La GenLa Genéética del Asma in Hispanos tica del Asma in Hispanos

Juan C. CeledJuan C. Celedóón, M.D., Dr.P.H.n, M.D., Dr.P.H.Channing Laboratory Division of Pulmonary and Critical Care MedicineBrigham and Women’s HospitalDepartment of MedicineHarvard Medical School

BRIGHAM AND WOMEN’S HOSPITAL

HARVARDMEDICAL SCHOOL

• Definition of the term Hispanic• Asthma among Hispanics in the United States• The Genetics of Asthma in Hispanics • Study of Asthma Genetics in Costa Rica

– Heritability Analysis – Genome-wide linkage analysis of asthma and

airway responsiveness– Future Directions

Outline

Definition of the Term “Hispanic”

1.

Introduction

• 2000 Census: ~32.8 million Hispanics in the United States (~12% of the population)

• “Hispanic”: Common heritage of language and culture

Asthma in Hispanics

2.

Asthma in Hispanics

• Asthma is a significant cause of morbidity in certain Hispanic subgroups in the U.S. (e.g., Puerto Ricans) and Hispanic America (e.g., Costa Ricans)

• Little is known about genetic and environmental influences on asthma in Hispanics

Hunninghake GM, Weiss ST, Celedón JC. State of the Art: Asthma in Hispanics. Am J Respir Crit Care Med 2006; 173:143-163.

The Genetics of Asthma in Hispanics

3.

The Genetics of Asthma in Hispanics

• To date, genome-wide analyses for linkage to asthma phenotypes have been conducted in 18 distinct populations– Until recently, only one genome scan included any

Hispanic individuals

Results of Selected CandidateResults of Selected Candidate--Gene Association Studies for Gene Association Studies for Asthma and/or AsthmaAsthma and/or Asthma--Related Phenotypes in HispanicsRelated Phenotypes in Hispanics

Raby B, et al3

No association between any of the SNPs tested and asthma or asthma-related phenotypes.

17

47 Hispanic families of children with asthma

Lind D, et al2

No association between any of the SNPs tested and asthma or asthma-related phenotypes.

6

Mexican (n=265) and Puerto Rican (n=318) families of individuals with asthma

Howard T, et al1

Two SNPs in ADAM33 were associated with asthma and allergen sensitization.

8

Hispanics with (n=112) and without (n=126) asthma

ADAM33

ReferenceResultsNumber of SNPsStudied

Study SampleGene

Candidate-Gene Association Studies in Hispanics: Limitations

• Small sample size• Inadequate definition of Hispanic subgroups• Heterogeneity of populations studied• Lack of correction for multiple comparisons• Lack of adequate data on environmental

exposures• (For case-control studies): lack of detection and

control of potential population stratification

The Genetics of Asthma in Costa Rica

4.

Rationale and Study Design

4.a.

• Prosperous Latin American Country– Universal access to health care– High literacy rate (~95%)

• High prevalence of childhood asthma in Costa Rica (~23%)

Asthma in Costa Rica

• 1697 Census–2,100 Spaniards–1,400 Amerindians–300 individuals of African descent–160 Mestizos

Costa Rica

• Most Costa Ricans live in a Central Valley separated from the coasts by mountain ranges

• Minimal migration in and out of the Central Valley until the late 1800s

The Central Valley of Costa Rica

Family-based Study of Asthma Genetics in Costa Rica

•• Rationale:Rationale:– To study “complex” diseases, a useful

strategy is to study communities that are relatively isolated genetically

•• Study HypothesisStudy Hypothesis:– A small number of chromosomal regions

contribute to the expression of asthma in Costa Ricans

• Funded by the NHLBI since March of 2001• Phenotypic and genotypic data collected in

2,471 individuals– 671 members of large families of children with

asthma– 1,800 members of nuclear families of children with

asthma

Family-based Study of Asthma Genetics in Costa Rica

Data Collected in Members of Family Pedigrees

• Children and adults:– Questionnaire information– Spirometry with B.D.R.– Methacholine challenge testing– Total and specific serum IgE– Eosinophil count– Skin test reactivity to allergens – Blood sample for DNA extraction

Subject Characteristics and Heritability Analysis

4.b.

Table 1. Characteristics of Family Members (n=687)

99.9 (45.8-115.9)% Predicted Post-BD FEV1/FVC, mean (range)

102.4 (33.6-152.4) % Predicted Post-BD FEV1, mean (range)

2 (0 to 9)Number of positive skin tests, median (range)

208.4 (30 to 7,500) Eosinophil count (cells/mm3), geometric mean (range)

97.0 (0-5,000)Total serum IgE (IU/ml), geometric mean (range)

29.5 (6.4-92.6)Age in years, mean (range)

368 (52.7)Female sex, n(%)

Table 1. Characteristics of Family Members (n=687)

47 (6.8)Current smoker, n (%)

109 (15.9) Ever smoker, n (%)

52 (29.9)87 (17.0)

Physician-diagnosed asthma and current wheezing, n (%)Age <13 yearsAge >=13 years

64 (36.8)166 (32.4)

Current wheezing, n (%)Age <13 yearsAge >=13 years

69 (39.7)128 (25.0)

Physician-diagnosed asthma, n(%)Age <13 yearsAge >=13 years

Table 2. Heritability Analysis of Asthma-Related Traits

14.3% (6.3%)Post-BD FEV1/FVC*

23.0% (6.5%)Post-BD FEV1 (L)*

49.9% (9.2%)Airway responsiveness (d.r.s. to methacholine)*

29.3% (7.6%)Eosinophil count (cells/mm3)&

56.6% (6.5%)Total serum IgE (IU/ml)&

h2N (standard error)

& Variance components analysis adjusted for age and sex.*Variance components analysis adjusted for age, sex, and height.Note: All heritability (h2

N) values > 0 with p<0.0001

Genome-wide Linkage Analysis

4.c.

Overview of Complex Trait Genetics

Demonstrate Familial Aggregation and/or Heritability of Trait

Localize Susceptibility Gene(s)

Identify Disease Susceptibility Gene and Functional Variants

Positional Cloning Candidate Gene Approach

Diagnostics Determine Fraction of Explained Variation Treatment

How to find a disease-susceptibility gene?

page

‘chromosomal region’

This is a sentence in a paragraph…

‘gene’

This it a sentence in a paragraph…

‘mutation’

• Difficult challenge: like finding a misspelled word in a set of encyclopedias!

Plausible candidate genes>100 !

‘which chromosome?’

Vol 7

A1

Z247

•• DefinitionDefinition:: A group of methods that analyze the distribution of DNA markers within families to determine if a particular region of the genome contains a gene related to the phenotype of interest.

•• LOD ScoreLOD Score:: Result of a statistical test to determine if genetic loci are linked, expressed as log10 of the odds that the loci are linked. LOD scores of 3-3.6 correspond to highly significant linkage.

Linkage Analysis

Whole Genome Scans

• A genome scan consists of ~400 STR markers spread across all 23 human chromosomes. These markers are like a dragnet, enabling us to pinpoint the location of genomic regions containing disease susceptibility genes.

• Shown on the right is a map of human chromosome 1 with different type of markers (SNPs and STR or microsatellite markers) overlaid on the map.

• Genome scan was completed in November of 2004 at the Genome Quebec Innovation Centre in 671 individuals– 380 short-tandem repeat (STR) markers, with an average

spacing of 8.2 cM (ranging from 6.2 cM on chromosome22 to 9 cM on chromosome 17)

- Marker locations were determined on the basis of the deCODE map

Genome Scan for Asthma and Asthma-related Phenotypes in Costa Rica

Short-Tandem Repeats: Repetitive elements flanked by unique sequence

GTC GTC GTC GTC

CAG CAG CAG CAG

1 2 3 4

5’ 3’

CAG CAG CAG CAG CAG CAG CAG

1 2 3 4 5 6 7

GTC GTC GTC GTC GTC GTC GTC

GF GM Mom Dad Son Ladder8765432

7/4 8/5

7/5 8/2

7/2

• Phenotype:– Dose-response slope to methacholine, which was log-

transformed (residual kurtosis <0.9)

• Variance component linkage analysis conducted with the SOLAR program– Age, sex, height, smoking (ever), pack-years, and quadratic

terms were considered for inclusion in final models

• All analyses were repeated in nonsmokers only

Genome-wide Linkage Analysis of Airway Responsiveness

1.01

1.32

1.75

All subjects(n=488)

Maximum LOD score

15

12

10

6

Chromosome

119

146

23

147

134

cM

1.04

2.33

1.50

1.41

Nonsmokers(n=423)

Table 1. Genome-wide Linkage Analysis of Airway Responsiveness

Staged Genomic Search

• A genome scan (Stage 1 or “Random Genome Scan”) is carried out at a low marker density and then those regions which may be linked to the trait of interest are investigated by typing more markers (Stage 2 or “Fine Mapping Linkage Analysis”)

• First genome-wide linkage analysis of airway responsiveness in a Hispanic population

• Chromosome 12q24 likely contains a locus (or loci) that influence airway responsiveness, a critical intermediate phenotype of asthma

Discussion

Sex Differences in Total Serum IgE in Costa Rican Pedigrees

p=0.003

Comparisons adjusted for age, age2, ever smoking, pack-years (py) and py2.

Narrow-sense heritability of log10IgE=0.556 (S.E=0.065), p=10-35

Genome-wide Linkage Analysis of Total Serum IgE in Costa Rican Pedigrees

Sex-specific Linkage to Total Serum IgE in Costa Rican Pedigrees

• First demonstration of a distinct genetic architecture of total serum IgE in males and females

• Chromosome 20p12 likely contains a gene(s) that influence total serum IgE in males

Discussion

Future Directions

4.d.

• Fine-mapping linkage analysis to confirm and narrow regions linked to asthma and/or asthma-related traits – Currently on chromosomes 1p, 7q, 20p

• Fine-mapping association analysis to identify gene(s) associated with asthma and/or its intermediate phenotypes (in nuclear families)– First region: chromosome 12q24

Family-based Study of Asthma Genetics in Costa Rica

• Channing Laboratory, Brigham and Women’s Hospital(Boston, MA): Jody Senter, Barbara Klanderman, Craig Hersh, Matt Hunninghake, Ngoc Ly, Ross Lazarus, Steve Lake, Catherine Liang, Dan Laskey, Jessica Su, Ed Silverman, and Scott T. Weiss

• Dept. of Biostatistics, Harvard School of Public Health(Boston, MA): Christoph Lange and Nan Laird

Collaborators

• Genome Quebec Innovation Centre (Montreal, Canada): Thomas Hudson

• Hospital Nacional de Niños (San José, Costa Rica): Manuel Soto-Quiros, Lydiana Avila, Eduardo Fournier,and Mitzi Spesny

Collaborators

The Genetics of The Genetics of Asthma in Costa Asthma in Costa

RicaRica

HL66289, HL04370HL66289, HL04370

The Genetics of COPD in The Genetics of COPD in Costa RicaCosta Rica

HL073373HL073373

Genes, Allergens, and Genes, Allergens, and Asthma in Puerto RicansAsthma in Puerto Ricans

HL079966HL079966

Genome Scan/Linkage →Flanking Markers/Linkage →Positional Candidates/Association →Animal Models/Function

Genetic Studies of Asthma and COPD in Hispanics at BWH