Transcript Presentation

Genetics Journal Club
Robert 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?
• 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
GLGC, Nat Genet, 2013
SNPs in the FTO genic region associate
with Obesity, BMI, and Type-2 Diabetes
Obesity Plot
Giant Consortium, 2013 (From LocusZoom)
GWAS Identifying the FTO locus
• 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
• 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 weight
in 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
Overexpression
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
SUMMARY
• 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
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?