Assessing the Impact of Candidate Gene Variation on Quantitative

Phenotypes

Dana C. Crawford, PhDUniversity of Washington

March 21, 2006

C-reactive protein and CRP

• Importance of C-reactive protein

• Genetics of C-reactive protein

• Genetic variation and C-reactive protein

C-reactive Protein

• Pentraxin family of proteins

• Produced in liver

• Acute phase plasma protein

• General marker for infection and inflammation

Volanakis 2001

Serum CRP and Risk of MI

Ridker et al NEJM 1997;336:937-979

Willerson, J. T. et al. Circulation 2004;109:II-2-10II-

Prognostic value of various cardiovascular biomarkers in healthy women in the Women's Health Study

CRP Better Predictor than LDL

Lowering CRP Prevents Recurrent Events

Ridker et al NEJM 2005;352:20-28

The Genetics of C-reactive ProteinA Quantitative Trait

• High heritability (40-60%)

• CRP located on 1q21-q23

5' 3'

UTR UTR

~1.9kb

Sequence variation in CRP

31 SNPs (6.8kb):

30 SNPs in African-Americans

13 SNPs in European-Americans

No amino acid changes

SeattleSNPs (pga.gs.washington.edu)

• Population-based epidemiological survey

• Genotype CRP SNPs

• Link genotypes to phenotypes

• Estimate SNP effects controlling for covariates

Percentiles of C-reactive protein concentration

0

14

mg/L

???

Ford et al Clin Chem 2003;49:686-690

Are CRP Variants Associated with CRPLevels in the General Population?

CDC population-based survey since 1960’s

• Demographic information

• Lifestyle questionnaire

• Laboratory measures (>100)

• Physical exam

NHANESNational Health and Nutrition Examination Survey

NHANES Participants

• Non-institutionalized civilians

• Both Sexes

• All racial/ethnic groups

• Ages 2 months to 85+

• Regardless of health

The Health ExaminationAnd

The Mobile Exam Center (MEC)

Data from NHANES

NCHS 2002

NHANES III

7,296 DNAs released August 2002:

White, not Hispanic n = 2,630

Black, not Hispanic n = 2,108

Mexican-American n = 2,073

Other n = 348

Choosing SNPs for Genotyping

Average gene (~30kb)126 SNPs46 common SNP (MAF >5%)5 coding SNPs

Crawford, Akey, and Nickerson Ann Rev Genomics Hum Genet 2005;6:287-312

CRP (6.8kb)31 SNPs17 common1 coding

Genotype at one site can predict genotype at another site

Proportion of sites are correlated

Genotypes Are Correlated(aka linkage disequilibrium)

“the nonindependence of alleles at different sites.” Pritchard and Przeworski AJHG 2001;69:1-14

Choosing tagSNPs for CRP

Common SNPs (>10%)LDSelect (r2>0.64)

13 SNPs 3 tagSNPs

European-Americans African-Americans

tagSNPs are Population Specific

tagSNP selection for CRP

Synonymous SNP(2667)“Promoter” SNPs

(790, 1440) Intron SNP(1919) Downstream SNPs

(3872, 5237)3’ UTR SNP(3006)

6 “cosmopolitan” tagSNPs1 rare synonymous SNP

CRP tagSNP Allele FrequenciesEuropean-Americans

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

790 1440 1919 2667 3006 3872 5237

SeattleSNPs (n=23)

CARDIA* (n=1,820)

NHANES III (n=2,630)

*Carlson et al AJHG 2005;77:64-77

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

790 1440 1919 2667 3006 3872 5237

SeattleSNPs (n=24)

CARDIA* (n=1,557)

NHANES III (n=2,108)

CRP tagSNP Allele FrequenciesAfrican-Americans

*Carlson et al AJHG 2005;77:64-77

CRP tagSNP Allele FrequenciesMexican-Americans

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

790 1440 1919 2667 3006 3872 5237

NHANES III (MA; n=2,037)

NHANES III (EA; n=2,630)

NHANES III (AA; n=2,108)

*Carlson et al AJHG 2005;77:64-77

r2=0.984

r2=0.784 (r2=0.767)

0

5

10

15

20

25

30

Min Q1

Med

ian Q3

Max

Quantiles of CRP in NHANES III

mg

/dL

0.21 mg/dL

NHANES III: DNA linked to phenotypes

Sample Characteristics

White Black Mexican(n=2,630) (n=2,108) (n=2,037)

% Female 51.5 54.8 48.6Mean age 42.5 37.1 33.1Mean BMI 26.1 27.4 26.9% Current Smokers 28.8 32.0 21.1Mean CRP 0.38 0.48 0.43Mean DBP 78.5 80.7 75.1Mean SBP 125.5 126.7 119.6Mean cholesterol 198.6 193.1 191.3Mean HDL 49.5 54.2 47.8Mean LDL 122.0 116.9 113.9Mean triglycerides 142.9 110.7 149.9Mean glucose 97.2 101.4 100.5Mean HbA1C 5.35 5.59 5.45

Multivariate Model• Continuous phenotype

ln(CRP)• Stratified by race/ethnicity• Covariates

SexAgeSmoking statusln(BMI)Diastolic blood pressureGlycosylated hemoglobinLipids [ln(cholesterol), ln(HDL), ln(triglycerides)]

• InteractionsSex x triglyceridesSex x diastolic

Multivariate Single SNP for CRP

Dominant model Beta p-value Sample

790 (promoter SNP)

AAAT/TT 0.23 <0.0001 Blacks

2667 (synonymous SNP)

GGCG/CC -0.12 0.0337 Mexican-Americans

3872 (flanking SNP)

GGAG/AA -0.15 0.0005 Blacks

5237 (flanking SNP)

AAAG/GG -0.09 0.0012 Blacks

Controlling for covariates

Multivariate Single SNP for CRP

Additive model 1440 (promoter SNP) Beta p-value Sample

AA 0.45 0.0339 Whites 0.17 0.0037 Blacks

TT 0.33 0.0001 Blacks 0.08 0.0100 Mexican-

Americans

AC 0.23 0.0004 Blacks

AT 0.14 0.0002 Blacks

CT 0.07 0.0151 Mexican-Americans

CC genotype is REFControlling for covariates

Inferring Haplotypes from tagSNPs

Haplotypes: assigning alleles to a chromosome

CRP tagSNP Haplotypes

7 SNPs inferred:

# haplotypes H

White 26 0.73

Black 25 0.83

Mexican-American 19 0.78

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

H1 H2 H3 H4 H5 H6 H7 H8

White

Black

Mexican-American

Common CRP tagSNP Haplotypes(>5% frequency)

Beta p-value Sample

ATTGCGA(H5*) 0.19 0.0020 Blacks

0.10 0.0244 Mexican-Americans

TTAGCGA (H6*) 0.51 <0.0001 Blacks

AAAGCGA (H7*) 0.33 0.0256 Mexican-Americans

AAAGAGA(H8*) 0.20 0.0067 Blacks

Multivariate Haplotype Results

*Carlson et al 2005Controlling for covariates ACAGCAA is REF

0

0.1

0.2

0.3

0.4

0.5

0.6

H2 H5 H6 H7 H8

Ch

ang

e in

ln

(CR

P)

per

co

py

rela

tive

to

H2

Black

Mexican-American

Carlson et al. AJHG 2005;77:64-77

Results Consistent with CARDIA

Haplotype Frequencies for CRPWhite Black Mexican

ACAGCCA 0.31 0.20 0.33(REF)

ATTGCGA 0.30 0.12 0.34(H5)

TTAGCGA <0.05 0.17 <0.05(H6)

AAAGCGA 0.06 <0.05 <0.05(H7)

AAAGAGA <0.05 0.23 <0.05(H8)

CRP variance explained by CRP variants

% Variance ExplainedWhite Black Mexican

Full model 18.1 23.8 18.8

-haplotype 17.7 21.1 18.0

CRP Associated HaplotypesA C A G C A A

A T T G C G A

T T A G C G A

A A A G C G A

A A A G A G A

(H6)

(H7)

(H5)

1440

(H8)

3872790(1421)

1919 3006

REF

CRP Associated SNPs in Promoter

• 790 is a tagSNP for 1421

• 1421 and 1440 alter transcription factor binding site

ecrbrowser.dcode.org

Common vs Rare SNPs in CRP

• Discovery sample identifies common SNPs48 chromosomes99% detection rate for MAF >5%

• Need more to identify rare SNPs192 chromosomes99% detection rate for MAF >1%

Do rare coding SNPs contribute to CRP levels?

• No known nonsynonymous SNPs in CRP

African-American(n=24)

European-American(n=23)

Asians(n=24)

Mexicans(n=10)

Indo-Pakistanis(n=6)

And PDR450(not shown)

~1,000 chromosomes:

68 SNPs4 synonymous

3 nonsynonymous

Rare coding CRP variation

Computational Predictions:

• 2314 Tyr67HisTolerated/benign

• 2513 Pro133LysTolerated/possibly damaging

• 2612 Gly166GluTolerated/possibly damaging

• 2513 (Pro133Lys)1 Indo-Pakistani0 NHANES III

• 2612 Gly166Glu1 PDR sample2 NHANES III

Impact of rare cSNPs inconclusive in NHANES III

cSNP Frequency in>14,000 chromosomes

Summary of CRP Results

Rare variant:

• CRP nsSNPs are rare in general population

Common variant:

• Common CRP SNPs associated with CRP levels

• CRP haplotypes associated with CRP levels

• CRP haplotypes account for 2-11% of CRP variation

Acknowledgements

University of Washington:Debbie NickersonMark RiederJosh SmithMike EberleNickerson laboratory

Alex Reiner

CDC/NCHS:Geraldine McQuillanChris SandersXiaoting QinLisa Mirel

NIH/NHLBIProgram for Genomic

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