Genetics and Allergic Diseases

GENETICS AND
ALLERGIC DISEASES
Presented by Wat Mitthamsiri, MD
Allergy and Clinical Immunology Fellow
King Chulalongkorn Memorial Hospital

Outline
 Introduction
 Genetic models of diseases
 Gene expression regulation
 Genetic code, Epigenetics , Functional genomics
 Roles of genetics in allergic diseases
 Genetic studies in allergic diseases
 Hypothesis-dependent/independent
 Genetics of allergic diseases
 Missing heritability in allergic diseases
 Epigenetics in allergic diseases
 Functional genomics in allergic diseases

Genetics: Definition
 1: A branch of biology that deals with the
heredity and variation of organisms
 2: The genetic makeup and phenomena of an
organism, type, group, or condition
"Genetics." Merriam-Webster.com. Merriam-Webster, n.d. Web. 15 Oct. 2014. <http://www.merriam-webster.com/dictionary/genetics>

Genetics: Origin
 Study of heredity in
general and of genes in
particular
 Modern genetics
began in the 19th
century with the work
of Gregor Mendel, who
formulated the basic
concepts of heredity
Image from: http://www.dnalc.org/content/c16/16163/16163_075prelate.jpg

Genetics: Origin
 1909: the word gene was coined by Wilhelm
Johannsen, thus giving genetics its name
Image from: http://izquotes.com/quotes-pictures/quote-it-appears-as-most-simple-to-use-the-last-syllable-gen-taken-from-darwin-s-well-known-word-wilhelm-
ludvig-johannsen-307122.jpg

Importance of genetic
knowledge in allergy
 Explication of disease pathogenesis
 By identification of genes and molecular pathways
 Generating novel pharmacologic targets
 Identification of environmental-genetic
interactions and prevention of disease through
environmental modification
JW Halloway, Middleton’s Allergy 8th edition, 2013, 343-363.

Importance of genetic
knowledge in allergy
 Detection of susceptible individuals
 Screening early in life
 Allowing targeted interventions
 Subclassification of disease by genetics
 Enabling tailor-made therapies
 Determination of the likelihood of a therapeutic
response
 For individualized treatment plans

Complex genetic disorder
Adapted from figure avialable at http://www.nature.com/ni/journal/v11/n7/carousel/ni.1892-F1.jpg
Information: Adkinson NF, et al. Middleton's allergy : principles and practice. 8. ed.; 2014.

Gene expression process
Image from: http://www.ncbi.nlm.nih.gov/probe/docs/applexpression/

Gene expression process
Nucleus
Cytoplasm

Expression regulation
DNA modification
Transcription control
RNA processing
control
RNA transportation
control
RNA translation
control
Phenotype

DNA modification
Nucleotide
sequence
modification
• Insertion
• Deletion
• Substitution
• Recombination
Mutation
• Loss of function
• Gain of function
Loewe, L. (2008) Genetic mutation. Nature Education 1(1):113
Clancy, S. (2008) Genetic mutation. Nature Education 1(1):187

DNA modification
Structural and chemical
modification
• DNA folding/coiling
• Phosphorylation
• Methylation
• Histone acetylation
Bell JT, PaiAA, Pickrell JK, Gaffney DJ, Pique-Regi R, Degner JF, GiladY, Pritchard JK (2011). Genome Biology 12 (1)

 RNA polymerase specificity factors
 Alter the specificity for given promoter(s) = more
or less likely to bind to them
 Repressors
 Bind to the Operator
 = Impeding the expression of the gene
 Transcription factors
Hoopes, L. (2008) Introduction to the gene expression and regulation topic room. Nature Education 1(1):160

Hoopes, L. (2008) Introduction to the gene expression and regulation topic room. Nature Education 1(1):160
Austin S, Dixon R (June 1992).. EMBO J. 11 (6): 2219–28.
 Activators
 Enhance the interaction between RNA
polymerase and a particular promoter
 = Encouraging the expression of the gene
 Enhancers
 Sites on the DNA helix that are bound by
activators in order to loop the DNA bringing a
specific promoter to the initiation complex
 Silencers
 Regions of DNA sequences that, when bound by
particular transcription factors, can silence
expression of the gene

Post-transcription control
 Capping
 Changes 5’-end of mRNA to a 3’-end
 Protects mRNA from 5' exonuclease
 Splicing
 Removes the introns
 The 2 ends of the exons are then joined together
 Polyadenylation (addition of poly(A) tail)
 Acts as a buffer to the 3' exonuclease
 Increase the half life of mRNA
 RNA editing
 Results in sequence variation in the RNA molecule
 mRNA Stability
 To control its half-life

Translation control
 Control of ribosome recruitment on the
initiation codon
 Modulation of the elongation or termination
of protein synthesis
 Modification of specific RNA secondary
structures on the mRNA
Kozak M (1999). Gene 234 (2): 187–208.
Malys N, McCarthy JEG (2010). Cellular and Molecular Life Sciences 68 (6): 991–1003.

Roles of genetics in
allergic diseases

Does genetic have role?
 Want to know?
 Look at heritability
 = The proportion of observed variation in a trait
that can be attributed to inherited genetic
factors rather than environmental influences

Heritability evidences
 Evidence for a heritable component in allergic
disease has been confirmed by:
 Family studies
 Segregation analysis
 Twin and adoption studies
 Heritability studies
 Population-based relative risk for relatives of
probands

So… What’s the role?
 Susceptibility
 Target organ determination
 Interaction of environmental factors
with disease
 Modification of disease severity
 Therapeutics

Susceptibility
 Th2 genes
 IgE switch genes (e.g., α chain of the high-affinity
IgE receptor associated with
sensitization and serum IgE levels)

Target organ determination
 Asthma-susceptibility genes
 OPN3, CHML
 Genes that regulate propensity of lung
epithelium and fibroblasts for remodeling in
response to allergic inflammation
 ADAM33
 Atopic dermatitis–susceptibility genes
 COL6A5, OVOL1
 Genes that regulate dermal barrier function
 FLG

Interactions
 Genes that determine responses to factors that
drive Th1/Th2 polarization
 CD14 and TLR4 polymorphisms vs early childhood
infection
 Genes that modulate the effect of exposures and
disease
 Glutathione S-transferase genes vs oxidant stresses
such as tobacco smoke and air pollution on asthma
susceptibility
 Genes that alter interactions between
environmental factors and established disease
 Genetic polymorphisms regulating responses to RSV
infection vs asthma symptoms

Severity and Rx
 Allele prevalence and risk of disease severity
 TNF-α polymorphisms and asthma
 Genetic variation and response to therapy
 β2-adrenergic receptor polymorphism and response
to β2-agonists

How to study genetics
of allergic diseases?

Image from: http://www.koonec.com/wp-content/uploads/2010/06/Slide1.jpg

Hypothesis-dependent
 Candidate gene association studies
IJ Kullo and K Ding, Nature Clinical Practice Cardiovascular Medicine (2007) 4, 558-569

 Candidate gene association studies:
Advantages
 Able to identify genetic variations with relatively
small effects on disease susceptibility
 More efficient in recruiting subjects and cost
 Candidate genes have biologic plausibility
  often display known functional consequences that
have potentially important implications

 Candidate gene association studies:
Limitations
 Choice of controls can be difficult
 Subjects ideally need to be matched for variables that
may confound the results, such as age, sex, and ethnic
background
 Genes are limited to those with known or postulated
involvement in the disease
  Excluding the discovery of novel genes that
influence the disease

Hypothesis-independent
 Genome-wide linkage studies

 Genome-wide linkage studies: Advantage
 Potential for discovery of new genes and pathways
relevant to disease of interest
D Vercelli, Nature Reviews Immunology 8, 169-182 (March 2008)
 Genome-wide linkage studies: Limitations
 Slow and expensive
 Because of the need to recruit and obtain phenotypes
for large cohorts of families.
 Most linkage studies were underpowered for
identifying susceptibility genes for complex
diseases, despite recruiting several hundred families.

Linkage study

 Genome-wide association studies (GWAS)
 Able to localizes the susceptibility locus to much
smaller region (10-500 kb) than is typically possible
in linkage study
 Provided compelling statistical associations for
hundreds of loci in the human genome
 Giving insight into the physiologic parameters and
biologic processes that underlie these phenotypes
and diseases

 Genome-wide association studies (GWAS)
 Successful in the identification of genetic factors
underlying allergic disease
 May identify novel genes and pathways
 Unlike traditional candidate gene association studies
 Can identify genes with small effects
 Unlike linkage studies

GWAS

 Genome-wide association studies (GWAS) :
Limitations
 Large number of false-positive results
 Replication of positive findings in additional
populations is crucial
 Accurate phenotypes must be obtained so that
genetic contributions to disease status can be
properly analyzed
 Because of the great expense and difficulties in
performing such studies in thousands of subjects

Limitations
 Study populations must be carefully characterized
 To select patient who are likely to share a genetic cause
of disease
 Thousands of cases and controls may be needed to
have sufficient statistical power to identify the
alleles of interest
 Some relevant statistical strategies are still being
developed
 Heterogeneity in environmental exposures1

Limitations
 Need to test enormous amount of DNA variants in
thousands of subjects
 Challenges in bioinformatics
 How to identify true positives in a sea of false positives?
 Technological challenges
 Finding the specific mutation may not be
straightforward without in-depth functional
studies

Possible error

Genetics of allergic
diseases

Genetics of allergic diseases

 By GWAS

Park SM, et al. Allergy, asthma & immunology research. 2013 Sep;5(5):258-76.
 In AERD

Genes related to allergy:
Remarks
 From heritability studies:
 Genes that predispose to atopy overlap with those
that predispose to asthma
 But… the overlap between loci identified as
predisposing to serum IgE levels and allergic
disease is so small

Genes related to allergy:
Remarks
 Is there evidence of those overlap foci?
 Study by the GABRIEL Consortium
 Designed to identify the genetic and environmental
causes of asthma in the European community enrolled
10,365 subjects with physician-diagnosed asthma and
16,110 controls
 Loci strongly associated with IgE levels were not
associated with asthma
 Except those for IL-13 and HLA region
 Supporting studies: No relationship between atopic
sensitization and asthma in many populations

GWAS in asthma: Remarks
 Study results have not fully explained the
heritability patterns
 Despite including 4 large-scale population
analyses
 European
 American (including European-American, African-
American, African- Caribbean, and Latino ancestry)
 Australian
 Japanese

 Why GWAS can not find all of the genetic
factors underlying asthma susceptibility?
 May be explained by limitations of GWASs
 Presence of other variants in the genome not
captured by the current genotyping platforms
 Analyses not being adjusted for gene-environment
and gene-gene interactions
 Epigenetic changes in gene expression

 Genes encoding proteins involved in Th2-
mediated immune responses are not the only
or the most important factors underlying
asthma susceptibility

Groups of genes in asthma
 Genes that directly modulate the response to
environmental exposures
 Genes that maintain epithelial barrier integrity
and cause the epithelium to signal the immune
system after environmental exposure
 Genes that regulate immune responses
 Genes involved in determining the tissue
response to chronic inflammation
 Genes that alter phenotypes related to disease
progression

Genes in asthma: Remarks
 Genetic studies of asthma have reinforced
observations about the importance of early-life
events in determining asthma susceptibility
 Overall
 Variations in genes regulating atopic immune
responses are not the major factor in determining
susceptibility to asthma
 Most of the asthma-susceptibility loci identified
were not associated with serum IgE levels.

Atopic dermatitis (AD)
 Filaggrin gene (FLG)
 Has a key role in epidermal barrier function
 One of the strongest genetic risk factors for atopic
dermatitis
 Located on chromosome 1q21 in the epidermal
differentiation complex
 40-80% of subjects carrying >/= 1 FLG null
mutations will develop AD

 AD patients have increased risk of atopic
sensitization and atopic asthma
FLG mutation
Deficit in epidermal barrier function
Initiate systemic allergy by allergen
exposure through the skin
Start the atopic progression in
susceptible individuals

 COL6A5 (formerly COL29A1)
 SNP C11orf30
 Adjacent to a locus of unknown function on
chromosome 11q13.5
 Strongly associated with susceptibility to AD
 Other 7 SNPs were identified as susceptibility
factors to AD
 Those loci are near genes that have been implicated
in epidermal proliferation and differentiation
 So… gene for allergic disease might acts at the
mucosal surface rather than by modulating the
level or type of immune response

Rhinitis
 Several genome-wide linkage studies have
identified potential disease susceptibility loci
 HLA regions
 C11orf30 or LRRC32 locus
 MRPL4 and BCAP loci in Chinese ethnicity
 Several candidate gene studies have shown
association with polymorphisms in
inflammatory genes such as IL13

Food allergy
 Polymorphisms of
 CD14
 STAT6
 Serine peptidase inhibitor, kazal type 5 (SPINK5)
 IL10
 Fillagrin gene (FLG)
 Functional SNPs in the NACHT protein domain
of the NLR family, pyrin domain–containing 3
gene (NLRP3)
 Strongly associated with food-induced anaphylaxis
and ASA-intolerant asthma

Missing heritability
in allergic diseases

 A large proportion of heritability remains
unaccounted for because of small size of SNP
effects (OR about 1.05-1.3)
 Genetic markers alone is not useful to predict
disease susceptibility
  Little or no diagnostic utility

 Missing heritability
 = The finding that loci identified through GWASs fail
to account for all heritability of those conditions
 Missing heritability may be due to:
 Gene-gene interactions
 Gene-environment interactions
 Epigenetic phenomena
 Other types of genetic variation

Gene-Gene Interactions

 Example: Asthma: IL-13/IL-4 cytokine pathway
 IL4RA and IL13 gene interaction markedly increases
asthma susceptibility
 A case-control study:
 SNP S478P in IL4RA vs −1112C/T promoter
polymorphism in IL13
 Individuals with risk genotype for both genes  5x risk
for asthma (P = .0004)

 Example: Asthma: IL-13/IL-4 cytokine pathway
 A cross-sectional study: 1120 children (9-11 yrs old)
 Combinations of genetic variations are significantly
related to development of atopy and childhood asthma

Gene-Environment Interactions
 Different genotypes = different sensitivities to
environmental exposures
 Passive smoking increases airway responsiveness
and incident asthma
 SNPs in susceptibility locus on chromosome 17q21,
which encompasses the ORMDL3 and GSDMB
genes, are confined to early-onset asthma
 esp. in those who exposed to environmental tobacco
smoke in early life
 Association of these 17q21 variants with asthma is
enhanced in children who have respiratory
infections before 2 years of age
 esp. in those also exposed to tobacco smoke

 Some components of the innate immune
response, such as the CD14 and TLR4 receptors,
are involved in the recognition and clearance of
bacterial endotoxin
 SNPs that alter the biology of these receptors can
influence the early-life origins of allergic disease by
modifying the effect of microbial exposure on the
developing immune system
 Studies have shown interactions between a
polymorphism of CD14 and measures of microbial
exposure, such as living on a farm, consumption of
raw (unpasteurized) farm milk, and household dust
endotoxin levels, in determining serum IgE levels,
sensitization, and asthma

 Tool for study: genome-wide interaction
studies (GWISs)
 Data on 500,000 SNPs were assessed for interaction
with 7 farm-related exposures
 1,708 children
 GWIS did not reveal any significant interactions with
common SNPs
 Among less common SNPs, 15 genes with crossover
interactions or effect concentrations were identified in
the exposed group for asthma or atopy in relation to
farming, consumption of farm milk, and contact with
cows and straw
 Many showed a flip-flop pattern of association

 Tool for study: genome-wide interaction
studies (GWISs)
 No interactions were observed involving SNPs in
genes previously identified as interacting with
farming exposures such as CD14 and TLR4
 Issues with exposure assessment?
 Endotoxin levels were not directly measured in the
population, and with farming exposure, which
correlated with endotoxin exposure but is nonetheless
a surrogate measure of exposure
 Accurate exposure assessment is needed

 Advantage of this knowledge:
 Proof that environmental exposure is truly causal
 Identify at-risk groups who could benefit from
preventative strategies that include environmental
modification
 Identification of at-risk groups, the degree of their
sensitivity to exposures, and their frequency in the
population
 Aid the cost-benefit analysis of safe exposure levels
in the public health setting

Other Sources of Variation
 Rare variants (mutations that occur in <5% of
the population)
 May be specific to different ethnic groups, isolates,
families, or individuals
 Harbors multiple penetrant mutations conferring
medium to high risk of disease
 May play a significant role in individual with the
severe end of the phenotype spectrum
 i.e. filaggrin in atopic dermatitis

Other Sources of Variation
 Unexpectedly heterogeneous structural
variation in the human genome = copy number
variations (CNVs)
 i.e., deletions, duplications, inversions, and
translocations
 Associated with a range of disease phenotypes
 Genome-wide studies of CNVs in allergic disease
have yet to be undertaken
 Examples of CNVs in candidate genes such as the
GSTM1 and GSTT1 genes show that this class of
genetic variant may be relevant to allergic disease

Epigenetics in
allergic diseases

Epigenetics in allergy
 Histone acetylation and methylation
 Alters the rate of transcription
 Alters protein expression
 DNA methylation
 Adding a methyl group to specific cytosine bases in
DNA
 Suppresses gene expression

 Causes of histone changes and DNA
methylation
 Environmental exposures
 Tobacco smoke
 Traffic pollution
 Alterations in early-life environment
 Maternal nutrition

 Transgenerational epigenetic effects mediated
by DNA methylation
 Grandmaternal smoking increasing the risk of
childhood asthma in their grandchildren
 Sex-specific transmission
 Paternal allergic disease predisposing male offspring
to development of allergic disease
 Maternal disease predisposing female offspring

 Animal models
 Mice exposed to in utero supplementation with methyl
donors exhibit enhanced airway inflammation after
allergen challenge, a phenotype that persists in the
second generation despite the absence of further
exposure
 Effect of environmental exposures relevant to
allergic disease
 Prospective studies of large birth cohorts with
information on maternal environmental exposures
during pregnancy are likely to provide important insights
into the role of epigenetic factors in the heritability of
allergic disease.

Functional genomics
in allergic diseases

Functional genomics
 Hypothesis-independent approaches
 => identification of genes of unknown function as
susceptibility factors for disease
 The variations in these genes -> affect function
or expression
 Indicate the importance of the encoded proteins in
disease pathogenesis
 But how?
 The mechanisms of action are often unclear

Functional genomics
 Hypothesis-independent approaches
 => identification of genes of unknown function as
susceptibility factors for disease
Functional genomics is a
measure to answer this!
 The variations in these genes -> affect function
or expression
 Indicate the importance of the encoded proteins in
disease pathogenesis
 But how?
 The mechanisms of action are often unclear

Functional genomics
Image from: http://www.ifcc.org/ifccfiles/images/4_1.gif

Functional genomics
 Experimental approaches that can be used to
understand the role of novel susceptibility
genes in disease biology
 Animal models
 Provide insights into gene function
 By comparing responses in gene-knockout and wild-type
mice

Functional genomics
 Experimental approaches that can be used to
understand the role of novel susceptibility genes in
disease biology
 Identification of commonalities in genetic susceptibility
and pathogenesis between complex diseases
 These and other functional studies of disease-susceptibility
genes = effort to close the gap
between gene identification and disease biology

Commonality identification
17q21 locus containing several genes, including ORMDL3,
has been associated with several inflammatory conditions,
such as IBD and rheumatoid arthritis, in addition to asthma
ORMDL3 regulates endoplasmic reticulum (ER) stress,
and several additional ER stress–associated genes have
been identified as risk factors for IBD
Intestinal epithelium of these patients commonly exhibits
marked ER stress
Because of the commonality in genetic association, ER
stress may also be an important pathogenetic factor in
asthma

Genetics and Allergic Diseases

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