The study population consisted of 1486 apparently healthy individuals from 427 families with premature CAD. Study participants were siblings (n = 594) of probands who had documented CAD before age 60, adult offspring of the probands or the siblings (n = 743), or the co-parent of the offspring (n = 149). The average age of subjects was 45 ± 13 years (range 21 to 79); 56% were female; 62.4 % reported their ethnicity as white and 37.6% reported that they were African American. Self-reported ethnicity was verified as correct in 96% of cases by a 32 STR ancestry panel, based on >60% of genotypes in the appropriate ancestry cluster (CEU, YRI, or CHB+JPT).24
Of the 427 families, 264 were white and 163 were African American (mean family size 3.51 ± 2.7 and 3.43 ± 2.7, respectively). Compared to white subjects, African Americans were more often female, and had higher prevalences of hypertension, current smoking, and diabetes (). Mean body mass index and serum fibrinogen were higher, and LDL cholesterol lower in African Americans ().
Demographics of study population by ethnicity N=1486
At baseline, 6.4% of subjects had no measurable platelet aggregation to arachidonic acid. Although 14.4% of subjects were taking aspirin regularly prior to the study, all reported complete cessation for at least 2 weeks prior to the baseline measurement. However, we suspect that those with zero baseline platelet aggregation were either non-compliant or were unknowingly exposed to aspirin or non-steroidal anti-inflammatory agents. Based on questionnaires and pill counts, all subjects took aspirin for two weeks according to protocol, except that 12% of subjects admitted to missing one or two of the 14 doses. All participants had taken a dose in the 24 hours preceding the post-aspirin measurements.
Of the 10 SNPs genotyped in PEAR1, 5 SNPs in whites and 7 SNPs in African Americans were polymorphic. Linkage disequilibrium (LD) patterns and the positions of the selected SNPs are shown by ethnicity group in . The genotype frequency of those SNPs successfully genotyped for each ethnic group are shown in Supplementary Table 1
(available on-line). A SNP in PEAR1, rs2768759, was found to be associated with nearly all platelet aggregation phenotypes. This SNP was not in LD with any of the other SNPs genotyped in PEAR1 (). The SNP was in Hardy Weinberg Equilibrium within each ethnic group (whites, p=0.73, and African Americans, p =0.87). The prevalence of the homozygous CC genotype was more than 25 fold higher in whites (). We tested whether the CC genotype in African Americans was related to European admixture using the deCODE 32 STR marker ancestry panel. European admixture in African Americans was on average 20%. A significantly greater percentage of European admixture was associated with the CC genotype, compared to the AC heterozygote and the AA homozygote in African Americans (40%, 22%, and 17% respectively, p<0.0001). European admixture is thus the likely origin of the C allele in African Americans.
PEAR1 SNPs successfully genotyped in whites (A) and African Americans (B) with associated LD blocks. The values provided are d′ estimates from the HapMap database. The SNP of interest, rs2768759, is noted by an arrow.
Prevalence of genotypes for rs2768759 by ethnicity
In unadjusted analyses, genotype for the rs2768759 SNP in PEAR 1 was associated with maximal aggregation to collagen (in whites), epinephrine (in whites and African Americans), and ADP (in African Americans) prior to aspirin, and these associations were generally stronger and more significant after aspirin (). There was no relation between genotype and arachidonic acid-induced aggregation at baseline. Removal from the analysis of subjects with zero aggregation to arachidonic acid at baseline (who presumably had known or inadvertent exposure to aspirin or non-steroidal anti-inflammatory agents) had no effect on the results except that the association between the genotype and aggregation to the lower dose of collagen and the lower dose of epinephrine were no longer significant in African Americans. In general, platelet aggregation prior to aspirin was highest in people with the CC genotype, least in those with the AA genotype, and intermediate in those with the AC genotype in both ethnic groups.
Relationship of native and post-aspirin platelet aggregation phenotypes by PEAR 1 rs2768759 genotype within ethnic groups N= 1486
Post-aspirin, maximal platelet aggregation to collagen and epinephrine was reduced substantially for all genotypes in both ethnic groups. Maximal aggregation to ADP was also reduced, but by a lesser amount. However, residual aggregation to all three agonists was consistently higher with the CC genotype, intermediate with the AC genotype, and lowest with the AA genotype in both ethnicities (). As expected, aggregation to arachidonic acid was markedly reduced after aspirin, with only 7.9% of subjects exhibiting measurable aggregation. However, the percent of subjects who did aggregate to arachidonic acid was significantly higher with the CC genotype in African Americans (aggregation occurred in 31.3% with CC, 9.1% with AC, 9.5% with AA, p=0.016). In whites, the trend was similar but the differences by genotype were not statistically significant (aggregation occurred in 8.2% with CC, 4.6% with AC, 6.9% with AA, p=0.13). In summary, these results are consistent with greater platelet reactivity both at baseline and after aspirin with the CC genotype.
To examine the effect of the PEAR1 genotype on aspirin responsiveness, we examined associations with a platelet response phenotype, expressed as post aspirin maximal aggregation adjusted for pre aspirin maximal aggregation, separately for each agonist and concentration. The associations of the rs2768759 genotype with aspirin responsiveness were significant for both collagen and epinephrine in both ethnicities, and borderline significant for ADP in whites. This analysis could not be done for arachidonic acid because of the dichotomous results for arachidonic acid-induced aggregation post aspirin. These results are consistent with an association between the genetic variant and reduced aspirin responsiveness, ie greater platelet aggregation post aspirin, even after adjustment for pre aspirin aggregation.
Consistent with the markedly higher C allele frequency we observed in whites, native platelets from white subjects demonstrated (in unadjusted analyses) greater aggregation to collagen, epinephrine, ADP, and arachidonic acid than platelets from African Americans (collagen 2μg/ml, 64.4 ± 29.2% vs 58.4 ± 33.2%, p<0.0001; collagen 5μg/ml, 82.2 ± 17.0% vs 78.9 ± 21.9%, p=0.062; epinephrine 2μM, 57.03 ± 33.5% vs 53.3 ± 36.1%, p=0.051; epinephrine 10μM, 72.9 ± 26.6% vs 66.3 ± 33.0%, p<0.0001; ADP, 80.1 ± 13.8% vs 78.2 ± 16.4%, p=0.028; arachidonic acid, 73.4 ± 24.7% vs. 67.0 ± 31.6%, p<0.0001). Post-aspirin, platelets from white subjects continued to be more aggregable to both collagen and epinephrine (but not ADP) than platelets from African Americans (collagen 2μg/ml, 13.7 ± 12.8% vs 12.9 ± 15.9%, p=0.55; collagen 5μg/ml, 28.7 ± 20.5% vs 25.8 ± 22.4%, p=0.051; epinephrine 2μM, 22.7 ± 12.3% vs 20.0 ± 14.3%, p=0.0002; epinephrine 10μM, 30.0 ± 14.6% vs 26.0 ± 16.3%, p<0.0001; ADP, 68.7 ± 12.9% vs 67.8 ± 13.6%, p=0.204).
In multivariable adjusted analyses, pre-aspirin phenotypes for collagen aggregation were not associated with the rs2768759 SNP in either ethnic group, but the post-aspirin phenotypes for collagen aggregation were significant and were similar by ethnicity (). In contrast, epinephrine aggregation, both before and after aspirin, was significantly and independently associated with the variant in both ethnicities. For ADP aggregation, the variant was significantly associated with the pre-aspirin phenotype in African Americans but not in whites, while the opposite was true for the post-aspirin phenotype (significant in whites, not in African Americans). Finally, for arachidonic acid-induced aggregation only the baseline phenotype could be analyzed, and for this, the variant was independently associated with aggregation in African Americans, but not in whites. Thus, although there was more variability in baseline platelet aggregation, almost all post-aspirin platelet aggregation measures in both ethnicities were significantly associated with the rs2768759 SNP in PEAR1.
Significance of associations of PEAR1 rs2768759 with pre-aspirin and post-aspirin platelet aggregation within ethnic groups adjusted for covariates*
Genetic contribution to phenotypic variance
We also conducted variance-components analysis to estimate the genetic component of aggregation phenotypes to collagen, epinephrine, and ADP, pre and post aspirin (). The phenotypic variance was higher in African Americans than in whites, and higher for baseline aggregation. The pre-aspirin platelet aggregation phenotypes were generally less heritable than those post-aspirin. The portion of genetic variance explained by the rs2768759 locus in native platelet phenotypes among African Americans varied from 0.22% for the lower concentration of epinephrine, to 5.3% for the higher concentration of collagen. In whites, the proportion of the genetic variance explained by the locus in native platelet phenotypes also ranged widely from 0.43 to 3.7%. Following aspirin, however, a greater proportion of the genetic variance was explained by the locus, and this proportion was higher and more consistent in African Americans, ranging from 1.7 to 6.9%, while in whites the values ranged from 1.3 to 2.5%.
Variance components analysis by ethnicity N=1486