A non-synonymous coding polymorphism, rs16969968, of the CHRNA5 gene which encodes the alpha-5 subunit of the nicotinic acetylcholine receptor (nAChR) has been found to be associated with nicotine dependence (20). The goal of the present study is to examine the association of this variant with cocaine dependence.
Genetic association analysis in two, independent samples of unrelated cases and controls; 1.) 504 European-American participating in the Family Study on Cocaine Dependence (FSCD); 2.) 814 European Americans participating in the Collaborative Study on the Genetics of Alcoholsim (COGA).
In the FSCD, there was a significant association between the CHRNA5 variant and cocaine dependence (OR = 0.67 per allele, p = 0.0045, assuming an additive genetic model), but in the reverse direction compared to that previously observed for nicotine dependence. In multivariate analyses that controlled for the effects of nicotine dependence, both the protective effect for cocaine dependence and the previously documented risk effect for nicotine dependence were statistically significant. The protective effect for cocaine dependence was replicated in the COGA sample. In COGA, effect sizes for habitual smoking, a proxy phenotype for nicotine dependence, were consistent with those observed in FSCD.
The minor (A) allele of rs16969968, relative to the major G allele, appears to be both a risk factor for nicotine dependence and a protective factor for cocaine dependence. The biological plausibility of such a bidirectional association stems from the involvement of nAChRs with both excitatory and inhibitory modulation of dopamine-mediated reward pathways.
Smoking; Nicotine dependence; Addiction; Substance-use disorders; Genetics; Receptors; nicotinic; Cocaine
Nicotine dependence is a highly heritable disorder associated with severe medical morbidity and mortality. Recent meta-analyses have found novel genetic loci associated with cigarettes per day (CPD), a proxy for nicotine dependence. The aim of this paper is to evaluate the importance of phenotype definition (i.e. CPD versus Fagerström Test for Cigarette Dependence (FTCD) score as a measure of nicotine dependence) on genome-wide association studies of nicotine dependence.
Genome-wide association study
A total of 3,365 subjects who had smoked at least one cigarette were selected from the Study of Addiction: Genetics and Environment (SAGE). Of the participants, 2,267 were European Americans,999 were African Americans.
Nicotine dependence defined by FTCD score ≥4, CPD
The genetic locus most strongly associated with nicotine dependence was rs1451240 on chromosome 8 in the region of CHRNB3 (OR=0.65, p=2.4×10−8). This association was further strengthened in a meta-analysis with a previously published dataset (combined p=6.7 ×10−16, total n=4,200).When CPD was used as an alternate phenotype, the association no longer reached genome-wide significance (β=−0.08, p=0.0007).
Daily cigarette consumption and the Fagerstrom Test for Cigarette Dependence (FTCD) show different associations with polymorphisms in genetic loci.
This study demonstrates a novel approach to test associations between highly heterogeneous genetic loci and complex phenotypes. Previous investigations of the relationship between Cytochrome P450 2A6 (CYP2A6) genotype and smoking phenotypes made comparisons by dividing subjects into broad categories based on assumptions that simplify the range of function of different CYP2A6 alleles, their numerous possible diplotype combinations and non-additive allele effects. A predictive model that translates CYP2A6 diplotype into a single continuous variable was previously derived from an in vivo metabolism experiment in 189 European Americans. Here, we apply this model to assess associations between genotype, inferred nicotine metabolism and smoking behaviors in larger samples without direct nicotine metabolism measurements. CYP2A6 genotype is not associated with nicotine dependence, as defined by the Fagerström Test of Nicotine Dependence, demonstrating that cigarettes smoked per day (CPD) and nicotine dependence have distinct genetic correlates. The predicted metric is significantly associated with CPD among African Americans and European American dependent smokers. Individual slow metabolizing genotypes are associated with lower CPD, but the predicted metric is the best predictor of CPD. Furthermore, optimizing the predictive model by including additional CYP2A6 alleles improves the fit of the model in an independent data set and provides a novel method of predicting the functional impact of alleles without direct metabolism measurements. Lastly, comprehensive genotyping and in vivo metabolism data are used to demonstrate that genome-wide significant associations between CPD and single nucleotide polymorphisms are the result of synthetic associations.
Genome-wide studies of psychiatric conditions frequently fail to explain a substantial proportion of variance, and replication of individual SNP effects is rare. We demonstrate a selective scoring approach, in which variants from several genes known to directly affect the dopamine system are considered concurrently to explain individual differences in cocaine dependence symptoms. 273 SNPs from eight dopamine-related genes were tested for association with cocaine dependence symptoms in an initial training sample. We identified a four-SNP score that accounted for 0.55% of the variance in a separate testing sample (p = 0.037). These findings suggest that 1) limiting investigated SNPs to those located in genes of theoretical importance improves the chances of identifying replicable effects by reducing statistical penalties for multiple testing, and 2) considering top-associated SNPs in the aggregate can reveal replicable effects that are too small to be identified at the level of individual SNPs.
candidate gene; cocaine dependence; dopamine
Mood disorders substantially increase risk of cardiovascular disease, though the mechanisms are unclear. We assessed for a dose-dependent relationship between course of illness or treatment with vasculopathy in a well-characterized cohort.
Participants with mood disorders were recruited for the National Institute of Mental Health Collaborative Depression Study (CDS) and followed prospectively. A cross-sectional metabolic and vascular function evaluation was performed on a sub-sample near completion after a mean follow-up of 27 years.
A total of 35 participants from the University of Iowa (33) and Washington University (2) sites of the CDS consented to a metabolic and vascular function assessment at the Iowa site. In multivariate linear regression, controlling for age, gender, and smoking, manic/hypomanic, but not depressive, symptom burden was associated with lower flow-mediated dilation (FMD). Cumulative exposure to antipsychotics and mood stabilizers was associated with elevated augmentation pressure and mean aortic systolic blood pressure. This appeared specifically related to first generation antipsychotic exposure and mediated by increases in brachial systolic pressure. Although second generation antipsychotics were associated with dyslipidemia and insulin resistance, they were not associated with vasculopathy.
These results provide evidence that chronicity of mood symptoms contribute to vasculopathy in a dose-dependent fashion. Patients with more manic/hypomanic symptoms had poorer endothelial function. First generation antipsychotic exposure was associated with arterial stiffness, evidenced by higher augmentation pressure, perhaps secondary to elevated blood pressure. Vascular phenotyping methods may provide a promising means of elucidating the mechanisms linking mood disorders to vascular disease.
adult; antipsychotics; major depression; bipolar disorder; cardiovascular mortality; mania
Chromosome 7 has shown consistent evidence of linkage with a variety of phenotypes related to alcohol dependence in the Collaborative Study on the Genetics of Alcoholism (COGA) project. Using a sample of 262 densely affected families, a peak lod score for alcohol dependence of 2.9 was observed at D7S1799 (Wang et al., 2004, Hum Mol Genet). The lod score in the region increased to 4.1 when a subset of the sample was genotyped with the Illumina Linkage III panel for the Genetic Analysis Workshop 14 (GAW14; Dunn et al., 2005, BMC Genetics). To follow-up on this linkage region, we systematically screened SNPs across a 2 LOD support interval surrounding the alcohol dependence peak.
SNPs were selected from the HapMap Phase I CEPH data to tag linkage disequilibrium bins across the region. 1340 across the 18Mb region, genotyped by the Center for Inherited Disease Research (CIDR), were analyzed. Family-based association analyses were performed on a sample of 1172 individuals from 217 Caucasian families. Results: Eight SNPs showed association with alcohol dependence at p<0.01. Four of the eight most significant SNPs were located in or very near the ACN9 gene. We conducted additional genotyping across ACN9 and identified multiple variants with significant evidence of association with alcohol dependence.
These analyses suggest that ACN9 is involved in the predisposition to alcohol dependence. Data from yeast suggest that ACN9 is involved in gluconeogenesis and the assimilation of ethanol or acetate into carbohydrate.
genetics; association; linkage disequilibrium; alcohol dependence; ACN9
Decades of genome-wide association studies (GWAS) have accumulated large volumes of genomic data that can potentially be reused to increase statistical power of new studies, but different genotyping platforms with different marker sets have been used as biotechnology has evolved, preventing pooling and comparability of old and new data. For example, to pool together data collected by 550K chips with newer data collected by 900K chips, we will need to impute missing loci. Many imputation algorithms have been developed, but the posteriori probabilities estimated by those algorithms are not a reliable measure the quality of the imputation. Recently, many studies have used an imputation quality score (IQS) to measure the quality of imputation. The IQS requires to know true alleles to estimate. Only when the population and the imputation loci are identical can we reuse the estimated IQS when the true alleles are unknown.
Here, we present a regression model to estimate IQS that learns from imputation of loci with known alleles. We designed a small set of features, such as minor allele frequencies, distance to the nearest known cross-over hotspot, etc., for the prediction of IQS. We evaluated our regression models by estimating IQS of imputations by BEAGLE for a set of GWAS data from the NCBI GEO database collected from samples from different ethnic populations.
We construct a ν-SVR based approach as our regression model. Our evaluation shows that this regression model can accomplish mean square errors of less than 0.02 and a correlation coefficient close to 0.75 in different imputation scenarios. We also show how the regression results can help remove false positives in association studies.
Reliable estimation of IQS will facilitate integration and reuse of existing genomic data for meta-analysis and secondary analysis. Experiments show that it is possible to use a small number of features to regress the IQS by learning from different training examples of imputation and IQS pairs.
Background. Sphincter of Oddi manometry is a highly specialized procedure associated with an increased risk of procedural complications. Published studies have typically been performed in large volume manometry centers. Objective. To examine the outcomes and complication rate of SOM when performed in small volumes. Design. Retrospective analysis at a tertiary care referral hospital that infrequently performs Sphincter of Oddi manometry. Patient records were reviewed for procedural details, patient outcomes, and complications after sphincter of Oddi manometry. Results. 36 patients, 23 (23 type II sphincter of Oddi dysfunction (SOD), 13 type III SOD) underwent sphincter of Oddi manometry and were followed up for mean of 16 months. Nine Type II patients (90%) with elevated basal sphincter pressures noted symptom improvement after sphincterotomy compared with only 3 patients (43%) of the patients with normal basal pressures. In type III SOD, 7 patients had elevated basal SO pressure and underwent sphincterotomy. Three patients (43%) improved. There were six
(16%) procedure-related complications. There were four cases
of post ERCP pancreatitis (11%), all of which were mild.
Conclusion. In low numbers, sphincter of Oddi
manometry can be performed successfully and safely by experienced
biliary endoscopists with results that are comparable to large
Event-related brain oscillations (EROs) represent highly heritable neuroelectrical correlates of human perception and cognitive performance that exhibit marked deficits in patients with various psychiatric disorders. We report the results of the first genome-wide association study (GWAS) of an ERO endophenotype – frontal theta ERO evoked by visual oddball targets during P300 response in 1,064 unrelated individuals drawn from a study of alcohol dependence. Forty-two SNPs of the Illumina HumanHap 1M microarray were selected from the theta ERO GWAS for replication in family-based samples (N = 1,095), with four markers revealing nominally significant association. The most significant marker from the two-stage study is rs4907240 located within ARID protein 5A gene (ARID5A) on chromosome 2q11 (unadjusted, Fisher’s combined P = 3.68 × 10−6). However, the most intriguing association to emerge is with rs7916403 in serotonin receptor gene HTR7 on chromosome 10q23 (combined P = 1.53 × 10−4), implicating the serotonergic system in the neurophysiological underpinnings of theta EROs. Moreover, promising SNPs were tested for association with diagnoses of alcohol dependence (DSM-IV), revealing a significant relationship with the HTR7 polymorphism among GWAS case-controls (P = 0.008). Significant recessive genetic effects were also detected for alcohol dependence in both case-control and family-based samples (P = 0.031 and 0.042, respectively), with the HTR7 risk allele corresponding to theta ERO reductions among homozygotes. These results suggest a role of the serotonergic system in the biological basis of alcohol dependence and underscore the utility of analyzing brain oscillations as a powerful approach to understanding complex genetic psychiatric disorders.
serotonin receptor gene (HTR7); serotonin receptor (5-HT7); event-related oscillation (ERO); alcohol dependence; genome-wide association study (GWAS)
We performed a case control study of 1,000 cases and 1,028 controls on 1,509 markers, 1,139 of which were located in a 8 Mb region on chromosome 6 (105-113 Mb). This region has shown evidence of involvement in BP in a number of other studies. We find association between BP and two SNPs in the gene LACE1. SNP rs9486880 and rs11153113 (both have p-values of 2 × 10-5). Both p-values are in the top 5% of the distribution derived from null simulations (p=0.02 and 0.01 respectively). LACE is a good candidate for BP; it is an ATPase. We genotyped 173 other markers in 17 other positional and/or functional loci but found no further evidence of association with BP.
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
Endophenotypes reflect more proximal effects of genes than diagnostic categories, hence providing a more powerful strategy in searching for genes involved in complex psychiatric disorders. There is strong evidence suggesting the P3 amplitude of the event-related potential (ERP) as an endophenotype for the risk of alcoholism and other disinhibitory disorders. Recent studies demonstrated a crucial role of corticotropin releasing hormone receptor 1 (CRHR1) in the environmental stress response and ethanol self-administration in animal models. The aim of the present study was to test the potential associations between single-nucleotide polymorphisms (SNPs) in the CRHR1 gene and the quantitative trait, P3 amplitude during the processing of visual target signals in an oddball paradigm, as well as alcohol dependence diagnosis.
We analyzed a sample from the Collaborative Study on the Genetics of Alcoholism (COGA) comprising 1049 Caucasian subjects from 209 families (including 472 alcohol-dependent individuals). Quantitative transmission disequilibrium test (QTDT) and family-based association test (FBAT) were used to test the association, and false discovery rate (FDR) was applied to correct for multiple comparisons.
Significant associations (p < 0.05) were found between the P3 amplitude and alcohol dependence with multiple SNPs in the CRHR1 gene.
Our results suggest that CRHR1 may be involved in modulating the P3 component of the ERP during information processing and in vulnerability to alcoholism. These findings underscore the utility of electrophysiology and the endophenotype approach in the genetic study of psychiatric disorders.
P3; Disinhibition; Endophenotype; Stress; Corticotropin Releasing Factor (CRF)
Recent studies suggest that the traditional case-control study design does not have sufficient power to discover rare risk variants. Two different methods—collapsing and family data—are suggested as alternatives for discovering these rare variants. Compared with common variants, rare variants have unique characteristics. In this paper, we assess the distribution of rare variants in family data. We notice that a large number of rare variants exist only in one or two families and that the association result is largely shaped by those families. Therefore we explore the possibility of integrating both the collapsing method and the family data method. This combinational approach offers a potential power boost for certain causal genes, including VEGFA, VEGFC, SIRT1, SREBF1, PIK3R3, VLDLR, PLAT, and FLT4, and thus deserves further investigation.
Several independent studies show that the chromosome 15q25.1 region, which contains the CHRNA5-CHRNA3-CHRNB4 gene cluster, harbors variants strongly associated with nicotine dependence, other smoking behaviors, lung cancer, and chronic obstructive pulmonary disease.
We investigated whether variants in other cholinergic nicotinic receptor subunit (CHRN) genes affect risk for nicotine dependence in a new sample of African-Americans (N = 710). We also analyzed this African-American sample together with a European-American sample (N=2062, 1608 of which have been previously studied), allowing for differing effects in the two populations. Cases are current nicotine-dependent smokers and controls are non-dependent smokers.
Variants in or near CHRND-CHRNG, CHRNA7, and CHRNA10 show modest association with nicotine dependence risk in the African-American sample. In addition, CHRNA4, CHRNB3-CHRNA6, and CHRNB1 show association in at least one population. CHRNG and CHRNA4 harbor SNPs that have opposite directions of effect in the two populations. In each of the population samples, these loci substantially increase the trait variation explained, although no loci meet Bonferroni-corrected significance in the African-American sample alone. The trait variation explained by three key associated SNPs in CHRNA5-CHRNA3-CHRNB4 is 1.9% in European-Americans and also 1.9% in African-Americans; this increases to 4.5% in EAs and 7.3% in AAs when we add six variants representing associations at other CHRN genes.
Multiple nicotinic receptor subunit genes outside of chromosome 15q25 are likely to be important in the biological processes and development of nicotine dependence, and some of these risks may be shared across diverse populations.
genetic association; smoking; cholinergic nicotinic receptors; nicotinic acetylcholine receptors
Owing to the clinical relationship between bipolar disorder and nicotine dependence, we investigated two research questions: (i) are genetic associations with nicotine dependence different in individuals with bipolar disorder as compared with individuals without bipolar disorder, and (ii) do loci earlier associated with nicotine dependence have pleiotropic effects on these two diseases.
Our study consisted of 916 cases with bipolar disorder and 1028 controls. On the basis of known associations with nicotine dependence, we genotyped eight single-nucleotide polymorphisms (SNPs) on chromosome 8 (three bins) in the regions of CHRNB3 and CHRNA6, and six SNPs on chromosome 15 (three bins) in the regions of CHRNA5 and CHRNA3.
To determine whether the genetic associations with nicotine dependence are different in bipolar disorder than in the general population, we compared allele frequencies of candidate SNPs between individuals with nicotine dependence only and individuals with both nicotine dependence and bipolar disorder. There were no statistical differences between these frequencies, indicating that genetic association with nicotine dependence is similar in individuals with bipolar disorder as in the general population. In the investigation of pleiotropic effects of these SNPs on bipolar disorder, two highly correlated synonymous SNPs in CHRNB3, rs4952 and rs4953, were significantly associated with bipolar disorder (odds ratio 1.7, 95% confidence interval: 1.2–2.4, P = 0.001). This association remained significant both after adjusting for a smoking covariate and analyzing the association in nonsmokers only.
Our results suggest that (i) bipolar disorder does not modify the association between nicotine dependence and nicotinic receptor subunit genes, and (ii) variants in CHRNB3/CHRNA6 are independently associated with bipolar disorder. Psychiatr Genet 00:000–000.
analyses; CHRNA3; CHRNA5; CHRNA6; genetic association; nicotine; tobacco use disorder
Nicotine withdrawal (NW) is both an important contributor to difficulty quitting cigarettes and because of mood-related withdrawal symptoms a problem of particular relevance to psychiatry. Twin-studies suggest that genetic factors influence NW (heritability= 45%). Only one previous linkage study has published findings on NW (Swan et al., 2006; LOD=2.7; Chr. 6 at 159 cM). As part of an international consortium, genome-wide scans (using 381 autosomal microsatellite markers) and telephone diagnostic interviews were conducted on 289 Australian (AUS) and 161 Finnish (FIN, combined (COMB) N=450 families) families ascertained from twin registries through index-cases with a lifetime history of cigarette smoking. The statistical approach used an affected-sib pair design (at least two adult full siblings reported a history of DSM-IV NW) and conducted the linkage analyses using MERLIN. Linkage signals with LOD scores greater than 1.5 were found on two chromosomes: 6 (FIN: LOD= 1.93 at 75 cM) and 11 at two different locations (FIN: LOD= 3.55 at 17 cM, and AUS: LOD= 1.68 with a COMB: LOD= 2.30 at 123 cM). The multipoint LOD score of 3.55 on chromosome 11p15 in FIN met genomewide significance (p = .013 with 1000 simulations). At least four strong candidate genes lie within or near this peak on chromosome 11: DRD4, TPH, TH, and CHRNA10. Other studies have reported that chromosome 11 may harbor genes associated with various aspects of smoking behavior. This study adds to that literature by highlighting evidence for nicotine withdrawal.
genetics; linkage; nicotine; withdrawal
Genome-wide association studies often incorporate information from public biological databases in order to provide a biological reference for interpreting the results. The dbSNP database is an extensive source of information on single nucleotide polymorphisms (SNPs) for many different organisms, including humans. We have developed free software that will download and install a local MySQL implementation of the dbSNP relational database for a specified organism. We have also designed a system for classifying dbSNP tables in terms of common tasks we wish to accomplish using the database. For each task we have designed a small set of custom tables that facilitate task-related queries and provide entity-relationship diagrams for each task composed from the relevant dbSNP tables. In order to expose these concepts and methods to a wider audience we have developed web tools for querying the database and browsing documentation on the tables and columns to clarify the relevant relational structure. All web tools and software are freely available to the public at http://cgsmd.isi.edu/dbsnpq. Resources such as these for programmatically querying biological databases are essential for viably integrating biological information into genetic association experiments on a genome-wide scale.
Several studies have found replicable associations between nicotine dependence and specific variants in the nicotinic receptor genes CHRNA5(rs16969968) and CHRNA3(rs3743078). How these newly identified genetic risks combine with known environmental risks is unknown. This study examined whether the level of parent monitoring during early adolescence modified the risk of nicotine dependence associated with these genetic variants.
In a cross-sectional case control study of US-based community sample of 2027 subjects, we use a systematic series of regression models to examine the effect of parent monitoring on risk associated with two distinct variants in the nicotinic receptor genes CHRNA5(rs16969968) and CHRNA3(rs3743078).
Low parent monitoring as well as the previously identified genetic variants were associated with an increased risk of nicotine dependence. An interaction was found between the SNP(rs16969968) and parent monitoring (p=0.034). The risk for nicotine dependence increased significantly with the risk genotype of SNP(rs16969968) when combined with lowest quartile parent monitoring. In contrast, there was no evidence of an interaction between SNP(rs3743078) and parent monitoring (p=0.80).
The genetic risk of nicotine dependent associated with rs16969968 was modified by level of parent monitoring, while the genetic risk associated with rs3743078 was not, suggesting that the increased risk due to some genes may be mitigated by environmental factors such as parent monitoring.
nicotine dependence; parent monitoring; phenotype; gene-environmental interaction; nicotinic receptor genes; case control study
Genetic association studies have demonstrated the importance of variants in the CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit gene cluster on chromosome 15q24-25.1 in risk for nicotine dependence, smoking, and lung cancer in populations of European descent. We have now carried out a detailed study of this region using dense genotyping in both European- and African-Americans.
We genotyped 75 known single-nucleotide-polymorphisms (SNPs) and one sequencing-discovered SNP in an African-American (AA) sample (N = 710) and European-American (EA) sample (N = 2062). Cases were nicotine-dependent and controls were non-dependent smokers.
The non-synonymous CHRNA5 SNP rs16969968 is the most significant SNP associated with nicotine dependence in the full sample of 2772 subjects (p = 4.49×10−8, OR 1.42 (1.25–1.61)) as well as in AAs only (p = 0.015, OR = 2.04 (1.15–3.62)) and EAs only (p = 4.14×10−7, OR = 1.40 (1.23–1.59)). Other SNPs that have been shown to affect mRNA levels of CHRNA5 in EAs are associated with nicotine dependence in AAs but not in EAs. The CHRNA3 SNP rs578776, which has low correlation with rs16969968, is associated with nicotine dependence in EAs but not in AAs. Less common SNPs (frequency ≤ 5%) also are associated with nicotine dependence.
In summary, multiple variants in this gene cluster contribute to nicotine dependence risk, and some are also associated with functional effects on CHRNA5. The non-synonymous SNP rs16969968, a known risk variant in European-descent populations, is also significantly associated with risk in African-Americans. Additional SNPs contribute in distinct ways to risk in these two populations.
genetic association; smoking; cholinergic nicotinic receptors; nicotinic acetylcholine receptors
Alcohol abuse and dependence are major causes of morbidity and mortality worldwide, and have a strong familial component. Several linkage and association studies have identified chromosomal regions and/or genes that affect alcohol consumption, notably in genes involved in the two-stage pathway of alcohol metabolism.
Here, we use multiple regression models to test for associations and interactions between two alcohol related phenotypes and SNPs in 17 genes involved in alcohol metabolism in the U.S. Caucasian subset of the Collaborative Genetic Study of Nicotine Dependence (COGEND) participants.
Several SNPs across six genes showed evidence for association with either maximum number of drinks consumed in a 24-hour period or DSM-IV symptom count. The strongest evidence for association was between rs1229984, a non-synonymous coding SNP in ADH1B, and DSM-IV symptom count (P = 0.0003). This SNP was also associated with maximum drinks (P = 0.0004). Each minor allele at this SNP predicts 45% fewer DSM-IV symptoms and 18% fewer max drinks. Another SNP in a splice site in ALDH1A1 (rs8187974) showed evidence for association with both phenotypes as well. Minor alleles at this SNP predict greater alcohol consumption. In addition, pairwise interactions were observed between SNPs in several genes (P = 0.00002).
We replicated the large effect of rs1229984 on alcohol behavior, and although not common (MAF = 4%), this polymorphism may be highly relevant from a public health perspective in European Americans. Another SNP, rs8187974, may also affect alcohol behavior but requires replication. Also, interactions between polymorphisms in genes involved in alcohol metabolism are likely determinants of the parameters that ultimately affect alcohol consumption.
Alcoholism; Alcohol Metabolism; Genetic Association
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.
SPOT (http://spot.cgsmd.isi.edu), the SNP prioritization online tool, is a web site for integrating biological databases into the prioritization of single nucleotide polymorphisms (SNPs) for further study after a genome-wide association study (GWAS). Typically, the next step after a GWAS is to genotype the top signals in an independent replication sample. Investigators will often incorporate information from biological databases so that biologically relevant SNPs, such as those in genes related to the phenotype or with potentially non-neutral effects on gene expression such as a splice sites, are given higher priority. We recently introduced the genomic information network (GIN) method for systematically implementing this kind of strategy. The SPOT web site allows users to upload a list of SNPs and GWAS P-values and returns a prioritized list of SNPs using the GIN method. Users can specify candidate genes or genomic regions with custom levels of prioritization. The results can be downloaded or viewed in the browser where users can interactively explore the details of each SNP, including graphical representations of the GIN method. For investigators interested in incorporating biological databases into a post-GWAS SNP selection strategy, the SPOT web tool is an easily implemented and flexible solution.
As the amount of data from genome wide association studies grows dramatically, many interesting scientific questions require imputation to combine or expand datasets. However, there are two situations for which imputation has been problematic: (1) polymorphisms with low minor allele frequency (MAF), and (2) datasets where subjects are genotyped on different platforms. Traditional measures of imputation cannot effectively address these problems.
We introduce a new statistic, the imputation quality score (IQS). In order to differentiate between well-imputed and poorly-imputed single nucleotide polymorphisms (SNPs), IQS adjusts the concordance between imputed and genotyped SNPs for chance. We first evaluated IQS in relation to minor allele frequency. Using a sample of subjects genotyped on the Illumina 1 M array, we extracted those SNPs that were also on the Illumina 550 K array and imputed them to the full set of the 1 M SNPs. As expected, the average IQS value drops dramatically with a decrease in minor allele frequency, indicating that IQS appropriately adjusts for minor allele frequency. We then evaluated whether IQS can filter poorly-imputed SNPs in situations where cases and controls are genotyped on different platforms. Randomly dividing the data into “cases” and “controls”, we extracted the Illumina 550 K SNPs from the cases and imputed the remaining Illumina 1 M SNPs. The initial Q-Q plot for the test of association between cases and controls was grossly distorted (λ = 1.15) and had 4016 false positives, reflecting imputation error. After filtering out SNPs with IQS<0.9, the Q-Q plot was acceptable and there were no longer false positives. We then evaluated the robustness of IQS computed independently on the two halves of the data. In both European Americans and African Americans the correlation was >0.99 demonstrating that a database of IQS values from common imputations could be used as an effective filter to combine data genotyped on different platforms.
IQS effectively differentiates well-imputed and poorly-imputed SNPs. It is particularly useful for SNPs with low minor allele frequency and when datasets are genotyped on different platforms.
A previous association analysis identified polymorphisms in GABRA4 and GABRA2 to be associated with nicotine dependence, as assessed by a score of 4 or more on the Fagerström Test for Nicotine Dependence (FTND). In the present report, we extend the previous study by significantly expanding our genotyping efforts for these two genes.
In 1,049 cases (FTND of 4 or more) and 872 controls (smokers with FTND of 0), from the U.S. and Australia, we examine the association between 23 GABRA4 and 39 GABRA2 recently genotyped single nucleotide polymorphisms (SNPs) and nicotine dependence using logistic regression-based association analyses in PLINK.
Two and 18 additional SNPs in GABRA4 and GABRA2 respectively were associated with nicotine dependence. The SNPs identified in GABRA4 (p value = 0.002) were restricted to introns 1 and 2, exon 1 and the 5’ end of the gene, while those in GABRA2 localized to the 3’ end of the gene and spanned introns 9 through 3, and were in moderate to high linkage disequilibrium (as measured by r2) with each other and with previously studied polymorphisms.
Our findings consistently demonstrate the role of GABRA4 and GABRA2 in nicotine dependence. However, further research is needed to identify the biological influence of these intronic variations and to isolate functionally relevant polymorphisms neighboring them.
Association; nicotine dependence; GABRA2; NICSNP
Markers for individual genotyping can be selected using quantitative genotyping of pooled DNA. This strategy saves time and money.
To determine the efficacy of this approach, we investigated the bivariate distribution of association test statistics from pooled and individual genotypes. We used a sample of approximately 1,000 samples with individual and pooled genotyping on 40,000 SNPs.
Results and Conclusions
We found that the distribution of the joint test statistics can be modelled as a mixture of two bivariate normal distributions. One distribution has a correlation of zero, and is probably due to SNPs whose pooled genotyping was unsuccessful. The other distribution has a correlation of approximately 0.65 in our data. This latter distribution is probably accounted for by SNPs whose pooled genotyping accurately predicts the underlying allele frequency. Approximately 87% of the data belongs to this distribution. We also derived a method to investigate the effect of both the correlation and selection cut-off on the relative power of pooling studies. We demonstrate that pooled genotyping has good power to detect SNPs that are truly associated with disease-causing variants for SNPs showing good correlation between pooled and individual genotyping. Therefore, this approach is a cost effective tool for association studies.
Pooling; Power; Association