After excluding subjects with atrial fibrillation or poor quality PWV tracings, a total of 4,221 and 1,828 Sardinians were considered for the initial GWA analysis and for the internal replication (SardiNIA stage 2), respectively. The demographic and clinical characteristics of the study cohort are shown in . The mean age was 43.7±17.6 years (range 14–102 years); 58% were women, 20% reported a history of smoking, 29% were hypertensive, 5% were diabetics, and only 1% reported a clinical history of myocardial infarction. As expected2
, PWV increased with advancing age in a quadratic fashion ().
Demographic and clinical characteristics of the SardiNIA cohort (overall, and stratified according to the phase of genotyping), and of the Old Order Amish study cohort
The relationship of pulse wave velocity (PWV) and age in men and women in the SardiNIA study cohort who were genotyped with the 500K/10K Gene Array Set. The best fit regression curves (quadratic) are shown.
We first conducted GWAS to survey the genome for common variants associated with PWV. graphically summarizes the associations of PWV with the >329,129 SNPs with a minor allele frequency >5% that passed quality control checks and transmission disequilibrium testing. The top 100 hits are shown in Table S1
. Promising findings were noted on chromosomes 1, 2, 4, 12–14, 17, 20, 21, encompassing 18 SNPs with p<2×10−5
Figure 2 Summary of genome-wide association studies (GWAS) for pulse wave velocity (PWV) in the SardiNIA study cohort. The −log10 of the p-values for the associations of PWV with 362,169 SNPs that passed quality control filters are plotted according to (more ...)
To further evaluate these initial results, a secondary custom chip from Affymetrix was devised based on the top findings from the GWAS. For PWV, the top 85 SNPs were considered; of these, 43 were not in strong linkage disequilibrium and were included in the custom chip. The 1,828 individuals typed with this custom chip were genetically independent from those genotyped in the initial analysis. Thus, the custom chip helped validate the initial findings, and provided an “internal” replication within the Sardinian cohort. Of the 43 SNPs that were included in the custom designed chip, two SNPs, rs3742207 and rs1495448 in the COL4A1 and MAGI1 genes respectively, were significantly associated with PWV with the same directionality of effect as in the GWAS (). Furthermore, when the results of the initial GWAS study group and the Stage 2 study group were combined in a meta-analysis (), the association of the C allele of rs3742207 with increased PWV was strengthened (p=7.08×10−7), whereas the association of the T allele of rs1495448 with increased PWV was weakened (p=1.07×10−5).
Summary of association results for rs3742207 (A) and rs1495448 (B)
We repeated the association analyses using models that adjusted for mean arterial pressure, creatinine and the use of blood-pressure lowering medications, which are important covariates of arterial stiffness, in addition to age, age2
and sex which were adjusted for in the base models. The association of rs3742207 with PWV was slightly strengthened, whereby the p-value decreased from 5.94×10−5
. Next, we excluded subjects on antihypertensive medications (N=544) and subjects on dialysis (N=10), and repeated the analyses adjusting for age, age2
, sex, mean arterial pressure and creatinine. The p-value for the association of rs3742207 with PWV was 2.48×10−5
. In this last model, the p-value was 3.19 ×10−5
when diabetic individuals (N=50) were excluded. However, the genomic control parameters for these 3 additional models were higher than the one for the base model (lambda =1.16, 1.17, 1.17 respectively, compared to 1.14 of the initial model). Conversely, the association of PWV with rs1495448 (in the MAGI1 gene) was weakened when the analyses were repeated using these three sets of additional adjustments (p=0.0014, p=0.0038, p=0.0081 respectively).
The associations of the two loci rs3742207 and rs1495448 with PWV were further evaluated in the Amish population, a genetically distant founder population of European ancestry. The HAPI Heart Study and the ALS study participants underwent both assessment of PWV and genotyping using the Affymetrix 500K chip. We confirmed that allele C of the SNP rs3742207 (the more frequent allele in the Amish population) was associated with PWV (p=0.02) with a comparable effect size (), whereas rs1495448 was not (p=0.49, ). Combining SardiNIA, SardiNIA stage 2, and the Old Order Amish yielded an overall p=5.16×10−8 for the association between rs3742207 and PWV.
The heritability of PWV in the SardiNIA study is 0.226 (adjusted for age, sex and age by sex interaction10
. The proportion of variance in PWV (which is equivalent to the proportion of the heritable fraction) that is explained by rs3742207 is 0.87%. The overall effect size, is 18.9 cm/s (calculated as a weighted average of the effect observed in each of the 3 study cohorts, where the weights correspond to those used in the meta-analysis).
The replicated SNP rs3742207 is a common nonsynonymous coding polymorphism located in exon 45 of the Col4A1
gene. This polymorphism involves a substitution of adenine by cytosine resulting in an amino acid change from Glycine to Histidine at position 1334, which is located in a central region of the protein that consists of multiple triple-helix repeat domains. Nonetheless, further studies are necessary to determine whether the true causal variant is this SNP or another one, which, according to linkage disequilibrium structure ()12, 22, 23
, appears to lie in exon 45 or nearby.
Figure 3 A) Summary of the association with PWV in the COL4A1 region in the SardiNIA study. Dots represent all the markers in this region that were analyzed, colored according to their linkage disequilibrium (r2) with rs3742207, which is represented by a red square. (more ...)