Genetics Journal Club

Sumeet A. Khetarpal

13 November 2014

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 SxOR

• 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?

Complications of T2DM

T2DM Pathophysiology

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

Insulin Resistance and Dysfunctional Adipose Tissue

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.

PPARγ Activation Promotes Insulin Sensitization

Tontonoz & Spiegelman. Ann Rev Biochem. 2008.

Many Causes of Insulin Resistance

Are any of the genetic causes of T2DM related to PPARG and

insulin sensitivity?

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.

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?

Approach

• Exome sequencing in 19752 subjects

– 9070 T2DM cases vs. 10682 controls

3284

3625

109511

249981146

2812

2859

881

1928

European CasesEuropean ControlsSouth Asian CasesSouth Asian ControlsEast Asian CasesEast Asian Controls Hispanic CasesHispanic ControlsAfrican American CasesAfrican American Controls

Ethnicity Distribution of Subjects Sequenced

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

Tools for Predicting ‘Functionality’ of Nonsynonymous Coding Variants

Tool DeveloperYear of

Development Basis for Scoring Reference

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-BLASTKumar et al. Nat Protocols. 2009.

Likelihood Ratio Test

(LRT)Justin Fay 2009 Conservation among 32 vertebrate

speciesChun & Fay.

Genome Res. 2009.

MutationTaster

Dominik Seelow 2010

Evolutionary conservation, splice-site changes, Polyadenylation signal

analysis, Kozak consensus analysis, protein annotations (SwissProt)

Schwarz et al. Nat Methods. 2010.

CondelNuria

Lopez-Bigas

2011Combination 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!

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

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 Rare PPARG Variants Lower Adipocyte Differentiation In Vitro

Variant segregating with

known lipodystrophy

Some PPARG Variants Still Allow Differentiation in Response to

Thiazolidinediones

Rare Variant Association with T2DM - Revisited

52 50

2 14

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

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

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.

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

Variant Deleteriousness Prediction Tools – You get what you pay for –

Rees et al. Hum Mol Genet. 2014

Genetic Architecture of T2DMLoci identified by GWAS and other common variant studies

Lots of common variants with small effect sizes

Thank you for your attention

• Acknowledgements– Dept. of Genetics

• Sadie Robinson• Robert Bauer