Validating absolute lymphocyte count and plasma sphingosine-1-phosphate as disease biomarkers of sphingosine phosphate lyase insufficiency syndrome in anticipation of a pyridoxine clinical trial

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Sphingosine phosphate lyase insufficiency syndrome (SPLIS) is an inherited metabolic childhood syndrome caused by recessive mutations in SGPL1. Affected children exhibit steroid-resistant nephrotic syndrome with rapid progression to end stage renal disease, adrenal insufficiency, neurological defects and lymphopenia. A range of severity has been observed, with the most severely affected patients dying in utero, while others live to adulthood, albeit requiring dialysis or kidney transplantation. Importantly, there is no specific cure for SPLIS, and thus there is a dire need for the development of novel safe and effective treatments. SGPL1 encodes sphingosine phosphate lyase (SPL), an essential intracellular enzyme responsible for the irreversible breakdown of sphingosine-1-phosphate (S1P) in the final step of sphingolipid metabolism. S1P is a bioactive lipid that signals through its five G protein coupled S1P receptors to regulate lymphocyte trafficking and other physiological processes. SGPL1 mutations impair SPL activity and cause sphingolipid accumulation leading to organ dysfunction and failure. In addition, aberrant S1P signaling in SPLIS children leads to lymphopenia due to a defect in lymphocyte egress. SPL activity depends on vitamin B6 as a cofactor. Independent of its role as a coenzyme in over 160 enzymatic reactions, vitamin B6 can also function as a chaperone, stabilizing vitamin B6-dependent enzymes. Pharmacological doses of vitamin B6 in the form of the B6 vitamer pyridoxine have proven effective in inborn errors of metabolism involving B6-dependent enzymes. Based on encouraging preliminary findings in two SPLIS patients treated with vitamin B6 supplementation, we are preparing to evaluate the safety and efficacy of vitamin B6 supplementation as the first specific treatment for SPLIS. Challenges to developing a reliable assay for monitoring SPL activity in blood samples from SPLIS patients make reliance on disease biomarkers a critical component of any clinical trial in SPLIS. We hypothesize that absolute lymphocyte count (ALC) and plasma S1P levels will serve as reliable biomarkers that reflect SPLIS disease status. Unfortunately, pediatric reference ranges for plasma S1P and other major sphingolipids have not been reported to date. Further, no study comprehensively profiling circulating immune cell populations in SPLIS has been reported. To confirm our hypothesis and overcome these obstacles to advancing treatments for children with SPLIS and other sphingolipid metabolic disorders, we will characterize the major plasma sphingolipids and blood markers of immune function in children with SPLIS and a healthy pediatric and young adult control cohort. In accomplishing our Specific Aims, we will validate plasma S1P and ALC as robust SPLIS disease biomarkers. We will establish reference ranges for a comprehensive set of plasma sphingolipids in a healthy pediatric and young adult cohort that will be useful in the future for diagnosis, monitoring and conducting clinical trials in a broad range of sphingolipid disorders. Our results will specifically facilitate clinical trial readiness for testing the safety and efficacy of vitamin B6 cofactor supplementation to treat SPLIS.