We performed an extensive SNP discovery effort of the CACNA1C coding region and intron/exon junctions and a clinical association study using the INVEST-GENES to assess the impact of CACNA1C genetic variation on outcomes and treatment response. Of eight SNPs tested in the genetic association study, we identified one SNP with a significant interaction with treatment strategy. This effect of this interaction was such that individuals homozygous for the major allele (A/A) randomized to verapamil SR-based treatment regimens had a 45% reduced risk of the primary outcome compared to A/A individuals randomized to atenolol-based regimens. In contrast, individuals homozygous for the minor allele (G/G) randomized to verapamil SR-based treatment had a 4.5-fold increase in the primary outcome compared to G/G individuals randomized to atenolol-based treatment. These findings suggest that individuals with rs1051375 A/A would benefit from treatment with a calcium channel blocker, those with the G/G genotype would benefit from treatment with a beta blocker, and in those with the heterozygous genotype it would not matter which treatment was chosen.
Given the study design of INVEST, it is difficult to determine whether the differences in treatment outcomes observed are due to differences in blood pressure response. In our post hoc analysis, we observed overall average treatment blood pressures that were higher among those with the G/G genotype. Additionally, patients with the A/A genotype who were randomized to the atenolol treatment strategy were more likely to require four or more drugs for blood pressure control than A/A patients randomized to the verapamil SR treatment strategy, suggesting that blood pressure response differences may be playing a role in differences in treatment outcomes.
Although the mechanism of the SNP*treatment interaction is unclear at this time, one explanation is that variants in CACNA1C result in reduced function of the L-type calcium channel. If this were the case, individuals with these genotypes might gain more benefit from a treatment approach involving a mechanism of action not dependent on the L-type calcium channel (e.g. beta-blocker instead of calcium channel blocker). In contrast, the major alleles have greater L-type calcium channel function and thus benefit more from treatment with calcium channel blockade. Last, it is possible that variation in CACNA1C might directly influence treatment response through interactions between calcium signaling and beta-adrenergic signaling pathways since protein kinase A activation via the beta-1-adrenergic receptor results in activation of the L-type calcium channel.
Two of the SNPs we found to be associated with outcomes or treatment response, rs1051375 and rs10848683, are in a fair degree of LD (D’=0.84 and r2
=0.36 in Caucasians). Therefore, it is unclear whether either or both of these SNPs are functional, or both might be tagSNPs for the actual functional SNP. The results of our haplotype analysis as well as the more significant p values in individual SNP analysis suggest that rs1051375 is the more likely causative SNP of the two or in stronger LD with the causative SNP. Rs1051375 is synonymous, Thr1835Thr, so the functional relevance of this SNP is not immediately clear. It was selected for analysis because it is located at putative ESE sites for SC35 and SF2/ASF. In addition, other synonymous SNPs have recently been identified as having functional importance in the ABCB1
Whether one or both of these SNPs are functional or whether they are linked to another functional SNP needs to be elucidated.
Although we have not yet discovered the mechanisms underlying our observed associations, we have made substantial progress toward eliminating several possible functional mechanisms. Based on our work presented here and previously published, we can now exclude differences in expression levels in the myocardium by genotype and that rs1051375 alters splicing in myocardial tissue as potential explanations for the functional basis.12
It is still possible, however, that expression or splicing differences in vasculature smooth muscle may exist.
We were unable to replicate the findings of Bremer et al in which rs2239050, rs2238032, and rs2239128 were found to be associated with calcium channel blocker antihypertensive response. However, rs2238032 was associated with a main effect on outcomes in our study, although it did not meet our predefined level for significance after adjustment for multiple comparisons. An additional study published during the revision of this manuscript found one SNP in CACNA1C
, although it was not one of the SNPs we assessed, and two SNPs in CACNA1D
to be associated with response to dihydropyridine CCBs among 161 Japanese individuals.5
One of the major strengths of our study is the fact that our population came from a randomized clinical trial. This study design greatly reduces possible biases that can be introduced into observational studies where many factors influence treatment decisions. Additionally, all endpoints in the clinical trial were adjudicated by a blinded endpoints committee which also strengthens our phenotype.
Our study has some limitations that should be addressed. First, although we have eliminated some possible functional mechanisms, we do not know the functional mechanism underlying the association we observed between CACNA1C variants and cardiovascular outcomes. These mechanistic studies will be important in order to understand how we might use this information in the future for genotype-guided treatment decisions. A second limitation of our study is that we do not have a replication cohort for our findings. Unfortunately, it is very challenging to find replication cohorts for pharmacogenetic studies with detailed drug phenotype data and similar patient populations, particularly when the phenotype is adverse cardiovascular outcomes. However, we are working toward replicating these findings in other cardiovascular outcomes studies.
We have identified a SNP in CACNA1C, the binding site for calcium channel blockers, with a significant interaction with treatment strategy in a group of hypertensive patients with CAD. Individuals homozygous for the major allele had a reduction in the occurrence of death, nonfatal MI, or nonfatal stroke when treated with a calcium channel blocker-based treatment regimen compared to those treated with a beta-blocker-based treatment regimen. In contrast, individuals homozygous for the minor allele had a reduction in adverse outcomes when treated with beta-blocker-based regimens. If validated, these findings might be used in the future to help guide choice of therapy in the treatment of hypertension. Of great interest, our findings suggest the potential of targeting an individual’s underlying molecular mechanism of disease to improve clinical outcomes.