Evidence for the role of EPHX2 gene variants in anorexia nervosa.

Anorexia nervosa (AN) and related eating disorders are complex, multifactorial neuropsychiatric conditions with likely rare and common genetic and environmental determinants. To identify genetic variants associated with AN, we pursued a series of sequencing and genotyping studies focusing on the coding regions and upstream sequence of 152 candidate genes in a total of 1205 AN cases and 1948 controls. We identified individual variant associations in the Estrogen Receptor-ß (ESR2) gene, as well as a set of rare and common variants in the Epoxide Hydrolase 2 (EPHX2) gene, in an initial sequencing study of 261 early-onset severe AN cases and 73 controls (P=0.0004). The association of EPHX2 variants was further delineated in: (1) a pooling-based replication study involving an additional 500 AN patients and 500 controls (replication set P=0.00000016); (2) single-locus studies in a cohort of 386 previously genotyped broadly defined AN cases and 295 female population controls from the Bogalusa Heart Study (BHS) and a cohort of 58 individuals with self-reported eating disturbances and 851 controls (combined smallest single locus P<0.01). As EPHX2 is known to influence cholesterol metabolism, and AN is often associated with elevated cholesterol levels, we also investigated the association of EPHX2 variants and longitudinal body mass index (BMI) and cholesterol in BHS female and male subjects (N=229) and found evidence for a modifying effect of a subset of variants on the relationship between cholesterol and BMI (P<0.01). These findings suggest a novel association of gene variants within EPHX2 to susceptibility to AN and provide a foundation for future study of this important yet poorly understood condition.

Genome-wide meta-analyses of smoking behaviors in African Americans.

The identification and exploration of genetic loci that influence smoking behaviors have been conducted primarily in populations of the European ancestry. Here we report results of the first genome-wide association study meta-analysis of smoking behavior in African Americans in the Study of Tobacco in Minority Populations Genetics Consortium (n = 32,389). We identified one non-coding single-nucleotide polymorphism (SNP; rs2036527[A]) on chromosome 15q25.1 associated with smoking quantity (cigarettes per day), which exceeded genome-wide significance (ß = 0.040, s.e. = 0.007, P = 1.84 × 10(-8)). This variant is present in the 5'-distal enhancer region of the CHRNA5 gene and defines the primary index signal reported in studies of the European ancestry. No other SNP reached genome-wide significance for smoking initiation (SI, ever vs never smoking), age of SI, or smoking cessation (SC, former vs current smoking). Informative associations that approached genome-wide significance included three modestly correlated variants, at 15q25.1 within PSMA4, CHRNA5 and CHRNA3 for smoking quantity, which are associated with a second signal previously reported in studies in European ancestry populations, and a signal represented by three SNPs in the SPOCK2 gene on chr10q22.1. The association at 15q25.1 confirms this region as an important susceptibility locus for smoking quantity in men and women of African ancestry. Larger studies will be needed to validate the suggestive loci that did not reach genome-wide significance and further elucidate the contribution of genetic variation to disparities in cigarette consumption, SC and smoking-attributable disease between African Americans and European Americans.

Varenicline for smoking cessation: nausea severity and variation in nicotinic receptor genes.

This study evaluated association between common and rare sequence variants in 10 nicotinic acetylcholine receptor subunit genes and the severity of nausea 21 days after initiating the standard, Food and Drug Administration-approved varenicline regimen for smoking cessation. A total of 397 participants from a randomized clinical effectiveness trial with complete clinical and DNA resequencing data were included in the analysis (mean age=49.2 years; 68.0% female). Evidence for significant association between common sequence variants in CHRNB2 and nausea severity was obtained after adjusting for age, gender and correlated tests (all P(ACT)<0.05). Individuals with the minor allele of CHRNB2 variants experienced less nausea than did those without the minor allele, consistent with previously reported findings for CHRNB2 and the occurrence of nausea and dizziness as a consequence of first smoking attempt in adolescents, and with the known neurophysiology of nausea. As nausea is the most common reason for discontinuance of varenicline, further pharmacogenetic investigations are warranted.

Gender-stratified gene and gene-treatment interactions in smoking cessation.

We conducted gender-stratified analyses on a systems-based candidate gene study of 53 regions involved in nicotinic response and the brain-reward pathway in two randomized clinical trials of smoking cessation treatments (placebo, bupropion, transdermal and nasal spray nicotine replacement therapy). We adjusted P-values for multiple correlated tests, and used a Bonferroni-corrected α-level of 5 × 10(-4) to determine system-wide significance. Four single-nucleotide polymorphisms (rs12021667, rs12027267, rs6702335, rs12039988; r2 > 0.98) in erythrocyte membrane protein band 4.1 (EPB41) had a significant male-specific marginal association with smoking abstinence (odds ratio (OR) = 0.5; 95% confidence interval (CI): 0.3-0.6) at end of treatment (adjusted P < 6 × 10(-5)). rs806365 in cannabinoid receptor 1 (CNR1) had a significant male-specific gene-treatment interaction at 6-month follow-up (adjusted P = 3.9 × 10(-5)); within males using nasal spray, rs806365 was associated with a decrease in odds of abstinence (OR = 0.04; 95% CI: 0.01-0.2). While the role of CNR1 in substance abuse has been well studied, we report EPB41 for the first time in the nicotine literature.

Epstein-Barr virus microRNAs and lung cancer.

BACKGROUND: We conducted the first analysis of viral microRNAs (miRNAs) in lung cancer, with a focus on Epstein-Barr virus (EBV). METHODS: We evaluated viral miRs with a two-channel oligo-array targeting mature, anti-sense miRNAs in 290 cases. In 48 cases, we compared microarray and real-time quantitative PCR (qPCR) expression for three EBV miRNAs. We tested for EBV DNA, RNA, and protein in tumour tissue from six cases with and six cases without strong qPCR-based evidence of EBV miRNAs. RESULTS: The EBV miRNAs strongly differentiated between adenocarcinoma and squamous cell carcinoma using the microarray (P<0.01 for 9 out of 16 EBV miRNAs). However, microarray and qPCR measurements of BART1, BART2, and BHRF1-3 expression were not significantly correlated (P=0.53, 0.94, and 0.47, respectively). Although qPCR provided substantial evidence of EBV miRNAs in 7 out of 48 cases, only 1 of these 7 cases had detectable EBV DNA in tumour tissue. None had detectable EBV RNA or protein by histochemical stains. CONCLUSION: In a comprehensive evaluation of EBV miRNA, DNA, RNA, and protein in lung cancer, we found little evidence of EBV in lung tumour tissue. Discrepancies between microarray- and qPCR-based strategies highlight the difficulty of validating molecular markers of disease. Our results do not support a role of EBV in lung cancer.

Inherited polymorphisms in the RNA-mediated interference machinery affect microRNA expression and lung cancer survival.

BACKGROUND: MicroRNAs (miRs) have an important role in lung carcinogenesis and progression. Single-nucleotide polymorphisms (SNPs) in genes involved in miR biogenesis may affect miR expression in lung tissue and be associated with lung carcinogenesis and progression. METHODS: we analysed 12 SNPs in POLR2A, RNASEN and DICER1 genes in 1984 cases and 2073 controls from the Environment And Genetics in Lung cancer Etiology (EAGLE) study. We investigated miR expression profiles in 165 lung adenocarcinoma (AD) and 125 squamous cell carcinoma tissue samples from the same population. We used logistic and Cox regression models to examine the association of individual genotypes and haplotypes with lung cancer risk and with lung cancer-specific survival, respectively. SNPs-miR expression associations in cases were assessed using two-sample t-tests and global permutation tests. RESULTS: a haplotype in RNASEN (Drosha) was significantly associated with shorter lung cancer survival (hazard ratio=1.86, 95% CI=1.19-2.92, P=0.007). In AD cases, a SNP within the same haplotype was associated with reduced RNASEN mRNA expression (P=0.013) and with miR expression changes (global P=0.007) of miRs known to be associated with cancer (e.g., let-7 family, miR-21, miR-25, miR-126 and miR15a). CONCLUSION: inherited variation in the miR-processing machinery can affect miR expression levels and lung cancer-specific survival.

Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies.

When cytobrush buccal cell samples have been collected as a genomic DNA (gDNA) source for an epidemiological study, whole genome amplification (WGA) can be critical to maintain sufficient DNA for genotyping. We evaluated REPLI-g WGA using gDNA from two paired cytobrushes (cytobush 'A' kept in a cell lysis buffer, and 'B' dried and kept at room temperature for 3 days, and frozen until DNA extraction) in a pilot study (n=21), and from 144 samples collected by mail in a breast cancer study. WGA success was assessed as the per cent completion/concordance of STR/SNP genotypes. Locus amplification bias was assessed using quantitative PCR of 23 human loci. The pilot study showed > 98% completion but low genotype concordance between cytobrush wgaDNA and paired blood gDNA (82% and 84% for cytobrushes A and B, respectively). Substantial amplification bias was observed with significantly lower human gDNA amplification from cytobrush B than A. Using cytobrush gDNA samples from the breast cancer study (n =20), an independent laboratory demonstrated that increasing template gDNA to the REPLI-g reaction improved genotype performance for 49 SNPs; however, average completion and concordance remained below 90%. To reduce genotype misclassification when cytobrush wgaDNA is used, inclusion of paired gDNA/wgaDNA and/or duplicate wgaDNA samples is critical to monitor data quality.

A genome-wide search for loci contributing to smoking and alcoholism.

Using the Collaborative Study on the Genetics of Alcoholism (COGA) data, we performed a sib-pair linkage analysis of two smoking-related traits and one alcoholism phenotype. The first trait, EVRNVR, was a dichotomous one we constructed based on epidemiological definitions of smoking. The second trait, PKYRS, used the quantitative pack-year history provided, and the third trait was the COGA alcoholism classification, ALDX1. There was some evidence for linkage of the EVRNVR trait to regions-on chromosomes 6, 9, and 14. Smaller numbers of loci provided nominal evidence for linkage to PKYRS, although some candidate gene regions were identified. The number of loci identified using EVRNVR suggests that a threshold-based phenotype may better identify loci affecting smoking history. Approximately one-third of the loci that showed evidence for linkage to EVRNVR at a nominal significance level (p < 0.01) also showed evidence for linkage to ALDX1. Some of these regions may represent loci increasing vulnerability to both smoking and alcoholism.

Sib-pair linkage analyses of alcoholism: dichotomous and quantitative measures.

We hypothesized that a quantitative alcoholism trait would have greater power than the Collaborative Study on the Genetics of Alcoholism (COGA) dichotomous alcoholism traits, ALDX1 and ALDX2, to detect putative alcoholism loci. To test this, we performed nonparametric sib-pair linkage analysis to screen 285 polymorphic autosomal markers for evidence of linkage to ALDX1, ALDX2, and a quantitative trait, QUANT, defined from the 11 COGA latent class variables. We also examined the effects on the analyses of including covariates (sex, age, and pack-years of smoking) and of transforming QUANT (log and square root). ALDX1 and ALDX2 showed the greatest evidence for linkage to markers on chromosome 1, by both the affected sib-pair and the Haseman-Elston tests. Regions of interest were also identified on chromosomes 4, 8, 16, and 17. QUANT showed little evidence for linkage to any chromosomal region, having no more significant results than were expected by chance. Including covariates or transforming QUANT had little effect on the analyses. A quantitative trait based on all 37 latent class variables, with each variable appropriately weighted, may have had more power than QUANT to detect genomic regions of relevance to alcoholism.

Cigarette smoking.

Cigarette smoking is the largest preventable risk factor for morbidity and mortality in developed countries. Dramatic changes in the prevalence of cigarette smoking in the second half of this century in the United States (i.e., a reduction among men and an increase among women) have reduced current smoking levels to approximately one quarter of the adult population and have reduced differences in smoking prevalence and smoking-attributable diseases between the sexes. Current smoking in the United States is positively associated with younger age, lower income, reduced educational achievement, and disadvantaged neighborhood environment. Daily smokers smoke cigarettes to maintain nicotine levels in the brain, primarily to avoid the negative effects of nicotine withdrawal, but also to modulate mood. Regular smokers exhibit higher and lower levels of stress and arousal, respectively, than nonsmokers, as well as higher impulsivity and neuroticism trait values. Nicotine dependence is the single most common psychiatric diagnosis in the United States, and substance abuse, major depression, and anxiety disorders are the most prevalent psychiatric comorbid conditions associated with nicotine dependence. Studies in twins have implicated genetic factors that explain most of the variability in vulnerability to smoking and in persistence of the smoking phenotype. Future research into the causes of smoking must take into account these associated demographics, social factors, comorbid psychiatric conditions, and genetic factors to understand this complex human behavior.

The polydeoxyadenylate tract of Alu repetitive elements is polymorphic in the human genome.

To identify DNA polymorphisms that are abundant in the human genome and are detectable by polymerase chain reaction amplification of genomic DNA, we tested the hypothesis that the polydeoxyadenylate tract of the Alu family of repetitive elements is polymorphic among human chromosomes. We analyzed the 3' ends of three specific Alu sequences and found that two (in the adenosine deaminase gene and the beta-globin pseudogene) were polymorphic. This novel class of polymorphisms, termed AluVpA [Alu variable poly(A)] may represent one of the most useful and informative group of DNA markers in the human genome.