Cholesteryl Ester Transfer Protein (CETP) polymorphisms affect mRNA splicing, HDL levels, and sex-dependent cardiovascular risk

Abstract

Polymorphisms in and around the Cholesteryl Ester Transfer Protein (CETP) gene have been associated with HDL levels, risk for coronary artery disease (CAD), and response to therapy. The mechanism of action of these polymorphisms has yet to be defined. We used mRNA allelic expression and splice isoform measurements in human liver tissues to identify the genetic variants affecting CETP levels. Allelic CETP mRNA expression ratios in 56 human livers were strongly associated with several variants 2.5-7 kb upstream of the transcription start site (e.g., rs247616 p = 6.4 × 10(-5), allele frequency 33%). In addition, a common alternatively spliced CETP isoform lacking exon 9 (Δ9), has been shown to prevent CETP secretion in a dominant-negative manner. The Δ 9 expression ranged from 10 to 48% of total CETP mRNA in 94 livers. Increased formation of this isoform was exclusively associated with an exon 9 polymorphism rs5883-C>T (p = 6.8 × 10(-10)) and intron 8 polymorphism rs9930761-T>C (5.6 × 10(-8)) (in high linkage disequilibrium with allele frequencies 6-7%). rs9930761 changes a key splicing branch point nucleotide in intron 8, while rs5883 alters an exonic splicing enhancer sequence in exon 9.The effect of these polymorphisms was evaluated in two clinical studies. In the Whitehall II study of 4745 subjects, both rs247616 and rs5883T/rs9930761C were independently associated with increased HDL-C levels in males with similar effect size (rs247616 p = 9.6 × 10(-28) and rs5883 p = 8.6 × 10(-10), adjusted for rs247616). In an independent multiethnic US cohort of hypertensive subjects with CAD (INVEST-GENE), rs5883T/rs9930761C alone were significantly associated with increased incidence of MI, stroke, and all-cause mortality in males (rs5883: OR 2.36 (CI 1.29-4.30), p = 0.005, n = 866). These variants did not reach significance in females in either study. Similar to earlier results linking low CETP activity with poor outcomes in males, our results suggest genetic, sex-dependent CETP splicing effects on cardiovascular risk by a mechanism independent of circulating HDL-C levels.

Figure 1. Association of CETP SNPs with Allelic Expression Ratios.

Panel A. Association of each SNP with RNA absolute allelic ratios, as measured using rs5882 as an indicator. * Since rs5882 is used as the indicator in the assay, the p value is not applicable. The allelic mRNA ratios were normalized to the overall mean allelic gDNA ratios (there was no indication of a gene dosage effect requiring normalization to the gDNA for each individual). The data are mean ± S.D. (n = 3–6). Panel B. Log2 AEI absolute values in rs247616 genotypes. There would not be a detectable difference in allelic expression of homozygous samples if the SNP is functional.

Figure 2. Association of CETP SNPs with the Δsplice variant.

Panel A: Association p values assessing relationship between CETP SNPs and Δ9 splice variant formation in human livers (n = 94). Only rs9930761 and rs5883 in exon 8–10 region can account for increased formation of the Δ9 splice variant. rs289714 (intron 9), rs5882 (I405V), and rs1801706 (G84A) show varying degrees of LD with rs9930761, accounting for the observed association p values. Details for the SNPs in this study are provided in Table S3. Panel B: Percent Δ9 splice variant of total CETP mRNA as a function of rs5883T>C and rs9930761C>T. Homozygous minor allele carriers for rs5883/rs9930761 were not observed. All livers were heterozygous for both rs9930761 and rs5883, except for two livers heterozygous only for rs9930761, indicating that rs5883 is necessary for enhanced splicing. Using ANOVA with Dunnett's post-test, p values for both homozygous vs. rs9930761 and vs. rs5883 are P<0.01.

Figure 3. Schematics of the genomic CETP region spanning exons 8–10.

The exonic enhancer site (ESE) in exon 9 is disrupted by rs5883. Splice site sequences and the predicted splice branch point with rs9930761 are also depicted.