WW-domain-containing oxidoreductase is associated with low plasma HDL-C levels

Abstract

Low serum HDL-cholesterol (HDL-C) is a major risk factor for coronary artery disease. We performed targeted genotyping of a 12.4 Mb linked region on 16q to test for association with low HDL-C by using a regional-tag SNP strategy. We identified one SNP, rs2548861, in the WW-domain-containing oxidoreductase (WWOX) gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent (p = 6.9 x 10(-7)). We extended our investigation to the population level by using two independent unascertained population-based Finnish cohorts, the cross-sectional METSIM cohort of 4,463 males and the prospective Young Finns cohort of 2,265 subjects. The combined analysis provided p = 4 x 10(-4) to 2 x 10(-5). Importantly, in the prospective cohort, we observed a significant longitudinal association of rs2548861 with HDL-C levels obtained at four different time points over 21 years (p = 0.003), and the T risk allele explained 1.5% of the variance in HDL-C levels. The rs2548861 resides in a highly conserved region in intron 8 of WWOX. Results from our in vitro reporter assay and electrophoretic mobility-shift assay demonstrate that this region functions as a cis-regulatory element whose associated rs2548861 SNP has a specific allelic effect and that the region forms an allele-specific DNA-nuclear-factor complex. In conclusion, analyses of 9,798 subjects show significant association between HDL-C and a WWOX variant with an allele-specific cis-regulatory function.

Figure 1

Association Results of Stage 1 and Combined Analyses of Stages 1 and 2 The −log10 of the p values obtained from the stage 1 FBAT −e analysis and the combined analysis of stage 1 and 2 study samples are shown. Black circles represent the 25 stage 1 SNPs that were re-examined in the stage 2 study samples. Results of the combined analysis of stage 1 and 2 family-based and case-control samples are shown in larger open circles (for details of analysis model, see Table 1). The open triangle indicates the result of the overall combined analysis of all dyslipidemic samples (n = 3,070). Small gray circles are the 1293 stage 1 SNPs that were not re-examined in the stage 2 samples. p = 3.7 × 10⁻⁵ indicates the Bonferroni corrected significance threshold.

Figure 2

A Profile Plot of the General Linear Model Showing the Longitudinal Effect of rs2548861 on HDL-C Levels in the Young Finns Cohort The circles represent the estimated marginal means of the standardized HDL-C residual values of measurements obtained in the corresponding year for the GG genotype carriers and the squares represent the TX carriers. TX indicates carriers of the T risk allele, i.e., genotypes TT and TG. The p value was obtained by RANOVA analysis for the standardized HDL-C residuals corrected for age, sex, and BMI.

Figure 3

Evidence of rs2548861 Allele-Specific Cis-Regulatory Function (A) A luciferase reporter assay demonstrates a cis-regulatory function for the 691 bp region surrounding rs2548861, and a significant allelic effect is observed. H1–H3 represent the common haplotypes of the SNPs rs2738572 and rs2548861 with frequency greater than 0.05 that were identified in the region by resequencing. The H1 haplotype is composed of the C-G alleles, H2 of A-T, and H3 of C-T for the rs2738572–rs2548861 haplotypes, respectively. The haplotypes H2 and H3 contain the T risk allele of rs2548861, and the haplotype H1 has the nonrisk G allele. Error bars represent the SD. (B) A comparative EMSA demonstrates the formation of a DNA-nuclear-factor complex in the 25 bp sequence surrounding the rs2548861 SNP with preferential nuclear-factor binding for the G allele. The lanes T and G indicate the allele of rs2548861 contained in the 25 bp biotin-labeled probe. The position of the unbound probe is indicated. An arrowhead indicates the position of the DNA-nuclear factor complex; −/−, the negative control; +/−, the addition of nuclear extract; and +/+, the addition of nuclear extract and a 200-fold excess of unlabeled competitor probe in the binding reactions, respectively.