Common SNPs in HMGCR in micronesians and whites associated with LDL-cholesterol levels affect alternative splicing of exon13

Abstract

Background- Variation in LDL-cholesterol (LDL-C) among individuals is a complex genetic trait involving multiple genes and gene-environment interactions.

Methods and results: In a genome-wide association study (GWAS) to identify genetic variants influencing LDL-C in an isolated population from Kosrae, we observed associations for SNPs in the gene encoding 3hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase (HMGCR). Three of these SNPs (rs7703051, rs12654264, and rs3846663) met the statistical threshold of genome-wide significance when combined with data from the Diabetes Genetics Initiative GWAS. We followed up the association results and identified a functional SNP in intron13 (rs3846662), which was in linkage disequilibrium with the SNPs of genome-wide significance and affected alternative splicing of HMGCR mRNA. In vitro studies in human lymphoblastoid cells demonstrated that homozygosity for the rs3846662 minor allele was associated with up to 2.2-fold lower expression of alternatively spliced HMGCR mRNA lacking exon13, and minigene transfection assays confirmed that allele status at rs3846662 directly modulated alternative splicing of HMGCR exon13 (42.9+/-3.9 versus 63.7+/-1.0%Deltaexon13/total HMGCR mRNA, P=0.02). Further, the alternative splice variant could not restore HMGCR activity when expressed in HMGCR deficient UT-2 cells.

Conclusions: We identified variants in HMGCR that are associated with LDL-C across populations and affect alternative splicing of HMGCR exon13.

Figure 1

LD plot (r² values) in HapMap CEU individuals for SNPs at the HMGCR gene locus demonstrating LD between Affymetrix 500k SNPs with genome-wide significance (green) and the functional intronic SNP rs3846662 (red). Coding exons are shown as blue boxes. Exon13, which is subject to alternative splicing is double framed.

Figure 2

HMGCR mRNA expression in lymphoblastoid cells of major and minor allele homozygotes at rs3846662 (n=9 per genotype). (A) Total HMGCR mRNA expression normalized to β-actin over 48h. (B) Alternatively spliced HMGCR mRNA (Δex13 mRNA) as percentage of total HMGCR mRNA expression. *p<0.01 and †p<0.05 between major and minor allele samples.

Figure 3

Absolute amounts of full-length and Δexon13 HMGCR mRNA expression in cDNA pools of various human tissues (Clontech). HMGCR transcripts were normalized to β-actin as housekeeping gene.

Figure 4

HMGCR minigene splicing in transfected HEK cells. Alternatively spliced RNA (Δex13) from minigene constructs is expressed as percentage of total HMGCR minigene RNA. rs3846662/G: minor allele minigene, rs3846662/A: major allele minigene, mut A→G: major allele minigene after site directed mutagenesis, *p<0.05 rs3846662/G vs rs3846662/A; ^†p<0.05 rs3846662/A vs mut A→G

Figure 5

Characterization of UT-2 cells with stable expression of human full-length (UT-2+FL) and Δexon13 (UT-2+ex13) HMGCR: (A) HMGCR mRNA expression; (B) HMGCR activity normalized to wild-type CHO cells; (C) Growth characteristics of UT-2, UT-2+ex13 and UT-2+FL cells in the presence and absence of 0.2 mM mevalonate