Admixture Mapping for Admixture Mapping for Atherosclerosis Loci in African Atherosclerosis Loci in African

AmericansAmericans

the NHLBI Family Heart Studythe NHLBI Family Heart Study

Q.Y. Zhang and M.A. Province Q.Y. Zhang and M.A. Province

Division of Statistical GenomicsDivision of Statistical GenomicsCenter for Genome SciencesCenter for Genome Sciences

Department of GeneticsDepartment of Genetics

Washington University School of MedicineWashington University School of Medicine

Feb. 24, 2006Feb. 24, 2006

Family Heart Study - Subclinical Coronary Family Heart Study - Subclinical Coronary Atherosclerosis NetworkAtherosclerosis Network

  A multi-center studyA multi-center study

Primary purpose :Primary purpose :

to identify familial, genetic, and non-genetic to identify familial, genetic, and non-genetic factors that contribute to atherosclerosis and factors that contribute to atherosclerosis and inflammatory response. inflammatory response.

FHS SCANFHS SCAN

(AA Field Center)

FHS-

SCAN

BirminghamBirmingham

HoustonHouston(DNA Lab)

(CT Reading)

Participants with Clinic Visits

NC(1)

MN(2)

MA(3)

UT(4)

AL(5)

Total

N % N % N % N % N % N %

N 607 100.0 745 100.0 518 100.0 897 100.0 622 100.0 3389 100.0

Coronary CT Scan 600 98.5 741 99.4 511 99.8 897 100.0 621 100.0 3370 99.8

Inflammation 607 100.0 745 100.0 518 100.0 897 100.0 622 100.0 3389 100.0

Sitting Blood Pressure

607 100.0 745 100.0 517 99.8 897 100.0 622 100.0 3388 100.0

Anthropometry 607 100.0 745 100.0 517 99.8 897 100.0 622 100.0 3388 100.0

Demographics 607 100.0 745 100.0 518 100.0 897 100.0 622 100.0 3389 100.0

Pedigree 607 100.0 745 100.0 518 100.0 897 100.0 622 100.0 3389 100.0

Medical Hx 607 100.0 745 100.0 518 100.0 897 100.0 622 100.0 3389 100.0

Medication Inventory

607 100.0 745 100.0 518 100.0 897 100.0 622 100.0 3389 100.0

Personal Hx 606 99.8 745 100.0 518 100.0 897 100.0 622 100.0 3388 100.0

Blood Assays 606 99.8 744 99.9 518 100.0 896 99.9 621 99.8 3385 99.9

Previously FHS Visit (ECG, Carotid Ultrasound, Pulmonary)

423 69.7 574 77.0 326 62.9 607 67.7 382 61.4 2312 68.2

FHS-SCAN Data 2001-2004

Population(s)Population(s)

Sample(s)Sample(s)

Genotypes Phenotype(s)Genotypes Phenotype(s)

Statistical Method(s)Statistical Method(s)

ResultsResults

General Considerations of QTL General Considerations of QTL Mapping Mapping

African AmericansAfrican Americans

622 Subjects from 211 families622 Subjects from 211 families

Admixture MappingAdmixture Mapping

CAC LociCAC Loci

400 microsatellite 400 microsatellite markers markers Average distance 10 Average distance 10

cMcM

Coronary and aortic Coronary and aortic artery calcium (CAC) artery calcium (CAC)

Quantified by CTQuantified by CT

calcified plaque

What is Admixture What is Admixture Mapping ?Mapping ?

Founding Population 2

Caucasians

Founding Population 1

Africans

Admixed Population African Americans Admixture Mapping

Admixture Information (Ancestry Analysis)

Only for admixed population

Based on admixture information (ancestry analysis)

Proportion of genetic materials descending from Proportion of genetic materials descending from each founding population each founding population

Population levelPopulation level : population admixture proportion : population admixture proportion

Individual levelIndividual level: individual admixture proportion: individual admixture proportion

Individual-locus levelIndividual-locus level: locus-specific ancestry: locus-specific ancestry

AncestryAncestry

If a disease has some genetic factors, and the disease gene If a disease has some genetic factors, and the disease gene frequency in pop 2 is higher than in pop 1. After the admixture frequency in pop 2 is higher than in pop 1. After the admixture of pop 1 and 2, the diseased individuals in admixed of pop 1 and 2, the diseased individuals in admixed generations will carry disease genes/alleles that have more generations will carry disease genes/alleles that have more ancestry from pop 2 than from pop 1. ancestry from pop 2 than from pop 1.

If a marker is linked with disease genes, because of linkage If a marker is linked with disease genes, because of linkage disequilibrium, the diseased individuals will also carry the disequilibrium, the diseased individuals will also carry the marker copies that have more ancestry from pop 2 than from marker copies that have more ancestry from pop 2 than from pop 1.pop 1.

Inversely, if we find a marker/locus whose ancestry from pop Inversely, if we find a marker/locus whose ancestry from pop 2 in diseased group is significantly different from that in non-2 in diseased group is significantly different from that in non-diseased group, we consider this marker/locus to be linked with diseased group, we consider this marker/locus to be linked with (or a part of ) disease gene.(or a part of ) disease gene.

Rationale of Admixture Rationale of Admixture MappingMapping

ReferencesReferences

D.C.Rife. Populations of hybrid origin as source material for the D.C.Rife. Populations of hybrid origin as source material for the detection of linkage. Am.J.Hum.Genet. detection of linkage. Am.J.Hum.Genet. 19541954, (6):26-33, (6):26-33

R.Chakraborty et al. Adimixture as a tool for finding linked genes R.Chakraborty et al. Adimixture as a tool for finding linked genes and detecting that difference from allelic association between loci. and detecting that difference from allelic association between loci. Proc.Natl.Acad.Sci. Proc.Natl.Acad.Sci. 19881988,Vol.85:9119-9123,Vol.85:9119-9123

N. Risch. Mapping genes for complex disease using association N. Risch. Mapping genes for complex disease using association studies with recently admixed populations. Am.J.Hum.Genet.Suppl. studies with recently admixed populations. Am.J.Hum.Genet.Suppl. 19921992, 51:13, 51:13......

P.M.McKeigue. Prospects for admixture mapping of complex traits. P.M.McKeigue. Prospects for admixture mapping of complex traits. Am.J.Hum.Genet. Am.J.Hum.Genet. 20052005, Vol.76:1-7, Vol.76:1-7

X.Zhu et al. Admixture mapping for hypertention loci with genome-X.Zhu et al. Admixture mapping for hypertention loci with genome-scan markers. Nature Genetics. scan markers. Nature Genetics. 20052005,Vol.37(2): 177-181,Vol.37(2): 177-181

STRUCTURESTRUCTURE Falush D, Stephens M, Pritchard JK (2003) Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164:1567–1587.

ADMIXMAPADMIXMAP Hoggart CJ, Parra EJ, Shriver MD, Bonilla C, Kittles RA, Clayton DG, McKeigue PM (2003) Control of confounding of genetic associations in stratified populations. Am J Hum Genet 72:1492–1504.

ANCESTRYMAPANCESTRYMAP Patterson N, Hattangadi N, Lane B, Lohmueller KE, Hafler DA, Oksenberg JR, Hauser SL, Smith MW, O’Brien SJ, Altshuler D, Daly MJ, Reich D (2004) Methods for high-density admixture mapping of disease genes. Am J Hum Genet 74:979–1000

SoftwaresSoftwares

Advantages of Admixture Advantages of Admixture MappingMappingAdmixed population has more genetic variation and Admixed population has more genetic variation and polymorphism than relatively pure ancestral populations.polymorphism than relatively pure ancestral populations.

Admixture produces new LD in admixed Admixture produces new LD in admixed population.Compared with ancestral populations, shorter population.Compared with ancestral populations, shorter genetic history of admixture population keeps more LD genetic history of admixture population keeps more LD (long genetic history will destroy LD), In admixed (long genetic history will destroy LD), In admixed population, LD could be detected for realtively loose population, LD could be detected for realtively loose linkage.linkage.

Ancestry infromation can be used to control population Ancestry infromation can be used to control population stratification caused by genetic admixture. stratification caused by genetic admixture.

According to simulation, admixture mapping According to simulation, admixture mapping demonstrates higher power than regular methods, needs demonstrates higher power than regular methods, needs less sample size. less sample size.

Flexible design: case-control or case-only, qualitative or Flexible design: case-control or case-only, qualitative or quantitative traits, no need of pedigree informationquantitative traits, no need of pedigree information

DataData

SamplesSamples 1672 subjects from 3 populations: 1672 subjects from 3 populations:622 African Americans (211 families) from622 African Americans (211 families) fromFHS-SCANFHS-SCAN893 Caucasians (320 families) from FHS-SCAN893 Caucasians (320 families) from FHS-SCAN157 Africans (unrelated) from Marshfield 157 Africans (unrelated) from Marshfield CenterCenter

GenotypesGenotypes302 microsatellite Loci of all subjects 302 microsatellite Loci of all subjects Average marker distance 11.9cM Average marker distance 11.9cM

PhenotypePhenotypeCoronary and aortic artery calcium (CAC) of Coronary and aortic artery calcium (CAC) of 622 African Americans, BLOM 622 African Americans, BLOM transformationtransformation

Statisticl ProcedureStatisticl Procedure

Step 1Step 1Randomly draw one subject from each family to create a Randomly draw one subject from each family to create a sample of 688 unrelated subjects which comprises : sample of 688 unrelated subjects which comprises : 211 African Americans from 211 families (FHS-SCAN) 211 African Americans from 211 families (FHS-SCAN) 320 whites from 320 families (FHS-SCAN)320 whites from 320 families (FHS-SCAN)157 unrelated Africans (Marshfield Center)157 unrelated Africans (Marshfield Center)Step 2Step 2Ancestry estimation, STRUCTURE 2.1 Ancestry estimation, STRUCTURE 2.1 Step 3Step 3Ancestry-CAC association analysis, regress 211 African Ancestry-CAC association analysis, regress 211 African Americans’ CAC scores on their locus-specific ancestries from Americans’ CAC scores on their locus-specific ancestries from Africans.Africans.Step 4Step 4Repeat step1~step3 (100 times), obtain the average p-value of Repeat step1~step3 (100 times), obtain the average p-value of each locus each locus Step 5Step 5For each locus: permutation test on average p-value For each locus: permutation test on average p-value Number of random permutations: 10000 Number of random permutations: 10000

RESULTSRESULTS

Sources of Variation of Ancestry-from-AfricansSources of Variation of Ancestry-from-Africans

Sources of variationSources of variation Variance Variance componentscomponents

Percent(%)Percent(%)

FamiliesFamilies

Subjects within familySubjects within family

Loci within subjectLoci within subject

Replications within Replications within locuslocus

0.010540.01054

0.004920.00492

0.005990.00599

0.000420.00042

48.1948.19

22.5022.50

27.3927.39

1.921.92

48%

23%

2%

27% Var(families)

Var(subjects/family)

Var(loci/subject)

Var(replications/locus)

RESULTSRESULTS

Ancestry Analysis at Population LevelAncestry Analysis at Population Level

Population Admixture Proportions in African Population Admixture Proportions in African AmericansAmericans

Founding populationFounding population Ancestry(%)Ancestry(%)

From CaucasiansFrom Caucasians 22.0422.04

From AfricansFrom Africans 77.9677.96

Individual Ancestry Distribution of 622 African Individual Ancestry Distribution of 622 African AmericansAmericans

Ancestry-from-Africans: average 77.96% (3.1%~96.9%)

RESULTSRESULTS

Ancestry Analysis at Individual LevelAncestry Analysis at Individual Level

RESULTSRESULTSAncestry Analysis at Individual-locus Ancestry Analysis at Individual-locus LevelLevel

Distribution of Locus-specific Ancestries from AfricansAn Example African American

Ance

stry

fro

m A

fric

ans

302 Microsatellite Loci

ordered by chromosome and position

from Chrom. 1 (4.22cM) to Chrom. 23 (104.83cM)

RESULTSRESULTS Locus-specific Ancestry-CAC association analysisLocus-specific Ancestry-CAC association analysis

    No.No. LociLoci Chr# Chr# Pos.Pos. Permu. p Permu. p Reg. coeff. Reg. coeff. RR22

11 AFM063XF4 AFM063XF4 1010 19 .0 (10p14)19 .0 (10p14) 0.00210.0021 -1.2442-1.2442 0.03100.0310

22 GATA64D02 GATA64D02 66 80.45 (6q12)80.45 (6q12) 0.00240.0024 -2.2112-2.2112 0.02050.0205

33 GATA42H02 GATA42H02 44 181.93 (4q32)181.93 (4q32) 0.00830.0083 2.79962.7996 0.01980.0198

44 AFMB337ZH9 AFMB337ZH9 2222 60.6160.61 0.01200.0120 1.15941.1594 0.01940.0194

55 GGAA20G10 GGAA20G10 22 27.627.6 0.01330.0133 0.72710.7271 0.01660.0166

66 GATA73H09 GATA73H09 1212 78.1478.14 0.01700.0170 -1.4403-1.4403 0.01500.0150

77 GGAA3F06 GGAA3F06 77 41.6941.69 0.01730.0173 1.66521.6652 0.01630.0163

88 UT1307 UT1307 2020 69.569.5 0.01780.0178 -1.1565-1.1565 0.01750.0175

99 UT7136 UT7136 2222 52.6152.61 0.01940.0194 1.94571.9457 0.01620.0162

1010 GATA163B10 GATA163B10 66 42.2742.27 0.02670.0267 -1.3473-1.3473 0.01650.0165

1111 GATA88F09 GATA88F09 1010 4.324.32 0.03150.0315 -2.1781-2.1781 0.01530.0153

1212 GATA26D02 GATA26D02 1212 83.1983.19 0.03190.0319 -2.0880-2.0880 0.01300.0130

1313 ATA1B07 ATA1B07 1111 54.0954.09 0.03390.0339 1.15401.1540 0.01430.0143

1414 ATA4E02 ATA4E02 11 192.05192.05 0.03940.0394 0.78290.7829 0.01220.0122

1515 GATA137H02 GATA137H02 77 29.2829.28 0.04180.0418 1.31681.3168 0.01210.0121

1616 GATA4D07 GATA4D07 22 145.08145.08 0.04550.0455 -1.0065-1.0065 0.01250.0125

1717 ATA31G11 ATA31G11 1010 28.3128.31 0.04610.0461 -1.2933-1.2933 0.01340.0134

-log(p value) of Markers on Chromosome -log(p value) of Markers on Chromosome 4 4

Chromosome 4(20 markers)

0

0.5

1

1.5

2

2.5

0 20 40 60 80 100 120 140 160 180 200 220

Distance (cM)

-lo

g(p

)

GATA42H02

-log(p value) of Markers on Chromosome -log(p value) of Markers on Chromosome 66

Chromosome 6(16 markers)

0

0.5

1

1.5

2

2.5

3

0 20 40 60 80 100 120 140 160 180 200

Distance (cM)

-lo

g(p

)

GATA64D02

-log(p value) of Markers on Chromosome -log(p value) of Markers on Chromosome 10 10

Chromosome 10(14 markers)

0

0.5

1

1.5

2

2.5

3

0 20 40 60 80 100 120 140 160 180

Distance (cM)

-lo

g(p

)

AFM063XF4

Other Evidences Other Evidences ??

Provided By Aldi Kraja

Chromosome 6(16 markers)

0

0.5

1

1.5

2

2.5

3

0 20 40 60 80 100 120 140 160 180 200

Distance (cM)

-lo

g(p

)

0 50 100 150 2000

1

2

3

4

Multipoint Empirical LOD Scores on Chromosome 6 for CAC by Smoking Exposure

Combined Sample

Excluding Current smokers

Position in Centimorgans

Ever smokers

Provided by Kari North, UNCProvided by Kari North, UNC

Supportive evidences of linkage on corresponding regions of chromosomes 6 from other genome wide scans of CAC related traits

Our Findings Supporting Evidences

Location

1-LOD Unit Support Interval Phenotype

P Value or LOD

Nearest Marker, Location in Centimorgans, or Cytogenic Location Reference

LOD=3.3

LOD=2.2

P=0.0024

99 cM (6q15)

82 cM(6q12)

80 cM(6q12)

6p21.1 - 6q16.1

CACCarotid IMTAge of onset for CHDEarly Onset CAD with atherogenic dyslipidemia

2.21.4 - 2.11.31.0 - 1.2

D6S1031 (6q14.1)6p21.31 - 6q12.36q156p12

Lange LA et al., 2002Wang D et al., 2005Francke et al., 2001Hauser et al., 2001

Provided by Kari North, UNCProvided by Kari North, UNC

Genes underlying the 1 LOD unit support interval of the chromosome 6 linkage peakChromosomal Location

Gene Symbol

Gene Name Function of protein product Association studies with atherosclerosis related phenotypes

6p21.1 - 6q16.1

IL17 interleukin-17 Cytokine whose functions include inflammation, neutrophil recruitment, and cytokine secretion [14]

[15]

IL17F interleukin-17F Inhibits the angiogenesis of endothelial cells and induces endothelial cells to produce IL2, TGF-β, and MCP-1 [16].

PLA2G7 phospholipase A2, group VII

Encodes the platelet activating factor acethylhydrolase protein which is primarily bound to LDL and has both pro- and anti-inflammatory properties [17]

[18-20] [21, 22]

BMP5 Bone morphogenic protein 5

Is part of the transforming growth factor-beta superfamily [23, 24] and likely plays a critical role in calcification in plaques [25, 26].

COL21A1 collagen, type XX1, alpha 1

Functions to maintain the integrity of the extracellular matrix [27]

VEGF vascular endothelial growth factor

Has been implicated in both angiogenesis and ischemia

[28]

Provided by Kari North, UNCProvided by Kari North, UNC

Statistical Methods Statistical Methods for Locus-specific Ancestry for Locus-specific Ancestry AnalysisAnalysisObserved G : genotypes of admixed and ancestral populations

Unknown Z : admixed individuals’ locus specific ancestries from ancestral populations

Poblem: How to estimate Z ?

Maximum Likelihood Estimate(MLE):

How to obtain a Z that maximizes Pr(G|Z) ?

Z is a huge space of parameters, in which search is difficult for likelihood method.

Bayesian and Markov Chain Monte Carlo (MCMC) methods• Assume ancestral population number K• Define prior distribution Pr(Z) under K• Use MCMC to sample from posterior distribution Pr(Z|G) = Pr(Z)∙ Pr(G|Z) • Average over large number of MCMC samples to obtain estimate of Z

Genes/QTLs at/around the 3 Significant CAC Genes/QTLs at/around the 3 Significant CAC LociLoci ( p value < 0.01 )( p value < 0.01 )

(Based on NCBI Web Map Viewer, Marshfield Map)(Based on NCBI Web Map Viewer, Marshfield Map)

LociLoci Chr# Chr# Pos.Pos. GenesGenes

AFM063XF4 AFM063XF4 1010 (10p14) 10p14-p15.1(10p14) 10p14-p15.1 BP6: Blood pressure QTL 6 BP6: Blood pressure QTL 6

GATA64D02 GATA64D02 66 (6q12) (6q12) 6p12-p126p12-p12 6p21 6p21

ARCC1 : Age-related cortical cataract 1ARCC1 : Age-related cortical cataract 1

PSORS1: psoriasis susceptibility 1 PSORS1: psoriasis susceptibility 1

GATA42H02 GATA42H02 44 (4q32) (4q32) 4q27-q314q27-q31 FOP: Fibrodysplasia ossificans FOP: Fibrodysplasia ossificans progressiva progressiva

Marker Information Content (MIC ) DistributionMarker Information Content (MIC ) DistributionUsed for Simulation (300 Loci)Used for Simulation (300 Loci)

Mean=0.22

Std Dev=0.1003

(MIC)

n

k

Bik

Wik

i

ffMIC

1 2

Freqency of allele k at locus i in Caucasians Freqency of allele k at

locus i in Africans

Allele number of locus i

Provided By Aldi Kraja

AcknowledgemeAcknowledgementsnts

Michael.A. Province, PhD, DSG of WUSTLMichael.A. Province, PhD, DSG of WUSTLAvril Adelman, DSG of WUSTLAvril Adelman, DSG of WUSTLJun Wu, DSG of WUSTLJun Wu, DSG of WUSTLAldi Kraja, PhD, DSG of WUSTLAldi Kraja, PhD, DSG of WUSTL

Kari North, PhD, UNC Kari North, PhD, UNC Collaborators of other centers in SCANCollaborators of other centers in SCAN