Loss of Cardioprotective Effects at the ADAMTS7 Locus as a Result of Gene-Smoking Interactions

BACKGROUND: Common diseases such as coronary heart disease (CHD) are complex in etiology. The interaction of genetic susceptibility with lifestyle factors may play a prominent role. However, gene-lifestyle interactions for CHD have been difficult to identify. Here, we investigate interaction of smoking behavior, a potent lifestyle factor, with genotypes that have been shown to associate with CHD risk. METHODS: We analyzed data on 60 919 CHD cases and 80 243 controls from 29 studies for gene-smoking interactions for genetic variants at 45 loci previously reported to be associated with CHD risk. We also studied 5 loci associated with smoking behavior. Study-specific gene-smoking interaction effects were calculated and pooled using fixed-effects meta-analyses. Interaction analyses were declared to be significant at a P value of <1.0x10(-3) (Bonferroni correction for 50 tests). RESULTS: We identified novel gene-smoking interaction for a variant upstream of the ADAMTS7 gene. Every T allele of rs7178051 was associated with lower CHD risk by 12% in never-smokers (P= 1.3x10(-16)) in comparison with 5% in ever-smokers (P= 2.5x10(-4)), translating to a 60% loss of CHD protection conferred by this allelic variation in people who smoked tobacco (interaction P value= 8.7x10(-5)). The protective T allele at rs7178051 was also associated with reduced ADAMTS7 expression in human aortic endothelial cells and lymphoblastoid cell lines. Exposure of human coronary artery smooth muscle cells to cigarette smoke extract led to induction of ADAMTS7. CONCLUSIONS: Allelic variation at rs7178051 that associates with reduced ADAMTS7 expression confers stronger CHD protection in never-smokers than in ever-smokers. Increased vascular ADAMTS7 expression may contribute to the loss of CHD protection in smokers.Peer reviewe

RNA sequencing data from blood demonstrates increased expression and abnormal splicing characterized by intron 14 retention in carriers of the splice region variant rs72658867-A.

<p><b>A.</b> Normalized average <i>LDLR</i> exon coverage for non-carriers (<i>N</i> = 238, in blue) and heterozygotes (<i>N</i> = 15, in red) of rs72658867-A demonstrates increased expression of <i>LDLR</i> transcripts in heterozygotes by ~22%, <i>P</i> = 0.0075. The X-axis is the exon number corresponding to RefSeq transcript NM_000527 for <i>LDLR</i>. The Y-axis shows the median normalized coverage (normalized for each individual to the total number of aligned reads). The error bars are based on the median absolute deviation within each group and is calculated separately for each exon. <b>B.</b> Using the same samples as in a) preferential intron 14 retention is observed in heterozygous carriers of rs72658867-A (shown in red). The X-axis is the genomic position in Mb (hg18/Build36). The Y-axis is the median count of normalized reads as in a). The structure of all <i>LDLR</i> RefSeq transcript variants is shown. The upper panel shows the full length gene whereas the lower panel shows the exons 13, 14 and 15 and the intron retention in intron 14. <b>C.</b> Quantitation of the proportion of transcripts with intron 14 retention in heterozygotes. The Y-axis corresponds to the proportion of RNA sequencing reads that are spliced from exon 14 to exon 15 (correctly spliced) out of the total number of reads that cover the last base of exon 14 (individuals that do not have coverage at this position are omitted). Median proportion: 1.00 (non-carriers); 0.70 (heterozygotes). Mean proportion: 0.95 (non-carriers); 0.71 (heterozygotes). Mann-Whitney test for location shift gives <i>P</i> = 6.0×10⁻⁹.</p

Association of <i>LDLR</i> sequence variants with CAD and age at diagnosis of CAD in Iceland.

<p>Association results for rs17248720, rs17248748, rs200238879 and rs72658867 with CAD (coronary artery disease) and age at diagnosis of CAD. Association results for each variant is presented with and without adjusting for the other three variants in the table.</p><p>^aFreq A1 = allellic frequency of A1.</p><p>^bOR is given with respect to allele A1.</p><p>^cEffect (β) is given in years with respect to allele A1.</p><p>Association of <i>LDLR</i> sequence variants with CAD and age at diagnosis of CAD in Iceland.</p

Overview of non-HDL-C associations in the region around <i>LDLR</i>.

<p>Plot <b>A</b> is a 0.8Mb overview centered on <i>LDLR</i> and plot <b>B</b> is a 70kb overview around the <i>LDLR</i> gene. Black circles show-log₁₀<i>P</i> as a function of build 36 coordinates for associations with non-HDL-C and red crosses correspond to non-HDL-C associations after adjusting for the four variants rs17248720, rs72658867, rs200238879 and rs17248748 that are indicated by vertical broken lines in plot b. Genes are shown in blue and recombination rates are reported in cM/Mb.</p

Association of <i>LDLR</i> splice region variant rs72658867-A and intronic variant rs17248748-T with non-HDL-C in Denmark, Netherlands and Iran.

<p>Shown are association results for rs72658867-A and rs17248748-T with non-HDL-C, TG and HDL-C in replication samples from Denmark, Netherlands and Iran.</p><p>^aAssociation results are adjusted for the variants rs17248748-T and rs6511720-T (r² = 0.96 with rs17248720-T in Europeans in the 1000G Phase 3 data).</p><p>^bAssociation results are adjusted for the variants rs72658867-A and rs6511720-T (r² = 0.96 with rs17248720-T in Europeans in the 1000G Phase 3 data).</p><p>^cAll replication samples combined for each trait.</p><p>^dReplication samples combined with Icelandic samples, # non-HDL-C = 139,385, # TG = 100,350, # HDL = 139,753.</p><p>^eEffect (β) in mmol/l given with respect to allele A for rs72658867 and allele T for rs17248748.</p><p>^fEffect (β) in % change is given with respect to the allele A for rs72658867 and allele T for rs17248748.</p><p>^g<i>P</i>_<i>het</i> = <i>P</i>-value for a test of heterogeneity in the combined effect estimate.</p><p>Association of <i>LDLR</i> splice region variant rs72658867-A and intronic variant rs17248748-T with non-HDL-C in Denmark, Netherlands and Iran.</p

Schematic overview of the study.

<p>Schematic overview of the study.</p

Regional plots illustrating conditional analyses of loci with more than one independent association signal for serum B₁₂ (<i>CUBN</i>, <i>TCN1</i> and <i>TCN2</i>) or serum folate (<i>MTHFR</i>).

<p>Genotyped and imputed SNVs passing quality control measures in the Icelandic data are plotted with their <i>P</i>-values (as −log10 values) as a function of genomic position (NCBI Build 36). Only SNVs with <i>P</i><10⁻⁵ in at least one of the models are shown. The analyses were performed in 25,960 and 20,717 chip-genotyped Icelanders for B₁₂ and folate, respectively. Data points illustrated by open circles represent unconditional analyses (M0); blue dots are results of analyses conditional on the most significant SNV in M0 (M1) and orange dots are results of analyses conditional on most significant SNVs in M0 and M1. Estimated recombination rates (HapMap CEU) are plotted to reflect the local LD structure. Gene annotations were obtained from RefGene.</p

Novel and previously reported genomic loci that associate with serum B₁₂ levels at <i>P</i><2.2×10⁻⁹.

<p>Association results for serum B₁₂ in Icelandic and Danish study samples separately and combined. The effect allele is the allele associated with increased serum B₁₂ levels. The effect is on a quantile normalized scale. Data were combined in fixed effect meta-analyses based on <i>P</i>-value and direction of effect adjusted for the number of individuals in each sample. Values of <i>I</i>² are percentages.</p><p>Chr., chromosome; EAF, effect allele frequency; HET, heterogeneity; SNV, single nucleotide variant.</p>1<p>The annotation is based on the RefSeq hg18.</p>2<p>The reference alleles based on Build 36 hg18 are shown in bold.</p>3<p>In the Icelandic data the strongest signal at the <i>FUT6</i> locus is for rs708686 located 5′ to the <i>FUT6</i> gene (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003530#pgen.1003530.s005" target="_blank">Table S3</a>).</p>4<p>Danish data are given for the perfect proxy rs4267943 (1000 Genomes data: <i>r</i>² = 1.0).</p

Novel and previously reported genomic loci that associate with serum folate levels at <i>P</i><2.2×10⁻⁹.

<p>Association results for serum folate in Icelandic and Danish study samples separately and combined. The effect allele is the allele associated with increased serum folate levels. The effect is on a quantile normalized scale. Data were combined in fixed effect meta-analyses based on <i>P</i>-value and direction of effect adjusted for the number of individuals in each sample. Values of <i>I</i>² are percentages. Association between serum folate levels and <i>MTHFR</i> rs1801133 in the Inter99 cohort has been published previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003530#pgen.1003530-Thuesen1" target="_blank">[14]</a>. Chr., chromosome; EAF, effect allele frequency; HET, heterogeneity; SNV, single nucleotide variant.</p>1<p>The annotation is based on the RefSeq hg18.</p>2<p>The reference allele based on Build 36 hg18 is shown in bold.</p>3<p>In the Icelandic data a 2 bp INDEL in exon 3 of <i>FOLR3</i> associated more strongly with serum folate levels. As only SNVs were analyzed in the Danish data this data was not available for the Danish samples.</p>4<p>The rs652197 variant was initially discovered in the Icelandic samples but subsequently genotyped in Danish samples to confirm the association.</p

Novel secondary association signals at the serum B₁₂ or serum folate loci that associate at <i>P</i><5×10⁻⁸.

<p>Conditional analyses were performed using imputed sequence data from chip-genotyped Icelanders with information on serum B₁₂ or folate levels. Results for SNV #1 (lead SNVs) at each loci are unconditional on other SNVs. Analysis of SNV #2 is conditional on SNV #1 and SNV #3 is conditional on SNV #1 and #2. The LD between the SNVs at each locus was estimated from the sequence information of the 1,179 whole genome sequenced Icelanders.</p