Alternative splicing of 3-hydroxy-3-methylglutaryl coenzyme A reductase is associated with plasma low-density lipoprotein cholesterol response to simvastatin

Abstract

Background: HMGCR(3-Hydroxy-3-methylglutaryl coenzyme A reductase), the direct target of statin inhibition, undergoes alternative splicing of exon 13, which encodes part of the statin-binding domain of the enzyme. We hypothesized that HMGCR alternative splicing might be related to the interindividual variation in plasma low-density lipoprotein cholesterol response to statin treatment.

Methods and results: We measured mRNA expression of both the full-length and the alternatively spliced HMGCR transcript lacking exon 13 (HMGCRv_1) in 170 simvastatin-incubated immortalized lymphocyte cell lines derived from participants in the Cholesterol and Pharmacogenetics (CAP) study who were treated with simvastatin 40 mg/d for 6 weeks. Greater upregulation of HMGCRv_1 in vitro was significantly correlated (P<or=0.0001) with smaller in vivo reductions of plasma total cholesterol, low-density lipoprotein cholesterol, apoprotein B, and triglycerides and explained 6% to 15% of the variation in their response to treatment. In contrast, no significant relationship was found between expression of the full-length HMGCR transcript and in vivo response. By siRNA knockdown of the full-length transcript, we found that HMGCR enzyme activity measured in cells enriched in HMGCRv_1 was relatively resistant to statin inhibition, consistent with the association of increased alternative splicing with reduced statin response in the CAP study. In addition, we found that a common HMGCR single-nucleotide polymorphism (rs3846662) located within intron 13 was associated with variation in the proportion of HMGCR mRNA that is alternatively spliced.

Conclusions: Variation in the production of an HMGCR isoform with reduced statin sensitivity is a determinant of interindividual differences in low-density lipoprotein cholesterol, apolipoprotein B, and triglyceride response to statin treatment.

Trial registration: ClinicalTrials.gov NCT00451828.

Conflict of interest statement

Disclosures

Dr Krauss has received grant support from Merck, Merck-Schering Plough, and Pfizer and has consulted for Merck and Merck-Schering Plough. The other authors report no conflicts.

Figures

Figure 1

A, HMGCR exon 13 alternative splicing patterns. B, Full-length HMGCR and HMGCRv_1 expression in immortalized lymphocyte cell lines. Reverse-transcription PCR with primers spanning exon 12 to 14 was used to amplify cDNA from 10 different immortalized lymphocyte cell lines from CAP subjects.

Figure 2

Associations between in vitro HMGCR alternative splicing after statin treatment and in vivo percentage response of total cholesterol, LDL-C, apoB, triglycerides, and HDL-C. Results are for the combined analysis of 170 subjects whose cells were incubated with 1.8 µmol/L (n=119) or 14.5 µmol/L (n=51) (see Methods). P values are for multiple regression models including age, race, smoking status, sex, and body mass index.

Figure 3

HMGCR gene expression (A) and enzyme activity (B) after knockdown of the full-length HMGCR transcript via siRNA. A, HMGCR gene expression measured by real-time PCR in 293 kidney cells (n=8) after 16 hours of transfection with 2 siRNA duplexes targeting exon 13. B, Comparison of HMGCR enzyme activity measured in the presence of 3 concentrations of simvastatin from siRNA-transfected vs control cells (n=8). No changes in either HMGCR transcript levels or enzyme activity were seen after transfection with a nontargeting control siRNA duplex (data not shown).

Figure 4

Association of the HMGCR SNP rs3846662 genotype with statin-induced expression of the unspliced and alternatively spliced HMGCR transcripts. HMGCR gene expression was measured in immortalized lymphocytes derived from CAP subjects with 3 TaqMan assays specific for the total HMGCR transcripts, the full-length HMGCR transcript (including exon 13), and HMGCRv_1 (lacking exon 13) after 24 hours of incubation with 1.8 µmol/L simvastatin (n=119). HMGCR SNP rs3846662 was genotyped with Ampliflor primers, and associations between baseline and statin-treated HMGCR gene expression with SNP rs3846662 genotype were tested with a dominant model in JMP 6.0. Ratios of the fold changes in both the HMGCRv_1 and full-length HMGCR transcripts to the fold change in total HMGCR transcripts were used to adjust the measurements of exon 13 alternative splicing for overall changes in HMGCR gene expression.