assessing the impact of candidate gene variation on quantitative phenotypes dana c. crawford, phd...
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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
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
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
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
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
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