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

<i>Anks3</i> expression knock-down <i>in vivo</i> stimulates expression of genes involved in the vasopressin signaling pathway.

<p>(<b>A</b>-<b>B</b>) Renal mRNA expression of <i>Vit32</i>, <i>Aqp1</i>, <i>Aqp2</i> and <i>Aqp3</i> was evaluated by quantitative RT-PCR in mice four days (n = 3)(<b>A</b>) and twelve days (n = 5)(<b>B</b>) after the final injection of Locked Nucleic Acid modified AntiSense Oligonucleotides (LNA ASO). Quantification of each cDNA was performed in duplicate and normalized to <i>Gusb</i> gene expression level. (<b>C</b>) AQP2 expression in kidney tissues. Representative Western blots performed with protein extracts from 5 kidneys treated with scrambled (SCR) or ANKS3 ASO (top) and quantitative analysis of blots normalized to β-actin expression (bottom) show that AQP2 expression is significantly increased in mice twelve days after the final injection of ANKS3 ASO when compared to mice injected with SCR ASO. (<b>D</b>, <b>E</b>) Immunofluorescence staining of the papilla for AQP2 (<b>D</b>) and AVPR2 (<b>E</b>) show a dramatic increase of the proteins in kidneys of ANKS3 ASO treated mice twelve days after the final injection compared to SCR ASO controls. Scale bar: 300 μm. Data are means ± SEM. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and control mice injected with saline (Ctr)- or SCR ASO. *P<0.05; **P<0.01 significantly different to controls.</p

Identification of ANKS3, a partner of ANKS6.

<p>(<b>A</b>) Schematic representation of ANKS6 protein and the bait construct (pGBD-B-Anks6-SAM) used to perform the yeast two hybrid screen. (<b>B</b>) Schematic representation of ANKS3 protein for comparison with cDNA sequences of prey 1, 2, 3 and 4, recovered from the yeast two hybrid screen. (<b>C</b>) Interaction of ANKS6 with the 4 preys in interaction assays in yeast. The bait constructions containing either the Ankyrin repeats region of ANKS6 (pGBD-B-Anks6-ANK), or the Serine Rich region (pGBD-B-Anks6-MID), or the SAM domain (pGBD-B-Anks6-SAM) are shown on the left. The ability of these domains to interact with the preys is shown on the right. (<b>D</b>) Effect of the mutation R823W in the SAM domain of ANKS6 on the interaction between the preys and ANKS6. The construction pGBD-ANKS6-SAM and its mutated version (pGBD-ANKS6-SAM-R823W) are shown on the left. The ability of the SAM and SAM-R823W domains of the ANKS6 protein to interact with the preys is shown on the right.</p

Induction of apoptosis and proliferation in <i>in vivo Anks3</i> knock-down mice.

<p>(<b>A</b>) Immunofluorescence TUNEL was used to evaluate the percentage of apoptotic cells in mouse kidneys four days (D4 post inj.) and twelve days (D12 post inj.) after the final injection of scrambled (SCR) or ANKS3 AntiSense Oligonucleotides (ASO). Apoptosis was significantly increased in SCR ASO kidneys and in ANKS3ASO kidneys 12 days after the last injection.(<b>B</b>, <b>C</b>) Abundance of caspase 9 (<b>B</b>) and caspase 3 (<b>C</b>) proteins was evaluated by Western blot twelve days after final Locked Nucleic Acid modified (LNA) ASO injection (n = 5). Protein quantification was tested in duplicate and normalized to the level of ß-actin protein. (<b>D</b>) Percentage of PCNA positive cells in kidneys from mice that received SCR and ANKS3 ASO, 4 and 12 days after the last injection. (<b>E</b>) <i>p53</i> mRNA expression was evaluated by quantitative RT-PCR four (n = 3) and twelve days (n = 5) post treatment in mice injected with LNA ASO, saline (CTR) and SCR ASO. Data are means ± SEM. Quantification of each cDNA was performed in duplicate and normalized to <i>Gusb</i> gene expression. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and control mice.*P<0.05 significantly different to control mice.</p

<i>In vivo</i> LNA ASO-induced downregulation of<i>Anks3</i> expression in mouse kidney.

<p>(<b>A</b>) Presence of ANKS3 in kidneys treated with saline (CTR) and scrambled AntiSense Oligonucleotides (SCR ASO) 12 days post injection and with ANKS3 Locked Nucleic Acid modified AntiSense Oligonucleotides (ANKS3 LNA ASOs). Representative photomicrographs of kidney sections from 5 mice four and twelve days after the last injection of LNA ASOs (D4 post inj., D12 post inj.). Kidneys were stained with ANKS3 (revealed by Alexa Fluor 488 in green) and the LNA ASOs were localised with the Alexa Fluor 647 marker in red. (<b>B-D</b>) <i>Anks3</i> renal decreased expression does not affect expression of <i>Anks6</i>. Abundance of <i>Anks3</i> transcripts (<b>B</b>) and protein (<b>C</b>), and <i>Anks6</i> transcripts (<b>D</b>) were evaluated in mouse kidneys four days (D4 post inj., n = 3) and twelve days (D12 post inj., n = 5) after the final injection. cDNA quantification was performed in duplicate and normalized to <i>Actb</i> gene expression level. (<b>C</b>) Western blots (upper panel) and quantitative protein analysis (lower panel) performed with extracts from kidneys of 5 mice treated with saline (Ctr), scramble ASO (SCR ASO) or ANKS3 ASO four and twelve days after the last injection showed that expression of ANKS3 normalized to that of β-actin is significantly decreased in mice treated with ANKS3 ASO. Data are mean ± SEM. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and SCR ASO treated mice.**P<0.01; *P<0.05 significantly different to control mice treated with saline (Ctr) or SCR ASO.</p

Organ expression, physiological interaction and renal location of ANKS3 and ANKS6 in the mouse.

<p>ANKS3 and ANKS6 are strongly expressed in the mouse kidney.<i>Anks3</i> (<b>A</b>) and <i>Anks6</i> (<b>B</b>) mRNA expression in kidney, liver, pancreas and spleen was evaluated by quantitative RT-PCR. Data are shown as means ± SEM. Quantification of cDNA from 3 mice was normalized to <i>Actb</i> gene expression level. One-way ANOVA was applied to assess statistical significance. P<0.0001 significantly different to renal expression of <i>Anks3</i> and <i>Anks6</i>. (<b>C</b>) Co-localisation of ANKS3 and ANKS6 in the kidney. Representative photomicrographs of 6 kidney sections. ANKS3 staining (revealed by Alexa Fluor 488 in green, left column) co-localises (merge staining in central column) with ANKS6 (right vertical column, revealed by Alexa Fluor 594 in red) in glomeruli (upper panel, scale bar: 90μm) and in tubules (lower panel, scale bar: 300μm. (<b>D</b>) Immunoprecipitation experiments performed with goat anti-ANKS6 antibody, goat anti-ANKS3 antibody or goat IgGs on protein lysates and followed by an immunoblot performed with rabbit anti-ANKS3 antibody (top) and rabbit anti-ANKS6 antibody (bottom). Note that ANKS6 antibody co-immunoprecipitates ANKS3 in kidney protein lysates.</p

The mutation (R823W) in PKD/Mhm(<i>cy/cy</i>) rats does not alter ANKS6-ANKS3 renal co-localisation.

<p>Immunohistochemical staining of renal cortex and medulla was performed in 3 week-old wild-type and PKD/Mhm(<i>cy/cy</i>) rats for ANKS3 (upper panel) and ANKS6 (lower panel).</p

Biological relationships between insulin resistance and miR-125a expression.

<p>MiR-125a expression levels were quantified by q (quantitative) RT-PCR in adipose tissue of mice fed HFD for 1 week (A) or 15 weeks (B). The effect of <i>in vitro</i> insulin resistance (C) on miR-125a expression (D) was tested by qRT-PCR in 3T3-L1 adipocytes. Expression of miR-125a was normalized with small nucleolar (sno) RNA202 in adipose tissue and U6 in cultured cells. Data are expressed as mean ± SEM for 4–5 mice per group and for 3 replicated experiments in cultured cells. *p<0.05 significant differential expression of miR-125a between HFD-fed and control mice of the same strain. ^£££p<0.001 significant differences in glucose transport stimulated by insulin in 3T3-L1 adipocytes. ∧∧p<0.01 significant differences in miR-125a expression between IR-3T3-L1 adipocytes and control-3T3-L1 adipocytes.</p

MiR-125a target gene expression regulation in adipose tissue of obese patients.

<p>Expression of <i>Tef</i>, <i>Masp1</i>, <i>Rtn2</i>, <i>Ube2l3</i> and <i>Adam9</i> was quantified by qRT-PCR in subcutaneous (A) and visceral (B) adipose tissue of obese patients (Ob) with diabetes (Diab) or without (NonDiab) and non obese subjects (Controls). Expression levels were normalized with human <i>β-Actin</i>. ^$p<0.05, ^$$p<0.01, ^$$$p<0.001 significantly different to non obese subjects.</p

Patient physiological parameters (F, Female; M, Male; BMI, Body mass index; TGL, Triglyceride; γGT, γ-glutamyltransferase; IL6, Interleukin 6; ApoB, Apolipoprotein B;QUICKI, Quantitative insulin sensitivity index).

<p>Patient physiological parameters (F, Female; M, Male; BMI, Body mass index; TGL, Triglyceride; γGT, γ-glutamyltransferase; IL6, Interleukin 6; ApoB, Apolipoprotein B;QUICKI, Quantitative insulin sensitivity index).</p