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 Sx
T2DM
is chronically elevated blood glucose
OR
Why is this bad, and what causes it?
• 2-hr postprandial glucose > 200 mg/dl after consuming
75 grams carbohydrate (i.e. Oral Glucose Tolerance
Test, OGTT)
OR
• HbA1c > 6.5%
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.
What can we learn about a known
complex trait by searching for rare
mutations
in a known candidate gene
Hypotheses
• Rare
Loss-of-function
(LoF)
nonsynonymous
variants in
for
this
trait in the
general
population?
PPARG underlie predisposition to T2DM in the general
population
• Not all the identified PPARG coding variants would be
functionally deleterious
Approach
• Exome sequencing in 19752 subjects
– 9070 T2DM cases vs. 10682 controls
Ethnicity Distribution of Subjects Sequenced
881
1928
European Cases
3284
European Controls
South Asian Cases
2859
3625
South Asian Controls
East Asian Cases
2812
1095
1146
998
1124
East Asian Controls
Hispanic Cases
Hispanic Controls
African American Cases
African American Controls
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
PolyPhen2
Developer
Shamil
Sunyaev
Year of
Development
2010
Basis for Scoring
Reference
Sequence features (Uniprot
annotations), structural tolerance of
Adzhubei et al. Nat
the substitution, comparison to
Methods. 2010.
known human disease-causing
mutations (HumVar)
Conservation among closely related Kumar et al. Nat
sequences from PSI-BLAST
Protocols. 2009.
Most groups use some, all, or
none of these tools to annotate
discovered variants!
Sift
J. Craig
Venter Inst.
2009
Likelihood
Ratio Test
(LRT)
Justin Fay
2009
MutationTast
er
Dominik
Seelow
2010
Condel
Nuria
LopezBigas
2011
Conservation among 32 vertebrate
Chun & Fay.
species
Genome Res. 2009.
Evolutionary conservation, splice-site
changes, Polyadenylation signal
Schwarz et al. Nat
analysis, Kozak consensus analysis, Methods. 2010.
protein annotations (SwissProt)
Combination of 5 tools (Logre, Gonzalez-Perez &
MAPP, Mutation Assessor,
Lopez-Bigas.
Polyphen2, Sift)
AJHG. 2011.
Functional Assay to Test Novel Variants
SGBS Human Pre-adipocytes
PPARγ
Variants
Automated
Image Analysis
Christancho & Lazar. Nat Rev Mol Cell Bio. 2011.
% 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 T2DM
Loci 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