Gregory T. Jones

A Variant Associated with Nicotine Dependence, Lung Cancer and Peripheral Arterial Disease

Smoking is a leading cause of preventable death, causing about 5 million premature deaths worldwide each year. Evidence for genetic influence on smoking behaviour and nicotine dependence (ND) has prompted a search for susceptibility genes. Furthermore, assessing the impact of sequence variants on smoking-related diseases is important to public health. Smoking is the major risk factor for lung cancer (LC) and is one of the main risk factors for peripheral arterial disease (PAD). Here we identify a common variant in the nicotinic acetylcholine receptor gene cluster on chromosome 15q24 with an effect on smoking quantity, ND and the risk of two smoking-related diseases in populations of European descent. The variant has an effect on the number of cigarettes smoked per day in our sample of smokers. The same variant was associated with ND in a previous genome-wide association study that used low-quantity smokers as controls, and with a similar approach we observe a highly significant association with ND. A comparison of cases of LC and PAD with population controls each showed that the variant confers risk of LC and PAD. The findings provide a case study of a gene–environment interaction, highlighting the role of nicotine addiction in the pathology of other serious diseases.

The same sequence variant on 9p21 associates with myocardial infarction, abdominal aortic aneurysm and intracranial aneurysm

Recently, two common sequence variants on 9p21, tagged by rs10757278-G and rs10811661-T, were reported to be associated with coronary artery disease (CAD) and type 2 diabetes (T2D), respectively. We proceeded to further investigate the contributions of these variants to arterial diseases and T2D. Here we report that rs10757278-G is associated with, in addition to CAD, abdominal aortic aneurysm (AAA; odds ratio (OR) = 1.31, P = 1.2 × 10−12) and intracranial aneurysm (OR = 1.29, P = 2.5 × 10−6), but not with T2D. This variant is the first to be described that affects the risk of AAA and intracranial aneurysm in many populations. The association of rs10811661-T to T2D replicates in our samples, but the variant does not associate with any of the five arterial diseases examined. These findings extend our insight into the role of the sequence variant tagged by rs10757278-G and show that it is not confined to atherosclerotic diseases.

Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction

Eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of inflammatory responses and thus have important roles in the pathogenesis of inflammatory diseases. Here we describe a genome-wide association scan for sequence variants affecting eosinophil counts in blood of 9,392 Icelanders. The most significant SNPs were studied further in 12,118 Europeans and 5,212 East Asians. SNPs at 2q12 (rs1420101), 2q13 (rs12619285), 3q21 (rs4857855), 5q31 (rs4143832) and 12q24 (rs3184504) reached genome-wide significance (P = 5.3 × 10−14, 5.4 × 10−10, 8.6 × 10−17, 1.2 × 10−10 and 6.5 × 10−19, respectively). A SNP at IL1RL1 associated with asthma (P = 5.5 × 10−12) in a collection of ten different populations (7,996 cases and 44,890 controls). SNPs at WDR36, IL33 and MYB that showed suggestive association with eosinophil counts were also associated with atopic asthma (P = 4.2 × 10−6, 2.2 × 10−5 and 2.4 × 10−4, respectively). We also found that a nonsynonymous SNP at 12q24, in SH2B3, associated significantly (P = 8.6 × 10−8) with myocardial infarction in six different populations (6,650 cases and 40,621 controls).

Calcium Regulates Key Components of Vascular Smooth Muscle Cell–Derived Matrix Vesicles to Enhance Mineralization

Rationale: Matrix vesicles (MVs) are specialized structures that initiate mineral nucleation during physiological skeletogenesis. Similar vesicular structures are deposited at sites of pathological vascular calcification, and studies in vitro have shown that elevated levels of extracellular calcium (Ca) can induce mineralization of vascular smooth muscle cell (VSMC)–derived MVs. Objectives: To determine the mechanisms that promote mineralization of VSMC-MVs in response to calcium stress. Methods and Results: Transmission electron microscopy showed that both nonmineralized and mineralized MVs were abundantly deposited in the extracellular matrix at sites of calcification. Using cultured human VSMCs, we showed that MV mineralization is calcium dependent and can be inhibited by BAPTA-AM. MVs released by VSMCs in response to extracellular calcium lacked the key mineralization inhibitor matrix Gla protein and showed enhanced matrix metalloproteinase-2 activity. Proteomics revealed that VSMC-MVs share similarities with chondrocyte-derived MVs, including enrichment of the calcium-binding proteins annexins (Anx) A2, A5, and A6. Biotin cross-linking and flow cytometry demonstrated that in response to calcium, AnxA6 shuttled to the plasma membrane and was selectively enriched in MVs. AnxA6 was also abundant at sites of vascular calcification in vivo, and small interfering RNA depletion of AnxA6 reduced VSMC mineralization. Flow cytometry showed that in addition to AnxA6, calcium induced phosphatidylserine exposure on the MV surface, thus providing hydroxyapatite nucleation sites. Conclusions: In contrast to the coordinated signaling response observed in chondrocyte MVs, mineralization of VSMC-MVs is a pathological response to disturbed intracellular calcium homeostasis that leads to inhibitor depletion and the formation of AnxA6/phosphatidylserine nucleation complexes.

Functional matrix metalloproteinase-9 polymorphism (C-1562T) associated with abdominal aortic aneurysm

PURPOSE Matrix metalloproteinase-9 (MMP-9) is a potent endopeptidase with activity against both collagens and elastin. Expression of MMP-9 is elevated in vascular disease, and in particular within aneurysm tissues. This study tested the hypothesis that the functionally more active T allele of the MMP9 C-1562T polymorphism may be overrepresented in patients with abdominal aortic aneurysm (AAA) compared with control subjects and patients with atherosclerotic peripheral vascular disease (PVD). METHODS Seven hundred eighty-nine unrelated persons (AAA, n = 414; control subjects, n = 203; PVD, n = 172) were genotyped for the common C-1562T functional promoter polymorphism of the MMP9 gene. RESULTS Genotypes containing the T allele of this polymorphism were significantly more common in patients with AAA compared with both control subjects and patients with PVD (adjusted odds ratio, 2.41 and 2.94, respectively). The greatest shift between groups was observed in male patients, with a difference of 20.6% in CT/TT genotypes. and 12.1% in T allele frequency between patients with AAA compared with patients with PVD. CONCLUSIONS This study provides further evidence to support the role of MMP-9 in the pathogenesis of AAA, and indicates that the MMP9 C-1562T functional polymorphism may represent a genetic component contributing to susceptibility to this vascular disease.

Apolipoprotein(a) genetic sequence variants associated with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism.

OBJECTIVES The purpose of this study is investigate the effects of variants in the apolipoprotein(a) gene (LPA) on vascular diseases with different atherosclerotic and thrombotic components. BACKGROUND It is unclear whether the LPA variants rs10455872 and rs3798220, which correlate with lipoprotein(a) levels and coronary artery disease (CAD), confer susceptibility predominantly via atherosclerosis or thrombosis. METHODS The 2 LPA variants were combined and examined as LPA scores for the association with ischemic stroke (and TOAST [Trial of Org 10172 in Acute Stroke Treatment] subtypes) (effective sample size [n(e)] = 9,396); peripheral arterial disease (n(e) = 5,215); abdominal aortic aneurysm (n(e) = 4,572); venous thromboembolism (n(e) = 4,607); intracranial aneurysm (n(e) = 1,328); CAD (n(e) = 12,716), carotid intima-media thickness (n = 3,714), and angiographic CAD severity (n = 5,588). RESULTS LPA score was associated with ischemic stroke subtype large artery atherosclerosis (odds ratio [OR]: 1.27; p = 6.7 × 10(-4)), peripheral artery disease (OR: 1.47; p = 2.9 × 10(-14)), and abdominal aortic aneurysm (OR: 1.23; p = 6.0 × 10(-5)), but not with the ischemic stroke subtypes cardioembolism (OR: 1.03; p = 0.69) or small vessel disease (OR: 1.06; p = 0.52). Although the LPA variants were not associated with carotid intima-media thickness, they were associated with the number of obstructed coronary vessels (p = 4.8 × 10(-12)). Furthermore, CAD cases carrying LPA risk variants had increased susceptibility to atherosclerotic manifestations outside of the coronary tree (OR: 1.26; p = 0.0010) and had earlier onset of CAD (-1.58 years/allele; p = 8.2 × 10(-8)) than CAD cases not carrying the risk variants. There was no association of LPA score with venous thromboembolism (OR: 0.97; p = 0.63) or intracranial aneurysm (OR: 0.85; p = 0.15). CONCLUSIONS LPA sequence variants were associated with atherosclerotic burden, but not with primarily thrombotic phenotypes.

Interleukin-6 receptor pathways in abdominal aortic aneurysm

Methods We conducted a systematic review and meta-analysis of studies reporting circulating IL-6 in AAA, and new investigations of the association between a common non-synonymous functional variant (Asp358Ala) in the IL-6R gene (IL6R) and AAA, followed the analysis of the variant both in vitro and in vivo. Inflammation may play a role in the development of abdominal aortic aneurysms (AAA). Interleukin-6 (IL-6) signalling through its receptor (IL-6R) is one pathway that could be exploited pharmacologically. We investigated this using a Mendelian randomization approach. Results Up to October 2011, we identified seven studies (869 cases, 851 controls). Meta-analysis demonstrated that AAA cases had higher levels of IL-6 than controls [standardized mean difference (SMD) = 0.46 SD, 95% CI = 0.25–0.66, I2 = 70%, P = 1.1 × 10–5 random effects]. Meta-analysis of five studies (4524 cases/15 710 controls) demonstrated that rs7529229 (which tags the non-synonymous variant Asp358Ala, rs2228145) was associated with a lower risk of AAA, per Ala358 allele odds ratio 0.84, 95% CI: 0.80–0.89, I2 = 0%, P = 2.7 × 10–11). In vitro analyses in lymphoblastoid cell lines demonstrated a reduction in the expression of downstream targets (STAT3, MYC and ICAM1) in response to IL-6 stimulation in Ala358 carriers. Conclusions A Mendelian randomization approach provides robust evidence that signalling via the IL-6R is likely to be a causal pathway in AAA. Drugs that inhibit IL-6R may play a role in AAA management.

The Level of Serum Anti-Müllerian Hormone Correlates with Vitamin D Status in Men and Women But Not in Boys

CONTEXT Anti-Müllerian hormone (AMH) is a gonad-specific hormone, which is extensively used as a marker of gonadal status. The level of serum AMH has a high variance in similar individuals for reasons that are unknown. The AMH gene promoter contains a vitamin D response element that may cause vitamin D status to influence serum AMH levels. AIM The objective of the study was to determine whether serum levels of AMH are related to 25-hydroxyitamin D [25(OH)D)] status. SETTING This was a correlative and intervention study. PARTICIPANTS Three cohorts of participants were analyzed; mature men (n = 113), premenopausal women (n = 33), and 5- to 6-yr-old boys (n = 74). Women were given a daily supplement of ergocalciferol, cholecalciferol, or a placebo for 6 months and provided baseline and posttreatment blood samples. MAIN OUTCOME MEASURES Serum AMH and 25(OH)D were measured and analyzed for covariation. RESULTS Serum AMH positively correlated with 25(OH)D in men (r = 0.22, P = 0.02) but not boys. Both 25(OH)D and AMH levels exhibited seasonal variation in women, with an 18% decrease in AMH levels in winter compared with summer (P = 0.01). Change in AMH level correlated with the initial AMH level and the magnitude of change in vitamin D levels (r = 0.36, P = 0.004). Cholecalciferol supplementation prevented seasonal AMH change. CONCLUSION Vitamin D may be a positive regulator of AMH production in adults, and vitamin D deficiency may confound clinical decisions based on AMH. Vitamin D deficiency should be considered when serum AMH levels are obtained for diagnosis.

Angiotensin II Type 1 Receptor 1166C Polymorphism Is Associated With Abdominal Aortic Aneurysm in Three Independent Cohorts

Objectives—Although polymorphic variations in genes of the RAS system have previously been associated with susceptibility to AAA, such studies have been significantly limited by small sample sizes. This study was undertaken, using the largest case series yet reported, to determine whether common genetic variants of the RAS are associated with either susceptibility or severity of AAA. Methods and Results—The frequencies of 4 common genetic variants of genes related to the renin-angiotensin system were investigated in 3 geographically distinct, but ethnically similar, case-control cohorts, resulting in comparison of 1226 AAA cases with 1723 controls. In all 3 the AGTR1 1166C allele was significantly more common in AAA patients than controls (overall adjusted OR 1.60, 95% CI 1.32 to 1.93, P=1.1×10−6). Overall, the ACE ID genotype was associated with AAA (OR 1.33, 95% CI 1.06 to 1.67, P<0.02). The AGT 268T allele appeared to have an epistatic effect on large aneurysm size. Conclusion—This study has identified a strong and repeated association between the AGTR1 1166C allele and susceptibility to AAA, and a weaker effect associated with the ACE deletion allele, in 3 geographically distinct, but ethnically similar, case-control cohorts. This study highlights the key role of the RAS in AAA and emphasizes the need for replication and validation of results in suitable independent cohorts.

A sequence variant associated with sortilin-1 (SORT1) on 1p13.3 is independently associated with abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a common human disease with a high estimated heritability (0.7); however, only a small number of associated genetic loci have been reported to date. In contrast, over 100 loci have now been reproducibly associated with either blood lipid profile and/or coronary artery disease (CAD) (both risk factors for AAA) in large-scale meta-analyses. This study employed a staged design to investigate whether the loci for these two phenotypes are also associated with AAA. Validated CAD and dyslipidaemia loci underwent screening using the Otago AAA genome-wide association data set. Putative associations underwent staged secondary validation in 10 additional cohorts. A novel association between the SORT1 (1p13.3) locus and AAA was identified. The rs599839 G allele, which has been previously associated with both dyslipidaemia and CAD, reached genome-wide significance in 11 combined independent cohorts (meta-analysis with 7048 AAA cases and 75 976 controls: G allele OR 0.81, 95% CI 0.76-0.85, P = 7.2 × 10(-14)). Modelling for confounding interactions of concurrent dyslipidaemia, heart disease and other risk factors suggested that this marker is an independent predictor of AAA susceptibility. In conclusion, a genetic marker associated with cardiovascular risk factors, and in particular concurrent vascular disease, appeared to independently contribute to susceptibility for AAA. Given the potential genetic overlap between risk factor and disease phenotypes, the use of well-characterized case-control cohorts allowing for modelling of cardiovascular disease risk confounders will be an important component in the future discovery of genetic markers for conditions such as AAA.