We identified variants in the HMGCR gene that were among our top hits for LDL-C in a GWAS in a population from the Island of Kosrae. We then conducted in vitro studies to follow-up the association signals and to identify a functional variant at the HMGCR locus. We present evidence that a common intronic SNP (rs384662) that is in linkage disequilibrium with the variants typed in the genome scan modulates alternative splicing of HMGCR mRNA. The resulting splice variant could not restore enzyme activity when expressed in HMGCR deficient UT-2 cells.
HMG-CoA reductase is a key enzyme in cholesterol homeostasis and catalyzes the rate limiting step in cholesterol biosynthesis 21
. In contrast to other well known determinants of cholesterol homeostasis, e.g. LDL-receptor or Apolipoprotein E, associations between variants in HMGCR and LDL-C have only recently emerged in the context of GWAS. As in the Kosrae study, the initial results of the DGI GWAS in 2758 Caucasians supported associations between SNPs in HMGCR and LDL-C, but did not meet the statistical threshold of genome-wide significance by themselves (best associated SNP rs12654264: p=4.09×10−4
. In this study, genome-wide significance was clearly established for HMGCR SNP rs12654264 after validation in three additional Caucasian cohorts, resulting in a combined p-value of 1×10−20
in a total of ~18000 subjects 13
. However in a separate study, the associations between SNPs in HMGCR and LDL-C that were observed in the DGI study were not strengthened by a meta analysis approach, consisting of the DGI and two other Caucasian GWAS (best associated SNP rs3846663: p=2.79×10−4
. This discrepancy might be attributable to some source of heterogeneity, e.g. differences in sample ascertainment or the impact of non-additive interactions with other genetic variants or unaccounted environmental exposures 22, 23
. Combining the association results from the Kosrae and DGI studies revealed three variants in LD (r2
>0.81) with genome-wide significance at the HMGCR locus, including the two SNPs mentioned above (rs12654264, rs3846663) and SNP rs7703051. Our data obtained in the Kosrae isolate thereby adds important evidence about the generalizibility of genetic associations at the HMGCR locus, demonstrating that these associations also extend to other ancestries. Interestingly, two pharmacogenetic studies investigating if genetic variants in HMGCR influence response to statin therapy demonstrated that common SNP haplotypes in HMGCR contribute to variation in statin response 24, 25
. These haplotypes included the SNPs that were associated with plasma LDL-C in the Kosrae and DGI studies and it is possible that the same underlying mechanisms contribute to variation in LDL-C levels and variation in statin response.
A major aspect of our study was to follow-up the findings from the GWAS and to identify the putative functional variant at the HMGCR locus. To address this question we used human lymphoblastoid cells from the HapMap CEU collection which have previously been established as a suitable model to study the regulation of cholesterol biosynthesis in normal subjects and subjects with genetic abnormalities in lipid metabolism 26
. Our efforts were facilitated by a near complete inventory (99%) of all common (>5% minor allele frequency) regional sequence variations, resulting from resequencing of the complete HMGCR locus in 23 Caucasians 19
. Since the only known common coding SNP in HMGCR (rs5908, I638V) is not in LD with any of the genotyped SNPs, we consider it to be unlikely that this variant is responsible for the association signal. Likewise, since we did not detect significant differences in total HMGCR mRNA expression, we consider it to be unlikely that the causal SNP is located in a regulatory element affecting HMGCR transcription. On the other hand, we provide mutually supportive evidence that a common intronic variant (rs3846662) in LD with the genotyped variants is functional and alters the efficiency of HMGCR exon13 alternative splicing: We could demonstrate that (1.) expression levels of alternatively spliced Δexon13 HMGCR mRNA were significantly lower in lymphoblastoid cells from homozygotes for the rs3846662 minor allele and (2.) allele status at rs3846662 directly modulated alternative splicing of HMGCR mRNA in minigene constructs. Further, alternative splicing of HMGCR appeared to be regulated and was present in vivo
, as we could detect Δexon13 HMGCR mRNA in all eleven human tissues that we studied.
HMGCR mRNA lacking exon13 was described in a survey of alternative pre-mRNA splicing by Johnson et al 20
, however its function and the underlying mechanisms remain unknown. The regulation of gene splicing in mammalians involves both cis- and trans-factors, which are composed of auxiliary element sequences in the pre-mRNA, known as splicing enhancers and silencers 27
and cellular splicing factors which include several protein families 28
. The most likely explanation for the observed differences in HMGCR mRNA splicing between major and minor allele homozygotes at rs3846662 is that this SNP is located in a binding motif for a splice auxiliary protein and allele status changes the binding affinity of this protein. Homozygosity for the major allele at rs3846662 increased the proportion of HMGCR mRNA lacking exon13. Skipping of exon 13 (159 bp) does not change the reading frame and the resulting protein lacks 53 amino acids in the catalytic domain. When we stably expressed both HMGCR variants in CHO cells deficient of endogenous HMGCR activity, the Δexon13 variant appeared to be non-functional and was not able to restore cell growth in the absence of mevalonate. At present, we can only speculate about the exact underlying mechanisms of this observation. Exon13 encodes parts of the catalytic domain and it contains the highly conserved sequence element ENVIGX3
I/LP which is thought to mediate dimerization of the enzyme’s monomers 29
. Thus, deletion of exon13 could potentially impact the stability of the enzyme, since experiments in which monomeric soluble proteins were fused to the HMGCR membrane domains illustrated that the protein was degraded faster when it was smaller than tetrameric 30
. In addition, exon13 contains the E559 residue which is located at the front of the active site and was proposed to directly participate in the reduction of HMG-CoA by serving as a proton donor to mevaldehyde 29
. Therefore, alternative splicing of HMGCR appears to result in altered enzymatic activity and could also lead to more rapid degradation of the protein. A decrease in HMGCR activity would lead to lower cellular cholesterol synthesis and subsequently a counter-regulatory increase of cholesterol uptake from the plasma via the LDL-receptor pathway to maintain intracellular cholesterol homeostasis. In accordance with this hypothesis the allele at rs3846662 that was causing higher levels of Δexon13 HMGCR mRNA in our in vitro studies was sharing a haplotype with the alleles that were associated with lower LDL-C in the genome-wide association studies. HMG-CoA reductase activity is subject to multivalent control on transcriptional and post-transcriptional levels and alternative splicing may be an additional regulatory mechanism.
Modulation of alternatively spliced HMGCR mRNA levels could be of pharmacologic interest with regard to response to statin therapy or as target for antisense-mediated exon skipping. Recently, an antisense oligonucleotide (AON)-mediated skipping approach related to lowering plasma cholesterol levels was applied by Khoo et al 31
. In their study, AON-mediated exon27 skipping of the Apolipoprotein B transcript specifically lowered the amount of functional ApoB100 protein, while maintaining ApoB48 levels 31
Therefore, identification of specific factors that regulate HMGCR alternative splicing and elucidating the underlying mechanism may lead to a better understanding of its impact on regulating cellular cholesterol homeostasis and plasma cholesterol levels.