Genetics and Allergic Diseases

GENETICS ANDALLERGIC DISEASES

Presented by Wat Mitthamsiri, MDAllergy and Clinical Immunology FellowKing 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

Introduction

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.


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 plansJW Halloway, Middleton’s Allergy 8th edition, 2013, 343-363.


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 plansJW Halloway, Middleton’s Allergy 8th edition, 2013, 343-363.

Phenotypes

Genetic models of diseases

Getting the diseases

Extrinsic Factors

Intrinsic (Host) Factors

Disease (Phenotype)


Extrinsic Factors


Disease (Phenotype)

• Pathogen factors• Type• Dosage• Route of infection• Virulence• Resistance• Etc.

• Environmental factors• Temperature• Humidity• Pollution• Hygiene• Etc.


Extrinsic Factors


Disease (Phenotype)

• Age• Genetic

factors• Sex• Ethnicity• Behavior• Epigenetic

process• Etc.

Single genetic disorder

Extrinsic Factors

Trisomy 21

Down’s syndrome

• Genes• ADAM33• DPP10• PHF11• HLA-G,• OPN3• NPSR1• Etc.

• Allergen induced• House dust mite• Pollen• Cochroach• Etc.

• Non-allergen induced• Cold air• Fuel particles• Infection• ASA• Exercise• Etc.

Complex genetic disorder

Extrinsic Factors

Intrinsic Factors

Asthma

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 regulation

Gene expression process

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

Gene expression process


Nucleus

Cytoplasm

Expression regulation


Transcription control

RNA processing control

RNA transportation

controlRNA translation

control

DNA modification

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, Pai AA, Pickrell JK, Gaffney DJ, Pique-Regi R, Degner JF, Gilad Y, Pritchard JK (2011). Genome Biology 12 (1)

DNA modification

Structural and chemical modification

• DNA folding/coiling• Phosphorylation• Methylation• Histone acetylation


Epigenetics


Hoopes, L. (2008) Introduction to the gene expression and regulation topic room. Nature Education 1(1):160


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

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

Kozak M (1999). Gene 234 (2): 187–208.

Malys N, McCarthy JEG (2010). Cellular and Molecular Life Sciences 68 (6): 991–1003.

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

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 symptomsJW Halloway, Middleton’s Allergy 8th edition, 2013, 343-363.

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


Hypothesis-dependent 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



Hypothesis-dependent 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

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.


D Vercelli, Nature Reviews Immunology 8, 169-182 (March 2008)

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


GWAS


GWAS


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



Genome-wide association studies (GWAS) : 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



Genome-wide association studies (GWAS) : 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









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


GWAS in asthma: Remarks 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


GWAS in asthma: Remarks 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 progressionJW Halloway, Middleton’s Allergy 8th edition, 2013, 343-363.

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 asthmaJW Halloway, Middleton’s Allergy 8th edition, 2013, 343-363.

Missing heritability in allergic diseases

Missing heritability 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 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 smokeJW Halloway, Middleton’s Allergy 8th edition, 2013, 343-363.


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 JW Halloway, Middleton’s Allergy 8th edition, 2013, 343-363.


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 JW Halloway, Middleton’s Allergy 8th edition, 2013, 343-363.


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

unclearJW Halloway, Middleton’s Allergy 8th edition, 2013, 343-363.

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

unclearJW Halloway, Middleton’s Allergy 8th edition, 2013, 343-363.

Functional genomics is a measure to

answer this!

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


Take home message

Take home message

Extrinsic Factors


Disease (Phenotype)

Take home message

Extrinsic Factors


Disease (Phenotype)

• Age• Genetic

factors• Genes per se.• Epigenetic

process• Etc.

Thank you

Health & Medicine

Genetics and Allergic Diseases