Distinct loci in the CHRNA5/CHRNA3/CHRNB4 gene cluster are associated with onset of regular smoking.

Neuronal nicotinic acetylcholine receptor (nAChR) genes (CHRNA5/CHRNA3/CHRNB4) have been reproducibly associated with nicotine dependence, smoking behaviors, and lung cancer risk. Of the few reports that have focused on early smoking behaviors, association results have been mixed. This meta-analysis examines early smoking phenotypes and SNPs in the gene cluster to determine: (1) whether the most robust association signal in this region (rs16969968) for other smoking behaviors is also associated with early behaviors, and/or (2) if additional statistically independent signals are important in early smoking. We focused on two phenotypes: age of tobacco initiation (AOI) and age of first regular tobacco use (AOS). This study included 56,034 subjects (41 groups) spanning nine countries and evaluated five SNPs including rs1948, rs16969968, rs578776, rs588765, and rs684513. Each dataset was analyzed using a centrally generated script. Meta-analyses were conducted from summary statistics. AOS yielded significant associations with SNPs rs578776 (beta = 0.02, P = 0.004), rs1948 (beta = 0.023, P = 0.018), and rs684513 (beta = 0.032, P = 0.017), indicating protective effects. There were no significant associations for the AOI phenotype. Importantly, rs16969968, the most replicated signal in this region for nicotine dependence, cigarettes per day, and cotinine levels, was not associated with AOI (P = 0.59) or AOS (P = 0.92). These results provide important insight into the complexity of smoking behavior phenotypes, and suggest that association signals in the CHRNA5/A3/B4 gene cluster affecting early smoking behaviors may be different from those affecting the mature nicotine dependence phenotype.

Examination of genetic variation in GABRA2 with conduct disorder and alcohol abuse and dependence in a longitudinal study.

Previous studies have shown associations between single nucleotide polymorphisms (SNPs) in gamma aminobutyric acid receptor alpha 2 (GABRA2) and adolescent conduct disorder (CD) and alcohol dependence in adulthood, but not adolescent alcohol dependence. The present study was intended as a replication and extension of this work, focusing on adolescent CD, adolescent alcohol abuse and dependence (AAD), and adult AAD. Family based association tests were run using Hispanics and non-Hispanic European American subjects from two independent longitudinal samples. Although the analysis provided nominal support for an association with rs9291283 and AAD in adulthood and CD in adolescence, the current study failed to replicate previous associations between two well replicated GABRA2 SNPs and CD and alcohol dependence. Overall, these results emphasize the utility of including an independent replication sample in the study design, so that the results from an individual sample can be weighted in the context of its reproducibility.

Familial aggregation of tobacco use behaviors among Amish men.

INTRODUCTION: Tobacco use is a complex behavior. The Old Order Amish community offers unique advantages for the study of tobacco use because of homogenous ancestral background, sociocultural similarity, sex-specific social norms regarding tobacco use, and large family size. Tobacco use in the Old Order Amish community is almost exclusively confined to males. METHODS: We examined characteristics of tobacco use and familial aggregation among 1,216 Amish males from cross-sectional prospectively collected data. Outcomes examined included ever using tobacco regularly, current use, quantity of use, duration of use, and frequency of use. RESULTS: Sixteen percent of Amish men were current tobacco users, with the majority reporting cigar use only. Higher rates of tobacco use were found among sons of fathers who smoked compared with sons of fathers who did not smoke (46% vs. 22%, p < .001) as well as among brothers of index cases who smoked compared with brothers of index cases who did not smoke (61% vs. 29%, p < .001). After controlling for shared household effects and age, heritability accounted for 66% of the variance in ever smoking regularly (p = .045). CONCLUSIONS: The familial patterns of tobacco use observed among Amish men highlight the important role of family in propagating tobacco use and support the usefulness of this population for future genetic studies of nicotine addiction.

Multiple independent loci at chromosome 15q25.1 affect smoking quantity: a meta-analysis and comparison with lung cancer and COPD.

Recently, genetic association findings for nicotine dependence, smoking behavior, and smoking-related diseases converged to implicate the chromosome 15q25.1 region, which includes the CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit genes. In particular, association with the nonsynonymous CHRNA5 SNP rs16969968 and correlates has been replicated in several independent studies. Extensive genotyping of this region has suggested additional statistically distinct signals for nicotine dependence, tagged by rs578776 and rs588765. One goal of the Consortium for the Genetic Analysis of Smoking Phenotypes (CGASP) is to elucidate the associations among these markers and dichotomous smoking quantity (heavy versus light smoking), lung cancer, and chronic obstructive pulmonary disease (COPD). We performed a meta-analysis across 34 datasets of European-ancestry subjects, including 38,617 smokers who were assessed for cigarettes-per-day, 7,700 lung cancer cases and 5,914 lung-cancer-free controls (all smokers), and 2,614 COPD cases and 3,568 COPD-free controls (all smokers). We demonstrate statistically independent associations of rs16969968 and rs588765 with smoking (mutually adjusted p-values<10(-35) and <10(-8) respectively). Because the risk alleles at these loci are negatively correlated, their association with smoking is stronger in the joint model than when each SNP is analyzed alone. Rs578776 also demonstrates association with smoking after adjustment for rs16969968 (p<10(-6)). In models adjusting for cigarettes-per-day, we confirm the association between rs16969968 and lung cancer (p<10(-20)) and observe a nominally significant association with COPD (p = 0.01); the other loci are not significantly associated with either lung cancer or COPD after adjusting for rs16969968. This study provides strong evidence that multiple statistically distinct loci in this region affect smoking behavior. This study is also the first report of association between rs588765 (and correlates) and smoking that achieves genome-wide significance; these SNPs have previously been associated with mRNA levels of CHRNA5 in brain and lung tissue.

Pharmacogenomics of anti-platelet and anti-coagulation therapy.

Arterial thrombosis is a major component of vascular disease, especially myocardial infarction (MI) and stroke. Current anti-thrombotic therapies such as warfarin and clopidogrel are effective in inhibiting cardiovascular events; however, there is great inter-individual variability in response to these medications. In recent years, it has been recognized that genetic factors play a significant role in drug response, and, subsequently, common variants in genes responsible for metabolism and drug action have been identified. These discoveries along with new diagnostic targets and therapeutic strategies hold promise for more effective individualized anti-coagulation and anti-platelet therapy.

The CHRNA5/A3/B4 gene cluster and tobacco, alcohol, cannabis, inhalants and other substance use initiation: replication and new findings using mixture analyses.

Multiple studies have provided evidence for genetic associations between single nucleotide polymorphisms (SNPs) located on the CHRNA5/A3/B4 gene cluster and various phenotypes related to Nicotine Dependence (Greenbaum et al. 2009). Only a few studies have investigated other substances of abuse. The current study has two aims, (1) to extend previous findings by focusing on associations between the CHRNA5/A3/B4 gene cluster and age of initiation of several different substances, and (2) to investigate heterogeneity in age of initiation across the different substances. All analyses were conducted with a subset of the Add Health study with available genetic data. The first aim was met by modeling onset of tobacco, alcohol, cannabis, inhalants, and other substance use using survival mixture analysis (SMA). Ten SNPs in CHRNA5/A3/B4 were used to predict phenotypic differences in the risk of onset, and differences between users and non-users. The survival models aim at investigating differences in the risk of initiation across the 5-18 age range for each phenotype separately. Significant or marginally significant genetic effects were found for all phenotypes. The genetic effects were mainly related to the risk of initiation and to a lesser extent to discriminating between users and non-users. To address the second goal, the survival analyses were complemented by a latent class analysis that modeled all phenotypes jointly. One of the ten SNPs was found to predict differences between the early and late onset classes. Taken together, our study provides evidence for a general role of the CHRNA5/A3/B4 gene cluster in substance use initiation that is not limited to nicotine and alcohol.

Externalizing behaviors are associated with SNPs in the CHRNA5/CHRNA3/CHRNB4 gene cluster.

There is strong evidence for shared genetic factors contributing to childhood externalizing disorders and substance abuse. Externalizing disorders often precede early substance experimentation, leading to the idea that individuals inherit a genetic vulnerability to generalized disinhibitory psychopathology. Genetic variation in the CHRNA5/CHRNA3/CHRNB4 gene cluster has been associated with early substance experimentation, nicotine dependence, and other drug behaviors. This study examines whether the CHRNA5/CHRNA3/CHRNB4 locus is correlated also with externalizing behaviors in three independent longitudinally assessed adolescent samples. We developed a common externalizing behavior phenotype from the available measures in the three samples, and tested for association with 10 SNPs in the gene cluster. Significant results were detected in two of the samples, including rs8040868, which remained significant after controlling for smoking quantity. These results expand on previous work focused mainly on drug behaviors, and support the hypothesis that variation in the CHRNA5/CHRNA3/CHRNB4 locus is associated with early externalizing behaviors.

PrecisionFDA Truth Challenge V2: Calling variants from short and long reads in difficult-to-map regions.

The precisionFDA Truth Challenge V2 aimed to assess the state of the art of variant calling in challenging genomic regions. Starting with FASTQs, 20 challenge participants applied their variant-calling pipelines and submitted 64 variant call sets for one or more sequencing technologies (Illumina, PacBio HiFi, and Oxford Nanopore Technologies). Submissions were evaluated following best practices for benchmarking small variants with updated Genome in a Bottle benchmark sets and genome stratifications. Challenge submissions included numerous innovative methods, with graph-based and machine learning methods scoring best for short-read and long-read datasets, respectively. With machine learning approaches, combining multiple sequencing technologies performed particularly well. Recent developments in sequencing and variant calling have enabled benchmarking variants in challenging genomic regions, paving the way for the identification of previously unknown clinically relevant variants.

Association of the 5'-upstream regulatory region of the alpha7 nicotinic acetylcholine receptor subunit gene (CHRNA7) with schizophrenia.

BACKGROUND: The alpha7 neuronal nicotinic acetylcholine receptor subunit gene (CHRNA7) is localized in a chromosomal region (15q14) linked to schizophrenia in multiple independent studies. CHRNA7 was selected as the best candidate gene in the region for a well-documented endophenotype of schizophrenia, the P50 sensory processing deficit, by genetic linkage and biochemical studies. METHODS: Subjects included Caucasian-Non Hispanic and African-American case-control subjects collected in Denver, and schizophrenic subjects from families in the NIMH Genetics Initiative on Schizophrenia. Thirty-five single nucleotide polymorphisms (SNPs) in the 5'-upstream regulatory region of CHRNA7 were genotyped for association with schizophrenia, and for smoking in schizophrenia. RESULTS: The rs3087454 SNP, located at position -1831 bp in the upstream regulatory region of CHRNA7, was significantly associated with schizophrenia in the case-control samples after multiple-testing correction (P=0.0009, African American; P=0.013, Caucasian-Non Hispanic); the association was supported in family members. There was nominal association of this SNP with smoking in schizophrenia. CONCLUSIONS: The data support association of regulatory region polymorphisms in the CHRNA7 gene with schizophrenia.

Heritability of plasma neopterin levels in the Old Order Amish.

BACKGROUND: We examined the heritability of neopterin, a biomarker for cell-mediated immunity and oxidative stress, and potentially for psychiatric disorders, in the Old Order Amish. METHODS: Plasma neopterin levels were determined in 2015 Old Order Amish adults. Quantitative genetic procedures were used to estimate heritability of neopterin. RESULTS: Heritability of log-neopterin was estimated at 0.07 after adjusting for age, gender, and household (p=0.03). The shared household effect was 0.06 (p<0.02). CONCLUSIONS: We found a low heritability of neopterin and small household effect, suggesting that non-household environmental factors are more important determinants of variance of neopterin levels in the Amish.

Chronotype and seasonality: morningness is associated with lower seasonal mood and behavior changes in the Old Order Amish.

BACKGROUND: Several studies documented that lower scores on the Morningness-Eveningness Questionnaire (MEQ) are associated with a higher global seasonality of mood (GSS). As for the Modern Man artificial lighting predominantly extends evening activity and exposure to light, and as evening bright light phase is known to delay circadian rhythms, this chronic exposure could potentially lead to both lower Morningness as well as higher GSS. The aim of the study was to investigate if the MEQ-GSS relationship holds in the Old Order Amish of Lancaster County, PA, a population that does not use network electrical light. METHODS: 489 Old Order Amish adults (47.6% women), with average (SD) age of 49.7 (14.2) years, completed both the Seasonal Pattern Assessment Questionnaire (SPAQ) for the assessment of GSS, and MEQ. Associations between GSS scores and MEQ scores were analyzed using linear models, accounting for age, gender and relatedness by including the relationship matrix in the model as a random effect. RESULTS: GSS was inversely associated with MEQ scores (p=0.006, adjusted). LIMITATIONS: include a potential recall bias associated with self-report questionnaires and no actual light exposure measurements. CONCLUSION: We confirmed the previously reported inverse association between MEQ scores and lower seasonality of mood, for the first time in a population that does not use home network electrical lighting. This result suggests that the association is not a byproduct of exposure to network electric light, and calls for additional research to investigate mechanisms by which Morningness is negatively associated with seasonality.

Seasonality shows evidence for polygenic architecture and genetic correlation with schizophrenia and bipolar disorder.

OBJECTIVE: To test common genetic variants for association with seasonality (seasonal changes in mood and behavior) and to investigate whether there are shared genetic risk factors between psychiatric disorders and seasonality. METHOD: Genome-wide association studies (GWASs) were conducted in Australian (between 1988 and 1990 and between 2010 and 2013) and Amish (between May 2010 and December 2011) samples in whom the Seasonal Pattern Assessment Questionnaire (SPAQ) had been administered, and the results were meta-analyzed in a total sample of 4,156 individuals. Genetic risk scores based on results from prior large GWAS studies of bipolar disorder, major depressive disorder (MDD), and schizophrenia were calculated to test for overlap in risk between psychiatric disorders and seasonality. RESULTS: The most significant association was with rs11825064 (P = 1.7 × 10⁻6, ß = 0.64, standard error = 0.13), an intergenic single nucleotide polymorphism (SNP) found on chromosome 11. The evidence for overlap in risk factors was strongest for schizophrenia and seasonality, with the schizophrenia genetic profile scores explaining 3% of the variance in log-transformed global seasonality scores. Bipolar disorder genetic profile scores were also associated with seasonality, although at much weaker levels (minimum P value = 3.4 × 10⁻³), and no evidence for overlap in risk was detected between MDD and seasonality. CONCLUSIONS: Common SNPs of large effect most likely do not exist for seasonality in the populations examined. As expected, there were overlapping genetic risk factors for bipolar disorder (but not MDD) with seasonality. Unexpectedly, the risk for schizophrenia and seasonality had the largest overlap, an unprecedented finding that requires replication in other populations and has potential clinical implications considering overlapping cognitive deficits in seasonal affective disorders and schizophrenia.

Seasonality of mood and behavior in the Old Order Amish.

BACKGROUND/OBJECTIVE: We examined seasonality and winter seasonal affective disorder (SAD) in the Old Order Amish of Lancaster County, Pennsylvania, a unique population that prohibits use of network electric light in their homes. METHODS: We estimated SAD using the seasonal pattern assessment questionnaire (SPAQ) in 1306 Amish adults and compared the frequencies of SAD and total SAD (i.e., presence of either SAD or subsyndromal-SAD) between men and women, young and old, and awareness of (ever vs. never heard about) SAD. Heritability of global seasonality score (GSS) was estimated using the maximum likelihood method, including a household effect to capture shared environmental effects. RESULTS: The mean (±SD) GSS was 4.36 (±3.38). Prevalence was 0.84% (95% CI: 0.36-1.58) for SAD and 2.59% (95% CI: 1.69-3.73) for total SAD. Heritability of GSS was 0.14±0.06 (SE) (p=0.002) after adjusting for age, gender, and household effects. LIMITATIONS: Limitations include likely overestimation of the rates of SAD by SPAQ, possible selection bias and recall bias, and limited generalizability of the study. CONCLUSIONS: In the Amish, GSS and SAD prevalence were lower than observed in earlier SPAQ-based studies in other predominantly Caucasian populations. Low heritability of SAD suggests dominant environmental effects. The effects of awareness, age and gender on SAD risk were similar as in previous studies. Identifying factors of resilience to SAD in the face of seasonal changes in the Amish could suggest novel preventative and therapeutic approaches to reduce the impact of SAD in the general population.

Multiple genes in the 15q13-q14 chromosomal region are associated with schizophrenia.

OBJECTIVE: The chromosomal region, 15q13-q14, including the α7 nicotinic acetylcholine receptor gene, CHRNA7, is a replicated region for schizophrenia. This study fine-mapped genes at 15q13-q14 to determine whether the association is unique to CHRNA7. METHODS: Family-based and case-control association studies were performed on Caucasian-non-Hispanic and African-American individuals from 120 families as well as 468 individual patients with schizophrenia and 144 well-characterized controls. Single-nucleotide polymorphism (SNP) markers were genotyped, and association analyses carried out for the outcomes of schizophrenia, smoking, and smoking in schizophrenia. RESULTS: Three genes were associated with schizophrenia in both ethnic populations: TRPM1, KLF13, and RYR3. Two SNPs in CHRNA7 were associated with schizophrenia in African-Americans, and a second SNP in CHRNA7 was significant for an association with smoking and smoking in schizophrenia in Caucasians. CONCLUSION: Results of these studies support association of the 15q13-q14 region with schizophrenia. The broad positive association suggests that more than one 15q gene may be contributing to the disorder, either in combination or through a regulatory mechanism.

Increased genetic vulnerability to smoking at CHRNA5 in early-onset smokers.

CONTEXT: Recent studies have shown an association between cigarettes per day (CPD) and a nonsynonymous single-nucleotide polymorphism in CHRNA5, rs16969968. OBJECTIVE: To determine whether the association between rs16969968 and smoking is modified by age at onset of regular smoking. DATA SOURCES: Primary data. STUDY SELECTION: Available genetic studies containing measures of CPD and the genotype of rs16969968 or its proxy. DATA EXTRACTION: Uniform statistical analysis scripts were run locally. Starting with 94,050 ever-smokers from 43 studies, we extracted the heavy smokers (CPD >20) and light smokers (CPD ≤10) with age-at-onset information, reducing the sample size to 33,348. Each study was stratified into early-onset smokers (age at onset ≤16 years) and late-onset smokers (age at onset >16 years), and a logistic regression of heavy vs light smoking with the rs16969968 genotype was computed for each stratum. Meta-analysis was performed within each age-at-onset stratum. DATA SYNTHESIS: Individuals with 1 risk allele at rs16969968 who were early-onset smokers were significantly more likely to be heavy smokers in adulthood (odds ratio [OR] = 1.45; 95% CI, 1.36-1.55; n = 13,843) than were carriers of the risk allele who were late-onset smokers (OR = 1.27; 95% CI, 1.21-1.33, n = 19,505) (P = .01). CONCLUSION: These results highlight an increased genetic vulnerability to smoking in early-onset smokers.

Mutational load analysis of unrelated individuals.

Evolutionary genetic models predict that the cumulative effect of rare deleterious mutations across the genome-known as mutational load burden-increases the susceptibility to complex disease. To test the mutational load burden hypothesis, we adopted a two-tiered approach: assessing the impact of whole-exome minor allele load burden and then conducting individual-gene screening. For our primary analysis, we examined various minor allele frequency (MAF) thresholds and weighting schemes to examine the overall effect of minor allele load on affection status. We found a consistent association between minor allele load and affection status, but this effect did not markedly increase within rare and/or functional single-nucleotide polymorphisms (SNPs). Our follow-up analysis considered minor allele load in individual genes to see whether only one or a few genes were driving the overall effect. Examining our most significant result-minor allele load of nonsynonymous SNPs with MAF < 2.4%-we detected no significantly associated genes after Bonferroni correction for multiple testing. After moderately significant genes (p < 0.05) were removed, the overall effect of rare nonsynonymous allele load remained significant. Overall, we did not find clear support for mutational load burden on affection status; however, these results are ultimately dependent on and limited by the nature of the Genetic Analysis Workshop 17 simulation.