Our findings suggest that in hypertensive patients with CAD, two nonsynonymous polymorphisms in the KCNMB1
gene, Glu65Lys and Val110Leu, may account for a component of the interpatient variability in BP response to verapamil SR. Additionally, our findings suggest that the Val110Leu polymorphism may be associated with adverse outcomes. These conclusions are based on results of several different types of analyses. First, we found that Lys65 variant carriers achieved BP goals more rapidly than Glu65 homozygote individuals in the verapamil SR monotherapy group and required fewer drugs to achieve BP control. These results are consistent with functional studies of this polymorphism indicating that the variant BK channel has enhanced calcium sensitivity [1
] and would be predicted to have enhanced responsiveness to calcium channel blockers. All of our BP response analyses (i.e. change in SBP at 6 weeks, time to BP control, and number of drugs needed for BP control) consistently identified Lys65 variant carriers to have the most favorable BP response to verapamil SR in patients on verapamil SR monotherapy. This effect was also consistently weaker when individuals being treated with other antihypertensive agents on stable background therapy were added to the analyses. This may be because the effect of KCNMB1
on antihypertensive response to verapamil SR is small and may be overcome by the addition of other antihypertensive medications. For example, individuals on multidrug regimens had lower BP at study entry and may be less dependent on vascular smooth muscle relaxation from the BK channel pathway because their other antihypertensive agents are decreasing vascular resistance through other mechanisms. On the other hand, patients on verapamil SR monotherapy might be more dependent on the BK channel pathway to decrease vascular tone, allowing us to observe the apparent pharmacogenetic effect of the Glu65Lys SNP. The second potential reason our findings with antihypertensive response to verapamil SR were only identified in the smaller group of patients is because of a type I error. We do not anticipate this to be the case given that our findings are in line with Fernandez–Fernandez and Senti and colleagues [1
The second major finding in our study is that the Leu110 variant allele is associated with a decreased risk of adverse outcome among patients with hypertension and CAD. Interestingly, this finding was significant only in the patients randomized to a verapamil SR-based treatment strategy, suggesting a pharmacogenetic effect, with the majority of the protective effect of the variant occurring only when the patients are treated with a calcium channel blocker. If these findings were replicated in an independent population, it would suggest that hypertensive CAD patients who are Leu110 carriers should receive a calcium channel blocker.
Our secondary analyses suggest that the protective effect of the Leu110 allele on the occurrence of adverse outcomes may be driven by a reduction in nonfatal MI. The mechanism for this reduction in risk, however, remains unclear. The subgroup analyses, suggested that greater protective effective effect associated with the variant allele was found in women, individuals less than 70 years, and Black individuals. Although these findings are hypothesis generating, they could be viewed as consistent with a previous study that found the association of codon 65 with hypertension to be strongest among older women [4
]. Thus, it is possible that the effects of this gene’s variants are greater in women and with increased age.
To our knowledge, this is the first report to date to evaluate the association of the Val110Leu polymorphism with either the response to verapamil SR or adverse outcomes. Although evidence for the functional role of the gene and the codon 65 SNP in hypertension exist (as discussed in the introduction), the functional significance of Val110Leu remains unclear. The codon 110 SNP represents a conservative amino acid substitution, but functional changes at the protein level cannot be ruled out. Alternatively, any nucleotide change in the transcribed region has the potential to affect mRNA folding, processing, turnover, and translation [11
]. Regulatory polymorphisms, including those affecting mRNA processing, are prevalent in determining human phenotypic variations [17
]. Therefore, we measured allelic KCNMB1 mRNA expression in human heart tissues from transplant patients, using the codon 110 SNP as the marker. Differential expression from the two alleles, or AEI, is a definitive indicator of the presence of a cis
-acting factor. This approach did not detect the presence of significant AEI, indicating that the codon 110 SNP does not affect mRNA expression levels in the myocardium. The use of human failing heart tissues obtained before transplantation, rather than vascular smooth muscle, however, could confound the interpretation of the results because mRNA expression (including allelic expression) may be tissue-dependent. Therefore, measuring myocardial allelic expression represents but a first step in clarifying the potential function of the Val110Leu polymorphism. Whether Val110Leu is functional owing to altered expression in vascular tissue, altered protein function owing to amino acid change or another mechanism, or is merely a tag for the true functional SNP, however, remains unknown. One other group has reported a genetic association with Val110Leu, specifically that Val110Leu heterozygote individuals had higher heart rate variability and baroreflex sensitivity in normotensive individuals [5
]. These results could be considered consistent with our findings given increased heart rate variability is associated with improved outcomes in epidemiological studies and Leu110 carriers had a better prognosis than Val110Val individuals in our study. Furthermore, because the r2
measures of LD between codons 65 and 110 were so low, it seems unlikely that the differences in genetic associations between these SNPs (i.e. codon 65 with BP response and codon 110 with adverse outcomes) are accounted for by LD between the two SNPs. Additionally, haplotype analysis considering the joint effects of these SNPs afforded no further improvement in defining the genetic association. In fact, the consideration of both SNPs only weakened the associations. Therefore, these data suggest that the mechanisms of association for the Glu65Lys and BP response and the Val110Leu and outcomes response to verapamil are different. As discussed above additional research is needed to define the functional basis for the codon 110 associations.
The association of the Glu65Lys polymorphism with the BP response to verapamil SR was not linked to a reduction in clinical outcomes. Given that the BP effect was only evident in those individuals on verapamil SR monotherapy (n = 163), it, however, is likely that this effect was not present in a large enough subset to be seen in the overall INVEST-GENES cohort.