Identification of a functional missense variant in the matrix metallopeptidase 10 (MMP10) gene in two families with premature myocardial infarction.

A positive family history is a major independent risk factor for atherosclerosis, and genetic variation is an important aspect of cardiovascular disease research. We identified a heterozygous missense variant p.L245P in the MMP10 gene in two families with premature myocardial infarction using whole-exome sequencing. The aim of this study was to investigate the consequences of this variant using in-silico and functional in-vitro assays. Molecular dynamics simulations were used to analyze protein interactions, calculate free binding energy, and measure the volume of the substrate-binding cleft of MMP10-TIMP1 models. The p.L245P variant showed an altered protein surface, different intra- and intermolecular interactions of MMP10-TIMP1, a lower total free binding energy between MMP10-TIMP1, and a volume-minimized substrate-binding cleft of MMP10 compared to the wild-type. For the functional assays, human THP-1 cells were transfected with plasmids containing MMP10 cDNA carrying the p.L245P and wild-type variant and differentiated into macrophages. Macrophage adhesion and migration assays were then conducted, and pro-inflammatory chemokine levels were evaluated. The p.L245P variant led to macrophages that were more adherent, less migratory, and secreted higher levels of the pro-inflammatory chemokines CXCL1 and CXCL8 than wild-type macrophages. Thus, the p.L245P variant in MMP10 may influence the pathogenesis of atherosclerosis in families with premature myocardial infarction by altering protein - protein interactions, macrophage adhesion and migration, and expression of pro-inflammatory chemokines, which may increase plaque rupture. These results could contribute to the development of selective MMP10 inhibitors and reduce the risk of atherosclerosis in families with a history of premature myocardial infarction.

MRAS in coronary artery disease-Unchartered territory.

Genome-wide association studies (GWAS) have identified coronary artery disease (CAD) susceptibility locus on chromosome 3q22.3. This locus contains a cluster of several genes that includes muscle rat sarcoma virus (MRAS). Common MRAS variants are also associated with CAD causing risk factors such as hypertension, dyslipidemia, obesity, and type II diabetes. The MRAS gene is an oncogene that encodes a membrane-bound small GTPase. It is involved in a variety of signaling pathways, regulating cell differentiation and cell survival (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase and phosphatidylinositol 3-kinase) as well as acute phase response signaling (tumor necrosis factor [TNF] and interleukin 6 [IL6] signaling). In this review, we will summarize the role of genetic MRAS variants in the etiology of CAD and its comorbidities with the focus on tissue distribution of MRAS isoforms, cell type/tissue specificity, and mode of action of single nucleotide variants in MRAS associated complex traits. Finally, we postulate that CAD risk variants in the MRAS locus are specific to smooth muscle cells and lead to higher levels of MRAS, particularly in arterial and cardiac tissue, resulting in MAPK-dependent tissue hypertrophy or hyperplasia.

Autoantibodies against the chemokine receptor 3 predict cardiovascular risk.

BACKGROUND AND AIMS: Chronic inflammation and autoimmunity contribute to cardiovascular (CV) disease. Recently, autoantibodies (aAbs) against the CXC-motif-chemokine receptor 3 (CXCR3), a G protein-coupled receptor with a key role in atherosclerosis, have been identified. The role of anti-CXCR3 aAbs for CV risk and disease is unclear. METHODS: Anti-CXCR3 aAbs were quantified by a commercially available enzyme-linked immunosorbent assay in 5000 participants (availability: 97.1%) of the population-based Gutenberg Health Study with extensive clinical phenotyping. Regression analyses were carried out to identify determinants of anti-CXCR3 aAbs and relevance for clinical outcome (i.e. all-cause mortality, cardiac death, heart failure, and major adverse cardiac events comprising incident coronary artery disease, myocardial infarction, and cardiac death). Last, immunization with CXCR3 and passive transfer of aAbs were performed in ApoE(-/-) mice for preclinical validation. RESULTS: The analysis sample included 4195 individuals (48% female, mean age 55.5 ± 11 years) after exclusion of individuals with autoimmune disease, immunomodulatory medication, acute infection, and history of cancer. Independent of age, sex, renal function, and traditional CV risk factors, increasing concentrations of anti-CXCR3 aAbs translated into higher intima-media thickness, left ventricular mass, and N-terminal pro-B-type natriuretic peptide. Adjusted for age and sex, anti-CXCR3 aAbs above the 75th percentile predicted all-cause death [hazard ratio (HR) (95% confidence interval) 1.25 (1.02, 1.52), P = .029], driven by excess cardiac mortality [HR 2.51 (1.21, 5.22), P = .014]. A trend towards a higher risk for major adverse cardiac events [HR 1.42 (1.0, 2.0), P = .05] along with increased risk of incident heart failure [HR per standard deviation increase of anti-CXCR3 aAbs: 1.26 (1.02, 1.56), P = .03] may contribute to this observation. Targeted proteomics revealed a molecular signature of anti-CXCR3 aAbs reflecting immune cell activation and cytokine-cytokine receptor interactions associated with an ongoing T helper cell 1 response. Finally, ApoE(-/-) mice immunized against CXCR3 displayed increased anti-CXCR3 aAbs and exhibited a higher burden of atherosclerosis compared to non-immunized controls, correlating with concentrations of anti-CXCR3 aAbs in the passive transfer model. CONCLUSIONS: In individuals free of autoimmune disease, anti-CXCR3 aAbs were abundant, related to CV end-organ damage, and predicted all-cause death as well as cardiac morbidity and mortality in conjunction with the acceleration of experimental atherosclerosis.

Homozygous frameshift variant in desmoglein 2 gene causes biventricular arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy caused by a homozygous frameshift variant (c.2358delA) in DSG2.

Autoimmune pre-disease.

Approximately 5% of the world-wide population is affected by autoimmune diseases. Overall, autoimmune diseases are still difficult to treat, impose a high burden on patients, and have a significant economic impact. Like other complex diseases, e.g., cancer, autoimmune diseases develop over several years. Decisive steps in the development of autoimmune diseases are (i) the development of autoantigen-specific lymphocytes and (often) autoantibodies and (ii) potentially clinical disease manifestation at a later stage. However, not all healthy individuals with autoantibodies develop disease manifestations. Identifying autoantibody-positive healthy individuals and monitoring and inhibiting their switch to inflammatory autoimmune disease conditions are currently in their infancy. The switch from harmless to inflammatory autoantigen-specific T and B-cell and autoantibody responses seems to be the hallmark for the decisive factor in inflammatory autoimmune disease conditions. Accordingly, biomarkers allowing us to predict this progression would have a significant impact. Several factors, such as genetics and the environment, especially diet, smoking, exposure to pollutants, infections, stress, and shift work, might influence the progression from harmless to inflammatory autoimmune conditions. To inspire research directed at defining and ultimately targeting autoimmune predisease, here, we review published evidence underlying the progression from health to autoimmune predisease and ultimately to clinically manifest inflammatory autoimmune disease, addressing the following 3 questions: (i) what is the current status, (ii) what is missing, (iii) and what are the future perspectives for defining and modulating autoimmune predisease.

Genetic and modifiable risk factors combine multiplicatively in common disease.

BACKGROUND: The joint contribution of genetic and environmental exposures to noncommunicable diseases is not well characterized. OBJECTIVES: We modeled the cumulative effects of common risk alleles and their prevalence variations with classical risk factors. METHODS: We analyzed mathematically and statistically numbers and effect sizes of established risk alleles for coronary artery disease (CAD) and other conditions. RESULTS: In UK Biobank, risk alleles counts in the lowest (175.4) and highest decile (205.7) of the distribution differed by only 16.9%, which nevertheless increased CAD prevalence 3.4-fold (p < 0.01). Irrespective of the affected gene, a single risk allele multiplied the effects of all others carried by a person, resulting in a 2.9-fold stronger effect size in the top versus the bottom decile (p < 0.01) and an exponential increase in risk (R > 0.94). Classical risk factors shifted effect sizes to the steep upslope of the logarithmic function linking risk allele numbers with CAD prevalence. Similar phenomena were observed in the Estonian Biobank and for risk alleles affecting diabetes mellitus, breast and prostate cancer. CONCLUSIONS: Alleles predisposing to common diseases can be carried safely in large numbers, but few additional ones lead to sharp risk increments. Here, we describe exponential functions by which risk alleles combine interchangeably but multiplicatively with each other and with modifiable risk factors to affect prevalence. Our data suggest that the biological systems underlying these diseases are modulated by hundreds of genes but become only fragile when a narrow window of total risk, irrespective of its genetic or environmental origins, has been passed.

The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets.

Platelets contribute to the regulation of tissue neovascularization, although the specific factors underlying this function are unknown. Here, we identified the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) on platelets as a negative regulatory mechanism of vessel formation. We showed that platelets expressing C5aR1 exert an inhibitory effect on endothelial cell functions such as migration and 2D and 3D tube formation. Growth factor- and hypoxia-driven vascularization was markedly increased in C5ar1-/- mice. Platelet-specific deletion of C5aR1 resulted in a proangiogenic phenotype with increased collateralization, capillarization and improved pericyte coverage. Mechanistically, we found that C5a induced preferential release of CXC chemokine ligand 4 (CXCL4, PF4) from platelets as an important antiangiogenic paracrine effector molecule. Interfering with the C5aR1-CXCL4 axis reversed the antiangiogenic effect of platelets both in vitro and in vivo.In conclusion, we identified a mechanism for the control of tissue neovascularization through C5a/C5aR1 axis activation in platelets and subsequent induction of the antiangiogenic factor CXCL4.

Studies in Zebrafish Demonstrate That CNNM2 and NT5C2 Are Most Likely the Causal Genes at the Blood Pressure-Associated Locus on Human Chromosome 10q24.32.

Background: Globally, high blood pressure (BP) is the most important risk factor for cardiovascular disease. Several genome-wide association studies (GWAS) have identified variants associated with BP traits at more than 535 chromosomal loci with genome-wide significance. The post-GWAS challenge is to annotate the most likely causal gene(s) at each locus. Chromosome 10q24.32 is a locus associated with BP that encompasses five genes: CYP17A1, BORCS7, AS3MT, CNNM2, and NT5C2 and warrants investigation to determine the specific gene or genes responsible for the phenotype. Aim: To identify the most likely causal gene(s) associated with BP at the 10q24.32 locus using zebrafish as an animal model. Results: We report significantly higher blood flow, increased arterial pulse, and elevated linear velocity in zebrafish larvae with cnnm2 and nt5c2 knocked down using gene-specific splice modification transcriptional morpholinos, compared with controls. No differences in blood-flow parameters were observed after as3mt, borcs7, or cyp17a1 knockdown. There was no effect on vessel diameter in animals with any of the four genes knocked down. At the molecular level, expression of hypertension markers (crp and ace) was significantly increased in cnnm2 and nt5c2 knockdown larvae. Further, the results obtained by morpholino knockdown were validated using zebrafish knockout (KO) lines with cnnm2 and nt5c2 deficiency, again resulting in higher blood flow, increased arterial pulse, and elevated linear velocity. Analysis of nt5c2a KO larvae demonstrated that lack of this gene resulted in reduced expression of cnnm2a, with reciprocal downregulation of nt5c2a in cnnm2a KO larvae. Staining of whole-blood smears from nt5c2 mutants revealed that KO of this gene might be associated with an acute lymphoblastic leukemia phenotype, consistent with literature reports. Additional experiments were designed based on previous literature on cnnm2a mutant zebrafish revealed impaired renal function, high levels of renin, and significantly increased expression of the ren gene, leading us to hypothesize that the observed elevated blood-flow parameters may be attributable to triggering of the renin-angiotensin-aldosterone signaling pathway. Conclusion: Our zebrafish data establish CNNM2 and NT5C2 as the most likely causal genes at the 10q24.32 BP locus and indicate that they trigger separate downstream mechanistic pathways.

Genetically modulated educational attainment and coronary disease risk.

AIMS: Genetic disposition and lifestyle factors are understood as independent components underlying the risk of multiple diseases. In this study, we aim to investigate the interplay between genetics, educational attainment-an important denominator of lifestyle-and coronary artery disease (CAD) risk. METHODS AND RESULTS: Based on the effect sizes of 74 genetic variants associated with educational attainment, we calculated a 'genetic education score' in 13 080 cases and 14 471 controls and observed an inverse correlation between the score and risk of CAD [P = 1.52 × 10-8; odds ratio (OR) 0.79, 95% confidence interval (CI) 0.73-0.85 for the higher compared with the lowest score quintile]. We replicated in 146 514 individuals from UK Biobank (P = 1.85 × 10-6) and also found strong associations between the 'genetic education score' with 'modifiable' risk factors including smoking (P = 5.36 × 10-23), body mass index (BMI) (P = 1.66 × 10-30), and hypertension (P = 3.86 × 10-8). Interestingly, these associations were only modestly attenuated by adjustment for years spent in school. In contrast, a model adjusting for BMI and smoking abolished the association signal between the 'genetic education score' and CAD risk suggesting an intermediary role of these two risk factors. Mendelian randomization analyses performed with summary statistics from large genome-wide meta-analyses and sensitivity analysis using 1271 variants affecting educational attainment (OR 0.68 for the higher compared with the lowest score quintile; 95% CI 0.63-0.74; P = 3.99 × 10-21) further strengthened these findings. CONCLUSION: Genetic variants known to affect educational attainment may have implications for a health-conscious lifestyle later in life and subsequently affect the risk of CAD.

Druggability of Coronary Artery Disease Risk Loci.

BACKGROUND: Genome-wide association studies have identified multiple loci associated with coronary artery disease and myocardial infarction, but only a few of these loci are current targets for on-market medications. To identify drugs suitable for repurposing and their targets, we created 2 unique pipelines integrating public data on 49 coronary artery disease/myocardial infarction-genome-wide association studies loci, drug-gene interactions, side effects, and chemical interactions. METHODS: We first used publicly available genome-wide association studies results on all phenotypes to predict relevant side effects, identified drug-gene interactions, and prioritized candidates for repurposing among existing drugs. Second, we prioritized gene product targets by calculating a druggability score to estimate how accessible pockets of coronary artery disease/myocardial infarction-associated gene products are, then used again the genome-wide association studies results to predict side effects, excluded loci with widespread cross-tissue expression to avoid housekeeping and genes involved in vital processes and accordingly ranked the remaining gene products. RESULTS: These pipelines ultimately led to 3 suggestions for drug repurposing: pentolinium, adenosine triphosphate, and riociguat (to target CHRNB4, ACSS2, and GUCY1A3, respectively); and 3 proteins for drug development: LMOD1 (leiomodin 1), HIP1 (huntingtin-interacting protein 1), and PPP2R3A (protein phosphatase 2, regulatory subunit b-double prime, α). Most current therapies for coronary artery disease/myocardial infarction treatment were also rediscovered. CONCLUSIONS: Integration of genomic and pharmacological data may prove beneficial for drug repurposing and development, as evidence from our pipelines suggests.

Etidronate prevents dystrophic cardiac calcification by inhibiting macrophage aggregation.

Cardiovascular calcification is associated with high risk of vascular disease. This involves macrophage infiltration of injured vascular tissue and osteoclast-related processes. Splenic monocytes from mice, that are predisposed (C3H) or resistant (B6) to calcification, were isolated and differentiated in vitro with M-CSF to generate macrophages, which aggregate to form multinucleated (MN) cells in the presence of RANKL. MN cell formation was significantly decreased in monocytes from resistant compared with calcifying mice. Conditioned media from C3H macrophages strongly induced calcification in vitro. However, medium from B6 macrophages inhibited calcification. An increase in ICAM-1 was detected in conditioned media from C3H macrophages compared with B6, suggesting a key role for this molecule in calcification processes. Due to natural genetic loss of Abcc6, the causal gene for cardiac calcification, C3H mice have reduced plasma levels of inorganic pyrophosphate (PPi), a potential calcification inhibitor. Supplementation of C3H mice with PPi or Etidronate prevented but did not completely reverse cardiac calcification. Our data provide strong evidence of the pathogenesis of macrophages and MNs during tissue calcification and suggest PPi or its analogue Etidronate as a potential inhibitor of MN formation and calcification. Furthermore, the adhesion molecule ICAM-1 was shown to play a key role in calcification.

CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis.

Intake of hemoglobin by the hemoglobin-haptoglobin receptor CD163 leads to a distinct alternative non-foam cell antiinflammatory macrophage phenotype that was previously considered atheroprotective. Here, we reveal an unexpected but important pathogenic role for these macrophages in atherosclerosis. Using human atherosclerotic samples, cultured cells, and a mouse model of advanced atherosclerosis, we investigated the role of intraplaque hemorrhage on macrophage function with respect to angiogenesis, vascular permeability, inflammation, and plaque progression. In human atherosclerotic lesions, CD163+ macrophages were associated with plaque progression, microvascularity, and a high level of HIF1α and VEGF-A expression. We observed irregular vascular endothelial cadherin in intraplaque microvessels surrounded by CD163+ macrophages. Within these cells, activation of HIF1α via inhibition of prolyl hydroxylases promoted VEGF-mediated increases in intraplaque angiogenesis, vascular permeability, and inflammatory cell recruitment. CD163+ macrophages increased intraplaque endothelial VCAM expression and plaque inflammation. Subjects with homozygous minor alleles of the SNP rs7136716 had elevated microvessel density, increased expression of CD163 in ruptured coronary plaques, and a higher risk of myocardial infarction and coronary heart disease in population cohorts. Thus, our findings highlight a nonlipid-driven mechanism by which alternative macrophages promote plaque angiogenesis, leakiness, inflammation, and progression via the CD163/HIF1α/VEGF-A pathway.

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 <1.0×10-3 (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=1.3×10-16) in comparison with 5% in ever-smokers (P=2.5×10-4), translating to a 60% loss of CHD protection conferred by this allelic variation in people who smoked tobacco (interaction P value=8.7×10-5). 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.

Pyrophosphate Supplementation Prevents Chronic and Acute Calcification in ABCC6-Deficient Mice.

Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6-/- mice fully inhibited the acute dystrophic cardiac calcification phenotype, whereas alendronate had no significant effect. We also found that daily injection of PPi to Abcc6-/- mice over several months prevented the development of pseudoxanthoma elasticum-like spontaneous calcification, but failed to reverse already established lesions. Furthermore, we found that the expression of low amounts of the human ABCC6 in liver of transgenic Abcc6-/- mice, resulting in only a 27% increase in plasma PPi levels, led to a major reduction in acute and chronic calcification phenotypes. This proof-of-concept study shows that the development of both acute and chronic calcification associated with ABCC6 deficiency can be prevented by compensating PPi deficits, even partially. Our work indicates that PPi substitution represents a promising strategy to treat ABCC6-dependent calcification disorders.

Systematic Evaluation of Pleiotropy Identifies 6 Further Loci Associated With Coronary Artery Disease.

BACKGROUND: Genome-wide association studies have so far identified 56 loci associated with risk of coronary artery disease (CAD). Many CAD loci show pleiotropy; that is, they are also associated with other diseases or traits. OBJECTIVES: This study sought to systematically test if genetic variants identified for non-CAD diseases/traits also associate with CAD and to undertake a comprehensive analysis of the extent of pleiotropy of all CAD loci. METHODS: In discovery analyses involving 42,335 CAD cases and 78,240 control subjects we tested the association of 29,383 common (minor allele frequency >5%) single nucleotide polymorphisms available on the exome array, which included a substantial proportion of known or suspected single nucleotide polymorphisms associated with common diseases or traits as of 2011. Suggestive association signals were replicated in an additional 30,533 cases and 42,530 control subjects. To evaluate pleiotropy, we tested CAD loci for association with cardiovascular risk factors (lipid traits, blood pressure phenotypes, body mass index, diabetes, and smoking behavior), as well as with other diseases/traits through interrogation of currently available genome-wide association study catalogs. RESULTS: We identified 6 new loci associated with CAD at genome-wide significance: on 2q37 (KCNJ13-GIGYF2), 6p21 (C2), 11p15 (MRVI1-CTR9), 12q13 (LRP1), 12q24 (SCARB1), and 16q13 (CETP). Risk allele frequencies ranged from 0.15 to 0.86, and odds ratio per copy of the risk allele ranged from 1.04 to 1.09. Of 62 new and known CAD loci, 24 (38.7%) showed statistical association with a traditional cardiovascular risk factor, with some showing multiple associations, and 29 (47%) showed associations at p < 1 × 10-4 with a range of other diseases/traits. CONCLUSIONS: We identified 6 loci associated with CAD at genome-wide significance. Several CAD loci show substantial pleiotropy, which may help us understand the mechanisms by which these loci affect CAD risk.

Cystatin C and Cardiovascular Disease: A Mendelian Randomization Study.

BACKGROUND: Epidemiological studies show that high circulating cystatin C is associated with risk of cardiovascular disease (CVD), independent of creatinine-based renal function measurements. It is unclear whether this relationship is causal, arises from residual confounding, and/or is a consequence of reverse causation. OBJECTIVES: The aim of this study was to use Mendelian randomization to investigate whether cystatin C is causally related to CVD in the general population. METHODS: We incorporated participant data from 16 prospective cohorts (n = 76,481) with 37,126 measures of cystatin C and added genetic data from 43 studies (n = 252,216) with 63,292 CVD events. We used the common variant rs911119 in CST3 as an instrumental variable to investigate the causal role of cystatin C in CVD, including coronary heart disease, ischemic stroke, and heart failure. RESULTS: Cystatin C concentrations were associated with CVD risk after adjusting for age, sex, and traditional risk factors (relative risk: 1.82 per doubling of cystatin C; 95% confidence interval [CI]: 1.56 to 2.13; p = 2.12 × 10(-14)). The minor allele of rs911119 was associated with decreased serum cystatin C (6.13% per allele; 95% CI: 5.75 to 6.50; p = 5.95 × 10(-211)), explaining 2.8% of the observed variation in cystatin C. Mendelian randomization analysis did not provide evidence for a causal role of cystatin C, with a causal relative risk for CVD of 1.00 per doubling cystatin C (95% CI: 0.82 to 1.22; p = 0.994), which was statistically different from the observational estimate (p = 1.6 × 10(-5)). A causal effect of cystatin C was not detected for any individual component of CVD. CONCLUSIONS: Mendelian randomization analyses did not support a causal role of cystatin C in the etiology of CVD. As such, therapeutics targeted at lowering circulating cystatin C are unlikely to be effective in preventing CVD.

Platelet-Related Variants Identified by Exomechip Meta-analysis in 157,293 Individuals.

Platelet production, maintenance, and clearance are tightly controlled processes indicative of platelets' important roles in hemostasis and thrombosis. Platelets are common targets for primary and secondary prevention of several conditions. They are monitored clinically by complete blood counts, specifically with measurements of platelet count (PLT) and mean platelet volume (MPV). Identifying genetic effects on PLT and MPV can provide mechanistic insights into platelet biology and their role in disease. Therefore, we formed the Blood Cell Consortium (BCX) to perform a large-scale meta-analysis of Exomechip association results for PLT and MPV in 157,293 and 57,617 individuals, respectively. Using the low-frequency/rare coding variant-enriched Exomechip genotyping array, we sought to identify genetic variants associated with PLT and MPV. In addition to confirming 47 known PLT and 20 known MPV associations, we identified 32 PLT and 18 MPV associations not previously observed in the literature across the allele frequency spectrum, including rare large effect (FCER1A), low-frequency (IQGAP2, MAP1A, LY75), and common (ZMIZ2, SMG6, PEAR1, ARFGAP3/PACSIN2) variants. Several variants associated with PLT/MPV (PEAR1, MRVI1, PTGES3) were also associated with platelet reactivity. In concurrent BCX analyses, there was overlap of platelet-associated variants with red (MAP1A, TMPRSS6, ZMIZ2) and white (PEAR1, ZMIZ2, LY75) blood cell traits, suggesting common regulatory pathways with shared genetic architecture among these hematopoietic lineages. Our large-scale Exomechip analyses identified previously undocumented associations with platelet traits and further indicate that several complex quantitative hematological, lipid, and cardiovascular traits share genetic factors.

Genome-wide association study and targeted metabolomics identifies sex-specific association of CPS1 with coronary artery disease.

Metabolites derived from dietary choline and L-carnitine, such as trimethylamine N-oxide and betaine, have recently been identified as novel risk factors for atherosclerosis in mice and humans. We sought to identify genetic factors associated with plasma betaine levels and determine their effect on risk of coronary artery disease (CAD). A two-stage genome-wide association study (GWAS) identified two significantly associated loci on chromosomes 2q34 and 5q14.1. The lead variant on 2q24 (rs715) localizes to carbamoyl-phosphate synthase 1 (CPS1), which encodes a mitochondrial enzyme that catalyses the first committed reaction and rate-limiting step in the urea cycle. Rs715 is also significantly associated with decreased levels of urea cycle metabolites and increased plasma glycine levels. Notably, rs715 yield a strikingly significant and protective association with decreased risk of CAD in only women. These results suggest that glycine metabolism and/or the urea cycle represent potentially novel sex-specific mechanisms for the development of atherosclerosis.

Systematic analysis of variants related to familial hypercholesterolemia in families with premature myocardial infarction.

Familial hypercholesterolemia (FH) is an oligogenic disorder characterized by markedly elevated low-density lipoprotein cholesterol (LDLC) levels. Variants in four genes have been reported to cause the classical autosomal-dominant form of the disease. FH is largely under-diagnosed in European countries. As FH increases the risk for coronary artery disease (CAD) and myocardial infarction (MI), it might be specifically overlooked in the large number of such patients. Here, we systematically examined the frequency of potential FH-causing variants by exome sequencing in 255 German patients with premature MI and a positive family history for CAD. We further performed co-segregation analyses in an average of 5.5 family members per MI patient. In total, we identified 11 potential disease-causing variants that co-segregate within the families, that is, 5% of patients with premature MI and positive CAD family history had FH. Eight variants were previously reported as disease-causing and three are novel (LDLR.c.811G>A p.(V271I)), PCSK9.c.610G>A (p.(D204N)) and STAP1.c.139A>G (p.(T47A))). Co-segregation analyses identified multiple additional family members carrying one of these FH variants and the clinical phenotype of either FH (n=2) or FH and premature CAD (n=15). However, exome sequencing also revealed that some variants in FH genes, which have been reported to cause FH, do not co-segregate with FH. The data reveal that a large proportion of FH patients escape the diagnosis, even when they have premature MI. Hence, systematic molecular-genetic screening for FH in such patients may reveal a substantial number of cases and thereby allow a timely LDLC-lowering in both FH/MI patients as well as their variant-carrying family members.