Alexandre F.R. Stewart

A Common Allele on Chromosome 9 Associated with Coronary Heart Disease

Coronary heart disease (CHD) is a major cause of death in Western countries. We used genome-wide association scanning to identify a 58-kilobase interval on chromosome 9p21 that was consistently associated with CHD in six independent samples (more than 23,000 participants) from four Caucasian populations. This interval, which is located near the CDKN2A and CDKN2B genes, contains no annotated genes and is not associated with established CHD risk factors such as plasma lipoproteins, hypertension, or diabetes. Homozygotes for the risk allele make up 20 to 25% of Caucasians and have a ∼30 to 40% increased risk of CHD.

A genome-wide meta-analysis identifies 22 loci associated with eight hematological parameters in the HaemGen consortium.

The number and volume of cells in the blood affect a wide range of disorders including cancer and cardiovascular, metabolic, infectious and immune conditions. We consider here the genetic variation in eight clinically relevant hematological parameters, including hemoglobin levels, red and white blood cell counts and platelet counts and volume. We describe common variants within 22 genetic loci reproducibly associated with these hematological parameters in 13,943 samples from six European population-based studies, including 6 associated with red blood cell parameters, 15 associated with platelet parameters and 1 associated with total white blood cell count. We further identified a long-range haplotype at 12q24 associated with coronary artery disease and myocardial infarction in 9,479 cases and 10,527 controls. We show that this haplotype demonstrates extensive disease pleiotropy, as it contains known risk loci for type 1 diabetes, hypertension and celiac disease and has been spread by a selective sweep specific to European and geographically nearby populations.

Identification of ADAMTS7 as a novel locus for coronary atherosclerosis and association of ABO with myocardial infarction in the presence of coronary atherosclerosis: two genome-wide association studies.

BACKGROUND We tested whether genetic factors distinctly contribute to either development of coronary atherosclerosis or, specifically, to myocardial infarction in existing coronary atherosclerosis. METHODS We did two genome-wide association studies (GWAS) with coronary angiographic phenotyping in participants of European ancestry. To identify loci that predispose to angiographic coronary artery disease (CAD), we compared individuals who had this disorder (n=12,393) with those who did not (controls, n=7383). To identify loci that predispose to myocardial infarction, we compared patients who had angiographic CAD and myocardial infarction (n=5783) with those who had angiographic CAD but no myocardial infarction (n=3644). FINDINGS In the comparison of patients with angiographic CAD versus controls, we identified a novel locus, ADAMTS7 (p=4·98×10(-13)). In the comparison of patients with angiographic CAD who had myocardial infarction versus those with angiographic CAD but no myocardial infarction, we identified a novel association at the ABO locus (p=7·62×10(-9)). The ABO association was attributable to the glycotransferase-deficient enzyme that encodes the ABO blood group O phenotype previously proposed to protect against myocardial infarction. INTERPRETATION Our findings indicate that specific genetic predispositions promote the development of coronary atherosclerosis whereas others lead to myocardial infarction in the presence of coronary atherosclerosis. The relation to specific CAD phenotypes might modify how novel loci are applied in personalised risk assessment and used in the development of novel therapies for CAD. FUNDING The PennCath and MedStar studies were supported by the Cardiovascular Institute of the University of Pennsylvania, by the MedStar Health Research Institute at Washington Hospital Center and by a research grant from GlaxoSmithKline. The funding and support for the other cohorts contributing to the paper are described in the webappendix.

Functional Analysis of the Chromosome 9p21.3 Coronary Artery Disease Risk Locus

Objectives—We have investigated the functional significance of conserved sequences within the 9p21.3 risk locus for coronary artery disease (CAD) and determined the relationship of 9p21.3 to expression of ANRIL and to whole genome gene expression. Methods and Results—We demonstrate that a conserved sequence within the 9p21.3 locus has enhancer activity and that the risk variant significantly increases reporter gene expression in primary aortic smooth muscle cells. Whole blood RNA expression of the short variants of ANRIL was increased by 2.2-fold whereas expression of the long ANRIL variant was decreased by 1.2-fold in healthy subjects homozygous for the risk allele. Expression levels of the long and short ANRIL variants were positively correlated with that of the cyclin-dependent kinase inhibitor, CDKN2B (p15) and TDGF1 (Cripto), respectively. Relevant to atherosclerosis, genome-wide expression profiling demonstrated upregulation of gene sets modulating cellular proliferation in carriers of the risk allele. Conclusion—These findings are consistent with the hypothesis that the 9p21.3 risk allele contains a functional enhancer, the activity of which is altered in carriers of the risk allele. 9p21.3 may promote atherosclerosis by regulating expression of ANRIL, which in turn is associated with altered expression of genes controlling cellular proliferation pathways.

Point-of-care genetic testing for personalisation of antiplatelet treatment (RAPID GENE): a prospective, randomised, proof-of-concept trial

BACKGROUND Prospective assessment of pharmacogenetic strategies has been limited by an inability to undertake bedside genetic testing. The CYP2C19*2 allele is a common genetic variant associated with increased rates of major adverse events in individuals given clopidogrel after percutaneous coronary intervention (PCI). We used a novel point-of-care genetic test to identify carriers of the CYP2C19*2 allele and aimed to assess a pharmacogenetic approach to dual antiplatelet treatment after PCI. METHODS Between Aug 26, 2010, and July 7, 2011, 200 patients were enrolled into our prospective, randomised, proof-of-concept study. Patients undergoing PCI for acute coronary syndrome or stable angina were randomly assigned to rapid point-of-care genotyping or to standard treatment. Individuals in the rapid genotyping group were screened for the CYP2C19*2 allele. Carriers were given 10 mg prasugrel daily, and non-carriers and patients in the standard treatment group were given 75 mg clopidogrel daily. The primary endpoint was the proportion of CYP2C19*2 carriers with high on-treatment platelet reactivity (P2Y12 reactivity unit [PRU] value of more than 234) after 1 week of dual antiplatelet treatment, which is a marker associated with increased adverse cardiovascular events. Interventional cardiologists and data analysts were masked to genetic status and treatment. Patients were not masked to treatment allocation. All analyses were by intention to treat. This study is registered with ClinicalTrials.gov, NCT01184300. FINDINGS After randomisation, 187 patients completed follow-up (91 rapid genotyping group, 96 standard treatment). 23 individuals in each group carried at least one CYP2C19*2 allele. None of the 23 carriers in the rapid genotyping group had a PRU value of more than 234 at day 7, compared with seven (30%) given standard treatment (p=0·0092). The point-of-care genetic test had a sensitivity of 100% (95% CI 92·3-100) and a specificity of 99·3% (96·3-100). INTERPRETATION Point-of-care genetic testing after PCI can be done effectively at the bedside and treatment of identified CYP2C19*2 carriers with prasugrel can reduce high on-treatment platelet reactivity. FUNDING Spartan Biosciences.

Rare Copy Number Variation Discovery and Cross-Disorder Comparisons Identify Risk Genes for ADHD

A high-resolution analysis of copy number variation in patients with ADHD reveals new gene associations, few de novo mutations, and overlap with genes implicated in other disorders such as autism. Complexities of Cognition: The Case of ADHD As for autism and schizophrenia, the closer we look at attention deficit hyperactivity disorder (ADHD), the more complicated it seems. Found in 4% of children, this syndrome of attention, hyperactivity, and impulsiveness is highly heritable, yet we know only a few of the responsible genetic variants. Here, Lionel et al. assessed a particularly well-defined population of 248 children with ADHD, plus many of their parents, for extra copies or deletions of genes. The 306 rare copy number variations (CNVs) found in these individuals were of various kinds—only 1.7% were de novo mutations in brain-specific genes, 7.7% were clearly inherited and occurred in genes known to be associated with ADHD or defined new culprit genes, and several were in genes already implicated in other disorders such as autism. To take a closer look at possible genes that confer risk for more than one developmental disorder, the authors examined the CNVs in a separate group of patients with autism. In four autism patients and two of the patients with ADHD, a cluster of rare disorder-associated CNVs occurred on chromosome 9 in and around two genes: ASTN2, necessary for mammalian brain development, and TRIM32, a neuronal stem cell–associated gene. This region has also been associated with CNVs in bipolar disorder, intellectual disability, and schizophrenia. In all, the authors found rare inherited CNVs at sites that had been previously implicated in ADHD or in other neurodevelopmental disorders in 8% of the individuals with ADHD. Their results implicate common genes and pathways for several neuropsychiatric disorders, which is consistent with the common clinical co-occurrence of ADHD with other such conditions. Attention deficit hyperactivity disorder (ADHD) is a common and persistent condition characterized by developmentally atypical and impairing inattention, hyperactivity, and impulsiveness. We identified de novo and rare copy number variations (CNVs) in 248 unrelated ADHD patients using million-feature genotyping arrays. We found de novo CNVs in 3 of 173 (1.7%) ADHD patients for whom we had DNA from both parents. These CNVs affected brain-expressed genes: DCLK2, SORCS1, SORCS3, and MACROD2. We also detected rare inherited CNVs in 19 of 248 (7.7%) ADHD probands, which were absent in 2357 controls and which either overlapped previously implicated ADHD loci (for example, DRD5 and 15q13 microduplication) or identified new candidate susceptibility genes (ASTN2, CPLX2, ZBBX, and PTPRN2). Among these de novo and rare inherited CNVs, there were also examples of genes (ASTN2, GABRG1, and CNTN5) previously implicated by rare CNVs in other neurodevelopmental conditions including autism spectrum disorder (ASD). To further explore the overlap of risks in ADHD and ASD, we used the same microarrays to test for rare CNVs in an independent, newly collected cohort of 349 unrelated individuals with a primary diagnosis of ASD. Deletions of the neuronal ASTN2 and the ASTN2-intronic TRIM32 genes yielded the strongest association with ADHD and ASD, but numerous other shared candidate genes (such as CHCHD3, MACROD2, and the 16p11.2 region) were also revealed. Our results provide support for a role for rare CNVs in ADHD risk and reinforce evidence for the existence of common underlying susceptibility genes for ADHD, ASD, and other neuropsychiatric disorders.

Design of the Coronary ARtery DIsease Genome-Wide Replication And Meta-Analysis (CARDIoGRAM) Study: A Genome-Wide Association Meta-analysis Involving More Than 22 000 Cases and 60 000 Controls

Background—Recent genome-wide association studies (GWAS) of myocardial infarction (MI) and other forms of coronary artery disease (CAD) have led to the discovery of at least 13 genetic loci. In addition to the effect size, power to detect associations is largely driven by sample size. Therefore, to maximize the chance of finding novel susceptibility loci for CAD and MI, the Coronary ARtery DIsease Genome-wide Replication And Meta-analysis (CARDIoGRAM) consortium was formed. Methods and Results—CARDIoGRAM combines data from all published and several unpublished GWAS in individuals with European ancestry; includes >22 000 cases with CAD, MI, or both and >60 000 controls; and unifies samples from the Atherosclerotic Disease VAscular functioN and genetiC Epidemiology study, CADomics, Cohorts for Heart and Aging Research in Genomic Epidemiology, deCODE, the German Myocardial Infarction Family Studies I, II, and III, Ludwigshafen Risk and Cardiovascular Heath Study/AtheroRemo, MedStar, Myocardial Infarction Genetics Consortium, Ottawa Heart Genomics Study, PennCath, and the Wellcome Trust Case Control Consortium. Genotyping was carried out on Affymetrix or Illumina platforms followed by imputation of genotypes in most studies. On average, 2.2 million single nucleotide polymorphisms were generated per study. The results from each study are combined using meta-analysis. As proof of principle, we meta-analyzed risk variants at 9p21 and found that rs1333049 confers a 29% increase in risk for MI per copy (P=2×10−20). Conclusion—CARDIoGRAM is poised to contribute to our understanding of the role of common genetic variation on risk for CAD and MI.

Disruption at the PTCHD1 locus on Xp22.11 in autism spectrum disorder and intellectual disability

Mutations of the X-linked gene PTCHD1 are associated with autism spectrum disorders and intellectual disability. A Patch in the Fabric of Autism What causes autism? This disabling disorder is characterized by severe language and social impairment and is now included under the umbrella term “autism spectrum disorder” (ASD), which also includes milder deficits in communication and social development. Numerous theories have been advanced as to its causes. These have ranged from discredited concepts—“refrigerator” mothers and vaccines—to the modern idea of gene-environment interactions. Although no one gene simply explains the predisposition of patients for ASD, these disorders are wellknown to have a strong genetic component. Here, Noor et al. report the results of genetic analysis in thousands of patients and control subjects: Mutations at the PTCHD1 (patched-related gene) locus are associated with the inheritance of ASD and with intellectual disability in a small fraction of cases. In this study, the authors analyzed the PTCHD1 gene from 1896 patients with ASD and 246 with intellectual disability, and compared these to more than 10,000 control individuals, and found mutations in various parts of this gene in 25 affected individuals in 20 different families, but not in any of the controls. Some patients had large deletions, in one case spanning the entire gene, and in others the culprit was a missense mutation. A result of this gene’s location on the X chromosome, the affected patients were almost all male, and most had unaffected mothers and other female relatives. The authors also present evidence that the PTCHD1 gene may be part of the Hedgehog signaling pathway, which is important in embryonic development. Autism and intellectual disability are not straightforward disorders that can be attributed to mutations in a single gene. Even when candidate genes such as PTCHD1 are known, differences in the gene sequence do not perfectly correlate with phenotype, because there are many as yet undefined additional genes and environmental influences that dictate the ultimate characteristics of the person. Identifying some of these genes, as Noor et al. have done in this study, allows a better understanding of the disorder and the development of ways to compensate for its disabilities. Autism is a common neurodevelopmental disorder with a complex mode of inheritance. It is one of the most highly heritable of the complex disorders, although the underlying genetic factors remain largely unknown. Here, we report mutations in the X-chromosome PTCHD1 (patched-related) gene in seven families with autism spectrum disorder (ASD) and in three families with intellectual disability. A 167-kilobase microdeletion spanning exon 1 was found in two brothers, one with ASD and the other with a learning disability and ASD features; a 90-kilobase microdeletion spanning the entire gene was found in three males with intellectual disability in a second family. In 900 probands with ASD and 208 male probands with intellectual disability, we identified seven different missense changes (in eight male probands) that were inherited from unaffected mothers and not found in controls. Two of the ASD individuals with missense changes also carried a de novo deletion at another ASD susceptibility locus (DPYD and DPP6), suggesting complex genetic contributions. In additional males with ASD, we identified deletions in the 5′ flanking region of PTCHD1 that disrupted a complex noncoding RNA and potential regulatory elements; equivalent changes were not found in male control individuals. Thus, our systematic screen of PTCHD1 and its 5′ flanking regions suggests that this locus is involved in ~1% of individuals with ASD and intellectual disability.

Homocysteine and Coronary Heart Disease: Meta-analysis of MTHFR Case-Control Studies, Avoiding Publication Bias

Robert Clarke and colleagues conduct a meta-analysis of unpublished datasets to examine the causal relationship between elevation of homocysteine levels in the blood and the risk of coronary heart disease. Their data suggest that an increase in homocysteine levels is not likely to result in an increase in risk of coronary heart disease.

Gene Dosage of the Common Variant 9p21 Predicts Severity of Coronary Artery Disease

OBJECTIVES The purpose of this study was to test the hypothesis that 9p21 gene dosage determines the severity of coronary artery disease (CAD). BACKGROUND The 9p21 locus is the first common genetic variant to associate with risk of CAD and/or myocardial infarction in multiple studies. METHODS A cross-sectional study examined nondiabetic patients with CAD defined by coronary angiography to have at least 1 epicardial stenosis >50%. In all, 950 patients with early onset CAD (age 56.1 +/- 9.6 years) and an independent sample of 764 patients with late onset CAD (age 70.0 +/- 8.0 years) were enrolled from the cardiac catheterization laboratories at the University of Ottawa Heart Institute from April 15, 2006, to August 15, 2008, and genotyped for the single nucleotide polymorphism rs1333049 9p21 risk variant. Angiographers were blinded to genotype. The association between 9p21 risk genotype and the proportion of patients with 3-vessel disease, 1-vessel disease, left main trunk disease, and coronary artery bypass graft surgery was tested, as was its association with the modified Gensini and Duke coronary scoring indexes. RESULTS Among younger CAD cases, 3-vessel disease demonstrated a strong, direct association with 9p21 gene dosage (p = 4.26 x 10(-4)). Conversely, 1-vessel disease demonstrated a strong inverse association with increasing gene dosage (p = 2.41 x 10(-5)). In the replication sample, gene dosage also predicted 3-vessel disease (p = 6.51 x 10(-6)). Left main trunk disease and coronary artery bypass graft surgery demonstrated a direct strong association with gene dosage (p = 3.66 x 10(-4)) and (p = 2.42 x 10(-2)), respectively. Gene dosage demonstrated a strong, direct association with both the modified Gensini (p < 0.0001) and modified Duke (p = 3 x 10(-4)) coronary scores. Risk variant 9p21 did not associate with myocardial infarction once stratified for disease severity. CONCLUSIONS Gene dosage of the common risk variant 9p21 predicts the severity of coronary atheromatous burden.