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.

Objectives

The voltage-dependent L-type calcium channel α-subunit 1c (Cav1.2, CACNA1C) undergoes extensive mRNA splicing, leading to numerous isoforms with different functions. L-type calcium channel blockers are used in the treatment of hypertension and arrhythmias, but response varies between individuals. We have studied the interindividual variability in mRNA expression and splicing of CACNA1C, in 65 heart tissue samples, taken from heart transplant recipients.

Methods

Splice variants were measured quantitatively by polymerase chain reaction in 12 splicing loci of CACNA1C mRNA. To search for functional cis-acting polymorphisms, we determined allelic expression ratios for total CACNA1C mRNA and several splice variants using marker single nucleotide polymorphisms in exon 4 and exon 30.

Results

Total CACNA1C mRNA levels varied ∼50-fold. Substantial splicing occurred in six loci generating two or more splice variants, some with known functional differences. Splice patterns varied broadly between individuals. Two heart tissues expressed predominantly the dihydropyridine-sensitive smooth muscle isoform of CACNA1C (containing exon 8), rather than the cardiac isoform (containing exon 8a). Lack of significant allelic expression imbalance, observed with total mRNA and several splice variants, argued against CACNA1C polymorphisms as a cause of variability. Taken together, highly variable splicing can cause profound phenotypic variations of CACNA1C function, potentially associated with disease susceptibility and response to L-type calcium channel blockers.