A novel genetic marker of decreased inflammation and improved survival after acute myocardial infarction.

The CHRNA5 gene encodes a neurotransmitter receptor subunit involved in multiple processes, including cholinergic autonomic nerve activity and inflammation. Common variants in CHRNA5 have been linked with atherosclerotic cardiovascular disease. Association of variation in CHRNA5 and specific haplotypes with cardiovascular outcomes has not been described. The aim of this study was to examine the association of CHRNA5 haplotypes with gene expression and mortality among patients with acute myocardial infarction (AMI) and explore potential mechanisms of this association. Patients (N = 2054) hospitalized with AMI were genotyped for two common variants in CHRNA5. Proportional hazard models were used to estimate independent association of CHRNA5 haplotype with 1-year mortality. Both individual variants were associated with mortality (p = 0.0096 and 0.0004, respectively) and were in tight LD (D' = 0.99). One haplotype, HAP3, was associated with decreased mortality one year after AMI (adjusted HR = 0.42, 95% CI 0.26, 0.68; p = 0.0004). This association was validated in an independent cohort (N = 637) of post-MI patients (adjusted HR = 0.23, 95% CI 0.07, 0.79; p = 0.019). Differences in CHRNA5 expression by haplotype were investigated in human heart samples (n = 28). Compared with non-carriers, HAP3 carriers had threefold lower cardiac CHRNA5 mRNA expression (p = 0.023). Circulating levels of the inflammatory marker hsCRP were significantly lower in HAP3 carriers versus non-carriers (3.43 ± 4.2 versus 3.91 ± 5.1; p = 0.0379). Activation of the inflammasome, an important inflammatory complex involved in cardiovascular disease that is necessary for release of the pro-inflammatory cytokine IL-1 ß, was assessed in bone marrow-derived macrophages (BMDM) from CHRNA5 knockout mice and wild-type controls. In BMDM from CHRNA5 knockout mice, IL-1ß secretion was reduced by 50% compared to wild-type controls (p = 0.004). Therefore, a common haplotype of CHRNA5 that results in reduced cardiac expression of CHRNA5 and attenuated macrophage inflammasome activation is associated with lower mortality after AMI. These results implicate CHRNA5 and the cholinergic anti-inflammatory pathway in survival following AMI.

Smoking Interacts With CHRNA5, a Nicotinic Acetylcholine Receptor Subunit Gene, to Influence the Risk of IBD-Related Surgery.

Background: Inflammatory bowel disease (IBD) is a chronic luminal disease with genetic and environmental factors affecting phenotype. This study evaluated the relationship between CHRNA5, a nicotinic receptor subunit gene, and smoking in predicting IBD-related surgery as well as the relationship between CHRNA5 and nicotine dependence. Methods: Participants completed a smoking questionnaire and were genotyped for CHRNA5 rs16969968. Demographic and clinical data were obtained from medical records. Wilcoxon, ANOVA, Chi square, and Fisher's exact tests were used for comparisons. Logistic regression was used to evaluate the effect of clinical and genetic predictors on surgery, stratified by disease subtype given paradoxical effects of smoking. Kaplan-Meier curves were used to examine the effect of smoking and genotype on time to surgery. (Significance: P < 0.05 for main effects; P < 0.2 for interaction terms). Results: 400 (65.8%) patients had Crohn's disease (CD) and 208 (34.2%) had ulcerative colitis (UC). 298 (49%) underwent an IBD-related surgery. There was a trend towards significance between rs16969968 and smoking behavior (smoking status [P = 0.05], nicotine dependence [AA > AG > GG; P = 0.08]). Smoking and genotype were not independently associated with surgery in UC or CD. However, interaction between rs16969968 and smoking in predicting surgery was observed for both UC (OR = 2.72; P = 0.05) and CD (OR = 2.88; P = 0.1). CHRNA5 genotype, but not smoking, predicted time to surgery in patients with UC (P = 0.007) but not in patients with CD. The interaction between smoking and genotype was not significantly associated with time to surgery in UC or CD. Conclusions: The CHRNA5 rs16969968 A variant interacts with smoking to influence IBD-related surgery. 10.1093/ibd/izx094_video1izx094.video15775248538001.

Cytochrome p450 gene variants, race, and mortality among clopidogrel-treated patients after acute myocardial infarction.

BACKGROUND: Clopidogrel is recommended after acute myocardial infarction but has variable efficacy and safety, in part related to the effect of cytochrome P450 (CYP) polymorphisms on its metabolism. The effect of CYP polymorphisms on cardiovascular events among clopidogrel-treated patients after acute myocardial infarction remains controversial, and no studies to date have investigated the association of CYP variants with outcomes in black patients. METHODS AND RESULTS: Subjects (2732: 2062 whites; 670 blacks) hospitalized with acute myocardial infarction enrolled in the prospective, multicenter TRIUMPH study were genotyped for CYP polymorphisms. The majority of whites (79%) and blacks (64.4%) were discharged on clopidogrel. Among whites, carriers of the loss-of-function CYP2C19*2 allele had significantly increased 1-year mortality (adjusted hazards ratio [HR]: 1.70; confidence interval [CI]: 1.01-2.86; P=0.046) and a trend toward increased rate of recurrent MI (adjusted HR: 2.10; CI: 0.95-4.63; P=0.066). Among blacks, increased 1-year mortality was associated with the gain-of-function CYP2C19*17 allele (adjusted HR for *1/*17 versus *1/*1: 2.02; CI: 0.92-4.44; *17/*17 versus *1/*1: 8.97; CI: 3.34-24.10; P<0.0001) and the CYP1A2*1C allele (adjusted HR for *1/*1C versus *1/*1: 1.89; CI: 0.85-4.22; *1C/*1C versus *1/*1: 4.96; CI: 1.69-14.56; P=0.014). Bleeding events were significantly more common among black carriers of CYP2C19*17 or CYP1A2*1C. CONCLUSIONS: Both loss-of-function and gain-of-function CYP polymorphisms affecting clopidogrel metabolism are associated with increased mortality among clopidogrel-treated patients after acute myocardial infarction; the specific polymorphism and the putative mechanism vary according to race.

Population-based rare variant detection via pooled exome or custom hybridization capture with or without individual indexing.

BACKGROUND: Rare genetic variation in the human population is a major source of pathophysiological variability and has been implicated in a host of complex phenotypes and diseases. Finding disease-related genes harboring disparate functional rare variants requires sequencing of many individuals across many genomic regions and comparing against unaffected cohorts. However, despite persistent declines in sequencing costs, population-based rare variant detection across large genomic target regions remains cost prohibitive for most investigators. In addition, DNA samples are often precious and hybridization methods typically require large amounts of input DNA. Pooled sample DNA sequencing is a cost and time-efficient strategy for surveying populations of individuals for rare variants. We set out to 1) create a scalable, multiplexing method for custom capture with or without individual DNA indexing that was amenable to low amounts of input DNA and 2) expand the functionality of the SPLINTER algorithm for calling substitutions, insertions and deletions across either candidate genes or the entire exome by integrating the variant calling algorithm with the dynamic programming aligner, Novoalign. RESULTS: We report methodology for pooled hybridization capture with pre-enrichment, indexed multiplexing of up to 48 individuals or non-indexed pooled sequencing of up to 92 individuals with as little as 70 ng of DNA per person. Modified solid phase reversible immobilization bead purification strategies enable no sample transfers from sonication in 96-well plates through adapter ligation, resulting in 50% less library preparation reagent consumption. Custom Y-shaped adapters containing novel 7 base pair index sequences with a Hamming distance of ≥2 were directly ligated onto fragmented source DNA eliminating the need for PCR to incorporate indexes, and was followed by a custom blocking strategy using a single oligonucleotide regardless of index sequence. These results were obtained aligning raw reads against the entire genome using Novoalign followed by variant calling of non-indexed pools using SPLINTER or SAMtools for indexed samples. With these pipelines, we find sensitivity and specificity of 99.4% and 99.7% for pooled exome sequencing. Sensitivity, and to a lesser degree specificity, proved to be a function of coverage. For rare variants (≤2% minor allele frequency), we achieved sensitivity and specificity of ≥94.9% and ≥99.99% for custom capture of 2.5 Mb in multiplexed libraries of 22-48 individuals with only ≥5-fold coverage/chromosome, but these parameters improved to ≥98.7 and 100% with 20-fold coverage/chromosome. CONCLUSIONS: This highly scalable methodology enables accurate rare variant detection, with or without individual DNA sample indexing, while reducing the amount of required source DNA and total costs through less hybridization reagent consumption, multi-sample sonication in a standard PCR plate, multiplexed pre-enrichment pooling with a single hybridization and lesser sequencing coverage required to obtain high sensitivity.

Adrenergic-pathway gene variants influence beta-blocker-related outcomes after acute coronary syndrome in a race-specific manner.

OBJECTIVES: Overcoming racial differences in acute coronary syndrome (ACS) outcomes is a strategic goal for U.S. health care. Genetic polymorphisms in the adrenergic pathway seem to explain some outcome differences by race in other cardiovascular diseases treated with ß-adrenergic receptor blockade (BB). Whether these genetic variants are associated with survival among ACS patients treated with BB, and if this differs by race, is unknown. BACKGROUND: ß-adrenergic receptor blockade after ACS is a measure of quality care, but the effectiveness across racial groups is less clear. METHODS: A prospective cohort of 2,673 ACS patients (2,072 Caucasian; 601 African-American) discharged on BB from 22 U.S. hospitals were followed for 2 years. Subjects were genotyped for polymorphisms in ADRB1, ADRB2, ADRA2C, and GRK5. We used proportional hazards regression to model the effect of genotype on mortality, stratified by race and adjusted for baseline factors. RESULTS: The overall 2-year mortality rate was 7.5% for Caucasians and 16.7% for African Americans. The prognosis associated with different genotypes in these BB-treated patients differed by race. In Caucasians, ADRA2C 322-325 deletion carriers had significantly lower mortality as compared with homozygous individuals lacking the deletion (hazard ratio: 0.46; confidence interval [CI]: 0.21 to 0.99; p = 0.047; race × genotype interaction p = 0.053). In African Americans, the ADRB2 16R allele was associated with significantly increased mortality (hazard ratio for RG vs. GG: 2.10; CI: 1.14 to 3.86; RR vs. GG: 2.65; CI: 1.38 to 5.08; p = 0.013; race × genotype interaction p = 0.096). CONCLUSIONS: Adrenergic pathway polymorphisms are associated with mortality in ACS patients receiving BB in a race-specific manner. Understanding the mechanism by which different genes impact post-ACS mortality differently in Caucasians and African Americans might illuminate opportunities to improve BB therapy in these groups.

PGC-1alpha and ERRalpha target gene downregulation is a signature of the failing human heart.

Heart failure is a cause of significant morbidity and mortality in developed nations, and results from a complex interplay between genetic and environmental factors. To discover gene regulatory networks underlying heart failure, we analyzed DNA microarray data based on left ventricular free-wall myocardium from 59 failing (32 ischemic cardiomyopathy, 27 idiopathic dilated cardiomyopathy) and 33 non-failing explanted human hearts from the Cardiogenomics Consortium. In particular, we sought to investigate cardiac gene expression changes at the level of individual genes, as well as biological pathways which contain groups of functionally related genes. Utilizing a combination of computational techniques, including Comparative Marker Selection and Gene Set Enrichment Analysis, we identified a subset of downstream gene targets of the master mitochondrial transcriptional regulator, peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), whose expression is collectively decreased in failing human hearts. We also observed decreased expression of the key PGC-1alpha regulatory partner, estrogen-related receptor alpha (ERRalpha), as well as ERRalpha target genes which may participate in the downregulation of mitochondrial metabolic capacity. Gene expression of the antiapoptotic Raf-1/extracellular signal-regulated kinase (ERK) pathway was decreased in failing hearts. Alterations in PGC-1alpha and ERRalpha target gene sets were significantly correlated with an important clinical parameter of disease severity - left ventricular ejection fraction, and were predictive of failing vs. non-failing phenotypes. Overall, our results implicate PGC-1alpha and ERRalpha in the pathophysiology of human heart failure, and define dynamic target gene sets sharing known interrelated regulatory mechanisms capable of contributing to the mitochondrial dysfunction characteristic of this disease process.