1. Introduction to the concept of functional genomics
David Meyre, Associate Professor, McMaster University
HRM 728 Graduate Course: Genetic Epidemiology – October, 24th 2014
Population Genomics Program

2. Introduction to the concept of functional genomics
What Is Functional Genomics?
The goal of functional genomics is to understand the relationship between an organism’s genome and its phenotype.
Functional genomics is a field of molecular biology that is attempting to make use of the vast wealth of data produced by genome sequencing projects to describe genome function. Functional genomics uses high-throughput techniques like DNA microarrays, proteomics, epigenomics, metagenomics, metabolomics and mutation analysis to describe the function and interactions of genes.

3. The genomic revolution High-throughput technologies
Human genome sequence
High-throughput technologies
GENES
Biostatistics & Bioinformatics
Large human biobanks
FUNCTIONAL
GENOMICS

4. Gene identification approaches Full exome / genome sequencing
Genome-wide linkage
Candidate gene
GENES
Homozygosity mapping
Genome-wide association
Full exome / genome sequencing

5. Classification of human genetic diseases
OBESITY
Syndromic disease
( < 0.004%)
Monogenic disease
( < 2 %)
Polygenic disease
(~ 20%)

6. SYNDROMIC / MONOGENIC DISEASE
Genes and causality
SYNDROMIC / MONOGENIC DISEASE
Beyond co-segregation studies, additional arguments are needed to demonstrate the causal role of a mutation in the disease
 functional genomics

7. POLYGENIC DISEASE (e.g. type 2 diabetes)
Genes and causality
POLYGENIC DISEASE (e.g. type 2 diabetes)
Beyond association studies, additional arguments are needed to demonstrate the causal role of a variant / gene in the disease
 functional genomics
Sladek et al., Nature 2007

8. Introduction to the concept of functional genomics
I-TRANS-ETHNIC FINE MAPPING APPROACH

9. Trans-ethnic fine mapping approach
.Linkage disequilibrium is the non-random association of alleles at two or more loci
. The human genome is composed of blocks of linkage disequilibrium
. The extent of linkage disequilibrium blocks varies according to the ethnic background

10. Trans-ethnic fine mapping approach
SNP1
SNP2
SNP3
SNP4
SNP5
Icelandic
French
Asian
African
Distance (Kb)
Disease-associated LD block
Causal SNP

11. Trans-ethnic fine mapping approach
. Large-scale resequencing and case control association studies in Icelandic, Danish, West African and American African subjects identified the rs as the likely causal type 2 diabetes-associated SNP

12. Introduction to the concept of functional genomics
II-EVOLUTIONARY GENETICS

13. Evolutionary genetics
Natural selection is the gradual, non-random process by which biological traits become either more or less common in a population as a function of differential reproduction of their bearers. It is a key mechanism of evolution. The term "natural selection" was popularized by Charles Darwin.
Evolutionary genetics (Huxley 1942)
-advantageous mutations have been positively selected in human populations during recent evolution
-disadvantageous mutations have been negatively selected in human populations during recent evolution

14. Evolutionary genetics
THRIFTY GENOTYPE HYPOTHESIS: the 'thrifty' genotype would have been advantageous for hunter-gatherer populations, especially child-bearing women, because it would allow them to fatten more quickly during times of abundance. Fatter individuals carrying the thrifty genes would thus better survive times of food scarcity.
 Obesity and type 2 diabetes predisposing mutations may show evidence of positive signature of evolution

15. Evolutionary genetics
.The LCT rs T variant confers lactase persistence
. The LCT rs T variant is associated with more milk / dairy products consumption and increased body mass index
. The LCT rs T variant has a selective advantage in milk-producing dairy farming populations and has been submitted to positive selection in relation with events of cattle domestication
. The LCT rs T allele frequency is more frequent in Northern (MAF: 0.7) than in Southern Europe (MAF: 0.1)

16. Evolutionary genetics LCT rs4988235 T allele frequency in UK
Davey-Smith et al., EJHG 2009

17. Evolutionary genetics
. Genome-wide approaches in diverse ethnic backgrounds have identified several hundreds of regions showing recent positive natural selection
. New methods are able to identify causal variants in regions with positive natural selection signature
. The amino-acid change Lys109Arg in the LEPR gene is as a causal variant submitted to positive selection
. The Lys109Arg variant is associated with body mass index variation
Grossman et al., Science 2010

18. Introduction to the concept of functional genomics
III-GENE VARIANT AND FUNCTION

19. Gene variant and function
Missense, nonsense, frameshift (indels) coding mutations: altered protein function
Intron / exon mutations: exon skipping
Copy Number Variants (CNV): modulation of gene expression, haplo-insufficiency
gene variant in the promoter (Transcription Factor Biding Site): change in gene expression
gene variant in 3’UTR: altered mRNA stability
gene variant in microRNAs: change in expression
gene variant in a long-range enhancer: change in expression of another gene
gene variant in a CpG methylation site: change in DNA methylation pattern
How to prove causality between a genetic variant and a biological effect?

20. In silico prediction studies
Mutations
PolyPhen-2
PANTHER
SIFT
SNAP
PMUT
K26E -
M125I +
T175M
N180S
Y181H
G226R
S325N
T558A NA
G593R
+: deleterious
-: neutral
Eight coding non-synonymous mutations in the PCSK1 gene have been identified in extreme obese patients: the Polyphen-2 software (conservation of the amino-acid across evolution + protein structure) is 100% concordant with in vitro studies
Creemers et al., Diabetes 2012

21. In vitro functional studies
68% of non-synonymous mutations found in obese patients are deleterious (test alpha-MSH)
Stutzmann et al., Diabetes 2008

22. Intron / exon mutations and exon skipping
. Extreme obesity cosegregates with homozygosity for a G/A substitution in the splice donor site of exon 16 of the LEPR gene
. The intron / exon mutation induces skipping of exon 16 and a truncated inactive leptin receptor
Clement et al., Nature 1998

23. CNVs are highly causal variants in mendelian diseases
a 600kb heterozygous deletion (~30 genes) on chromosome 16p11.2 explains 0.7% of morbid hyperphagic obesity and is associated with developmental delays
duplications in the same chromosomal region are associated with underweight and eating restrictive disorders
SH2B1, a key modulator of the response to the satiety hormone leptin, and a Mendelian hyperphagic obesity gene, is located in the deleted interval
Walters et al., Nature 2010; Jacquemont et al., Nature 2012

24. Gene variation in the promoter and gene expression
. The G>A variant in the promoter of the ACDC/adiponectin gene is associated with higher in vitro promoter activity and with higher plasma adiponectin level in lean and in obese children
Bouatia-Naji et al., Diabetes 2006

25. Gene variation in 3’UTR and mRNA stability Meyre et al., unpublished
.A>G TGA SNP is included in the ENPP1 risk haplotype associated with higher ENPP1 plasma level and risk of obesity / T2D
.A>G TGA forms a linkage disequilibrium block in 3’UTR with A>C TGA and C>T+1157 TGA
.In HLA cells transfected with either 3’UTR variant or wild-type cDNA, specific ENPP1 mRNA half-life was increased for those transfected with 3’UTR variant cDNA (t/2=4.35 vs h; p=0.001)
Meyre et al., unpublished

26. Gene variation and long-range enhancer
. The obesity-associated FTO intron 1 region directly interacts with the promoter of IRX3 gene (580 Kb downstream of FTO)
. The intron 1 SNP in FTO modulates IRX3 (but not FTO) expression
. Irx3-deficient mice display a leanness phenotype
Smemo et al., Nature 2014

27. Gene variation at a CpG methylation site
. Gene variant rs in intron 1 of FTO is the main contributor to polygenic obesity (Dina et al., Nat Genet 2007)
. Gene variant rs , in full linkage disequilibrium with rs , creates a CpG methylation site and is associated with increased methylation of a 7.7 kb regulatory region within FTO
. The 7.7 kb regulatory region encapsulates a Highly-Conserved non Coding Element that acts as a long range gene expression enhancer
Bell et al., PLOS One 2010

28. Introduction to the concept of functional genomics
IV-GENE CANDIDACY

29. Gene candidacy
Sometimes several genes are included in a same linkage disequilibrium block  how can we identify the causal gene(s)?
Sladek et al., Nature 2007

30. Gene candidacy
. Are genes in the disease-associated LD block involved in syndromic / monogenic forms of the same disease?
-loci associated with polygenic obesity: MC4R, BDNF, POMC, PCSK1, SIM1
-GWAS for complex traits: 20% of the GWAS loci include genes involved in mendelian disorders for the same trait
. Are genes in the disease-associated LD block involved in a corresponding phenotype in animal models (KO, Tg, SiRNA)?
-loci associated with polygenic obesity: MC4R, BDNF, POMC, PCSK1, SIM1, FTO, GIPR, NPC1, SH2B1, TBC1D1, NEGR1
- > 170 genes induce a phenotype of severe obesity in genetic mice models
. Gene function, biology
-function related to energy metabolism

31. Gene candidacy
In order to find the causal gene in a disease-associated linkage disequilibrium block, mRNA expression studies can be useful (microarrays, RT-PCR):
1-Is the gene expressed in target tissues for the disease (obesity: brain, adipocytes; T2D: pancreas)?
2-Is the gene mRNA expression modulated by the disease status in a relevant tissue?
3-Is the gene mRNA expression modulated by the disease-associated SNP in a relevant tissue?

32. Gene candidacy
. ORMDL3 is one of the 19 genes located in the asthma-associated LD block
. ORMDL3 is expressed in the lung
. ORMDL3 mRNA level is modulated by asthma disease status in lymphoblastoid cell lines
. ORMDL3 mRNA level is strongly modulated by the asthma-associated SNP in lymphoblastoid cell lines
ORMDL3 is a highly relevant candidate gene at this locus
Moffatt et al., Nature 2007

33. Cis versus Trans e-QTLs?
. The polymorphism rs is associated with T1D, modulates the expression of the cis-gene GPR183 and the expression of the IRF7 network genes
Heinig et al., Nature 2010

34. Gene candidacy . Combination of expression mRNA and GWAS studies
. 27 genes differentially regulated in adipose tissue of monozygotic twins discordant for obesity
. ‘Hypothesis driven’ GWAS analysis for these 27 genes followed by a replication in a second independent sample identified a novel obesity gene: F13A1
Naukkarinen et al., PLOS Genet 2010

35. Introduction to the concept of functional genomics
V-STUDY OF ENDOPHENOTYPES

36. Study of endophenotypes
. Rs near MC4R has been associated with BMI by GWAS
. Deleterious coding mutations in MC4R are the commonest form of monogenic obesity with hyperphagia and increased stature
. If the SNP modulates the expression / function of MC4R, we can predict associations with the same traits in an appropriate direction
. The SNP rs obesity predisposing allele is associated with more snacking and overeating and increased stature
 MC4R is a highly relevant candidate gene at this locus
Stutzmann et al., Int J Obes 2009

37. Introduction to the concept of functional genomics
I-TRANS-ETHNIC FINE MAPPING APPROACH
II-EVOLUTIONARY GENETICS
III-GENE VARIANT AND FUNCTION
IV-GENE CANDIDACY
V-STUDY OF ENDOPHENOTYPES

38. FTO, a good illustration of integrative approach
.Novel variants identified in African populations
.FTO SNP shows evidence of positive natural selection
.The SNP is associated with different patterns of methylation (demethylase)
. FTO complete deficiency leads to a polymalformative lethal syndrome in humans
. FTO partial deficiency does not relate to leanness/obesity in humans
. FTO knock-out mice are lean, FTO transgenic mice are obese
. FTO is highly expressed in hypothalamus and is regulated by fasting and feeding
. FTO SNP is associated with food intake in humans

39. Ichimura et al., Nature 2012

41. ANY QUESTIONS?
The French fair-play!