Genetics Journal ClubRobert C. Bauer

January 22nd, 2015

As of 01/21/15, the catalog includes 2100 publications and 15257 SNPs.

How to identify causal gene from a GWAS?

Global Lipids Genetics Consortium, Plasma Total Cholesterol Locus

GLGC, Nat Genet, 2013

How to identify causal gene from a GWAS?

GLGC, Nat Genet, 2013

• Closest Gene• Can be challenging with intergenic loci with many genes

• Most logical gene based on ontology/known function• Lots of completely unannotated genes

• In Vitro assays – overexpression/Knockdown• Cell type specific?

• Viral Somatic Overexpression• Is tissue readily accessible

• KO Animal Models• Costly to do multiple genes in mice• Zebrafish allow faster testing, may not recapitulate mammals• False Positives/Negatives

SNPs in the FTO genic region associate with Obesity, BMI, and Type-2 Diabetes

Obesity Plot

Giant Consortium, 2013 (From LocusZoom)

• SNPs in FTO locus associate with BMI and T2D (mediated by BMI)• Adults homozygous for risk alleles weighed +3kg

• SNPs in first intron of FTO associate with early onset obesity in both children and adults

GWAS Identifying the FTO locus

• SNPs in first intron of FTO associate with BMI, hip circumference, and weight

• Homozygotes for the minor allele are 1.3 BMI units heavier

FTO knockout mouse has reduced body weight

Fischer et al, Nature, 2009

FTO transgenic mouse has INCREASED body weightin a dose dependent manner

Church et al., Nature Genet, 2010

Multiple Mouse Models support role for FTO in obesity

Mouse model Body weight Fat/lean mass Energy expenditure Food intake Reference

Knockout Decreased Decreased Increased Increased Fischer, J. et al Nature 2009

Knockout Decreased Decreased Increased Not affected Church, C et al Plos Genet 2009

Over-expression Increased Increased Not affected Increased Church, C. et al.Nature

Genet 2010

Knockout Decreased Not affected Increased Increased Gao, X. et al. PLoS ONE 2010

Neural specific KO Decreased Not affected Increased Increased Gao, X. et al. PLoS

ONE 2010

The case against FTO – no eQTL

Wahlen et al., J Lipid Res, 2008

Grunnet et al., Diabetes, 2009

IS FTO THE GENE???

LD Block defines FTO “Association Interval”

Extended Data Figure 5.Human HapMap II Data

Circular Chromosome Conformation Capture (4C)-Seq

Stadhouders R et al. Nat Protoc. 2013

Figure 1A: The Association Interval physically interacts with promoter of Irx3 in Fetal Mouse

ED Fig. 1B: The Association Interval exclusively interacts with promoter of Irx3 in adult mouse brain

ED Fig. 2B: The IRX3 interaction replicates in human fibroblasts via Hi-C

• Also replicated in ENCODE ChIA-PET data in MCF-7 cells• Another replication in zebrafish embryos by 4C

Fig 1B: The Association Interval contains features of enhancer elements

In Vivo Enhancer Reporter Irx3 LacZ Knock-In

ED Fig 3: IRX3 promoter alone cannot recapitulate expression patterns while FTO promoter can.

• BAC with 162kb fragment of human FTO locus and Association Interval

Fig 2A: BMI associated SNPs have eQTL with IRX3 in human brain

• No SNPs showed association with FTO expression

Fig 2: IRX3 eSNPs are associated with BMI in brain but not adipose

Fig 3: Irx3 KO mice have decreased Body weight and adipocity

Fig 3: Irx3 KO mice have decreased adipocyte size, are glucose tolerant and insulin sensitive

Fig 3: Irx3 KO mice have increased energy expenditure and browning of WAT

Fig 3: Irx3 is expressed in mouse hypothalamus

Fig 4: Expression of DN Irx3 in hypothalamus phenocopies Irx3 KO mouse

Fig 4: Loss of Irx3 function in hypothalamus causes browning of WAT

• Irx3 promoter interacts with the obesity-associated interval – over 400kb away

• Obesity associated interval contains enhancer elements with activity pattern similar to Irx3

• BMI associated SNPs in the interval are associated with Irx3 expression level in human brain

• Irx3 knockout mice have a lean phenotype and higher energy expenditure which can be recapitulated by hypothalamus specific disruption of Irx3 function

SUMMARY

COMMENTS

• Mouse models not sufficient for validation of GWAS loci?

“We searched among 7,556 targeted mouse gene knockout models for evidence of alterations in body size, mass and growth (see Methods). Nearly one-third (2,166; 29%) of gene knockouts in mice show alteration of these phenotypes, underscoring that animal models alone are not sufficient to definitively establish a functional relationship between a given gene and long-range noncoding variants (Supplementary Table 4).”

GWAS Signal for Coronary Artery Diseases in ADAMTS7/MORF4L1 locus

Adamts7 KO mice have reduced atherosclerosis

COMMENTS

• Mouse models not sufficient for validation of GWAS loci?• Power of GWAS in identifying novel biology

FTO: Nuclear whose exact physiological is not known. Proteins with smiliar homology participate in oxidative demethylation of damaged DNA/RNA.

IRX3: Member of the Iroquois homeobox gene family involved in early steps of neural development. Members of this family appear to play multiple roles during pattern formation of vertebrate embryos.

NEITHER GENE IMPLICATED IN OBESITY PRIOR TO THESE STUDIES

COMMENTS

• Mouse models not sufficient for validation of GWAS loci?• Power of GWAS in identifying novel biology• Irx3 KO mouse – show the KO??

COMMENTS

• Mouse models not sufficient for validation of GWAS loci?• Power of GWAS in identifying novel biology• Irx3 KO mouse – show the KO??• Mechanism? Known hormones that regulate metabolism?