Common, low-frequency, and rare genetic variants associated with lipoprotein subclasses and triglyceride measures in Finnish men from the METSIM study

<div><p>Lipid and lipoprotein subclasses are associated with metabolic and cardiovascular diseases, yet the genetic contributions to variability in subclass traits are not fully understood. We conducted single-variant and gene-based association tests between 15.1M variants from genome-wide and exome array and imputed genotypes and 72 lipid and lipoprotein traits in 8,372 Finns. After accounting for 885 variants at 157 previously identified lipid loci, we identified five novel signals near established loci at <i>HIF3A</i>, <i>ADAMTS3</i>, <i>PLTP</i>, <i>LCAT</i>, and <i>LIPG</i>. Four of the signals were identified with a low-frequency (0.005LCAT. Gene-based associations (<i>P</i><10⁻¹⁰) support a role for coding variants in <i>LIPC</i> and <i>LIPG</i> with lipoprotein subclass traits. 30 established lipid-associated loci had a stronger association for a subclass trait than any conventional trait. These novel association signals provide further insight into the molecular basis of dyslipidemia and the etiology of metabolic disorders.</p></div

Comparison of METSIM association data between conventional lipid traits and subclass traits at established loci.

<p>Comparison of METSIM association data between conventional lipid traits and subclass traits at established loci.</p

Gene-based tests of association with HDL subclass traits for <i>LIPC</i> and <i>LIPG</i>.

<p>The distribution of the inverse normalized residuals of the trait values for all individuals (histogram) compared to individuals carrying variants included in the gene-based tests of association (triangles) (A) at <i>LIPC</i> with triglycerides in very large HDL and (B) at <i>LIPG</i> with phospholipids in medium HDL. The histograms indicate counts of individuals per trait bin in the METSIM study, and the dashed gray line below the histograms indicates the mean trait level. The rows of black and red triangles represent individuals that are heterozygous and homozygous, respectively for each variant indicated, and the solid black lines indicate the mean trait level for variant carriers. <i>P</i>_{<i>discovery</i>}, p-value for the individual variant-trait association; <i>P</i>_<i>gene</i>, p-value for the gene-based test of association; Annotation, functional annotation of the variants; Splice accept., splice acceptor variant. Figure created with VARV (<a href="https://github.com/shramdas/varv" target="_blank">https://github.com/shramdas/varv</a>).</p

Novel independent signal at <i>LIPG</i>.

<p>Association with phospholipids in medium HDL at the <i>LIPG</i> locus. The colors and shapes distinguish the association signals and are based on the LD (r²) in METSIM samples between each variant and a reference variant, rs538509310 or rs1943973, represented in red and blue, respectively. X-axis, genomic (GRCh37/hg19) position in Mb. Left y-axis, p- value of variant-trait association in–log₁₀. Right y-axis, local estimates of genomic recombination rate in cM/Mb, represented by blue lines. (A) Unconditional association with phospholipids in medium HDL. Black squares indicate the five coding variants (rs200435657, rs201922257, rs142545730, rs138438163, and rs77960347) used in the <i>LIPG</i> gene-based association tests. (B) Association with phospholipids in medium HDL after genome-wide conditional analysis of known lipid-associated variants (n = 885). (C) Association with phospholipids in medium HDL after conditioning on rs538509310. The association plots for four additional signals at <i>HIF3A</i>, <i>ALB</i>, <i>SYS1</i>, and <i>LCAT</i> are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007079#pgen.1007079.s004" target="_blank">S4 Fig</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007079#pgen.1007079.s005" target="_blank">S5 Fig</a>.</p

Newly identified signals associated with lipoprotein subclasses and triglyceride measures.

<p>Newly identified signals associated with lipoprotein subclasses and triglyceride measures.</p

Functional characterization of wild type and variant G6PC2 proteins.

<p>(A) Expression levels in HEK293 and (B) INS-1E cells were determined by western blot and densitometry analysis. The multiple bands on the western blot are likely to represent glycosylated G6PC2 protein products. Data are presented as mean ± standard error of the mean for at least three independent experiments. Significant differences are indicated as ** <i>P</i><0.01; *** <i>P</i><0.001; **** <i>P</i><0.0001. EV, empty vector; WT, wild type. (C) Expression levels in HEK293 and INS-1E cells in the presence of proteasomal inhibitor MG-132 or lysosomal inhibitor chloroquine were determined by western blot. (D) Cellular localization in HEK293 cells was assessed by immunofluorescence microscopy. Cells were double immunostained for FLAG tag (green) and calnexin (red), and merged images with a DNA stain (blue) are shown. Images were taken with laser settings that were optimized separately for each sample. Scale bar, 10µm.</p

<i>G6PC2</i> gene-based association with FG levels using SKAT and BURDEN test

<p><i>G6PC2</i> gene-based association with FG levels using SKAT and BURDEN test</p

Haplotypes of the lead non-coding GWAS SNP rs560887 and the three coding variants.

<p>rs138726309 (p.His177Tyr), rs2232323 (p.Tyr207Ser), and rs492594 (p.Val219Leu), obtained from 4,442 unrelated individuals from the Oxford Biobank. (A) Percentage minor allele frequency (MAF) and effect size estimates (<math><mi>β</mi><mo>^</mo></math>) of the four variants reported for the minor allele in mmol/L of FG after adjustment for age, sex, and BMI. (B) Haplotypes of the four associated variants in G6PC2 revealed that the glucose-lowering Leu219 allele was carried exclusively in cis with the glucose-raising allele at the GWAS SNP. Wild-type, glucose-raising alleles are circled in blue and the mutant, glucose-lowering alleles are circled in red. Diameter of the circle is proportional to the effect size estimates. Haplotype association was performed with FG derived residuals (after adjustment for age, sex, and BMI) using the most frequent haplotype as baseline.</p

Seven SNPs show sex difference.

a<p>Trait and sex for which the SNP was selected;</p>b<p>Gene labels state the nearest gene or the gene as published previously; details on all genes near the association signal can be found in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003500#pgen.1003500.s002" target="_blank">Figure S2</a>;</p>c<p>One-sided P-Values.</p>d<p>larger sample size due to one additional study that did not have hip circumference, and therefore could not contribute to WHRadjBMI.</p>e<p>smaller sample size as this SNP was not on Metabochip.</p><p>Shown are the seven SNPs with significant (at 5% false discovery rate) sex difference in the follow-up data. These seven SNPs exhibit genome-wide significant association in women (joint discovery and follow-up <i>P_women</i><5×10−8) and only two of these show nominally significant association in men (joint <i>P_men</i><0.05). The three loci MAP3K1, HSD17B4, and PPARG are shown here for the first time for their anthropometric trait association as well as for sex-difference.</p

Genome-wide scan for sex-specific genome-wide association highlights numerous loci.

<p>(a) Manhattan plot showing the men-specific (upward, up to 60,586 men) and women-specific (downward, up to 73,137 women) association P-values from the discovery with the 619 selected loci colored by the phenotype for which the locus was selected; (b) QQ-plot showing the sex-specific association P-values as observed against those expected under the null overall phenotypes (black) and for each phenotypes separately (colored).</p