Deep Sequencing in Extended Pedigrees Reveals a Major Rare Non-Synonymous Variant Influencing the De Novo Ceramide Synthesis Pathway. J. E. Curran1, P. J. Meikle2, J. M. Weir2, J. B. Jowett2, T. M. Teslovich3, G. Jun3, S. Kumar1, M. Almeida1, J. M. Peralta1, C. Fuchsberger3, A. R. Wood4, A. Manning5, T. M. Frayling4, P. Gingolani6, R. Sladek7, D. M. Lehman8, G. Abecasis3, M. C. Mahaney1, T. D. Dyer1, L. Almasy1, R. Duggirala1, J. Blangero1, T2D-GENES Consortium 1) Texas Biomedical Research Institute, San Antonio, TX; 2) Baker IDI Heart and Diabetes Institute, Melbourne, Vic, AU; 3) University of Michigan, Ann Arbor, MI; 4) University of Exeter, Exeter, UK; 5) Broad Institute, Boston, MA; 6) McGill University, Montreal, CA; 7) Montreal Diabetes Research Institute, Montreal, CA; 8) University of Texas Health Science Center at San Antonio, San Antonio, TX.

   Lipids play critical roles in many cellular processes; and a major role in health and disease. The biologically simple nature of lipid species suggests their determinants may reside closer to the action of genes than the classical lipid measures. Thus their potential for a simpler causal web makes them valuable endophenotypes for identifying genes in lipid metabolism. Using targeted lipidomic profiles and whole genome sequence (WGS) in 605 Mexican Americans from large pedigrees, we searched for causal variants influencing the de novo ceramide synthesis pathway. This pathway plays a role in obesity, diabetes and CVD. The core of this pathway is the generation of biologically active ceramide from its inactive precursor, dihydroceramide. We focused our initial search on ~9,000 missense and nonsense mutations predicted to be highly deleterious by PolyPhen-2 (score 0.80). We identified a rare functional variant (L175Q) in the DEGS1 gene on chr 1, observing 17 total copies in our pedigrees; it was only seen in 3 individuals in the 1000 Genomes Project. This rare variant is significantly associated with an increase in dihydroceramide levels (p=1.5710-6) by a biologically significant amount of 1.2 standard deviation units (SDU). Pleiotropy analyses revealed that this increase in dihydroceramide is accompanied by significant decreases in both ceramide (p=0.027, -0.45 SDU) and spingolipids including sphinogmyelin (p=0.0009, -0.75 SDU) and trihexosylceramide (p=0.003, -0.70 SDU). It was also associated with a significant decrease in waist/hip ratio (p=0.014, -0.45 SDU) and cholesterol esters (p=0.020, -0.53 SDU). DEGS1 is the fatty acid desaturase responsible for the desaturation of dihydroceramide to generate ceramide de novo. Studies have shown that silencing DEGS1 in cells focused on specific processes such as metabolic stress, oxidative stress, apoptosis and cancer, results in a decrease in ceramide and sphingomyelin synthesis, while increasing cell levels of dihydroceramide and sphinganine species. Additionally, DEGS1 knockdown results in down-regulation in almost all metabolic biosynthesis pathways, including cell-cycle growth. Our association results support these findings in that the L175Q variant results in a significant increase in dihydroceramide levels and a significant reduction in biologically active ceramide/sphingomyelin levels. Given the diverse role of ceramide in cellular processes, DEGS1 has excellent therapeutic potential for many diseases.

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