Maximum number of alcoholic drinks consumed in a 24-h period (maxdrinks) is a heritable (> 50%) trait and is strongly correlated with vulnerability to excessive alcohol consumption and subsequent alcohol dependence (AD). Several genome-wide association studies (GWAS) have studied alcohol dependence, but few have concentrated on excessive alcohol consumption. We performed two GWAS using maxdrinks as an excessive alcohol consumption phenotype: one in 118 extended families (N=2322) selected from the Collaborative Study on the Genetics of Alcoholism (COGA), and the other in a case-control sample (N=2593) derived from the Study of Addiction: Genes and Environment (SAGE). The strongest association in the COGA families was detected with rs9523562 (p = 2.1×10−6) located in an intergenic region on chromosome 13q31.1; the strongest association in the SAGE dataset was with rs67666182 (p = 7.1×10−7), located in an intergenic region on chromosome 8. We also performed a meta-analysis with these two GWAS and demonstrated evidence of association in both datasets for the LMO1 (p = 7.2×10−7) and PLCL1 genes (p = 4.1×10−6) with increased maxdrinks. A variant in AUTS2 and variants in INADL, C15orf32 and HIP1 that were associated with measures of alcohol consumption in a meta-analysis of GWAS studies and a GWAS of alcohol consumption factor score also showed nominal association in the current meta-analysis. The present study has identified several loci that warrant further examination in independent samples. Among the top SNPs in each of the dataset (p≤10−4) far more showed the same direction of effect in the other dataset than would be expected by chance (p = 2×10−3, 3×10−6), suggesting that there are true signals among these top SNPs, even though no SNP reached genome-wide levels of significance.
Alcohol consumption; maximum number of alcoholic drinks; GWAS; COGA; SAGE
Discrete time survival analysis (DTSA) was used to assess the age-specific association of event related oscillations (EROs) and CHRM2 gene variants on the onset of regular alcohol use and alcohol dependence. The subjects were 2938 adolescents and young adults ages 12 to 25. Results showed that the CHRM2 gene variants and ERO risk factors had hazards which varied considerably with age. The bulk of the significant age-specific associations occurred in those whose age of onset was under 16. These associations were concentrated in those subjects who at some time took an illicit drug. These results are consistent with studies which associate greater rates of alcohol dependence among those who begin drinking at an early age. The age specificity of the genetic and neurophysiological factors is consistent with recent studies of adolescent brain development, which locate an interval of heightened vulnerability to substance use disorders in the early to mid teens.
alcoholism; CHRM2; survival analysis; ERO; genetics; adolescents
Several studies have identified genes associated with alcohol use disorders, but the variation in each of these genes explains only a small portion of the genetic vulnerability. The goal of the present study was to perform a genome-wide association study (GWAS) in extended families from the Collaborative Study on the Genetics of Alcoholism (COGA) to identify novel genes affecting risk for alcohol dependence. To maximize the power of the extended family design we used a quantitative endophenotype, measured in all individuals: number of alcohol dependence symptoms endorsed (symptom count). Secondary analyses were performed to determine if the single nucleotide polymorphisms (SNPs) associated with symptom count were also associated with the dichotomous phenotype, DSM-IV alcohol dependence. This family-based GWAS identified SNPs in C15orf53 that are strongly associated with DSM-IV alcohol (p=4.5×10−8, inflation corrected p=9.4×10−7). Results with DSM-IV alcohol dependence in the regions of interest support our findings with symptom count, though the associations were less significant. Attempted replications of the most promising association results were conducted in two independent samples: non-overlapping subjects from the Study of Addiction: Genes and Environment (SAGE) and the Australian twin-family study of alcohol use disorders (OZALC). Nominal association of C15orf53 with symptom count was observed in SAGE. The variant that showed strongest association with symptom count, rs12912251 and its highly correlated variants (D′=1, r2≥ 0.95), has previously been associated with risk for bipolar disorder.
DSM-IV alcohol dependence symptoms; Family-based GWAS; C15orf53; Quantitative traits
Variants within the gene cluster encoding α3, α5, and β4 nicotinic receptor subunits are major risk factors for substance dependence. The strongest impact on risk is associated with variation in the CHRNA5 gene, where at least two mechanisms are at work: amino acid variation and altered mRNA expression levels. The risk allele of the non-synonymous variant (rs16969968; D398N) primarily occurs on the haplotype containing the low mRNA expression allele. In populations of European ancestry, there are approximately 50 highly correlated variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and the adjacent PSMA4 gene region that are associated with CHRNA5 mRNA levels. It is not clear which of these variants contribute to the changes in CHRNA5 transcript level. Because populations of African ancestry have reduced linkage disequilibrium among variants spanning this gene cluster, eQTL mapping in subjects of African ancestry could potentially aid in defining the functional variants that affect CHRNA5 mRNA levels. We performed quantitative allele specific gene expression using frontal cortices derived from 49 subjects of African ancestry and 111 subjects of European ancestry. This method measures allele-specific transcript levels in the same individual, which eliminates other biological variation that occurs when comparing expression levels between different samples. This analysis confirmed that substance dependence associated variants have a direct cis-regulatory effect on CHRNA5 transcript levels in human frontal cortices of African and European ancestry and identified 10 highly correlated variants, located in a 9 kb region, that are potential functional variants modifying CHRNA5 mRNA expression levels.
A large segment of the population suffers from addiction to alcohol, smoking, or illicit drugs. Not only do substance abuse and addiction pose a threat to health, but the consequences of addiction also impose a social and economic burden on families, communities, and nations. Genome-wide linkage and association studies have been used for addiction research with varying degrees of success. The most well-established genetic factors associated with alcohol dependence are in the genes encoding alcohol dehydrogenase (ADH), which oxidizes alcohol to acetaldehyde, and aldehyde dehydrogenase (ALDH2), which oxidizes acetaldehyde to acetate. Recently emerging genetic studies have linked variants in the genes encoding the α3, α5, and β4 nicotinic acetylcholine receptor subunits to smoking risk. However, the influence of these well-established genetic variants accounts for only a small portion of the heritability of alcohol and nicotine addiction, and it is likely that there are both common and rare risk variants yet to be identified. Newly developed DNA sequencing technologies could potentially advance the detection of rare variants with a larger impact on addiction risk.
addiction; alcoholism; smoking; nicotinic acetylcholine receptors; alcohol metabolizing genes
Excessive alcohol use is the third leading cause of preventable death and is highly correlated with alcohol dependence, a heritable phenotype. Many genetic factors for alcohol dependence have been found, but many remain unknown. In search of additional genetic factors, we examined the association between DSM-IV alcohol dependence and all common copy number variations (CNV) with good reliability in the Study of Addiction: Genetics and Environment (SAGE).
All participants in SAGE were interviewed using the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA), as a part of three contributing studies. 2,610 non-Hispanic European American samples were genotyped on the Illumina Human 1M array. We performed CNV calling by CNVpartition, PennCNV and QuantiSNP and only CNVs identified by all three software programs were examined. Association was conducted with the CNV (as a deletion/duplication) as well as with probes in the CNV region. Quantitative polymerase chain reaction (qPCR) was used to validate the CNVs in the laboratory.
CNVs in 6q14.1 (P= 1.04 × 10−6) and 5q13.2 (P= 3.37 × 10−4) were significantly associated with alcohol dependence after adjusting multiple tests. On chromosome 5q13.2 there were multiple candidate genes previously associated with various neurological disorders. The region on chromosome 6q14.1 is a gene desert that has been associated with mental retardation, and language delay. The CNV in 5q13.2 was validated whereas only a component of the CNV on 6q14.1 was validated by qPCR. Thus, the CNV on 6q14.1 should be viewed with caution.
This is the first study to show an association between DSM-IV alcohol dependence and CNVs. CNVs in regions previously associated with neurological disorders may be associated with alcohol dependence.
Copy Number Variations; Alcohol dependence; CNV Accuracy
Event-related oscillations (EROs) represent highly heritable neuroelectric correlates of cognitive processes that manifest deficits in alcoholics and in offspring at high risk to develop alcoholism. Theta ERO to targets in the visual oddball task has been shown to be an endophenotype for alcoholism. A family-based genome-wide association study was performed for the frontal theta ERO phenotype using 634583 autosomal single nucleotide polymorphisms (SNPs) genotyped in 1560 family members from 117 families densely affected by alcohol use disorders, recruited in the Collaborative Study on the Genetics of Alcoholism. Genome-wide significant association was found with several SNPs on chromosome 21 in KCNJ6 (a potassium inward rectifier channel; KIR3.2/GIRK2), with the most significant SNP at P = 4.7 × 10-10). The same SNPs were also associated with EROs from central and parietal electrodes, but with less significance, suggesting that the association is frontally focused. One imputed synonymous SNP in exon 4, highly correlated with our top three SNPs, was significantly associated with the frontal theta ERO phenotype. These results suggest KCNJ6 or its product GIRK2 account for some of the variations in frontal theta band oscillations. GIRK2 receptor activation contributes to slow inhibitory postsynaptic potentials that modulate neuronal excitability, and therefore influence neuronal networks.
Both linkage and association studies have been successfully applied to identify disease susceptibility genes with genetic markers such as microsatellites and Single Nucleotide Polymorphisms (SNPs). As one of the traditional family-based studies, the Transmission/Disequilibrium Test (TDT) measures the over-transmission of an allele in a trio from its heterozygous parents to the affected offspring and can be potentially useful to identify genetic determinants for complex disorders. However, there is reduced information when complete trio information is unavailable. In this study, we developed a novel approach to “infer” the transmission of SNPs by combining both the linkage and association data, which uses microsatellite markers from families informative for linkage together with SNP markers from the offspring who are genotyped for both linkage and a Genome-Wide Association Study (GWAS). We generalized the traditional TDT to process these inferred dosage probabilities, which we name as the dosage-TDT (dTDT). For evaluation purpose, we developed a simulation procedure to assess its operating characteristics. We applied the dTDT to the simulated data and documented the power of the dTDT under a number of different realistic scenarios. Finally, we applied our methods to a family study of alcohol dependence (COGA) and performed individual genotyping on complete families for the top signals. One SNP (rs4903712 on chromosome 14) remained significant after correcting for multiple testing Methods developed in this study can be adapted to other platforms and will have widespread applicability in genomic research when case-control GWAS data are collected in families with existing linkage data.
Cigarette smoking is a common addiction that increases the risk for many diseases, including lung cancer and chronic obstructive pulmonary disease. Genome-wide association studies (GWAS) have successfully identified and validated several susceptibility loci for nicotine consumption and dependence. However, the trait variance explained by these genes is only a small fraction of the estimated genetic risk. Pathway analysis complements single marker methods by including biological knowledge into the evaluation of GWAS, under the assumption that causal variants lie in functionally related genes, enabling the evaluation of a broad range of signals. Our approach to the identification of pathways enriched for multiple genes associated with smoking quantity includes the analysis of two studies and the replication of common findings in a third dataset. This study identified pathways for the cholinergic receptors, which included SNPs known to be genome-wide significant; as well as novel pathways, such as genes involved in the sensory perception of smell, that do not contain any single SNP that achieves that stringent threshold.
Copy number variations (CNVs) are a major source of alterations among individuals and are a potential risk factor in many diseases. Numerous diseases have been linked to deletions and duplications of these chromosomal segments. Data from genome-wide association studies and other microarrays may be used to identify CNVs by several different computer programs, but the reliability of the results has been questioned.
To help researchers reduce the number of false-positive CNVs that need to be followed up with laboratory testing, we evaluated the relative performance of CNVPartition, PennCNV and QuantiSNP, and developed a statistical method for estimating sensitivity and positive predictive values of CNV calls and tested it on 96 duplicate samples in our dataset.
We found that the positive predictive rate increases with the number of probes in the CNV and the size of the CNV, with the highest positive predicted rates in CNVs of at least 500 kb and at least 100 probes. Our analysis also indicates that identifying CNVs reported by multiple programs can greatly improve the reproducibility rate and the positive predicted rate.
Our methods can be used by investigators to identify CNVs in genome-wide data with greater reliability.
Accuracy; Copy number variations; False positives; Genome-wide association studies
Several genome-wide association and candidate gene studies have linked chromosome 15q24–q25.1 (a region including the CHRNA5-CHRNA3-CHRNB4 gene cluster) with alcohol dependence, nicotine dependence and smoking-related illnesses such as lung cancer and chronic obstructive pulmonary disease. To further examine the impact of these genes on the development of substance use disorders, we tested whether variants within and flanking the CHRNA5-CHRNA3-CHRNB4 gene cluster affect the transition to daily smoking (individuals who smoked cigarettes 4 or more days per week) in a cross sectional sample of adolescents and young adults from the COGA (Collaborative Study of the Genetics of Alcoholism) families. Subjects were recruited from families affected with alcoholism (either as a first or second degree relative) and the comparison families. Participants completed the SSAGA interview, a comprehensive assessment of alcohol and other substance use and related behaviors. Using the Quantitative trait disequilibrium test (QTDT) significant association was detected between age at onset of daily smoking and variants located upstream of CHRNB4. Multivariate analysis using a Cox proportional hazards model further revealed that these variants significantly predict the age at onset of habitual smoking among daily smokers. These variants were not in high linkage disequilibrium (0.28
CHRNA5, encoding the nicotinic α5 subunit, is implicated in multiple disorders, including nicotine addiction and lung cancer. Previous studies demonstrate significant associations between promoter polymorphisms and CHRNA5 mRNA expression, but the responsible sequence variants remain uncertain. To search for cis-regulatory variants, we measured allele-specific mRNA expression of CHRNA5 in human prefrontal cortex autopsy tissues and scanned the CHRNA5 locus for regulatory variants. A cluster of six frequent single nucleotide polymorphisms (rs1979905, rs1979906, rs1979907, rs880395, rs905740, and rs7164030), in complete linkage disequilibrium, fully account for a >2.5-fold allelic expression difference and a fourfold increase in overall CHRNA5 mRNA expression. This proposed enhancer region resides more than 13 kilobases upstream of the CHRNA5 transcription start site. The same upstream variants failed to affect CHRNA5 mRNA expression in peripheral blood lymphocytes, indicating tissue-specific gene regulation. Other promoter polymorphisms were also correlated with overall CHRNA5 mRNA expression in the brain, but were inconsistent with allelic mRNA expression ratios, a robust and proximate measure of cis-regulatory variants. The enhancer region and the nonsynonymous polymorphism rs16969968 generate three main haplotypes that alter the risk of developing nicotine dependence. Ethnic differences in linkage disequilibrium across the CHRNA5 locus require consideration of the upstream enhancer variants when testing clinical associations.
Nicotinic receptor; alpha5 subunit; gene expression; nicotine dependence; lung cancer; enhancer
CHRNA5, encoding the nicotinic α5 subunit, is implicated in multiple disorders, including nicotine addiction and lung cancer. Previous studies demonstrate significant associations between promoter polymorphisms and CHRNA5 mRNA expression, but the responsible sequence variants remain uncertain. To search for cis-regulatory variants, we measured allele-specific mRNA expression of CHRNA5 in human prefrontal cortex autopsy tissues and scanned the CHRNA5 locus for regulatory variants. A cluster of six frequent single-nucleotide polymorphisms (rs1979905, rs1979906, rs1979907, rs880395, rs905740, and rs7164030), in complete linkage disequilibrium (LD), fully account for a >2.5-fold allelic expression difference and a fourfold increase in overall CHRNA5 mRNA expression. This proposed enhancer region resides more than 13 kilobases upstream of the CHRNA5 transcription start site. The same upstream variants failed to affect CHRNA5 mRNA expression in peripheral blood lymphocytes, indicating tissue-specific gene regulation. Other promoter polymorphisms were also correlated with overall CHRNA5 mRNA expression in the brain, but were inconsistent with allelic mRNA expression ratios, a robust and proximate measure of cis-regulatory variants. The enhancer region and the nonsynonymous polymorphism rs16969968 generate three main haplotypes that alter the risk of developing nicotine dependence. Ethnic differences in LD across the CHRNA5 locus require consideration of upstream enhancer variants when testing clinical associations.
nicotinic receptor; α5 subunit; gene expression; nicotine dependence; lung cancer; enhancer
Results 1-14 (14)
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