Next Generation Sequencing based comprehensive molecular studies in young onset diabetes Aaron Chapla, Deny Varghese, Manika Varshney, Ridhi Dasgupta, Mercy Inbakumari, Asha HS,Deepak Abraham*, Thomas V Paul, Nihal Thomas. Departments Of Endocrinology1, Endocrine Surgery*, Christian Medical College, Vellore, India. BACKGROUND: T2DM pathophysiology which is characterized by insulin resistance and relative impairment in insulin secretion can be explained in individuals whose BMI is above 25 Kg/m2. In T2D, β cells via the feedback loop compensate to the increasing need of insulin and maintain a fine balance with the insulin sensitive tissues to sustain normoglycemia and the onset of T2D is usually above 35 years. HYPOTHESIS

STRATIFICATION OF STUDY SUBJECTS NEXT GENERATION SEQUENCING METHODS STRATIFICATION OF STUDY SUBJECTS NEXT GENERATION SEQUENCING BASED METHODOLOGY

MUTATIONS IDENTIFIED UTILIZING THE 2GDMODY PROTOCOL MEPN Sex Age AOD BMI TREATMENT GENE MODY Mutation Aminoacid change M85 F 37 27 21.7 Insulin HNF4A* MODY1 c.811G>A p.Glu271Lys M10 M 23 21 25 Metformin HNF4A c.505G>A c.493-4G>A (splice site mutation) c.493-20C>T p.Val169Ile BLK c.953G>A p.G318E M15 8   Diet GCK MODY2 c.1318G>T p.Glu440X M01 11 19.1 Insulin & Glibenclamide HNF1A c.1501G>T p.Ala501Ser ABCC8 c.1555G>A p.R519C M62 40 26 24.4 PDX1 MODY4 c.529G>A p.Val177Met M19 26.3 Metformin & Insulin HNF1B MODY5 c.274C>T p.Leu92Phe M26 47 28 22.8 Glimepiride NEUROD1 c.723C>G p.His241Gln c.670G>A p.Glu224Lys M47 35 24 39.7 Metformin & Glimepride MODY6 M18 30 19.3 c.175 G>C p.Glu59Gln M30 27.5 Metformin & Glipizide c.-162G>A 5'UTR M33 14 Glimepiride & Insulin PAX4 c.92G>T p.Arg31Leu c.487C>T p.G163S M05 52 32 Metformin & Glimepride  MODY12  M12 29 Insulin   MODY12 c.1005C>A p.Q335H M32 Metformin  c.3995G>A p.S1332L M43  Metformin MODY ?   c.2209C>T p.V737I M29 KCNJ11 MODY13  c.247A>G p.W83R

Summary of mutations in MODY subjects Group I- N =56 Clinically suspected MODY Group II- N =24 Young onset diabetes N=15 N=41 N=1 N=23 N=6 BMI-22.7±2.9 N=12 N=4 BMI-27.6±5 N=6 N=9

Summary and conclusions of the study Through this study NGS based cost-effective, scalable, accurate molecular method was established for comprehensive parallelized genetic testing in clinical settings We have identified 27% mutation positive rate in subjects with suspected MODY and 4% mutation positive rate in subjects with young onset diabetes when screened for a complete panel of all the known 13 MODY genes. First report of PDX1, HNF1Β, PAX4, KLF11, BLK11 novel mutations from India and also first report of novel digenic NEUROD1+PDX1, PAX4+ABCC8, HNF4A+ABCC8 mutations. The subjects with digenic mutations (6/15) had a mean BMI of 22.7kg/m2 and those subjects with single gene mutation had a BMI of 28kg/m2 . This data suggest that subjects with higher mutational burden could be predisposed to develop diabetes even with low BMI. Reference: Aaron Chapla, Mahesh Doddabelavangala Mruthyunjaya, Hesarghatta Shyamasunder Asha, Denny Varghese, Manika Varshney, Senthil K Vasan, Padmanaban Venkatesan, Veena Nair, Sarah Mathai, Thomas Vizhalil Paul and Nihal Thomas. Maturity onset diabetes of the young in india.-a distinctive mutation pattern identified through targeted next generation sequencing. Clinical Endocrinology, Volume 82, Issue 4, April 2015 Pages 533–542.