1. Genetics Journal Club Sumeet A. Khetarpal 13 November 2014

2. Type 2 Diabetes Mellitus (T2DM) Fasting plasma glucose > 126 mg/dl (7mmol/L) on two separate occasions OR Random glucose > 200 mg/dl with classic Sx OR 2-hr postprandial glucose > 200 mg/dl after consuming 75 grams carbohydrate (i.e. Oral Glucose Tolerance Test, OGTT) OR HbA1c > 6.5% T2DM is chronically elevated blood glucose Why is this bad, and what causes it?

3. Complications of T2DM

4. T2DM Pathophysiology T2DM is caused by insulin resistance – how does this manifest?

5. Insulin Resistance and Dysfunctional Adipose Tissue

6. Peroxisome Proliferator-Activated Receptor Gamma (PPARG) Nuclear hormone receptor Master regulator of adipocyte differentiation Receptor for thiazolidinediones – antidiabetic drugs increasing insulin sensitivity Heterodimerizes with RXR transcription factors to regulate gene expression Tontonoz & Spiegelman. Ann Rev Biochem. 2008.

7. PPARγ Activation Promotes Insulin Sensitization Tontonoz & Spiegelman. Ann Rev Biochem. 2008.

8. Many Causes of Insulin Resistance Are any of the genetic causes of T2DM related to PPARG and insulin sensitivity?

9. Genetic Variation in PPARG and T2DM Common Variation –Pro12Ala (rs1801282) Alters DBD 60 association studies suggest increased risk of T2DM MAF 2-25% (ethnicity dependent) Rare Variation –16 variants segregate with familial partial lipodystrophy type 3 Severe insulin resistance, hyperinsulinemia, hypertriglyceridemia, low HDL, hepatic steatosis, hypertension Autosomal dominant Jeninga et al. Trends Endocrinol Metab. 2009.

10. Hypotheses Rare Loss-of-function (LoF) nonsynonymous variants in PPARG underlie predisposition to T2DM in the general population Not all the identified PPARG coding variants would be functionally deleterious What can we learn about a known complex trait by searching for rare mutations in a known candidate gene for this trait in the general population?

11. Approach Exome sequencing in 19752 subjects –9070 T2DM cases vs. 10682 controls Ethnicity Distribution of Subjects Sequenced

12. What did they find? 53 nonsynonymous variants 52 rare – MAF < 1% 1 with MAF > 1% – P12A 49 novel, 3 previously reported with familial partial lipodystrophy 3 (FPLD3) 33 were singletons 2 nonsense mutations 120 individuals harbored a rare variant – aggregate frequency 0.6% All carriers were heterozygous

13. Tools for Predicting ‘Functionality’ of Nonsynonymous Coding Variants ToolDeveloper Year of Development Basis for ScoringReference PolyPhen2 Shamil Sunyaev 2010 Sequence features (Uniprot annotations), structural tolerance of the substitution, comparison to known human disease-causing mutations (HumVar) Adzhubei et al. Nat Methods. 2010. Sift J. Craig Venter Inst. 2009 Conservation among closely related sequences from PSI-BLAST Kumar et al. Nat Protocols. 2009. Likelihood Ratio Test (LRT) Justin Fay2009 Conservation among 32 vertebrate species Chun & Fay. Genome Res. 2009. MutationTast er Dominik Seelow 2010 Evolutionary conservation, splice-site changes, Polyadenylation signal analysis, Kozak consensus analysis, protein annotations (SwissProt) Schwarz et al. Nat Methods. 2010. Condel Nuria Lopez- Bigas 2011 Combination of 5 tools (Logre, MAPP, Mutation Assessor, Polyphen2, Sift) Gonzalez-Perez & Lopez-Bigas. AJHG. 2011. Most groups use some, all, or none of these tools to annotate discovered variants!

14. Functional Assay to Test Novel Variants Christancho & Lazar. Nat Rev Mol Cell Bio. 2011. SGBS Human Pre-adipocytes PPARγ Variants Automated Image Analysis % Differentiation = Adipocytes / Nulcei

15. Functional Assay to Test Novel Variants But what about endogenous (WT) PPARγ? SGBS Human Pre-adipocytes PPARγ Variants Automated Image Analysis % Differentiation = Adipocytes / Nulcei

16. 16 Rare PPARG Variants Lower Adipocyte Differentiation In Vitro Variant segregating with known lipodystrophy

17. Some PPARG Variants Still Allow Differentiation in Response to Thiazolidinediones

18. Rare Variant Association with T2DM - Revisited 52 50 2 14

19. Summary Sequencing the exomes in ~20,000 subjects (T2DM cases vs. controls) identified several novel, rare variants in PPARG In silico prediction alone did not demonstrate differing frequency of rare variants in cases vs. controls Functional testing of variants demonstrated higher frequency of rare PPARG variants in T2DM cases

20. Strengths Large, multi-ethnic cohorts comprising study group Stringent initial in silico criterion for ascertaining ‘functionality’ Robust + quantitative functional assay –Therapeutic insight from additional testing after rosiglitazone stimulation

21. Weaknesses For some ethnicities, N’s may not be big enough Functional assay –SGBS cells are NOT ‘true’ adipocytes –PPAR gamma has many functions beyond white adipose tissue differentiation Some variants with profound loss-of-function do not fit the FPLD3 paradigm Ahmadian et al. Nat Med. 2013.

22. Rare Variants for Common Diseases Can provide insight into the cumulative contribution of a gene/pathway to disease at the population level Many variants will ultimately not have an identifiable functional effect Requires careful dissection of ‘functionality’ for identified variants

23. Variant Deleteriousness Prediction Tools – You get what you pay for – Rees et al. Hum Mol Genet. 2014

24. Genetic Architecture of T2DM Loci identified by GWAS and other common variant studies Lots of common variants with small effect sizes

25. Thank you for your attention Acknowledgements –Dept. of Genetics Sadie Robinson Robert Bauer