Sclerostin Downregulation Globally by Naturally Occurring Genetic Variants, or Locally in Atherosclerotic Plaques, Does Not Associate With Cardiovascular Events in Humans.

Inhibition of sclerostin increases bone formation and decreases bone resorption, leading to increased bone mass, bone mineral density, and bone strength and reduced fracture risk. In a clinical study of the sclerostin antibody romosozumab versus alendronate in postmenopausal women (ARCH), an imbalance in adjudicated serious cardiovascular (CV) adverse events driven by an increase in myocardial infarction (MI) and stroke was observed. To explore whether there was a potential mechanistic plausibility that sclerostin expression, or its inhibition, in atherosclerotic (AS) plaques may have contributed to this imbalance, sclerostin was immunostained in human plaques to determine whether it was detected in regions relevant to plaque stability in 94 carotid and 50 femoral AS plaques surgically collected from older female patients (mean age 69.6 ± 10.4 years). Sclerostin staining was absent in most plaques (67%), and when detected, it was of reduced intensity compared with normal aorta and was located in deeper regions of the plaque/wall but was not observed in areas considered relevant to plaque stability (fibrous cap and endothelium). Additionally, genetic variants associated with lifelong reduced sclerostin expression were explored for associations with phenotypes including those related to bone physiology and CV risk factors/events in a population-based phenomewide association study (PheWAS). Natural genetic modulation of sclerostin by variants with a significant positive effect on bone physiology showed no association with lifetime risk of MI or stroke. These data do not support a causal association between the presence of sclerostin, or its inhibition, in the vasculature and increased risk of serious cardiovascular events. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Phenome-wide association studies across large population cohorts support drug target validation.

Phenome-wide association studies (PheWAS) have been proposed as a possible aid in drug development through elucidating mechanisms of action, identifying alternative indications, or predicting adverse drug events (ADEs). Here, we select 25 single nucleotide polymorphisms (SNPs) linked through genome-wide association studies (GWAS) to 19 candidate drug targets for common disease indications. We interrogate these SNPs by PheWAS in four large cohorts with extensive health information (23andMe, UK Biobank, FINRISK, CHOP) for association with 1683 binary endpoints in up to 697,815 individuals and conduct meta-analyses for 145 mapped disease endpoints. Our analyses replicate 75% of known GWAS associations (P < 0.05) and identify nine study-wide significant novel associations (of 71 with FDR < 0.1). We describe associations that may predict ADEs, e.g., acne, high cholesterol, gout, and gallstones with rs738409 (p.I148M) in PNPLA3 and asthma with rs1990760 (p.T946A) in IFIH1. Our results demonstrate PheWAS as a powerful addition to the toolkit for drug discovery.

Functional confirmation that the R1488* variant in SCN9A results in complete loss-of-function of Nav1.7.

BACKGROUND: Individuals with an extremely rare inherited condition, termed Congenital Insensitivity to Pain (CIP), do not feel pain in response to noxious stimuli. Variants in SCN9A, encoding the transmembrane voltage-gated sodium channel Nav1.7, have previously been reported in subjects with CIP accompanied by anosmia, which are typically transmitted in a recessive pattern. Functional characterisations of some of these SCN9A mutations show that they result in complete loss-of-function of Nav1.7. METHODS: In a consanguineous family we performed whole exome sequencing of three members who have a diagnosis of CIP and one unaffected family member. The functional effects of the segregating variant in SCN9A were determined using patch clamp electrophysiology in human embryonic kidney (HEK) 293 cells transfected with the variant. RESULTS: We found that each CIP subject was homozygous for a putatively nonsense variant, R1488*, in SCN9A. This variant was reported elsewhere in a subject with CIP, though the functional effect was not determined. Using electrophysiology, we confirm that this variant results in a complete loss-of-function of Nav1.7. CONCLUSIONS: We confirm through electrophysiological analysis that this R1488* variant in SCN9A results in complete loss-of-function of Nav1.7, which is consistent with reports on other variants in this gene in subjects with CIP.

Physiology of Hyperuricemia and Urate-Lowering Treatments.

Gout is the most common form of inflammatory arthritis and is a multifactorial disease typically characterized by hyperuricemia and monosodium urate crystal deposition predominantly in, but not limited to, the joints and the urinary tract. The prevalence of gout and hyperuricemia has increased in developed countries over the past two decades and research into the area has become progressively more active. We review the current field of knowledge with emphasis on active areas of hyperuricemia research including the underlying physiology, genetics and epidemiology, with a focus on studies which suggest association of hyperuricemia with common comorbidities including cardiovascular disease, renal insufficiency, metabolic syndrome and diabetes. Finally, we discuss current therapies and emerging drug discovery efforts aimed at delivering an optimized clinical treatment strategy.

Genetic variants in PPARGC1B and CNTN4 are associated with thromboxane A2 formation and with cardiovascular event free survival in the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT).

BACKGROUND AND AIMS: Elevated urinary 11-dehydro thromboxane B2 (TxB2), a measure of thromboxane A2 formation in vivo, predicts future atherothrombotic events. To further understand this relationship, the genetic determinants of 11-dehydro TxB2 and their associations with cardiovascular morbidity were investigated in this study. METHODS: Genome-wide and targeted genetic association studies of urinary 11-dehydro TxB2 were conducted in 806 Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) participants. RESULTS: The strongest associations were in PPARGC1B (rs4235745, rs32582, rs10515638) and CNTN4 (rs10510230, rs4684343), these 5 single nucleotide polymorphisms (SNPs) were independently associated with 11-dehydro TxB2 formation. Haplotypes of 11-dehydro TxB2 increasing alleles for both PPARGC1B and CNTN4 were significantly associated with 11-dehydro TxB2, explaining 5.2% and 4.5% of the variation in the whole cohort, and 8.8% and 7.9% in participants not taking aspirin, respectively. In a second ASCOT population (n = 6199), addition of these 5 SNPs significantly improved the covariate-only Cox proportional hazards model for cardiovascular events (chisq = 14.7, p=0.01). Two of the risk alleles associated with increased urinary 11-dehydro TxB2 were individually associated with greater incidences of cardiovascular events - rs10515638 (HR = 1.31, p=0.01) and rs10510230 (HR = 1.25, p=0.007); effect sizes were larger in those not taking aspirin. CONCLUSIONS: PPARGC1B and CNTN4 genotypes are associated with elevated thromboxane A2 formation and with an excess of cardiovascular events. Aspirin appears to blunt these associations. If specific protection of PPARGC1B and CNTN4 variant carriers by aspirin is confirmed by additional studies, PPARGC1B and CNTN4 genotyping could potentially assist in clinical decision making regarding the use of aspirin in primary prevention.

Impact of genetic variation in the 5-HT transporter and receptor on platelet function in patients with stable CAD taking aspirin.

BACKGROUND: Serotonin (5-HT) induces platelet aggregation by activating its 5-HT2A receptor. Platelet uptake is mediated by the 5-HT transporter (5-HTT). A common 5-HTT promoter (5-HTTLPR) splice variant results in long (L) and short (S) alleles. 5-HTTLPR genotype has been associated with increased platelet activation and risk of MI. Variation within HTR2A gene (C1354T) that encodes the 5-HT2A receptor has also been associated with enhanced platelet aggregation. We hypothesised that 5-HTT and/or HTR2A variation may influence platelet response to aspirin in patients with stable CAD. METHODS: Patients (n=144) with stable cardiovascular disease taking aspirin were genotyped for the 5-HTTLPR and HTR2A variants. Platelet inhibition was assessed by serum thromboxane and arachidonic acid-induced platelet aggregation assay. RESULTS: 5-HTT genotype (LL vs *S) was a significant determinant of serum TX level (8.9±2.6ng/ml vs 6.0±1.6ng/ml respectively; p<0.02) and 5-HTT LL genotype predicted an incomplete aspirin response (serum TXB2>2.2ng/ml) (p=0.04; OR=2.22, CI=1.03-4.79). Odds ratio of the effect of LL genotype on TX elevation was 3.8 (95% CI 1.2-11.6) in younger patients (under 64) compared to 1.0 (95% CI=0.3-3.8) in older subjects. LL genotype did not influence AA aggregation (p=0.83, OR=1.2, CI=0.3-4.1). The HTR2A variant had no effect on TX generation (p=0.70; OR=1.22, CI=0.45-3.26) nor AA aggregation (p=0.99; OR=1.0, CI=0.2-4.9). CONCLUSIONS: In younger patients with stable CAD 5HTT LL genotype carried by almost one third of our cohort is associated with a diminished response to aspirin that may increase cardiovascular risk. Genotypic variation in platelet activation may be a contributing mechanism.

Two further blood pressure loci identified in ion channel genes with a gene-centric approach.

BACKGROUND: Blood pressure (BP) is highly heritable, but our understanding of the genetic causes underlying variations in BP is incomplete. In this study, we explored whether novel loci associated with BP could be identified using a genecentric approach in 3 community-based cohorts with accurate BP measurements. METHODS AND RESULTS: Genotyping of 1857 single nucleotide polymorphisms (SNPs) in 91 ion channel genes was performed in a discovery cohort (n=358). Thirty-four SNPs associated with BP traits (P≤0.01) were followed up in an independent population (n=387); significant SNPs from this analysis were looked up in another independent population (n=1010) and meta-analyzed. Repeated clinic and ambulatory measurements were available for all but the discovery cohort (clinic only). Association analyses were performed, with systolic, diastolic, and pulse pressures as quantitative traits, adjusting for age and sex. Quantile-quantile plots indicated that the genecentric approach resulted in an inflation of association signals. Of the 29 SNPs taken forward from the discovery cohort, 2 SNPs were associated with BP phenotypes with the same direction of effect, with experiment-wide significance, in follow-up cohort I. These were rs2228291, in the chloride channel gene CLCN2, and rs10513488, in the potassium channel gene KCNAB1. Both associations were subsequently replicated in follow-up cohort II. CONCLUSIONS: Using a genecentric design and 3 well-phenotyped populations, this study identified 2 previously unreported, biologically plausible, genetic associations with BP. These results suggest that dense genotyping of genes, in pathways known to influence BP, could add to candidate-gene and Genome Wide Association studies in further explaining BP heritability.

Whole exome re-sequencing implicates CCDC38 and cilia structure and function in resistance to smoking related airflow obstruction.

Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality and, whilst smoking remains the single most important risk factor, COPD risk is heritable. Of 26 independent genomic regions showing association with lung function in genome-wide association studies, eleven have been reported to show association with airflow obstruction. Although the main risk factor for COPD is smoking, some individuals are observed to have a high forced expired volume in 1 second (FEV1) despite many years of heavy smoking. We hypothesised that these "resistant smokers" may harbour variants which protect against lung function decline caused by smoking and provide insight into the genetic determinants of lung health. We undertook whole exome re-sequencing of 100 heavy smokers who had healthy lung function given their age, sex, height and smoking history and applied three complementary approaches to explore the genetic architecture of smoking resistance. Firstly, we identified novel functional variants in the "resistant smokers" and looked for enrichment of these novel variants within biological pathways. Secondly, we undertook association testing of all exonic variants individually with two independent control sets. Thirdly, we undertook gene-based association testing of all exonic variants. Our strongest signal of association with smoking resistance for a non-synonymous SNP was for rs10859974 (P = 2.34 × 10(-4)) in CCDC38, a gene which has previously been reported to show association with FEV1/FVC, and we demonstrate moderate expression of CCDC38 in bronchial epithelial cells. We identified an enrichment of novel putatively functional variants in genes related to cilia structure and function in resistant smokers. Ciliary function abnormalities are known to be associated with both smoking and reduced mucociliary clearance in patients with COPD. We suggest that genetic influences on the development or function of cilia in the bronchial epithelium may affect growth of cilia or the extent of damage caused by tobacco smoke.

A polymorphism in ACE2 is associated with a lower risk for fatal cardiovascular events in females: the MORGAM project.

Angiotensin II, a vasoconstrictor and the main effector molecule of the renin-angiotensin system, is known to influence inflammation, thrombosis, low-density lipoprotein oxidation and growth factors, all of which contribute to cardiovascular disease. The associations of polymorphisms in the angiotensin-converting enzyme 2 (ACE2) gene with cardiovascular risk have not been fully determined. Single nucleotide polymorphisms (SNPs) in ACE2 were genotyped in participants of the prospective MORGAM study (n = 5092) from five cohorts: ATBC, FINRISK, Northern Sweden, PRIME/Belfast and PRIME/France. Using a case-cohort design, associations were sought between SNPs and haplotypes with cardiovascular events during follow-up (Cox proportional hazards model). The comparison group were a subset of all MORGAM participants who were selected to ensure similar age and sex distributions among the cases and controls. The A allele of the rs2285666 SNP (HR = 0.3, p = 0.04) was significantly associated with the risk of cardiovascular death in female subjects. These findings complement those found in other studies of SNPs in the ACE2 gene in relation to cardiovascular disease risk. As females carry two copies of the ACE2 gene, and given its plausible biological role in cardiovascular disease risk, further studies of ACE2 should be prioritized.

Confirmation that the renin gene distal enhancer polymorphism REN-5312C/T is associated with increased blood pressure.

BACKGROUND: Studies of knockout and transgenic mice have demonstrated key roles for genes encoding components of the renin angiotensin system in blood pressure regulation. However, whether polymorphisms in these genes contribute to the cause of essential hypertension in humans is still a matter of debate. METHODS AND RESULTS: We performed an experiment with dense tagging single-nucleotide polymorphism coverage of 4 genes encoding proteins that control the overall activity of the cascade, namely renin, angiotensinogen, angiotensin-converting enzyme, and angiotensin-converting enzyme 2, in 2 Irish populations. Both clinic and 24-hour ambulatory blood pressure measurements were available from population I (n=387), whereas just clinic blood pressure was measured in population II (n=1024). Of the 23 polymorphisms genotyped, only a single renin gene polymorphism, REN-5312C/T, showed consistent statistically significant associations with elevated diastolic pressures. Carriage of one REN-5312T allele was associated with the following age- and sex-adjusted increments in diastolic pressures (mean [95% CI]): population I, clinic, 1.5 mm Hg (0.3 to 2.8); daytime, 1.4 mm Hg (0.4 to 2.4); night-time, 1.3 mm Hg (0.4 to 2.3), and population II, clinic, 1.1 mm Hg (0.1 to 2.1). Haplotypic analyses and multivariate stepwise regression analyses were in concordance with individual single-nucleotide polymorphism analyses. CONCLUSIONS: The REN-5312T allele had been shown previously to result in increased in vitro expression of the renin gene. We have now shown, in 2 independent populations, that carriage of a REN-5312T allele is associated with elevated diastolic blood pressure. These data provide evidence that renin is an important susceptibility gene for arterial hypertension in whites.