Characterization of a Novel ASAH2 Variant Associated with Diabetes and Kidney Failure in Tongan and Samoan Patients

Background: Ceramides are bioactive lipids which modulate metabolic pathways to drive insulin resistance, apoptosis, and fibrosis. In search of novel genetic divers of high ceramides and their resulting contribution to heritable metabolic pathologies, we have identified a novel coding mutation in neutral ceramidase (ASAH2). The mutation is exclusive to patients of Tongan and/or Samoan ancestry, a population which faces the highest rates of obesity, diabetes, and end-stage renal disease (ESRD) of any racial or ethnic group in the United States. Methods: We profiled circulating ceramides in 5 founder ASAH2 point mutant (chr10:50236064C→G(hg38), c.G511C, p.V171L) carriers and characterized the effect of the mutation on neutral ceramidase activity and ASAH2 mRNA splicing in vitro with CRISPR Cas9 knock-in and/or mutant expression constructs in HEK293 cells. Lastly, we tested for renal and metabolic consequences of loss-of-ceramidase function in Asah2 knockout mice challenged with high-fat diet or streptozotocin-induced diabetic kidney disease (DKD). Results: Heterozygous carriers of the ASAH2 mutation have 29% higher circulating ceramides compared to Polynesian non-carriers with diabetes and ESRD (p=0.009). Sequencing of RNA from ASAH2c.G511C knock-in cells revealed that the point mutation located at the first base pair of exon 5 causes excision of exon 5, which encodes crucial amino acids within the ASAH2 catalytic domain. Functional studies in cells overexpressing ASAH2 lacking exon 5 confirm ablation of ceramidase activity. In vivo, both male and female Asah2-/- mice displayed worsened insulin sensitivity by insulin tolerance test compared to Asah2+/+littermate controls with diet-induced obesity. Additionally, female Asah2-/- mice had exacerbated albuminuria (473.3±83.6 (-/-) vs. 159.3±37.7 (+/+) ug/mg creatinine, p<0.0001), kidney hypertrophy (7.7 (-/-) vs. 5.9 (+/+) mg/g body weight, p<0.0001), and cortex expression of markers related to fibrosis (Tgfb1, Col1a1, Col3a1, Col4a1, Fn1) and kidney damage (Havcr1, Lcn2) compared to Asah2+/+littermate controls in the setting of DKD. Conclusion: These data reveal a novel risk allele for diabetes and kidney disease that is present in 1 in 10 individuals of Tongan or Samoan descent, which could be treated with precision therapies designed to lower ceramides. Funding: NIDDK Support