Few previous studies have examined multiple polymorphisms in relation to PH associated with COPD. In our study we examined seven SNPs in six candidate genes which encode mediators that act on pulmonary vessels. We tested whether these SNPs were associated with RVSP, which is a measure of PH. We found that patients with the NOS3
-VNTR 4aa or 4ab genotype had significantly higher RVSPs than those with the NOS3
-VNTR 4bb genotype. In contrast, a study of 42 COPD patients and 40 controls found that the NOS3
-VNTR 4bb genotype was associated with PH in COPD [13
]. The smaller number of patients in their study raises the possibility of a type I error Further studies are required to validate our findings.
There have been conflicting functional studies of the NOS3
-VNTR 4aa genotype and plasma nitrite and nitrate (NOx) levels. A study of 428 healthy Caucasian members of 108 nuclear families found significantly higher levels of plasma NOx associated with the 4aa genotype compared to the 4bb and 4ab genotypes [32
]. However another study found that there was a strong association between plasma NOx levels and the NOS3
-VNTR polymorphism in 413 healthy Japanese subjects, the subjects with the 4aa genotype having significantly lower NOx levels [33
]. The discordant results between these two studies may be due to ethnicity or methodological differences in measuring NOx levels. The functional results from this second study would support our finding of the 4aa genotype (with potentially lower nitric oxide levels and therefore less vasodilatation) being associated with higher RVSP (higher vascular resistance). The exact functional mechanisms of how the VNTR, or a nearby SNP which is in linkage disequilibrium with it, affects either nitric oxide or vascular remodelling, needs further elucidation.
We identified a number of clinical factors as being significantly associated with elevated RVSP on univariate analysis. These factors are well-known clinical markers of severity of COPD, and would be expected to correlate with elevated RVSP, since PH is related to COPD severity [31
]. The NOS3
-VNTR polymorphism was significantly associated with RVSP, and remained so when controlling for these factors.
Considering potential links between COPD severity and PH, we examined the effect of SNPs in relation to respiratory function tests. Patients with the ACE
II or ID genotypes showed a statistically significantly lower FEV1
% predicted, albeit a clinically small difference, than the ACE
DD genotypes. Analysis of the other genetic models for ACE
genotypes did not show associations. This is in contrast to a previous study of Caucasian Mediterraneans which found a higher frequency of ACE
DD genotypes in 74 male smokers with COPD than in 77 male smokers with normal lung function (odds ratio 2.2) [34
]. Additional studies are required to clarify this relationship.
Patients with the NOS3
-298 TT genotype had significantly lower KCO than those with the GG or GT genotypes. Sun and co-workers’ hypothesis [8
] supports our results in that the lower NO levels associated with the TT genotype would potentially predispose those patients to greater lung tissue damage from cigarette smoke reflected in a reduced KCO.
Potential limitations of this study should be considered. Two of the SNPs showed minor deviation in Hardy-Weinberg equilibrium, although these did not show positive associations. The reason for the minor deviation could include chance or differences in population sampling; we had performed repeat genotyping in 10% of samples and the results were concordant. In some COPD patients, RVSP measurement was not successful because of hyperinflated lungs causing a large retrosternal window, or because the tricuspid regurgitant jet was insufficient to enable calculation of RVSP. Use of echo contrast may have increased the yield of RVSP measurements in these cases. Given the exploratory nature of this study of 7 SNPs and RVSP in COPD, we did not correct for multiple comparisons, and the statistical significance of the results should be considered in light of this. Even with this relatively large cohort of patients, replication in other cohorts is needed. Functional analysis of polymorphisms in model systems and genome-wide association studies of PH in COPD would be worthwhile in the future.