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Introduction to the concept of functional genomics
David Meyre, Associate Professor, McMaster
University (meyred_at_mcmaster.ca) HRM 728 Graduate
Course Genetic Epidemiology October, 24th 2014
Population Genomics Program
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Introduction to the concept of functional genomics
What Is Functional Genomics? The goal of
functional genomics is to understand the
relationship between an organisms 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.
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The genomic revolution
Human genome sequence
High-throughput technologies
GENES
Biostatistics Bioinformatics
Large human biobanks
FUNCTIONAL GENOMICS
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Gene identification approaches
Genome-wide linkage
Candidate gene
GENES
Genome-wide association
Homozygosity mapping
Full exome / genome sequencing
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Classification of human genetic diseases
OBESITY
Syndromic disease ( lt 0.004)
Monogenic disease ( lt 2 )
Polygenic disease ( 20)
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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
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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
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Introduction to the concept of functional genomics
TRANS-ETHNIC FINE MAPPING APPROACH
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Candidate gene
Hypothesis free approaches
We are here
Fine mapping
Gene / locus Identification
Variant identification Functional prediction
(in silico)
Functional validation (in vitro, in vivo)
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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
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Trans-ethnic fine mapping approach
SNP2
SNP3
SNP4
SNP5
SNP1
Icelandic
French
Asian
African
Distance (Kb)
Disease-associated LD block
Causal SNP
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Trans-ethnic fine mapping approach
. Large-scale resequencing and case control
association studies in Icelandic, Danish, West
African and American African subjects identified
the rs903146 as the likely causal type 2
diabetes-associated SNP
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We are here
Candidate gene
Hypothesis free approaches
Fine mapping
Gene / locus Identification
Variant identification Functional prediction
(in silico)
Functional validation (in vitro, in vivo)
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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 - gt
170 genes induce a phenotype of severe obesity in
genetic mice models . Gene function,
biology -function related to energy metabolism
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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?
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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
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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
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Candidate gene
Hypothesis free approaches
Fine mapping
Gene / locus Identification
We are here
Functional prediction (in silico)
Functional validation (in vitro, in vivo)
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Introduction to the concept of functional genomics
GENE VARIANT AND FUNCTION
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Gene variant and function

• Two major types of variants
• 1- Variants that affect the protein structure and
  function of the gene in which they occur
• Missense, nonsense, frameshift (indels) coding
  mutations altered protein function
• Intron / exon mutations, splicing branch points
  exon skipping/adding

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Gene variant and function

• 2- Variants that affect expression and regulation
  of the gene in which they occur or other distal
  genes (eQTLs)
• gene variant in the promoter (Transcription
  Factor Biding Site) change in gene expression
• gene variant in 3UTR altered mRNA stability
• gene variant in microRNAs binding sites change
  in expression
• gene variant in enhancers / silencers/
  insulators change in expression in a distal gene
  (or a group of genes)
• gene variant in a CpG methylation site change
  in DNA methylation pattern
• Copy Number Variants (CNV) modulation of gene
  expression, haplo-insufficiency

How to prove causality between a genetic variant
and a biological effect?
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In silico prediction studies for coding variants
Mutations PolyPhen-2 PANTHER SIFT SNAP PMUT
K26E - - - - -
M125I - - - -
T175M
N180S -
Y181H -
G226R -
S325N -
T558A NA - - -
G593R NA
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
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prediction studies for regulatory variants

• Combine both in silico and indirect experimental
  data, ex ANOVAR, FunciSNP, PMCA, GWAS3D
• These tools attribute a score to each variant in
  the LD block of the genomic region thought to
  cause the phenotype and predict its functionalaty
  based on its proximity to

• Transcription factor binding sites and cis
  regulatory modules
• phylogenetically conserved sites
• specific epigenetic marks (ex enhancer
  /silencers /insulators specific proteins,
  promoter proteins , DNA methylation, DNAse
  hypersensitivity )

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Introduction to the concept of functional genomics
EVOLUTIONARY GENETICS
We are here
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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 -disadvantageo
us mutations have been negatively selected in
human populations during recent evolution
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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
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Evolutionary genetics
.The LCT rs4988235 T variant confers lactase
persistence . The LCT rs4988235 T variant is
associated with more milk / dairy products
consumption and increased body mass index . The
LCT rs4988235 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 rs4988235 T allele frequency is more
frequent in Northern (MAF 0.7) than in Southern
Europe (MAF 0.1)
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Evolutionary genetics
LCT rs4988235 T allele frequency in UK
Davey-Smith et al., EJHG 2009
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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
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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
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Introduction to the concept of functional genomics
Sources of data for variant functionality
prediction
We are here
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ENCODE PROJECT (ENCyclopedia of Dna Elements).

• Integrative analysis of
• 3545 biosamples (2441 in humans)
• from different cell lines/ tissues
• 971 epigenetic marks
• 5194 assays (Chip-seq, RNA-seq, IP, DNAse seq,
  transcription profiling )

• https//www.encodeproject.org/

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NIH Roadmap Epigenomics Mapping Consortium

• Integrative analysis of
• 111 reference human cells/tissues
• 40 epigenetic marks

• http//genomebrowser.wustl.edu/

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Genotype-Tissue Expression (GTEx) project 

• Correlations between genotype and tissue-specific
  gene expression levels in
• 42 cell lines/ tissues
• 100 - 200 RNA seq and genotyped samples

• http//www.gtexportal.org/home/

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Examples of coding and regulatory variants
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Gene variation in the promoter and gene
expression
. The -11391 GgtA 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
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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
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Gene variation at a CpG methylation site
. Gene variant rs1421085 in intron 1 of FTO is
the main contributor to polygenic obesity (Dina
et al., Nat Genet 2007) . Gene variant
rs7202116, in full linkage disequilibrium with
rs1421085, 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
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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
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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
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Gene variation in 3UTR and mRNA stability
.AgtG 1044 TGA SNP is included in the ENPP1 risk
haplotype associated with higher ENPP1 plasma
level and risk of obesity / T2D .AgtG 1044 TGA
forms a linkage disequilibrium block in 3UTR
with AgtC 1092 TGA and CgtT1157 TGA .In HLA
cells transfected with either 3UTR variant or
wild-type cDNA, specific ENPP1 mRNA half-life was
increased for those transfected with 3UTR
variant cDNA (t/24.35 vs. 2.55 h p0.001)
Meyre et al., unpublished
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Cis versus Trans e-QTLs?
. The polymorphism rs9585056 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
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Gene candidate
Hypothesis free approaches
Fine mapping
Gene / locus Identification
Functional prediction (in silico)
We are here
Functional validation (in vitro, in vivo)
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In vitro functional studies
68 of non-synonymous mutations found in obese
patients are deleterious (test
alpha-MSH)
Stutzmann et al., Diabetes 2008
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In vitro/ In vivo functional studies
CRISPR/Cas9 system, a powerful genetic tool for
genome
editing and the study of functional variants
?The rs1421085 variant of FTO identified by PMCA
has been proven to modulate the expression of
IRX3 and IRX5 genes using CRISPR/Cas9 method
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Introduction to the concept of functional genomics
STUDY OF ENDOPHENOTYPES
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Study of endophenotypes
. Rs17782313 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 rs17782313
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
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Gene candidate
Hypothesis free approaches
Fine mapping
Gene / locus Identification
Functional prediction (in silico)
Functional validation (in vitro, in vivo)
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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 SNPs in intron 1 affect the
expression of other genes (IRX3 and IRX5)
implicated in fat storage and energy expenditure
. 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
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Ichimura et al., Nature 2012
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