Deciphering mechanisms of disease associated with Sphingosine-1-phosphate lyase deficiency

Defects in the gene, sphingosine-1-phosphate lyase, SGPL1, are associated with a rare disease affecting multiple organs. Children affected with disease can have problems with their endocrine organs (including the adrenal glands, testes, and thyroid gland), kidneys and skin. They can have delay in their development and develop progressive neurological disease. They have a reduction in SGPL1, a key enzyme of an important lipid system, the sphingolipids, which are present throughout the body. Smaller sphingolipids have important roles in signalling whereas the larger sphingolipids have a structural role in the body's cells. Deficiency of SGPL1 results in a block in the system, likely to cause certain sphingolipids to accumulate. Whilst rare, SGPL1 deficiency is extremely debilitating and can significantly affect life expectancy. We will conduct a range of patient studies and cell experiments to try to gain a better understanding of the disease mechanism.
We will investigate disease progression in patients identified with SGPL1 deficiency. These individuals will be identified through genetic screening of patients with adrenal and kidney disorders. For the first time we will also be studying the effects of having a genetic defect in SGPL1 in children with delay in their development in an allied study.
We need to understand in what way the sphingolipid system is disturbed by deficiency of SGPL1. We will be investigating this by measurement of the sphingolipids in patient blood samples and in our adrenal cell model of disease. Our group primarily investigates adrenal insufficiency, where inherited genetic defects result in the adrenal glands failing to produce cortisol, an essential steroid in maintaining the body's appropriate response during times of physical and emotional stress. In our established adrenal cell model of SGPL1 deficiency we will test the effects of manipulating the sphingolipid system, in restoring the ability to produce cortisol.
Work in our group is underway to try to identify the disease mechanism in the adrenals at the cellular level. Our work to date indicates that SGPL1 deficiency affects mitochondria, regarded as the powerhouse of cells, reducing their normal ability to provide cells with energy. In this study we will look at the knock-on effects on another component of the cell, lysosomes, which are normally responsible for clearing defective mitochondria. In circumstances where there are increased mitochondrial abnormalities the lysosomes can become overwhelmed with deleterious effects. This may explain the overlap that patients with SGPL1 deficiency have with other disorders involving the sphingolipid system, which are known to specifically affect lysosome function.
Disease mechanisms identified in our adrenal studies may inform how SGPL1 deficiency affects other organs. We are establishing a consortium of experts with wide-ranging research interests to develop studies to investigate the disease mechanism in the other organ systems involved and identify new therapeutic targets.
Study of how the disease progresses will guide clinical screening to allow early treatment and inform genetic counselling for the families. It is crucial to our patients that we are able to discern the specific sphingolipid abnormalities and disease mechanism in order to identify where treatments need to be targeted. The study may be relevant to a wider population furthering our understanding of endocrine, kidney and neurological disease. It will provide better understanding of how the sphingolipid system affects cellular components (such as lysosomes and mitochondria) and how this can contribute towards disease. Furthermore, as strategies to alter SGPL1 activity are being explored in therapies for several conditions, understanding the impact of SGPL1 deficiency in human disease will provide important insights into the potential effects of manipulating this lipid system.

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS), secondary to biallelic loss of function mutations in SGPL1, is associated with endocrinopathy (primary adrenal insufficiency, hypothyroidism, gonadal failure), steroid resistant nephrotic syndrome, ichthyosis, and neurodevelopmental deficit.
SGPL1 is a key enzyme within the sphingolipid pathway and we aim to test the hypothesis that the disease manifests by perturbation of sphingolipid homeostasis, using patient studies and a CRISPR engineered SGPL1-knockout adrenal cell line.
We will gather prospective clinical information using Research Electronic Data Capture on patients identified through genetic screening of cohorts with primary adrenal insufficiency and congenital nephrotic syndrome. For the first time we will interrogate the effect of harbouring rare variants/ copy number variants in SGPL1 in a cohort with developmental delay, with comparative analysis of phenotype and available MRI Brain. It is critical that we understand the sphingolipid signature of SGPL1 deficiency by plasma sphingolipid and sphingolipid pathway product profiling by LCMS. As a research tool, we will establish a fluorogenic lyase activity assay for use in patient white cells.
In vitro, we will interrogate the sphingolipid and pathway product profile in a compartment specific manner following cellular fractionation. We will test whether steroidogenesis can be restored by chemically inhibiting de novo sphingolipid production and/or replacing the pathway product phosphatidylethanolamine, which has an established role in normal autophagy. Accordingly, we will assay expression of regulators of lysosomal biogenesis, the function of representative lysosomal enzymes and expression of autophagy markers.
Finally, beyond the study of adrenal disease, we will establish a consortium of experts to develop future studies of the disease mechanisms in this multi-systemic disorder and identify therapeutic targets.

Short term (0-3 years)
Patient and practitioner
The patient phenotype in Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is variable and our prospective clinical phenotyping will inform genetic counselling, screening for comorbidities and early management of the condition. We will be inviting referring clinicians as advocates for their patients to participate in our consortium meetings.
Patients primarily present with adrenal insufficiency and nephrotic syndrome. Presentation at national and international forums and publication of our findings will further inform practitioners dealing with primary adrenal insufficiency, critically allowing prompt recognition of the syndrome and coordinated multidisciplinary care. We have a collaboration with Professor Moin Saleem, leading the UKNephroS cohort and similarly aim for early identification of these patients within renal cohorts.
In this proposal annotation of the sphingolipid profile and lyase activity in patient samples will serve as a research tool alongside identification of SGPL1 genetic variants. There will be scope to plan future studies to develop these as a diagnostic adjunct.
Biomedical research
The sphingolipid pathway poses a novel biological system for the study of adrenal development and disease and will be of interest to other international groups detailing the processes involved in steroidogenesis. There is scope for deficiencies in other enzymes in the pathway to be identified in association with novel causes of primary adrenal insufficiency.
For researchers in disorders of sphingolipid metabolism, the work will further the understanding of the role of sphingolipids in human physiology and disease.
Commercial sector pharmaceuticals
More widely there has been rapidly growing interest in manipulation of the sphingolipid pathway in immune disease, oncology and best developed in multiple sclerosis. Our publications on the disease phenotype of SGPL1 deficiency will provide critical information on the potential effects of modulating the enzyme and bioactive sphingolipids therapeutically.

Long term 5-8 years
Other biomedical research
Within our established consortium we aim to delineate the disease mechanisms involved beyond the adrenal gland to the wider endocrinopathy, studying the effects on organ development and disease in the thyroid and gonads.
The nephropathy and neurological aspects of disease are akin to those seen in more commonly occurring conditions. Study of patients with SPLIS will generate hypotheses for genotype recall studies testing the effects of harbouring SGPL1 variants in other conditions. There is growing interest for instance in the sphingolipid signature seen in more commonly occurring disease such as diabetes and its associated complications.
Commercial sector pharmaceuticals
Annotation of the disturbance in sphingolipid homeostasis seen in the condition will hasten identification of therapeutic targets and development of drugs to modulate the sphingolipid pathway for patient benefit. This is of particular interest for neurological aspects of the disease, for which there is no current treatment, which in turn may benefit other neurodegenerative disease.

Longer term >10 years
We hope, with greater understanding of the disease and knowledge of the precise effect of SGPL1 deficiency on sphingolipid metabolism, that clinical trials of drugs successfully manipulating the pathway will be in place for patient benefit.