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

8 kHz synaptic cochleogram.

<p>Synaptic ribbon count measured at the 8 kHz tonotopic position within the cochlea. Despite the absence of a statistical difference in OHC counts, the wild-type mice demonstrated significantly greater post-noise exposure synaptic ribbon density per IHC (9A). Projections (60x, 3x zoom, oil immersed) of confocal stacks (9B) of immunostained pre and post-noise exposure mouse IHC synaptic ribbons (green, mouse anti-CtBP2).-/- = <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i>:-/+ = <i>Nox3</i>^<i>het</i>/+: +/+ = wild-type.</p

Regional plot of the 8 kHz ABR post noise-exposure at Chr 17 association in the HMDP centered on the lead SNP at the Nox3 locus (rs33652818).

<p>The blue diamond represents the most significant SNP (p = 9.63E-06) and SNPs are colored based on their LD with the most significant SNP being: red SNPs in LD at r²>0.8, orange SNPs in LD at r²>0.6 and green SNPs in LD at r²>0.4. The positions of all RefSeq genes are plotted using genome locations (NCBI’s Build37 genome assembly).</p

Genome-Wide Association Study Identifies <i>Nox3</i> as a Critical Gene for Susceptibility to Noise-Induced Hearing Loss

<div><p>In the United States, roughly 10% of the population is exposed daily to hazardous levels of noise in the workplace. Twin studies estimate heritability for noise-induced hearing loss (NIHL) of approximately 36%, and strain specific variation in sensitivity has been demonstrated in mice. Based upon the difficulties inherent to the study of NIHL in humans, we have turned to the study of this complex trait in mice. We exposed 5 week-old mice from the Hybrid Mouse Diversity Panel (HMDP) to a 10 kHz octave band noise at 108 dB for 2 hours and assessed the permanent threshold shift 2 weeks post exposure using frequency specific stimuli. These data were then used in a genome-wide association study (GWAS) using the Efficient Mixed Model Analysis (EMMA) to control for population structure. In this manuscript we describe our GWAS, with an emphasis on a significant peak for susceptibility to NIHL on chromosome 17 within a haplotype block containing NADPH oxidase-3 (<i>Nox3</i>). Our peak was detected after an 8 kHz tone burst stimulus. <i>Nox3</i> mutants and heterozygotes were then tested to validate our GWAS. The mutants and heterozygotes demonstrated a greater susceptibility to NIHL specifically at 8 kHz both on measures of distortion product otoacoustic emissions (DPOAE) and on auditory brainstem response (ABR). We demonstrate that this sensitivity resides within the synaptic ribbons of the cochlea in the mutant animals specifically at 8 kHz. Our work is the first GWAS for NIHL in mice and elucidates the power of our approach to identify tonotopic genetic susceptibility to NIHL.</p></div

Detailed analysis of the 8 kHz frequency stimulus.

<p>Dissection of the 8 kHz frequency by DPOAE I/O function (7A) and ABR wave 1 amplitudes (7B) consistently indicate more impairment in the <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i> and <i>Nox3</i>^<i>het</i>/+ than wild-type. One-way ANOVA (Tukey multiple comparisons). * = p < 0.05. Homozygous = <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i>: Heterozygous = <i>Nox3</i>^<i>het</i>/+: Wild-type = C57BL/6JEiJ strain.</p

Topographical analysis of the auditory pathway at different frequencies (post-noise exposure).

<p>Although no significant difference was seen for the DPOAE thresholds (6A), the wild-type controls show a higher wave 1 amplitude (p = 0.010) only at 8 kHz compared to <i>Nox3</i>^<i>het</i>/+ and <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i> (6B). One-way ANOVA (Tukey test for multiple comparisons). * = p < 0.05. Homozygous = <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i>: Heterozygous = <i>Nox3</i>^<i>het</i>/+: Wild-type = C57BL/6JEiJ strain.</p

Cytocochleogram of wild-type and mutant <i>Nox3</i> mice (8A).

<p>No significant difference in OHC counts were detected among wild-type, <i>Nox3</i>^<i>het</i>/+ and <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i>. OHC preparations of immunostained (40x) post-noise exposure cochleae (8B) at the 8 kHz tonotopic location (red, rabbit anti-myosin6) demonstrate a small impact with the loss of apical OHC (2 weeks post-noise exposure). Homozygous = <i>Nox3</i>^<i>het</i>/<i>Nox3</i>^<i>het</i>: Heterozygous = <i>Nox3</i>^<i>het</i>/+: Wild-type = C57BL/6JEiJ strain.</p

Genes within NIHL 5 association peaks regulated by local eQTL in the cochlea.

<p>^atxStart, location of transcription (NCBI Build37 genome assembly) start.</p><p>^btxEnd, location of transcription (NCBI Build37 genome assembly) end.</p><p>^cStatistically significant p value ≤ 5.1E-04 (Bonferroni corrected for the number of probes tested)</p><p>Genes within NIHL 5 association peaks regulated by local eQTL in the cochlea.</p

Genotypic effects of the peak SNP (rs33652818) at the <i>Nox3</i> locus.

<p>Comparison between alleles GG and AA across the various frequencies. There is a statistically significant difference between the alleles at 4 and 8 kHz. * = p value < 0.001. Error bars +/- 1 SE.</p

Characterization of post-exposure thresholds in the HMDP.

<p>Mean ± SEM for 8 kHz post-noise exposure hearing thresholds in 64 HMDP inbred mouse strains. The difference between the strains with the lowest and the highest values were 3.22-fold.</p