate operating systems include Windows, iOS, Oracle Solaris, Linux, IBM i, Unix, and others.

[0118] Aspects of the present disclosure further include non-transitory computer readable storage mediums having instructions for practicing the subject methods. Computer readable storage mediums may be employed on one or more computers for complete automation or partial automation of a system for practicing methods described herein. Tn certain embodiments, instructions in accordance with the method described herein can be coded onto a computer- readable medium in the form of “programming”, where the term "computer readable medium" as used herein refers to any non-transitory storage medium that participates in providing instructions and data to a computer for execution and processing. Non-transitory computer- readable media include all computer-readable media except for a transitory, propagating signal. Examples of suitable non-transitory storage media include a floppy disk, hard disk, optical disk, magneto-optical disk, CD-ROM, CD-R, magnetic tape, non-volatile memory card, ROM, DVD- ROM, Blue-ray disk, solid state disk, and network attached storage (NAS), whether or not such devices are internal or external to the computer. A file containing information can be “stored” on computer readable medium, where “storing” means recording information such that it is accessible and retrievable at a later date by a computer. The computer-implemented method described herein can be executed using programming that can be written in one or more of any number of computer programming languages. Such languages include, for example, Python, Java, Java Script, C, C#, C++, Go, R, Swift, PHP, as well as many others.

[0119] The non-transitory computer readable storage medium may be employed on one or more computer systems having a display and operator input device. Operator input devices may, for example, be a keyboard, mouse, or the like. The processing module includes a processor which has access to a memory having instructions stored thereon for performing the steps of the subject methods. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, memory storage devices, input-output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or it may be one of other processors that are or will become available. The processor executes the operating system and the operating system interfaces with firmware and hardware in a well-known manner, and facilitates the processor in coordinating and executing the functions of various computer programs that may be written in a variety of programming languages, such as those mentioned above, other high level or low-level languages, as well as combinations thereof, as is known in the art. The operating system, typically in cooperation with the processor, coordinates and executes functions of the other components of the computer. The operating system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques.

[0120] EXEMPLARY NON-LIMITING ASPECTS OF THE DISCLOSURE

[0121] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of ordinary skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below. It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

[0122] 1. A method for identifying a subject as having sphingosine phosphate lyase insufficiency syndrome (SPLIS), the method comprising; assaying in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-S IP, wherein a ratio of the SIP level to the dihydro-S IP level greater than a threshold ratio indicates the subject has SPLIS.

[0123] 2. The method of aspect 1, wherein the method further comprises assaying in a body fluid sample of the subject a level of one or more of ceramides, ceramide- 1 -phosphates, and hexosylceramides, wherein a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1 -phosphates higher than a threshold level of ceramide- 1 -phosphates, and / or a level of hexosylceramides higher than a threshold level of hexosylceramides, indicates the subject has SPLIS.

[0124] 3. The method of aspects 1 or 2, wherein the SGPL1 gene of the subject is sequenced.

[0125] 4. The method of aspect 3, wherein the subject has a mutation in the SGPL1 gene.

[0126] 5. The method of aspect 4, wherein the effect of the mutation on sphingosine phosphate lyase (SPL) activity is unknown.

[0127] 6. The method of aspect 4, wherein the mutation is a known SPLIS pathogenic variant. 7. The method of any one of aspects 1 -6, wherein the subject has one or more of lymphopenia, steroid resistant necrotic kidney disease, renal fibrosis, kidney failure, adrenal insufficiency, neurological defects, ichthyosis, and hypercholesterolemia.

[0128] 8. The method of aspect 7, wherein the subject has steroid resistant necrotic kidney disease, renal fibrosis, and / or kidney failure.

[0129] 9. The method of aspect 8, wherein a urine sample of the subject is assayed for protein levels in order to assess SPLIS severity.

[0130] 10. The method of aspect 7, wherein the subject has one or more neurological defects.

[0131] 11. The method of aspect 10, wherein a nerve conduction study is performed on the subject in order to assess SPLIS severity.

[0132] 12. The method of any one of aspects 1-11, further comprising treating the subject having SPLIS.

[0133] 13. The method of aspect 12, wherein the treatment comprises pyridoxine therapy and / or gene therapy.

[0134] 14. The method of any one of aspects 1-13, wherein the body fluid sample comprises blood, serum, or plasma.

[0135] 15. The method of any one of aspects 1-14, wherein assaying the level of SIP and dihydro- S1P comprises performing mass spectrometry.

[0136] 16. The method aspect 15, wherein the mass spectrometry comprises tandem mass spectrometry.

[0137] 17. The method of any one of aspects 1-14, wherein assaying the level of SIP and dihydro- S1P comprises contacting the sample with specific binding member of SIP and a specific binding member of dihydro-SIP.

[0138] 18. The method of any one of aspects 1-17, wherein the subject has previously been diagnosed with and / or is being treated for a sphingolipid disorder other than SPLIS and / or a kidney disease other than SPLIS.

[0139] 19. The method of aspect 18, wherein the sphingolipid disorder is dihydroceramide desaturase deficiency or ceramidase deficiency.

[0140] 20. The method of aspect 18, wherein the kidney disease is focal segmental glomerulosclerosis. 21 . The method of any one of aspects 18-20, wherein the ratio of the S 1 P level to the dihydro-SIP level greater than the threshold ratio, and wherein the method comprises altering the non-SPLIS treatment, wherein altering the treatment comprises tapering the dose and / or dosage of the treatment or terminating the treatment.

[0141] 22. A method comprising: assaying in a body fluid sample of a subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP in order to obtain a ratio of the SIP level to the dihydro-SIP level; generating a sphingosine phosphate lyase insufficiency syndrome (SPLIS) assessment for the subject using the ratio of the SIP level to the dihydro-SIP level; and providing a health evaluation for the subject based on the SPLIS assessment.

[0142] 23. The method of aspect 22, wherein the method further comprises assaying in the body fluid sample of the subject a level of one or more of ceramides, ceramide- 1 -phosphates, and hexosylceramides in order to generate the SPLIS assessment.

[0143] 24. The method of aspect 22 or 23, wherein the health assessment comprises a predicted clinical outcome based on the SPLIS assessment .

[0144] 25. The method of any one of aspects 22-24, wherein the SGPL1 gene of the subject is sequenced.

[0145] 26. The method of aspect 25, wherein the subject has a mutation in the SGPL1 gene.

[0146] 27. The method of aspect 26, wherein the health evaluation comprises a natural history study.

[0147] 28. The method of any one of aspects 22-26, wherein the subject is being treated for SPLIS.

[0148] 29. The method of aspect 28, wherein the SPLIS treatment comprises pyridoxine therapy, adeno-associated virus (AAV) mediated gene therapy, and / or induced pluripotent stem cell (iPSC) gene therapy.

[0149] 30. The method of aspect 28 or 29, wherein the health evaluation comprises an interventional clinical trial.

[0150] 31. A method for monitoring response to treatment for sphingosine phosphate lyase insufficiency syndrome (SPLIS) of a subject receiving the treatment, the method comprising: assaying in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-S IP, wherein a ratio of the SIP level to the dihydro-SIP level lower than a threshold ratio indicates that the subject is responding positively to the treatment. 32. A method for monitoring response to treatment for sphingosine phosphate lyase insufficiency syndrome (SPLIS) of a subject receiving the treatment, the method comprising: assaying in a body fluid sample of the subject a level of ceramides, ceramide- 1- phosphate, and hexosylceramides, wherein a level of ceramides lower than a threshold level of ceramides, a level of ceramide- 1 -phosphate lower than a threshold level of ceramide- 1 -phosphate, and a level of hexosylceramides lower than a threshold level of hexosylceramides, indicates that the subject is responding positively to the treatment.

[0151] 33. The method of aspects 31 or 32, further comprising assaying a protein level in a urine sample of the subject, wherein a level of protein lower than a threshold level of protein indicates that the subject is responding positively to the treatment.

[0152] 34. The method of any one of aspects 31-33, further comprising detecting specific markers of naive T cells in the subject, wherein detected specific markers are used to determine the response of the subject to the treatment.

[0153] 35. The method of aspect 34, wherein the detecting comprises measuring recent thymic emigrants using flow cytometry and / or performing T cell receptor excision circle testing.

[0154] 36. The method of any one of aspects 31-35, wherein the treatment comprises pyridoxine therapy.

[0155] 37. The method of any one of aspects 31-35, wherein the treatment comprises adeno- associated virus (AAV) mediated gene therapy.

[0156] 38. The method of any one of aspects 31-35, wherein the treatment comprises induced pluripotent stem cell (iPSC) gene therapy.

[0157] 39. The method of any one of aspects 31-38, wherein the body fluid sample comprises blood, serum, or plasma.

[0158] 40. The method of any one of aspects 31-39, wherein assaying the level of SIP and dihydro- S1P comprises performing mass spectrometry.

[0159] 41. The method aspect 40, wherein the mass spectrometry comprises tandem mass spectrometry.

[0160] 42. The method of any one of aspects 31-39, wherein assaying the level of SIP and dihydro- S1P comprises contacting the sample with specific binding member of SIP and a specific binding member of dihydro-SIP. 43. The method of any one of aspects 31 -42, wherein the ratio of the S 1 P level to the dihydro-SIP level is higher than the threshold ratio and wherein the method comprises altering the treatment, wherein altering the treatment comprises changing the treatment regimen or changing the active agent administered to the subject.

[0161] 44. The method of aspect 43, wherein changing the treatment regimen comprises increasing dose and / or dosage of the active agent administered to the subject.

[0162] 45. The method of any one of aspects 31-44, wherein the ratio of the SIP level to the dihydro-SIP level is higher than the threshold ratio and wherein the method comprises tapering the dose and / or dosage of the treatment or terminating the treatment.

[0163] 46. The method of any one of aspects 31-45, wherein the method further comprises assaying in a body fluid sample of the subject whose treatment has been terminated, a level of SIP and dihydro-SIP, wherein a ratio of the SIP level to the dihydro-SIP level higher than a threshold ratio indicates that the subject has a relapse of SPLIS symptoms.

[0164] 47. A method for monitoring relapse of sphingosine phosphate lyase insufficiency syndrome (SPLIS) symptoms in a subject treated for the SPLIS, the method comprising: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain a first ratio of SIP to dihydro-SIP; measuring at a second time point in a body fluid sample of the subject a level of S IP and dihydro-SIP to obtain a second ratio of SIP to dihydro-SIP, wherein the second time point is after the first time point; wherein an increased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP indicates relapse of the SPLIS, and wherein a lack of change between first and second ratios of SIP to dihydro-SIP or a decreased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of a stable disease state.

[0165] 48. The method of aspect 47, comprising measuring in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a ratio of SIP to dihydro-SIP at multiple points of time over a period, wherein a trend of increased ratio of SIP to dihydro-SIP is indicative of relapse of the SPLIS symptoms.

[0166] 49. The method of aspect 47 or 48, wherein the body fluid sample comprises blood, serum, or plasma. 50. The method of any one of aspects 47-49, wherein measuring the level of S 1 P and dihydro-SIP comprises performing mass spectrometry.

[0167] 51. The method aspect 50, wherein the mass spectrometry comprises tandem mass spectrometry.

[0168] 52. The method of any one of aspects 47-49, wherein measuring the level of SIP and dihydro-SIP comprises contacting the sample with specific binding member of SIP and a specific binding member of dihydro-SIP.

[0169] 53. The method of any one of aspects 47-52, wherein the method further comprises treating the subject having a relapse of the SPLIS symptoms.

[0170] 54. The method of aspect 53, wherein the treatment comprises pyridoxine therapy.

[0171] 55. The method of aspect 54, wherein the treatment comprises adeno-associated virus (AAV) mediated gene therapy.

[0172] 56. The method of aspect 54, wherein the treatment comprises induced pluripotent stem cell (iPSC) gene therapy.

[0173] 57. A method for monitoring progression of sphingosine phosphate lyase insufficiency syndrome (SPLIS) in a subject, the method comprising: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain a first ratio of S IP to dihydro-SIP; measuring at a second time point in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a second ratio of SIP to dihydro-SIP, wherein the second time point is after the first time point; wherein an increased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of increased severity of SPLIS, wherein a lack of change between first and second ratios of SIP to dihydro-SIP is indicative of lack of change in severity of SPLIS, and wherein a decreased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of improvement in SPLIS.

[0174] 58. The method of aspect 57, comprising measuring in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a ratio of SIP to dihydro-SIP at multiple points of time over a period, wherein a trend of increased ratio of SIP to dihydro-SIP is indicative of increased severity of SPLIS. 59. A method for monitoring responsiveness of a subject to a treatment for sphingosine phosphate lyase insufficiency syndrome (SPLIS), the method comprising: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain a first ratio of SIP to dihydro-SIP; measuring at a second time point in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a second ratio of SIP to dihydro-SIP, wherein the second time point is after the first time point; wherein a decreased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP indicates a positive response to the treatment, wherein an increased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of increased severity of SPLIS, and wherein a lack of change between first and second ratios of SIP to dihydro-SIP is indicative of lack of change in severity of SPLIS.

[0175] 60. The method of aspect 59, comprising measuring in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a ratio of SIP to dihydro-SIP at multiple points of time over a period, wherein a trend of decreased ratios of SIP to dihydro-SIP is indicative of positive response to the treatment.

[0176] 61. The method of aspect 59 or 60, further comprising: measuring at the first time point in a body fluid sample of the subject a level of ceramides, ceramide- 1-phosphates, and / or hexosylceramides to obtain a first level of ceramides, ceramide- 1 -phosphates, and / or hexosylceramides; measuring at the second time point in a body fluid sample of the subject a level of ceramides, ceramide- 1-phosphates, and / or hexosylceramides to obtain a second level of ceramides, ceramide- 1-phosphates, and / or hexosylceramides; wherein a decreased second level of ceramides compared to the first level of ceramides, a decreased second level of ceramide- 1-phosphates compared to the first level of ceramide- 1- phosphates, and / or a decreased second level of hexosylceramides compared to the first level of hexosylceramides, indicates a positive response to the treatment.

[0177] 62. A method for treating a subject diagnosed with sphingosine phosphate lyase insufficiency syndrome (SPLIS), the method comprising: administering to a subject an effective amount of: a vitamin B6 compound; and / or a recombinant adcno-associatcd viral (rAAV) virion comprising a nucleic acid encoding sphingosine- 1 -phosphate lyase (SPL), wherein the subject is identified as in need for treatment for SPLIS based on having, in a body fluid sample, a ratio of sphingosine- 1 -phosphate (SIP) to dihydro-SIP higher than a threshold ratio of SIP to dihydro-SIP.

[0178] 63. The method aspect 62, wherein the subject is identified as in need for treatment for SPLIS based on having, in a body fluid sample, a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1 -phosphates higher than a threshold level of ceramide- 1- phosphates, and / or a level of hexosylceramides higher than a threshold level of hexo sy Iceramides .

[0179] 64. A method for treating sphingosine phosphate lyase insufficiency syndrome (SPLIS) in a subject, the method comprising: administering a pyridoxine therapy and / or a genetic therapy to the subject; measuring a level of at least one biomarker selected from sphingosine- 1 -phosphate (SIP) to dihydro-SIP ratio, ceramides level, ceramide- 1-phosphates level, and hexosylceramides level in a biological sample of the subject after the administering; tapering or terminating the pyridoxine therapy and / or the genetic therapy if the level of the measured biomarker is below a threshold level of the biomarker; or continuing the pyridoxine therapy and / or the genetic therapy if the level of the measured biomarker is at or above a threshold level of the biomarker.

[0180] 65. The method of aspect 64, wherein continuing the pyridoxine therapy comprises changing the treatment regimen or changing the active agent administered to the subject to a different vitamin B6 vitamer.

[0181] 66. The method of aspect 64 or 65, wherein the measuring comprises measuring a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain the ratio of SIP to dihydro-SIP.

[0182] 67. The method of any one of aspects 64-66, wherein the measuring comprises measuring a level of ceramides, ceramide- 1-phosphates, and / or hexosylceramides.

[0183] 68. The method of any one of aspects 64-67, wherein the genetic therapy comprises adeno- associated virus (AAV) mediated gene therapy. 69. A method for treating sphingosine phosphate lyase insufficiency syndrome (SPLIS) in a subject, the method comprising: administering a pyridoxine therapy to the subject; measuring a level of at least sphingosine- 1 -phosphate (SIP) and dihydro-SIP in a biological sample of the subject to obtain a ratio of SIP to dihydro-SIP after the administering; if the ratio of SIP to dihydro-SIP is at or above a threshold ratio of SIP to dihydro-SIP changing the treatment regimen; or continuing the administering if the ratio of SIP to dihydro-SIP is below a threshold ratio of SIP to dihydro-SIP.

[0184] 70. The method of aspect 69, wherein changing the treatment regimen comprises increasing dose and / or dosage of the pyridoxine therapy or changing the active agent administered to the subject to a different vitamin B6 vitamer.

[0185] 71. The method of aspect 69 or 70, wherein the biological sample is serum, plasma, or blood.

[0186] 72. A method for diagnosing a subject as having phosphate lyase insufficiency syndrome (SPLIS), the method comprising: measuring a level of at least sphingosine- 1 -phosphate (SIP) and dihydro-SIP in a biological sample of the subject to obtain a ratio of SIP to dihydro-SIP, wherein a ratio of the measured SIP level and the measured dihydro-SIP level that is higher than a threshold ratio of SIP to dihydro-SIP indicates that the subject has SPLIS.

[0187] 73. The method of aspect 72, wherein the method further comprises measuring in a body fluid sample of the subject a level of one or more of ceramides, ceramide- 1 -phosphates, and hexosylceramides, wherein a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1 -phosphates higher than a threshold level of ceramide- 1 -phosphates, and / or a level of hexosylceramides higher than a threshold level of hexosylceramides, indicates the subject has SPLIS.

[0188] 74. The method of aspect 72 or 73, wherein the SGPL1 gene of the subject is sequenced.

[0189] 75. The method of aspect 74, wherein the subject has a mutation in the SGPL1 gene.

[0190] 76. The method of any one of aspects 72-75, further comprising administering a therapy for treatment of SPLIS. EXAMPLES

[0191] As demonstrated in the above disclosure, the present invention has a wide variety of applications. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, weights, concentrations, temperatures, etc.) but some experimental errors and deviations should be accounted for.

[0192] Example 1: An LC / MS / MS Method for Measurement of Sphingolipids in the Plasma of Pediatric Individuals with Disorders of Sphingolipid Metabolism

[0193] Introduction

[0194] In recent years, there has been a fast- growing body of evidence supporting the contribution of sphingolipids to the pathophysiology of a variety of common diseases. In addition, an ever-growing number of rare disorders of sphingolipid metabolism are being discovered by diagnostic next generation sequencing. In concert with the growing appreciation of the importance of sphingolipids to human health and disease is an exploding demand for fast and reliable quantitative determination of sphingolipids in plasma, and a need to overcome analytical challenges for confident profiling detection. An end-to-end workflow for the extraction, screening, identification, and quantitation of over 80 sphingolipids from 10 classes in plasma by Agilent 1290 Infinity II LC coupled to an Agilent 6495 triple quadrupole LC / MS system was developed. Nine internal standards that represent each of the sphingolipid class were selected to evaluate the method performance by accuracy and precision runs and typically achieving accuracy (70-130%) and precision (RSD < 20%) for all spiking levels, limits of quantitation (LOQ) of 2.5 to 25 nM in plasma, and linear calibration curves with R2> 0.99. The method was applied to quantify sphingolipids in pediatric patients with sphingosine phosphate lyase insufficiency syndrome (SPLIS), ceramidase deficiency, and dihydroceramide desaturase deficiency for comparative analysis of plasma sphingolipids. The collected data was applied to develop pediatric reference ranges, identify gender- specific differences in post-pubertal children and / or age-specific differences, and demonstrate the characteristic derangements in circulating sphingolipids found in three atypical sphingolipid disorders.

[0195] FIG. 3 illustrates a portion of the ceramide pathway with linkage to, e.g., ceramide-1- phosphate, ethanolamine- 1 -phosphate, sulfatide, glycosphingolipid, and sphingomyelin synthesis.

[0196] Materials and Methods

[0197] Sample Preparation'.

[0198] 1. Pipet 50 pL plasma into a 2mL microcentrifuge tube.

[0199] 2. Add 10 pL internal standard mix.

[0200] 3. Extraction: a. Add 1 mL ice cold methanol. b. Vortex and shake at 4°C for 5 min. c. Centrifuge and transfer the supernatant into a new tube; place the tube at -20 °C for Ihr to further precipitate the proteins. d. Centrifuge and dry down. c. Reconstitute and ready for injection.

[0201] Method Validation Procedure'.

[0202] Nine internal standards that represent each of the sphingolipid classes were selected to evaluate the method performance.

[0203] Three sets of standards (extracted matrix-matched standards, post-extraction matrix- matched standards and standards in solvent) were prepared to test the calibration curve linearity, limit of quantitation (LOQ), selectivity, accuracy, precision, and matrix effect.

[0204] Criteria to Accept the Validation Results

[0205] • The calibration curve constructed from extracted matrix-matched standards has a coefficient of determination (r2) of > 0.99.

[0206] • LOQ in plasma determination: o Accuracy on extraction efficiency within 70-130%. o Precision (n>3) within 30%. o S / N > 7.

[0207] Criteria to Accept the Validation Results

[0208] Blood was collected from 66 healthy pediatric subjects (55% male, 45% female) ages 4 months to 21 years in accordance with an approved UCSF Institutional Review Board protocol.

[0209] Subjects were undergoing elective surgery for benign conditions such as congenital skeletal deformity, gastric tube placement or take down, inguinal hernia, etc. Individuals with malignant, infectious, metabolic, hemolytic, or autoimmune diagnoses were excluded from the study. Plasma sphingolipid results were analyzed as a whole, as well as by comparison of results from pre-pubertal males, pre-pubertal females, post-pubertal males and post-pubertal females.

[0210] FIGS. 4A to 4B depict liquid chromatography (LC) (Agilent 1290 Infinity II LC; FIG. 4A) and LC / mass spectrometry (MS) (Agilent 6495 triple quadrupole LC / MS; FIG. 4B) systems.

[0211] FIG. 5 provides a table of specific conditions for different analytes (belonging to 10 classes). The sphingoid base for listed compounds arc dl8: 1 , except for those specified in the table.

[0212] Table 1: Chromatographic Conditions'.

[0213] Table 2: MS Conditions -Agilent 6495 Triple-Quadrupole'.

[0214] Results

[0215] The established LC / MS / MS method: establishes reference ranges for plasma sphingolipids in healthy pre- and post-pubertal children; helps reveal the mechanism underlying sphingolipid regulated diseases in children; and enables sphingolipid biomarker development needed for treating children with inborn errors of sphingolipid metabolism.

[0216] FIG. 6 provides an elution profile of the internal standards spiked in plasma.

[0217] FIG. 7 illustrates plasma SIP levels in a healthy pediatric cohort in comparison to children with two types of inborn errors of sphingolipid metabolism. Plasma SIP in post-pubertal females (>13, n = 10) were lower than in pre-pubertal females (12, n = 17), consistent with lower red cell mass after mcnarchc (red cells being the primary source of plasma SIP). High plasma SIP was observed in all individuals with SPLIS, caused by deficiency in the SIP degrading enzyme SIP lyase. In one young adult female SPLIS individual, plasma SIP levels fell in response to vitamin B6. Nearly undetectable SIP was observed in individuals lacking the ceramide desaturase encoded by DEGS1, consistent with inability to generate sphingosine, the precursor to SIP. SPLIS is a rare metabolic disorder caused by a deficiency in sphingosine- 1- phosphate lyase (SPL), the final enzyme in the sphingolipid degradative pathway. SPLIS leads to the accumulation of sphingosine- 1- phosphate (SIP). SPLIS presentations include fetal hydrops, steroid-resistant nephrotic syndrome (SRNS), primary adrenal insufficiency (PAI), rapid or insidious neurological deterioration, immunodeficiency, acanthosis, and endocrine abnormalities.

[0218] FIGS. 8A to 8B illustrates dihydroceramides (DHCer) and ceramides (Cer) in a healthy individual and in an individual with dihydroceramide desaturase (DES) deficiency. DES catalyzes the insertion of a double bond into DHCer to convert them to Cer, both of which are further metabolized to more complex (dihydro) sphingolipids. Deficiency in DES leads to the accumulation of DHCer with minimal formation of ceramides, sphingosine and SIP, causing oxidative stress, neuropathy, lipid toxicity, and a wide range of cellular processes including cell growth, cell death, autophagy, immune responses, and metabolic diseases.

[0219] Table 3: Method Evaluation Results In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0220] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0221] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0222] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0223] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

WHAT TS CLAIMED IS:

1. A method for identifying a subject as having sphingosine phosphate lyase insufficiency syndrome (SPLIS), the method comprising: assaying in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-S IP, wherein a ratio of the SIP level to the dihydro-S IP level greater than a threshold ratio indicates the subject has SPLIS.

2. The method of claim 1, wherein the method further comprises assaying in a body fluid sample of the subject a level of one or more of ceramides, ceramide- 1 -phosphates, and hexosylceramides, wherein a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1 -phosphates higher than a threshold level of ceramide- 1 -phosphates, and / or a level of hexosylceramides higher than a threshold level of hexosylceramides, indicates the subject has SPLIS.

3. The method of claims 1 or 2, wherein the SGPL1 gene of the subject is sequenced.

4. The method of claim 3, wherein the subject has a mutation in the SGPL1 gene.

5. The method of claim 4, wherein the effect of the mutation on sphingosine phosphate lyase (SPL) activity is unknown.

6. The method of claim 4, wherein the mutation is a known SPLIS pathogenic variant.

7. The method of any one of claims 1-6, wherein the subject has one or more of lymphopenia, steroid resistant necrotic kidney disease, renal fibrosis, kidney failure, adrenal insufficiency, neurological defects, ichthyosis, and hypercholesterolemia.

8. The method of claim 7, wherein the subject has steroid resistant necrotic kidney disease, renal fibrosis, and / or kidney failure.

9. The method of claim 8, wherein a urine sample of the subject is assayed for protein levels in order to assess SPLIS severity.

10. The method of claim 7, wherein the subject has one or more neurological defects.

11. The method of claim 10, wherein a nerve conduction study is performed on the subject in order to assess SPLIS severity.

12. The method of any one of claims 1-11, further comprising treating the subject having SPLIS.

13. The method of claim 12, wherein the treatment comprises pyridoxine therapy and / or gene therapy.

14. The method of any one of claims 1-13, wherein the body fluid sample comprises blood, serum, or plasma.

15. The method of any one of claims 1-14, wherein assaying the level of SIP and dihydro- S1P comprises performing mass spectrometry.

16. The method claim 15, wherein the mass spectrometry comprises tandem mass spectrometry.

17. The method of any one of claims 1-14, wherein assaying the level of SIP and dihydro- S1P comprises contacting the sample with specific binding member of SIP and a specific binding member of dihydro-SIP.

18. The method of any one of claims 1-17, wherein the subject has previously been diagnosed with and / or is being treated for a sphingolipid disorder other than SPLIS and / or a kidney disease other than SPLIS.

19. The method of claim 18, wherein the sphingolipid disorder is dihydroceramide desaturase deficiency or ccramidasc deficiency.

20. The method of claim 18, wherein the kidney disease is focal segmental glomerulosclerosis.

21. The method of any one of claims 18-20, wherein the ratio of the SIP level to the dihydro- S1P level greater than the threshold ratio, and wherein the method comprises altering the non- SPLIS treatment, wherein altering the treatment comprises tapering the dose and / or dosage of the treatment or terminating the treatment.

22. A method comprising: assaying in a body fluid sample of a subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP in order to obtain a ratio of the SIP level to the dihydro-SIP level; generating a sphingosine phosphate lyase insufficiency syndrome (SPLIS) assessment for the subject using the ratio of the SIP level to the dihydro-SIP level; and providing a health evaluation for the subject based on the SPLIS assessment.

23. The method of claim 22, wherein the method further comprises assaying in the body fluid sample of the subject a level of one or more of ceramides, ceramide- 1 -phosphates, and hexosylceramides in order to generate the SPLIS assessment.

24. The method of claim 22 or 23, wherein the health assessment comprises a predicted clinical outcome based on the SPLIS assessment .

25. The method of any one of claims 22-24, wherein the SGPL1 gene of the subject is sequenced.

26. The method of claim 25, wherein the subject has a mutation in the SGPL1 gene.

27. The method of claim 26, wherein the health evaluation comprises a natural history study.

28. The method of any one of claims 22-26, wherein the subject is being treated for SPLIS.

29. The method of claim 28, wherein the SPLIS treatment comprises pyridoxine therapy, adeno-associated virus (AAV) mediated gene therapy, and / or induced pluripotent stem cell (iPSC) gene therapy.

30. The method of claim 28 or 29, wherein the health evaluation comprises an interventional clinical trial.

31. A method for monitoring response to treatment for sphingosine phosphate lyase insufficiency syndrome (SPLIS) of a subject receiving the treatment, the method comprising: assaying in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP, wherein a ratio of the SIP level to the dihydro-SIP level lower than a threshold ratio indicates that the subject is responding positively to the treatment.

32. A method for monitoring response to treatment for sphingosine phosphate lyase insufficiency syndrome (SPLIS) of a subject receiving the treatment, the method comprising: assaying in a body fluid sample of the subject a level of ceramides, ceramide- 1- phosphate, and hexosylceramides, wherein a level of ceramides lower than a threshold level of ceramides, a level of ceramide- 1 -phosphate lower than a threshold level of ceramide- 1 -phosphate, and a level of hexosylceramides lower than a threshold level of hexosylceramides, indicates that the subject is responding positively to the treatment.

33. The method of claims 31 or 32, further comprising assaying a protein level in a urine sample of the subject, wherein a level of protein lower than a threshold level of protein indicates that the subject is responding positively to the treatment.

34. The method of any one of claims 31 -33, wherein the treatment comprises pyridoxine therapy.

35. The method of any one of claims 31-33, wherein the treatment comprises adeno- associated virus (AAV) mediated gene therapy.

36. The method of any one of claims 31-33, wherein the treatment comprises induced pluripotent stem cell (iPSC) gene therapy.

37. The method of any one of claims 31-36, wherein the body fluid sample comprises blood, serum, or plasma.

38. The method of any one of claims 31-37, wherein assaying the level of SIP and dihydro- S1P comprises performing mass spectrometry.

39. The method claim 38, wherein the mass spectrometry comprises tandem mass spectrometry.

40. The method of any one of claims 31-37, wherein assaying the level of SIP and dihydro- S1P comprises contacting the sample with specific binding member of SIP and a specific binding member of dihydro-SIP.

41. The method of any one of claims 31-40, wherein the ratio of the SIP level to the dihydro- SIP level is higher than the threshold ratio and wherein the method comprises altering the treatment, wherein altering the treatment comprises changing the treatment regimen or changing the active agent administered to the subject.

42. The method of claim 41, wherein changing the treatment regimen comprises increasing dose and / or dosage of the active agent administered to the subject.

43. The method of any one of claims 31 -42, wherein the ratio of the S 1 P level to the dihydro- S1P level is higher than the threshold ratio and wherein the method comprises tapering the dose and / or dosage of the treatment or terminating the treatment.

44. The method of any one of claims 31-43, wherein the method further comprises assaying in a body fluid sample of the subject whose treatment has been terminated, a level of SIP and dihydro-SIP, wherein a ratio of the SIP level to the dihydro-SIP level higher than a threshold ratio indicates that the subject has a relapse of SPLIS symptoms.

45. A method for monitoring relapse of sphingosine phosphate lyase insufficiency syndrome (SPLIS) symptoms in a subject treated for the SPLIS, the method comprising: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain a first ratio of SIP to dihydro-SIP; measuring at a second time point in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a second ratio of SIP to dihydro-SIP, wherein the second time point is after the first time point; wherein an increased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP indicates relapse of the SPLIS, and wherein a lack of change between first and second ratios of SIP to dihydro-SIP or a decreased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of a stable disease state.

46. The method of claim 45, comprising measuring in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a ratio of SIP to dihydro-SIP at multiple points of time over a period, wherein a trend of increased ratio of SIP to dihydro-SIP is indicative of relapse of the SPLIS symptoms.

47. The method of claim 45 or 46, wherein the body fluid sample comprises blood, serum, or plasma.

48. The method of any one of claims 45-47, wherein measuring the level of SI P and dihydro- S1P comprises performing mass spectrometry.

49. The method claim 48, wherein the mass spectrometry comprises tandem mass spectrometry.

50. The method of any one of claims 45-47, wherein measuring the level of SIP and dihydro- S1P comprises contacting the sample with specific binding member of SIP and a specific binding member of dihydro-S IP.

51. The method of any one of claims 45-50, wherein the method further comprises treating the subject having a relapse of the SPLIS symptoms.

52. The method of claim 51, wherein the treatment comprises pyridoxine therapy.

53. The method of claim 51, wherein the treatment comprises adeno-associated virus (AAV) mediated gene therapy.

54. The method of claim 51, wherein the treatment comprises induced pluripotent stem cell (iPSC) gene therapy.

55. A method for monitoring progression of sphingosine phosphate lyase insufficiency syndrome (SPLIS) in a subject, the method comprising: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain a first ratio of SIP to dihydro-SIP; measuring at a second time point in a body fluid sample of the subject a level of S IP and dihydro-SIP to obtain a second ratio of SIP to dihydro-SIP, wherein the second time point is after the first time point; wherein an increased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of increased severity of SPLIS,wherein a lack of change between first and second ratios of SIP to dihydro-S1P is indicative of lack of change in severity of SPLIS, and wherein a decreased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of improvement in SPLIS.

56. The method of claim 55, comprising measuring in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a ratio of S IP to dihydro-SIP at multiple points of time over a period, wherein a trend of increased ratio of SIP to dihydro-SIP is indicative of increased severity of SPLIS.

57. A method for monitoring responsiveness of a subject to a treatment for sphingosine phosphate lyase insufficiency syndrome (SPLIS), the method comprising: measuring at a first time point in a body fluid sample of the subject a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain a first ratio of SIP to dihydro-SIP; measuring at a second time point in a body fluid sample of the subject a level of S IP and dihydro-SIP to obtain a second ratio of SIP to dihydro-SIP, wherein the second time point is after the first time point; wherein a decreased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP indicates a positive response to the treatment, wherein an increased second ratio of SIP to dihydro-SIP compared to the first ratio of SIP to dihydro-SIP is indicative of increased severity of SPLIS, and wherein a lack of change between first and second ratios of SIP to dihydro-SIP is indicative of lack of change in severity of SPLIS.

58. The method of claim 57, comprising measuring in a body fluid sample of the subject a level of SIP and dihydro-SIP to obtain a ratio of SIP to dihydro-SIP at multiple points of time over a period, wherein a trend of decreased ratios of SIP to dihydro-SIP is indicative of positive response to the treatment.

59. The method of claim 58 or 59, further comprising: measuring at the first time point in a body fluid sample of the subject a level of ceramides, ceramide- 1-phosphates, and / or hexosylceramides to obtain a first level of ceramides, ceramide- 1 -phosphates, and / or hexosylceramides; measuring at the second time point in a body fluid sample of the subject a level of ceramides, ceramide- 1-phosphates, and / or hexosylceramides to obtain a second level of ceramides, ceramide- 1-phosphates, and / or hexosylceramides; wherein a decreased second level of ceramides compared to the first level of ceramides, a decreased second level of ceramide- 1-phosphates compared to the first level of ceramide- 1- phosphates, and / or a decreased second level of hexosylceramides compared to the first level of hexosylceramides, indicates a positive response to the treatment.

60. A method for treating a subject diagnosed with sphingosine phosphate lyase insufficiency syndrome (SPLIS), the method comprising: administering to a subject an effective amount of: a vitamin B6 compound; and / or a recombinant adeno-associated viral (rAAV) virion comprising a nucleic acid encoding sphingosine- 1 -phosphate lyase (SPL), wherein the subject is identified as in need for treatment for SPLIS based on having, in a body fluid sample, a ratio of sphingosine- 1 -phosphate (SIP) to dihydro-SIP higher than a threshold ratio of SIP to dihydro-SIP.

61. The method claim 60, wherein the subject is identified as in need for treatment for SPLIS based on having, in a body fluid sample, a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1-phosphates higher than a threshold level of ceramide-1- phosphates, and / or a level of hexosylceramides higher than a threshold level of hexosylceramides .

62. A method for treating sphingosine phosphate lyase insufficiency syndrome (SPLIS) in a subject, the method comprising: administering a pyridoxine therapy and / or a genetic therapy to the subject;measuring a level of at least one biomarker selected from sphingosine- 1 -phosphate (S IP) to dihydro-SIP ratio, ceramides level, ceramide- 1-phosphatcs level, and hcxosylccramidcs level in a biological sample of the subject after the administering; tapering or terminating the pyridoxine therapy and / or the genetic therapy if the level of the measured biomarker is below a threshold level of the biomarker; or continuing the pyridoxine therapy and / or the genetic therapy if the level of the measured biomarker is at or above a threshold level of the biomarker.

63. The method of claim 62, wherein continuing the pyridoxine therapy comprises changing the treatment regimen or changing the active agent administered to the subject to a different vitamin B6 vitamer.

64. The method of claim 62 or 63, wherein the measuring comprises measuring a level of sphingosine- 1 -phosphate (SIP) and dihydro-SIP to obtain the ratio of SIP to dihydro-SIP.

65. The method of any one of claims 62-64, wherein the measuring comprises measuring a level of ceramides, ceramide- 1 -phosphates, and / or hexosylceramides.

66. The method of any one of claims 62-65, wherein the genetic therapy comprises adeno- associated virus (AAV) mediated gene therapy.

67. A method for treating sphingosine phosphate lyase insufficiency syndrome (SPLIS) in a subject, the method comprising: administering a pyridoxine therapy to the subject; measuring a level of at least sphingosine- 1 -phosphate (SIP) and dihydro-SIP in a biological sample of the subject to obtain a ratio of SIP to dihydro-SIP after the administering; if the ratio of SIP to dihydro-SIP is at or above a threshold ratio of SIP to dihydro-SIP changing the treatment regimen; or continuing the administering if the ratio of SIP to dihydro-SIP is below a threshold ratio of SIP to dihydro-SIP.

68. The method of claim 67, wherein changing the treatment regimen comprises increasing dose and / or dosage of the pyridoxine therapy or changing the active agent administered to the subject to a different vitamin B6 vitamer.

69. The method of claim 67 or 68, wherein the biological sample is serum, plasma, or blood.

70. A method for diagnosing a subject as having phosphate lyase insufficiency syndrome (SPLIS), the method comprising: measuring a level of at least sphingosine- 1 -phosphate (SIP) and dihydro-SIP in a biological sample of the subject to obtain a ratio of SIP to dihydro-SIP, wherein a ratio of the measured SIP level and the measured dihydro-SIP level that is higher than a threshold ratio of SIP to dihydro-SIP indicates that the subject has SPLIS.

71. The method of claim 70, wherein the method further comprises measuring in a body fluid sample of the subject a level of one or more of ceramides, ceramide- 1 -phosphates, and hexosylceramides, wherein a level of ceramides higher than a threshold level of ceramides, a level of ceramide- 1 -phosphates higher than a threshold level of ceramide- 1 -phosphates, and / or a level of hexosylceramides higher than a threshold level of hexosylceramides, indicates the subject has SPLIS.

72. The method of claim 70 or 71, wherein the SGPL1 gene of the subject is sequenced.

73. The method of claim 72, wherein the subject has a mutation in the SGPL1 gene.

74. The method of any one of claims 70-73, further comprising administering a therapy for treatment of SPLIS.

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