Association Analysis Between Genetic Variation in GATA Binding Protein 4 (GATA4) and Alcohol Use Disorder

Association Analysis Between Genetic Variation in GATA Binding Protein 4 (GATA4) and Alcohol Use... Abstract Aims Previous genetic association studies have shown that variation in the GATA4 gene encoding the GATA binding protein 4, a binding protein that binds to the ANA sequence GATA, increase susceptibility for alcohol use disorder (AUD). In this study, we aimed to replicate those findings in an independent sample and analyze their association with anxiety. Methods Overall, 1044 individuals with AUD [534 European American (EA), 510 African Americans (AA)] and 645 controls [413 EA, 232 AA] were genotyped using 34 markers. Genotype and allele frequencies were compared between cases and controls using chi-square analysis. Other phenotype data were analyzed for possible associations with GATA4 single-nucleotide polymorphisms (SNPs) in individuals with AUD. Results Rs6601604 was nominally significantly associated with AUD in EA, and 3 SNPs (rs6990313, rs11250159 and rs17153694) showed trend-level significance (P < 0.10) in AA. However, none of the SNPs were significant after correcting for multiple testing. Haplotype analysis of the 34 SNPs did not find a significant association between haplotype blocks and AUD diagnosis after correcting for multiple testing. From the phenotype analysis, anxiety was associated with GATA4 SNP rs10112596 among the AA group with AUD after a correction for multiple testing. Conclusions Although previous studies have shown a relationship between variants of the GATA4 gene and a diagnosis of AUD, we did not replicate these findings in our sample. Additional studies of variation in this gene are needed to elucidate whether polymorphisms of the GATA4 gene are associated with AUD and other alcohol-related phenotypes. Short Summary GATA4 variants were not associated with AUD in either the European ancestry or African ancestry groups after correcting for multiple comparisons. Rs10112596 demonstrated a significant relationship with an anxiety measure among the African ancestry group with AUD. INTRODUCTION The prevalence, individual health risks and societal costs of excessive alcohol use demonstrate the importance of identifying underlying causes of pathological alcohol consumption and developing novel treatment approaches (Bouchery et al., 2011; Stahre et al., 2014; Grant et al., 2015). Genetic factors account for ~40–60% of the variance in risk of developing alcohol use disorder (AUD) (Rietschel and Treutlein, 2013; Tawa et al., 2016); however, AUD is a complex disorder, with many factors contributing to its onset and maintenance. Identification of the underlying genetic risk variants will further our understanding of the disorder’s neurobiology and may direct the development of novel individualized (i.e. pharmacogenetic) treatment options for AUD. Recent case-control genome-wide association studies (GWAS) implicate GATA binding protein 4 (GATA4), a gene located on chromosome 8, in associations with vulnerability to AUD diagnosis (Treutlein et al., 2009; Edenberg et al., 2010). While a previous candidate gene study found a significant association between GATA4 and AUD using gene-level testing (Karpyak et al., 2014), a more recent study by Degenhardt et al. (2016) failed to show an association between rare GATA4 variants and AUD. However, it should be noted that Degenhardt et al. (2016) attempted to identify only rare risk-associated variants, which precluded them from identifying likely protective variants in GATA4. The GATA4 gene encodes transcription factor GATA4, which regulates the expression of the atrial natriuretic peptide (ANP), among other proteins (McBride and Nemer, 2001). Importantly, GATA4 protein is expressed throughout cells in the central nervous system (CNS). Reduced ANP expression in the CNS is associated with the dysregulation of stress and anxiety mechanisms in the brain, suggesting a possible link between ANP and AUD (Jorde et al., 2014). ANP also influences hypothalamic–pituitary–adrenal (HPA) axis functioning, as well as amygdala activation, further supporting the relationship between ANP and AUD-related phenotypes (McBride and Nemer, 2001). Clinically, post-detoxification patients with AUD and decreased ANP plasma levels report increased craving and anxiety levels compared to both detoxified patients with AUD and higher ANP plasma levels, as well as controls (Kiefer et al., 2002). GATA4 also acts as a transcription factor for brain natriuretic peptide (BNP), a peptide involved in the regulation of the cardiovascular system. Interestingly, BNP is also involved in stress responses when found in the CNS (Amir et al., 2010). One study demonstrated a relationship between GATA4 binding site methylation and BNP expression among alcohol-dependent patients experiencing alcohol withdrawal (Glahn et al., 2016). Previous GWAS highlight an association between the specific intronic single-nucleotide polymorphism (SNP) rs13273672 in the GATA4 gene and AUD-related phenotypes (Kiefer et al., 2011; Jorde et al., 2014). In a randomized, double-blind, placebo-controlled study, Kiefer et al. (2011) showed that alcohol-dependent individuals with the rs13273672 G allele had a decreased time to relapse following Acamprosate treatment compared to A allele carriers with AUD. Furthermore, the G allele of this SNP was predictive of a significant decrease in variance in ANP plasma concentration compared to the A allele. Acamprosate is an FDA-approved pharmacological intervention for AUD that decreases cravings to reduce relapse risk; it is thought to primarily work through the glutamatergic system, although the exact mechanism of action remains unclear (Kiefer et al., 2011). Further research identified the AA genotype as associated with stronger alcohol cue-induced amygdala activation, and this association was predictive of a lower relapse risk (Jorde et al., 2014). Zois et al. (2016) expanded on this work by identifying an interaction between GATA4 genotype and gray matter volume on relapse risk, such that the AA genotype group showed an association between greater gray matter and a reduced relapse risk. This provides further support for the possible protective nature of the AA genotype. Furthermore, AUD has been shown to be associated with mood and anxiety disorders, neuroticism and alcohol withdrawal (Regier et al., 1990; Malouff et al., 2007; Becker and Mulholland, 2014), all of which may play a role in genetic predisposition to AUD (Tawa et al., 2016). In particular, much research has determined the significant co-occurrence of AUD and anxiety disorders, and it is likely that genetic variation influences this comorbidity (Poikolainen, 2000; Smith and Randall, 2012). According to the common factor model proposed by Smith , a third variable (e.g. genetic susceptibility) explains the presence of both AUD and anxiety disorders. In line with this model, Merikangas et al. (1996) found that relatives of patients with anxiety disorders had an increased risk for alcohol dependence. This result could be partly explained by shared genetic factors influencing susceptibility to AUD and anxiety disorders. Moreover, a review by Kenna et al. (2012) highlights more recent research that has found an association between 5-HTTLPR, a 5-HT transporter polymorphism, and both alcohol dependence and anxiety symptoms. Given the high degree of heritability of AUD and frequent comorbid occurrence of anxiety symptoms, identifying genetic risk factors that contribute to their shared pathophysiology may improve our understanding of comorbid AUD and anxiety, as well as inform the development of pharmacological treatments. Therefore, the present study aimed to explore associations between genetic variation in GATA4 and anxiety, as assessed by the Brief Scale for Anxiety. In summary, previous studies indicate a possible association between variation in the GATA4 gene and AUD. However, the underlying mechanisms of this relationship are still relatively unknown and poorly understood. Therefore, additional studies of SNPs within GATA4 and their association with AUD-related phenotypes are needed. In this case-control study, we sought to (a) replicate findings associating variants in GATA4 with increased susceptibility to AUD and (b) determine associations between GATA4 variants and alcohol-related clinical phenotypes, specifically anxiety, as assessed by the Brief Scale for Anxiety. Identifying genetic variants associated with AUD and related clinical phenotypes could be used to identify individuals at risk of developing AUD. Ultimately, this could inform the development of more targeted pharmacological prevention and treatment approaches for AUD. MATERIALS AND METHODS Participants This study was approved by the Institutional Review Board at the National Institutes of Health (NIH). All participants provided written informed consent and permission to use collected samples. Out of 1778 individuals with collected samples, 1044 individuals with AUD [534 European Americans (EA), 510 African Americans (AA)] and 645 controls [413 EA and 232 AA] took part in this study. The 89 missing participants were excluded because they did not have a completed SCID diagnosis. Study participants were recruited between 2005 and 2016 from the inpatient unit and outpatient clinic of the Laboratory of Clinical and Translational Studies at the National Institute on Alcohol Abuse and Alcoholism (NIAAA), NIH (Bethesda, MD). Participants were recruited from three screening protocols, all of which excluded those under 18 years of age. Two of the screening protocols included only those in good health without major medical problems, and excluded individuals that were under court-mandated or involuntary treatment. The third protocol excluded prisoners, as well as pregnant women. Alcohol-dependent subjects were diagnosed with alcohol dependence according to the Diagnostic and Statistical Manuel for Mental Disorders, 4th edn, Text-revised (DSM-IV-TR) (American Psychiatric Association, 2000). Participants were diagnosed using the Structured Clinical Interview (SCID-I) for DSM-IV-TR (First, et al., 2002). Given the overlap between the DSM-IV alcohol dependence criteria and the Diagnostic and Statistical Manual of Mental Disorders, 5th edn (DSM-5) (American Psychiatric Association, 2013) AUD criteria, all participants also met criteria for AUD; however, a separate clinical interview was not conducted. Informed consent was obtained from all subjects who participated in accordance with the Declaration of Helsinki. Genotyping and SNP selection Large-scale genotyping was performed at the NIAAA Laboratory of Neurogenetics using the Illumina OmniExpress BeadChip (Illumina, San Diego, CA). Data for all SNPs located within the GATA4 gene that were genotyped on the array were extracted using PLINK version 1.07 (Purcell et al., 2007) (http://pngu.mgh.harvard.edu/purcell/plink/), based on start and end base pair positions for the gene located on chromosome 8 (11561716, 1161750; GRCh37/hg19 assembly). This procedure resulted in genotype data for 34 SNPs. Ancestry informative markers (AIMs; n = 2500) were also extracted from the Illumina array to calculate ancestral proportions for all study participants. Using methods described previously for an AIM panel including 186 markers (Hodgkinson et al., 2008), which were not available for the current data set, the ancestry assessment identified six ethnic factors (Africa, Europe, Asia, Far East Asia, Oceania and Americas). An analysis of the 34 SNPs among the full sample (n = 1778) found that all were in Hardy–Weinberg equilibrium (HWE) except rs12550668 (P < 0.005) and rs3729856 (P < 0.033) in the EA group. In the AA group, all SNPs were in HWE except rs6601604 (P < 0.03), rs804280 (P < 0.004) and rs867858 (P < 0.003).The same analysis found that rs10105409 in the EA group and rs13275657, rs17153747, rs3729856, rs804290 and rs11785481 in the AA group all had minor allele frequencies (MAF) < 5%. All other SNPs had a MAF > 5%. Analysis Allele frequencies for each SNP were determined using PLINK for the sample as a whole, and then separately for subjects of European and African ancestry (based on self-report). Due to multiple differences in allele frequency across the 34 SNPs, subsequent analyses were conducted separately in each group. Single marker association analyses were conducted using frequency comparison by chi-square test, which is the standard case-control method in PLINK, with adjustment for multiple comparisons performed using the Benjamini–Hochberg method for false discovery rate (FDR) (Benjamini and Hochberg, 1995). The threshold for FDR was set at q = 0.05. Haplotype blocks were determined using HaploView software (Barrett et al., 2005), with haplotype blocks defined using the default D’/LOD method. Haplotype association tests using these defined blocks were conducted in PLINK, and were corrected for multiple comparisons using permutation tests (5000 permutations). Participants also completed a variety of clinical assessments, including the Alcohol Dependence Scale (ADS; Skinner and Allen, 1982), Montgomery Asberg Depression Rating Scale (MADRS; Montgomery and Asberg, 1979), Brief Scale for Anxiety (BSA; Tyrer et al., 1984), State-Trait Anxiety Inventory (STAI; Spielberger et al., 1970), Clinical Institute Withdrawal Assessment for Alcohol (CIWA; Sullivan et al., 1989) and NEO-PI-R (Costa and McCrae, 2002). Sample sizes for these assessments are inconsistent due to missing data, particularly among the control group who were not administered these assessments until later in the study’s recruitment. Single marker association and haplotype analyses were conducted for these continuous outcomes using linear regression models in PLINK. These analyses controlled for age, gender, and African and European ancestry via the AIMS scores for Africa and Europe, based on research showing age, gender and ethnicity differences in alcohol consumption and its related consequences (Delker et al. 2016). RESULTS Table 1 shows the demographic information of participants, as well as differences in the clinical assessments between groups. As expected, AUD participants had significantly greater scores in all alcohol-related phenotype measures when compared to controls in both the EA and AA group. To analyze the association between the 34 GATA4 SNPs and AUD diagnosis, single marker association analyses were conducted using frequency comparison by chi-square test with adjustment for multiple comparisons using the Benjamini–Hochberg method for FDR. Results revealed that one SNP (rs6601604) was nominally significantly associated with AUD in the EA group (P = 0.036). However, this SNP was not significant after adjusting for multiple comparisons (Table 2). Three SNPs (rs6990313, rs11250159 and rs17153694) trended towards a significant association with AUD in the AA group (Ps ≤ 0.065) (Table 3), but these SNPs failed to reach trend-level significance after adjusting for multiple comparisons. The a priori SNP of interest, rs13273672, was not significantly associated with AUD for either ancestry group in our sample. Table 1. Demographic and clinical assessment information   European ancestry  African ancestry  AUD (n = 534)  Controls (n = 413)  P-valuea  AUD (n = 510)  Controls (n = 232)  P-valuea  Gender count (female)  166 (31.1%)  175 (42.4%)  0.0003  140 (27.5%)  107 (46.1%)  <0.0001  Mean age (SD)  42.5 (11.4)  32.0 (12.1)  <0.0001  43.2 (10.1)  35.6 (11.0)  <0.0001  Mean ADS score (SD)  21.8 (8.3) [n = 451]  2.1 (4.1) [n = 95]  <0.0001  17.7 (8.6) [n = 345]  1.3 (3.9) [n = 94]  <0.0001  Mean MADRS score (SD)  15.3 (9.7) [n = 444]  1.5 (3.5) [n = 166]  <0.0001  11.2 (9.4) [n = 441]  1.2 (3.1) [n = 155]  <0.0001  Mean BSA score (SD)  11.1 (7.0) [n = 446]  1.3 (2.6) [n = 166]  <0.0001  8.7 (7.2) [n = 441]  1.1 (2.6) [n = 155]  <0.0001  Mean STAI score (SD)  43.2 (13.6) [n = 199]  33.6 (11.5) [n = 185]  <0.0001  41.1 (12.4) [n = 235]  32.4 (11.9) [n = 145]  <0.0001  Mean neuroticism score (SD)  56.3 (11.5) [n = 483]  44.8 (10.0) [n = 377]  <0.0001  54.6 (9.7) [n = 444]  44.9 (8.4) [n = 202]  <0.0001    European ancestry  African ancestry  AUD (n = 534)  Controls (n = 413)  P-valuea  AUD (n = 510)  Controls (n = 232)  P-valuea  Gender count (female)  166 (31.1%)  175 (42.4%)  0.0003  140 (27.5%)  107 (46.1%)  <0.0001  Mean age (SD)  42.5 (11.4)  32.0 (12.1)  <0.0001  43.2 (10.1)  35.6 (11.0)  <0.0001  Mean ADS score (SD)  21.8 (8.3) [n = 451]  2.1 (4.1) [n = 95]  <0.0001  17.7 (8.6) [n = 345]  1.3 (3.9) [n = 94]  <0.0001  Mean MADRS score (SD)  15.3 (9.7) [n = 444]  1.5 (3.5) [n = 166]  <0.0001  11.2 (9.4) [n = 441]  1.2 (3.1) [n = 155]  <0.0001  Mean BSA score (SD)  11.1 (7.0) [n = 446]  1.3 (2.6) [n = 166]  <0.0001  8.7 (7.2) [n = 441]  1.1 (2.6) [n = 155]  <0.0001  Mean STAI score (SD)  43.2 (13.6) [n = 199]  33.6 (11.5) [n = 185]  <0.0001  41.1 (12.4) [n = 235]  32.4 (11.9) [n = 145]  <0.0001  Mean neuroticism score (SD)  56.3 (11.5) [n = 483]  44.8 (10.0) [n = 377]  <0.0001  54.6 (9.7) [n = 444]  44.9 (8.4) [n = 202]  <0.0001  aChi-square test for gender; t-test for all remaining continuous variables. Table 1. Demographic and clinical assessment information   European ancestry  African ancestry  AUD (n = 534)  Controls (n = 413)  P-valuea  AUD (n = 510)  Controls (n = 232)  P-valuea  Gender count (female)  166 (31.1%)  175 (42.4%)  0.0003  140 (27.5%)  107 (46.1%)  <0.0001  Mean age (SD)  42.5 (11.4)  32.0 (12.1)  <0.0001  43.2 (10.1)  35.6 (11.0)  <0.0001  Mean ADS score (SD)  21.8 (8.3) [n = 451]  2.1 (4.1) [n = 95]  <0.0001  17.7 (8.6) [n = 345]  1.3 (3.9) [n = 94]  <0.0001  Mean MADRS score (SD)  15.3 (9.7) [n = 444]  1.5 (3.5) [n = 166]  <0.0001  11.2 (9.4) [n = 441]  1.2 (3.1) [n = 155]  <0.0001  Mean BSA score (SD)  11.1 (7.0) [n = 446]  1.3 (2.6) [n = 166]  <0.0001  8.7 (7.2) [n = 441]  1.1 (2.6) [n = 155]  <0.0001  Mean STAI score (SD)  43.2 (13.6) [n = 199]  33.6 (11.5) [n = 185]  <0.0001  41.1 (12.4) [n = 235]  32.4 (11.9) [n = 145]  <0.0001  Mean neuroticism score (SD)  56.3 (11.5) [n = 483]  44.8 (10.0) [n = 377]  <0.0001  54.6 (9.7) [n = 444]  44.9 (8.4) [n = 202]  <0.0001    European ancestry  African ancestry  AUD (n = 534)  Controls (n = 413)  P-valuea  AUD (n = 510)  Controls (n = 232)  P-valuea  Gender count (female)  166 (31.1%)  175 (42.4%)  0.0003  140 (27.5%)  107 (46.1%)  <0.0001  Mean age (SD)  42.5 (11.4)  32.0 (12.1)  <0.0001  43.2 (10.1)  35.6 (11.0)  <0.0001  Mean ADS score (SD)  21.8 (8.3) [n = 451]  2.1 (4.1) [n = 95]  <0.0001  17.7 (8.6) [n = 345]  1.3 (3.9) [n = 94]  <0.0001  Mean MADRS score (SD)  15.3 (9.7) [n = 444]  1.5 (3.5) [n = 166]  <0.0001  11.2 (9.4) [n = 441]  1.2 (3.1) [n = 155]  <0.0001  Mean BSA score (SD)  11.1 (7.0) [n = 446]  1.3 (2.6) [n = 166]  <0.0001  8.7 (7.2) [n = 441]  1.1 (2.6) [n = 155]  <0.0001  Mean STAI score (SD)  43.2 (13.6) [n = 199]  33.6 (11.5) [n = 185]  <0.0001  41.1 (12.4) [n = 235]  32.4 (11.9) [n = 145]  <0.0001  Mean neuroticism score (SD)  56.3 (11.5) [n = 483]  44.8 (10.0) [n = 377]  <0.0001  54.6 (9.7) [n = 444]  44.9 (8.4) [n = 202]  <0.0001  aChi-square test for gender; t-test for all remaining continuous variables. Table 2. Associations between GATA4 gene SNPs and AUD in EA samplea SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.10  0.09  1.37  1.21  0.243  0.836  rs10105409  G/A  0.01  0.00  0.05  1.16  0.818  0.904  rs6601604  A/G  0.29  0.34  4.42  0.81  0.036*  0.836  rs10112596  A/G  0.17  0.19  1.33  0.87  0.248  0.836  rs12550668  A/G  0.40  0.43  1.74  0.88  0.188  0.836  rs2898292  G/A  0.10  0.10  0.00  1.00  0.995  0.995  rs4840579  G/A  0.39  0.40  0.28  0.95  0.597  0.836  rs11250159  A/C  0.08  0.09  0.65  0.87  0.421  0.836  rs17153694  A/G  0.07  0.08  0.18  0.93  0.670  0.836  rs17153698  A/G  0.16  0.15  0.24  1.06  0.625  0.836  rs6983129  C/A  0.47  0.48  0.59  0.93  0.444  0.836  rs2898295  A/G  0.50  0.47  1.06  1.10  0.304  0.836  rs11250163  C/A  0.47  0.45  0.69  1.08  0.407  0.836  rs13275657  A/G  0.19  0.20  0.64  0.91  0.423  0.836  rs2029969  G/A  0.38  0.36  0.53  1.07  0.465  0.836  rs2173117  A/C  0.33  0.30  1.78  1.14  0.182  0.836  rs3779664  A/G  0.14  0.16  1.08  0.87  0.299  0.836  rs3735814  A/G  0.48  0.49  0.18  0.96  0.671  0.836  rs2740434  A/G  0.33  0.34  0.13  0.97  0.720  0.844  rs2645399  A/G  0.34  0.36  0.49  0.93  0.486  0.836  rs11784693  A/G  0.29  0.32  1.44  0.89  0.231  0.836  rs804283  G/A  0.29  0.31  1.24  0.89  0.265  0.836  rs17153747  G/A  0.13  0.12  0.75  1.13  0.386  0.836  rs804282  C/A  0.45  0.46  0.40  0.94  0.529  0.836  rs13264774  A/G  0.15  0.15  0.02  1.02  0.880  0.935  rs13273672  G/A  0.30  0.29  0.26  1.05  0.610  0.836  rs804280  C/A  0.43  0.44  0.32  0.95  0.574  0.836  rs3729856  G/A  0.14  0.14  0.16  1.06  0.689  0.836  rs867858  C/A  0.31  0.31  0.05  1.02  0.824  0.904  rs1062219  A/G  0.45  0.46  0.20  0.96  0.655  0.836  rs804290  A/G  0.24  0.22  1.31  1.13  0.253  0.836  rs11785481  A/G  0.14  0.15  0.26  0.93  0.607  0.836  rs12458  T/A  0.33  0.31  0.36  1.06  0.548  0.836  rs3203358  C/G  0.33  0.32  0.01  1.01  0.917  0.944  SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.10  0.09  1.37  1.21  0.243  0.836  rs10105409  G/A  0.01  0.00  0.05  1.16  0.818  0.904  rs6601604  A/G  0.29  0.34  4.42  0.81  0.036*  0.836  rs10112596  A/G  0.17  0.19  1.33  0.87  0.248  0.836  rs12550668  A/G  0.40  0.43  1.74  0.88  0.188  0.836  rs2898292  G/A  0.10  0.10  0.00  1.00  0.995  0.995  rs4840579  G/A  0.39  0.40  0.28  0.95  0.597  0.836  rs11250159  A/C  0.08  0.09  0.65  0.87  0.421  0.836  rs17153694  A/G  0.07  0.08  0.18  0.93  0.670  0.836  rs17153698  A/G  0.16  0.15  0.24  1.06  0.625  0.836  rs6983129  C/A  0.47  0.48  0.59  0.93  0.444  0.836  rs2898295  A/G  0.50  0.47  1.06  1.10  0.304  0.836  rs11250163  C/A  0.47  0.45  0.69  1.08  0.407  0.836  rs13275657  A/G  0.19  0.20  0.64  0.91  0.423  0.836  rs2029969  G/A  0.38  0.36  0.53  1.07  0.465  0.836  rs2173117  A/C  0.33  0.30  1.78  1.14  0.182  0.836  rs3779664  A/G  0.14  0.16  1.08  0.87  0.299  0.836  rs3735814  A/G  0.48  0.49  0.18  0.96  0.671  0.836  rs2740434  A/G  0.33  0.34  0.13  0.97  0.720  0.844  rs2645399  A/G  0.34  0.36  0.49  0.93  0.486  0.836  rs11784693  A/G  0.29  0.32  1.44  0.89  0.231  0.836  rs804283  G/A  0.29  0.31  1.24  0.89  0.265  0.836  rs17153747  G/A  0.13  0.12  0.75  1.13  0.386  0.836  rs804282  C/A  0.45  0.46  0.40  0.94  0.529  0.836  rs13264774  A/G  0.15  0.15  0.02  1.02  0.880  0.935  rs13273672  G/A  0.30  0.29  0.26  1.05  0.610  0.836  rs804280  C/A  0.43  0.44  0.32  0.95  0.574  0.836  rs3729856  G/A  0.14  0.14  0.16  1.06  0.689  0.836  rs867858  C/A  0.31  0.31  0.05  1.02  0.824  0.904  rs1062219  A/G  0.45  0.46  0.20  0.96  0.655  0.836  rs804290  A/G  0.24  0.22  1.31  1.13  0.253  0.836  rs11785481  A/G  0.14  0.15  0.26  0.93  0.607  0.836  rs12458  T/A  0.33  0.31  0.36  1.06  0.548  0.836  rs3203358  C/G  0.33  0.32  0.01  1.01  0.917  0.944  aN = 534 cases, 413 controls. bAlleles 1 and 2 refer to minor and major allele, respectively. cMAF = minor allele frequency. *P < 0.05. Table 2. Associations between GATA4 gene SNPs and AUD in EA samplea SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.10  0.09  1.37  1.21  0.243  0.836  rs10105409  G/A  0.01  0.00  0.05  1.16  0.818  0.904  rs6601604  A/G  0.29  0.34  4.42  0.81  0.036*  0.836  rs10112596  A/G  0.17  0.19  1.33  0.87  0.248  0.836  rs12550668  A/G  0.40  0.43  1.74  0.88  0.188  0.836  rs2898292  G/A  0.10  0.10  0.00  1.00  0.995  0.995  rs4840579  G/A  0.39  0.40  0.28  0.95  0.597  0.836  rs11250159  A/C  0.08  0.09  0.65  0.87  0.421  0.836  rs17153694  A/G  0.07  0.08  0.18  0.93  0.670  0.836  rs17153698  A/G  0.16  0.15  0.24  1.06  0.625  0.836  rs6983129  C/A  0.47  0.48  0.59  0.93  0.444  0.836  rs2898295  A/G  0.50  0.47  1.06  1.10  0.304  0.836  rs11250163  C/A  0.47  0.45  0.69  1.08  0.407  0.836  rs13275657  A/G  0.19  0.20  0.64  0.91  0.423  0.836  rs2029969  G/A  0.38  0.36  0.53  1.07  0.465  0.836  rs2173117  A/C  0.33  0.30  1.78  1.14  0.182  0.836  rs3779664  A/G  0.14  0.16  1.08  0.87  0.299  0.836  rs3735814  A/G  0.48  0.49  0.18  0.96  0.671  0.836  rs2740434  A/G  0.33  0.34  0.13  0.97  0.720  0.844  rs2645399  A/G  0.34  0.36  0.49  0.93  0.486  0.836  rs11784693  A/G  0.29  0.32  1.44  0.89  0.231  0.836  rs804283  G/A  0.29  0.31  1.24  0.89  0.265  0.836  rs17153747  G/A  0.13  0.12  0.75  1.13  0.386  0.836  rs804282  C/A  0.45  0.46  0.40  0.94  0.529  0.836  rs13264774  A/G  0.15  0.15  0.02  1.02  0.880  0.935  rs13273672  G/A  0.30  0.29  0.26  1.05  0.610  0.836  rs804280  C/A  0.43  0.44  0.32  0.95  0.574  0.836  rs3729856  G/A  0.14  0.14  0.16  1.06  0.689  0.836  rs867858  C/A  0.31  0.31  0.05  1.02  0.824  0.904  rs1062219  A/G  0.45  0.46  0.20  0.96  0.655  0.836  rs804290  A/G  0.24  0.22  1.31  1.13  0.253  0.836  rs11785481  A/G  0.14  0.15  0.26  0.93  0.607  0.836  rs12458  T/A  0.33  0.31  0.36  1.06  0.548  0.836  rs3203358  C/G  0.33  0.32  0.01  1.01  0.917  0.944  SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.10  0.09  1.37  1.21  0.243  0.836  rs10105409  G/A  0.01  0.00  0.05  1.16  0.818  0.904  rs6601604  A/G  0.29  0.34  4.42  0.81  0.036*  0.836  rs10112596  A/G  0.17  0.19  1.33  0.87  0.248  0.836  rs12550668  A/G  0.40  0.43  1.74  0.88  0.188  0.836  rs2898292  G/A  0.10  0.10  0.00  1.00  0.995  0.995  rs4840579  G/A  0.39  0.40  0.28  0.95  0.597  0.836  rs11250159  A/C  0.08  0.09  0.65  0.87  0.421  0.836  rs17153694  A/G  0.07  0.08  0.18  0.93  0.670  0.836  rs17153698  A/G  0.16  0.15  0.24  1.06  0.625  0.836  rs6983129  C/A  0.47  0.48  0.59  0.93  0.444  0.836  rs2898295  A/G  0.50  0.47  1.06  1.10  0.304  0.836  rs11250163  C/A  0.47  0.45  0.69  1.08  0.407  0.836  rs13275657  A/G  0.19  0.20  0.64  0.91  0.423  0.836  rs2029969  G/A  0.38  0.36  0.53  1.07  0.465  0.836  rs2173117  A/C  0.33  0.30  1.78  1.14  0.182  0.836  rs3779664  A/G  0.14  0.16  1.08  0.87  0.299  0.836  rs3735814  A/G  0.48  0.49  0.18  0.96  0.671  0.836  rs2740434  A/G  0.33  0.34  0.13  0.97  0.720  0.844  rs2645399  A/G  0.34  0.36  0.49  0.93  0.486  0.836  rs11784693  A/G  0.29  0.32  1.44  0.89  0.231  0.836  rs804283  G/A  0.29  0.31  1.24  0.89  0.265  0.836  rs17153747  G/A  0.13  0.12  0.75  1.13  0.386  0.836  rs804282  C/A  0.45  0.46  0.40  0.94  0.529  0.836  rs13264774  A/G  0.15  0.15  0.02  1.02  0.880  0.935  rs13273672  G/A  0.30  0.29  0.26  1.05  0.610  0.836  rs804280  C/A  0.43  0.44  0.32  0.95  0.574  0.836  rs3729856  G/A  0.14  0.14  0.16  1.06  0.689  0.836  rs867858  C/A  0.31  0.31  0.05  1.02  0.824  0.904  rs1062219  A/G  0.45  0.46  0.20  0.96  0.655  0.836  rs804290  A/G  0.24  0.22  1.31  1.13  0.253  0.836  rs11785481  A/G  0.14  0.15  0.26  0.93  0.607  0.836  rs12458  T/A  0.33  0.31  0.36  1.06  0.548  0.836  rs3203358  C/G  0.33  0.32  0.01  1.01  0.917  0.944  aN = 534 cases, 413 controls. bAlleles 1 and 2 refer to minor and major allele, respectively. cMAF = minor allele frequency. *P < 0.05. In addition to tests of single SNP associations, we ran haplotype analyses of the 34 SNPs. Interestingly, the haplotype structure differed in the EA and AA populations. There were nine haplotype blocks in the EA group (Supplementary Fig. S1), and seven haplotype bocks in the AA group (Supplementary Fig. S2). Two haplotype blocks (Blocks 1 and 5) were nominally significantly related to AUD in the EA group (P = 0.037, P = 0.015, respectively). Block 1 included rs6601604 (Supplementary Table S1), which was nominally significantly associated with AUD in the single SNP association (Table 2). One block (Block 9) reached trend-level significance in this group (P = 0.087) (Supplementary Table S1). Two haplotype blocks (Block 2 and Block 3) trended towards a significant relationship with AUD in the AA group (Ps ≤ 0.068). Block 3 contained both rs11250159 and rs17153694 (Supplementary Table S2), both of which trended towards significance in the single SNP association (Table 3). There was no significant association between haplotype blocks and AUD diagnosis after correction for multiple testing. Further analyses of continuous phenotype outcomes co-varied for age, gender, and African and European ancestry. These analyses showed no significant relationships that survived multiple comparisons between the GATA4 SNPs and scores on the ADS, MADRS, STAI, CIWA or NEO Neuroticism (data not shown). When analyzing only those with current AUD, one phenotype, anxiety, as measured by the BSA was significantly associated with GATA4 SNP rs10112596 when adjusting for multiple comparisons (P = 0.032) in the AA ancestry only (Table 4). This SNP was not in a haplotype block. Table 3. Associations between GATA4 gene SNPs and AUD in AA samplea SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.28  0.32  3.39  0.80  0.065  0.742  rs10105409  G/A  0.17  0.19  0.96  0.87  0.327  0.930  rs6601604  A/G  0.43  0.41  0.62  1.09  0.431  0.930  rs10112596  A/G  0.16  0.14  0.86  1.16  0.353  0.930  rs12550668  G/A  0.11  0.10  0.66  1.16  0.417  0.930  rs2898292  G/A  0.30  0.27  2.06  1.20  0.151  0.921  rs4840579  A/G  0.47  0.46  0.40  1.07  0.527  0.930  rs11250159  A/C  0.21  0.17  3.41  1.31  0.065  0.742  rs17153694  A/G  0.09  0.06  3.79  1.54  0.051  0.742  rs17153698  A/G  0.32  0.31  0.29  1.07  0.588  0.930  rs6983129  C/A  0.38  0.35  1.46  1.15  0.226  0.930  rs2898295  A/G  0.39  0.38  0.12  1.04  0.728  0.930  rs11250163  C/A  0.10  0.10  0.02  0.98  0.898  0.930  rs13275657  A/G  0.04  0.03  0.64  1.27  0.425  0.930  rs2029969  G/A  0.24  0.24  0.01  0.99  0.927  0.930  rs2173117  A/C  0.24  0.23  0.34  1.08  0.559  0.930  rs3779664  A/G  0.06  0.06  0.13  1.09  0.721  0.930  rs3735814  A/G  0.49  0.49  0.06  1.03  0.802  0.930  rs2740434  A/G  0.31  0.31  0.01  1.01  0.930  0.930  rs2645399  A/G  0.48  0.51  0.69  0.91  0.408  0.930  rs11784693  A/G  0.22  0.21  0.12  1.05  0.726  0.930  rs804283  G/A  0.13  0.11  1.72  1.26  0.190  0.921  rs17153747  G/A  0.05  0.04  0.27  1.15  0.605  0.930  rs804282  C/A  0.46  0.48  0.18  0.95  0.671  0.930  rs13264774  A/G  0.22  0.26  1.82  0.84  0.177  0.921  rs13273672  G/A  0.37  0.38  0.20  0.95  0.657  0.930  rs804280  C/A  0.42  0.41  0.20  1.05  0.655  0.930  rs3729856  G/A  0.02  0.02  0.63  0.74  0.428  0.930  rs867858  C/A  0.23  0.23  0.06  0.97  0.810  0.930  rs1062219  A/G  0.17  0.17  0.03  1.03  0.867  0.930  rs804290  A/G  0.05  0.03  2.20  1.54  0.138  0.921  rs11785481  A/G  0.03  0.03  0.03  0.94  0.852  0.930  rs12458  T/A  0.39  0.39  0.02  0.98  0.892  0.930  rs3203358  C/G  0.06  0.06  0.19  1.11  0.667  0.930  SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.28  0.32  3.39  0.80  0.065  0.742  rs10105409  G/A  0.17  0.19  0.96  0.87  0.327  0.930  rs6601604  A/G  0.43  0.41  0.62  1.09  0.431  0.930  rs10112596  A/G  0.16  0.14  0.86  1.16  0.353  0.930  rs12550668  G/A  0.11  0.10  0.66  1.16  0.417  0.930  rs2898292  G/A  0.30  0.27  2.06  1.20  0.151  0.921  rs4840579  A/G  0.47  0.46  0.40  1.07  0.527  0.930  rs11250159  A/C  0.21  0.17  3.41  1.31  0.065  0.742  rs17153694  A/G  0.09  0.06  3.79  1.54  0.051  0.742  rs17153698  A/G  0.32  0.31  0.29  1.07  0.588  0.930  rs6983129  C/A  0.38  0.35  1.46  1.15  0.226  0.930  rs2898295  A/G  0.39  0.38  0.12  1.04  0.728  0.930  rs11250163  C/A  0.10  0.10  0.02  0.98  0.898  0.930  rs13275657  A/G  0.04  0.03  0.64  1.27  0.425  0.930  rs2029969  G/A  0.24  0.24  0.01  0.99  0.927  0.930  rs2173117  A/C  0.24  0.23  0.34  1.08  0.559  0.930  rs3779664  A/G  0.06  0.06  0.13  1.09  0.721  0.930  rs3735814  A/G  0.49  0.49  0.06  1.03  0.802  0.930  rs2740434  A/G  0.31  0.31  0.01  1.01  0.930  0.930  rs2645399  A/G  0.48  0.51  0.69  0.91  0.408  0.930  rs11784693  A/G  0.22  0.21  0.12  1.05  0.726  0.930  rs804283  G/A  0.13  0.11  1.72  1.26  0.190  0.921  rs17153747  G/A  0.05  0.04  0.27  1.15  0.605  0.930  rs804282  C/A  0.46  0.48  0.18  0.95  0.671  0.930  rs13264774  A/G  0.22  0.26  1.82  0.84  0.177  0.921  rs13273672  G/A  0.37  0.38  0.20  0.95  0.657  0.930  rs804280  C/A  0.42  0.41  0.20  1.05  0.655  0.930  rs3729856  G/A  0.02  0.02  0.63  0.74  0.428  0.930  rs867858  C/A  0.23  0.23  0.06  0.97  0.810  0.930  rs1062219  A/G  0.17  0.17  0.03  1.03  0.867  0.930  rs804290  A/G  0.05  0.03  2.20  1.54  0.138  0.921  rs11785481  A/G  0.03  0.03  0.03  0.94  0.852  0.930  rs12458  T/A  0.39  0.39  0.02  0.98  0.892  0.930  rs3203358  C/G  0.06  0.06  0.19  1.11  0.667  0.930  aN = 510 cases, 232 controls. bAlleles 1 and 2 refer to minor and major allele, respectively. cMAF = minor allele frequency. Table 3. Associations between GATA4 gene SNPs and AUD in AA samplea SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.28  0.32  3.39  0.80  0.065  0.742  rs10105409  G/A  0.17  0.19  0.96  0.87  0.327  0.930  rs6601604  A/G  0.43  0.41  0.62  1.09  0.431  0.930  rs10112596  A/G  0.16  0.14  0.86  1.16  0.353  0.930  rs12550668  G/A  0.11  0.10  0.66  1.16  0.417  0.930  rs2898292  G/A  0.30  0.27  2.06  1.20  0.151  0.921  rs4840579  A/G  0.47  0.46  0.40  1.07  0.527  0.930  rs11250159  A/C  0.21  0.17  3.41  1.31  0.065  0.742  rs17153694  A/G  0.09  0.06  3.79  1.54  0.051  0.742  rs17153698  A/G  0.32  0.31  0.29  1.07  0.588  0.930  rs6983129  C/A  0.38  0.35  1.46  1.15  0.226  0.930  rs2898295  A/G  0.39  0.38  0.12  1.04  0.728  0.930  rs11250163  C/A  0.10  0.10  0.02  0.98  0.898  0.930  rs13275657  A/G  0.04  0.03  0.64  1.27  0.425  0.930  rs2029969  G/A  0.24  0.24  0.01  0.99  0.927  0.930  rs2173117  A/C  0.24  0.23  0.34  1.08  0.559  0.930  rs3779664  A/G  0.06  0.06  0.13  1.09  0.721  0.930  rs3735814  A/G  0.49  0.49  0.06  1.03  0.802  0.930  rs2740434  A/G  0.31  0.31  0.01  1.01  0.930  0.930  rs2645399  A/G  0.48  0.51  0.69  0.91  0.408  0.930  rs11784693  A/G  0.22  0.21  0.12  1.05  0.726  0.930  rs804283  G/A  0.13  0.11  1.72  1.26  0.190  0.921  rs17153747  G/A  0.05  0.04  0.27  1.15  0.605  0.930  rs804282  C/A  0.46  0.48  0.18  0.95  0.671  0.930  rs13264774  A/G  0.22  0.26  1.82  0.84  0.177  0.921  rs13273672  G/A  0.37  0.38  0.20  0.95  0.657  0.930  rs804280  C/A  0.42  0.41  0.20  1.05  0.655  0.930  rs3729856  G/A  0.02  0.02  0.63  0.74  0.428  0.930  rs867858  C/A  0.23  0.23  0.06  0.97  0.810  0.930  rs1062219  A/G  0.17  0.17  0.03  1.03  0.867  0.930  rs804290  A/G  0.05  0.03  2.20  1.54  0.138  0.921  rs11785481  A/G  0.03  0.03  0.03  0.94  0.852  0.930  rs12458  T/A  0.39  0.39  0.02  0.98  0.892  0.930  rs3203358  C/G  0.06  0.06  0.19  1.11  0.667  0.930  SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.28  0.32  3.39  0.80  0.065  0.742  rs10105409  G/A  0.17  0.19  0.96  0.87  0.327  0.930  rs6601604  A/G  0.43  0.41  0.62  1.09  0.431  0.930  rs10112596  A/G  0.16  0.14  0.86  1.16  0.353  0.930  rs12550668  G/A  0.11  0.10  0.66  1.16  0.417  0.930  rs2898292  G/A  0.30  0.27  2.06  1.20  0.151  0.921  rs4840579  A/G  0.47  0.46  0.40  1.07  0.527  0.930  rs11250159  A/C  0.21  0.17  3.41  1.31  0.065  0.742  rs17153694  A/G  0.09  0.06  3.79  1.54  0.051  0.742  rs17153698  A/G  0.32  0.31  0.29  1.07  0.588  0.930  rs6983129  C/A  0.38  0.35  1.46  1.15  0.226  0.930  rs2898295  A/G  0.39  0.38  0.12  1.04  0.728  0.930  rs11250163  C/A  0.10  0.10  0.02  0.98  0.898  0.930  rs13275657  A/G  0.04  0.03  0.64  1.27  0.425  0.930  rs2029969  G/A  0.24  0.24  0.01  0.99  0.927  0.930  rs2173117  A/C  0.24  0.23  0.34  1.08  0.559  0.930  rs3779664  A/G  0.06  0.06  0.13  1.09  0.721  0.930  rs3735814  A/G  0.49  0.49  0.06  1.03  0.802  0.930  rs2740434  A/G  0.31  0.31  0.01  1.01  0.930  0.930  rs2645399  A/G  0.48  0.51  0.69  0.91  0.408  0.930  rs11784693  A/G  0.22  0.21  0.12  1.05  0.726  0.930  rs804283  G/A  0.13  0.11  1.72  1.26  0.190  0.921  rs17153747  G/A  0.05  0.04  0.27  1.15  0.605  0.930  rs804282  C/A  0.46  0.48  0.18  0.95  0.671  0.930  rs13264774  A/G  0.22  0.26  1.82  0.84  0.177  0.921  rs13273672  G/A  0.37  0.38  0.20  0.95  0.657  0.930  rs804280  C/A  0.42  0.41  0.20  1.05  0.655  0.930  rs3729856  G/A  0.02  0.02  0.63  0.74  0.428  0.930  rs867858  C/A  0.23  0.23  0.06  0.97  0.810  0.930  rs1062219  A/G  0.17  0.17  0.03  1.03  0.867  0.930  rs804290  A/G  0.05  0.03  2.20  1.54  0.138  0.921  rs11785481  A/G  0.03  0.03  0.03  0.94  0.852  0.930  rs12458  T/A  0.39  0.39  0.02  0.98  0.892  0.930  rs3203358  C/G  0.06  0.06  0.19  1.11  0.667  0.930  aN = 510 cases, 232 controls. bAlleles 1 and 2 refer to minor and major allele, respectively. cMAF = minor allele frequency. DISCUSSION Previous studies have found evidence that implicates the GATA4 gene in susceptibility to alcohol dependence (Treutlein et al., 2009; Edenberg et al., 2010; Karpyak et al., 2014). In particular, the SNP rs13273672 has been found to be related to variance in ANP expression, alcohol-induced cue reactivity and relapse risk (Kiefer et al., 2011; Jorde et al., 2014; Zois et al., 2016). This case-control study aimed to replicate previous GWAS and candidate gene studies relating GATA4 and SNP rs13273672 with AUD and alcohol-related phenotypes. Although previous studies have shown a relationship between variants of this gene and a diagnosis of AUD, we did not replicate these findings in our sample. Table 4. Associations between GATA4 gene SNPs and Brief Scale for Anxiety (BSA) scores in AA sample with AUD SNP  A1a  N  BETA  STAT  P-value  FDR  rs6990313  A  414  1.14  2.09  0.037  0.418  rs10105409  G  415  1.55  2.43  0.015  0.262  rs6601604  A  415  0.24  0.47  0.642  0.845  rs10112596  A  415  −2.24  −3.33  0.001*  0.032*  rs12550668  G  415  −0.76  −0.95  0.344  0.828  rs2898292  G  415  0.94  1.72  0.086  0.418  rs4840579  A  415  0.30  0.58  0.560  0.828  rs11250159  A  415  1.07  1.73  0.085  0.418  rs17153694  A  412  1.03  1.17  0.241  0.746  rs17153698  A  415  0.83  1.58  0.115  0.489  rs6983129  C  414  0.34  0.65  0.516  0.828  rs2898295  A  415  0.22  0.43  0.671  0.845  rs11250163  C  415  −0.27  −0.32  0.753  0.883  rs13275657  A  411  −1.33  −0.94  0.346  0.828  rs2029969  G  415  −0.38  −0.63  0.532  0.828  rs2173117  A  415  −0.44  −0.74  0.461  0.828  rs3779664  A  415  −0.44  −0.43  0.665  0.845  rs3735814  A  415  0.92  1.86  0.064  0.418  rs2740434  A  415  0.42  0.77  0.440  0.828  rs2645399  A  414  0.42  0.83  0.405  0.828  rs11784693  A  414  0.33  0.53  0.598  0.845  rs804283  G  415  0.06  0.08  0.940  0.940  rs17153747  G  415  −0.72  −0.61  0.545  0.828  rs804282  C  414  −0.04  −0.09  0.927  0.940  rs13264774  A  415  0.42  0.67  0.506  0.828  rs13273672  G  415  0.20  0.36  0.717  0.870  rs804280  C  415  0.46  0.87  0.384  0.828  rs3729856  G  415  0.17  0.10  0.924  0.940  rs867858  C  415  −0.10  −0.15  0.877  0.940  rs1062219  A  412  −1.15  −1.78  0.076  0.418  rs804290  A  415  −1.60  −1.40  0.161  0.548  rs11785481  A  415  0.33  0.22  0.825  0.935  rs12458  T  413  −0.32  −0.61  0.541  0.828  rs3203358  C  415  −1.57  −1.43  0.153  0.548  SNP  A1a  N  BETA  STAT  P-value  FDR  rs6990313  A  414  1.14  2.09  0.037  0.418  rs10105409  G  415  1.55  2.43  0.015  0.262  rs6601604  A  415  0.24  0.47  0.642  0.845  rs10112596  A  415  −2.24  −3.33  0.001*  0.032*  rs12550668  G  415  −0.76  −0.95  0.344  0.828  rs2898292  G  415  0.94  1.72  0.086  0.418  rs4840579  A  415  0.30  0.58  0.560  0.828  rs11250159  A  415  1.07  1.73  0.085  0.418  rs17153694  A  412  1.03  1.17  0.241  0.746  rs17153698  A  415  0.83  1.58  0.115  0.489  rs6983129  C  414  0.34  0.65  0.516  0.828  rs2898295  A  415  0.22  0.43  0.671  0.845  rs11250163  C  415  −0.27  −0.32  0.753  0.883  rs13275657  A  411  −1.33  −0.94  0.346  0.828  rs2029969  G  415  −0.38  −0.63  0.532  0.828  rs2173117  A  415  −0.44  −0.74  0.461  0.828  rs3779664  A  415  −0.44  −0.43  0.665  0.845  rs3735814  A  415  0.92  1.86  0.064  0.418  rs2740434  A  415  0.42  0.77  0.440  0.828  rs2645399  A  414  0.42  0.83  0.405  0.828  rs11784693  A  414  0.33  0.53  0.598  0.845  rs804283  G  415  0.06  0.08  0.940  0.940  rs17153747  G  415  −0.72  −0.61  0.545  0.828  rs804282  C  414  −0.04  −0.09  0.927  0.940  rs13264774  A  415  0.42  0.67  0.506  0.828  rs13273672  G  415  0.20  0.36  0.717  0.870  rs804280  C  415  0.46  0.87  0.384  0.828  rs3729856  G  415  0.17  0.10  0.924  0.940  rs867858  C  415  −0.10  −0.15  0.877  0.940  rs1062219  A  412  −1.15  −1.78  0.076  0.418  rs804290  A  415  −1.60  −1.40  0.161  0.548  rs11785481  A  415  0.33  0.22  0.825  0.935  rs12458  T  413  −0.32  −0.61  0.541  0.828  rs3203358  C  415  −1.57  −1.43  0.153  0.548  aAllele 1 refers to minor allele. bAnalysis controls for age, gender, African ancestry informative markers and European ancestry informative markers. *P < 0.05. Table 4. Associations between GATA4 gene SNPs and Brief Scale for Anxiety (BSA) scores in AA sample with AUD SNP  A1a  N  BETA  STAT  P-value  FDR  rs6990313  A  414  1.14  2.09  0.037  0.418  rs10105409  G  415  1.55  2.43  0.015  0.262  rs6601604  A  415  0.24  0.47  0.642  0.845  rs10112596  A  415  −2.24  −3.33  0.001*  0.032*  rs12550668  G  415  −0.76  −0.95  0.344  0.828  rs2898292  G  415  0.94  1.72  0.086  0.418  rs4840579  A  415  0.30  0.58  0.560  0.828  rs11250159  A  415  1.07  1.73  0.085  0.418  rs17153694  A  412  1.03  1.17  0.241  0.746  rs17153698  A  415  0.83  1.58  0.115  0.489  rs6983129  C  414  0.34  0.65  0.516  0.828  rs2898295  A  415  0.22  0.43  0.671  0.845  rs11250163  C  415  −0.27  −0.32  0.753  0.883  rs13275657  A  411  −1.33  −0.94  0.346  0.828  rs2029969  G  415  −0.38  −0.63  0.532  0.828  rs2173117  A  415  −0.44  −0.74  0.461  0.828  rs3779664  A  415  −0.44  −0.43  0.665  0.845  rs3735814  A  415  0.92  1.86  0.064  0.418  rs2740434  A  415  0.42  0.77  0.440  0.828  rs2645399  A  414  0.42  0.83  0.405  0.828  rs11784693  A  414  0.33  0.53  0.598  0.845  rs804283  G  415  0.06  0.08  0.940  0.940  rs17153747  G  415  −0.72  −0.61  0.545  0.828  rs804282  C  414  −0.04  −0.09  0.927  0.940  rs13264774  A  415  0.42  0.67  0.506  0.828  rs13273672  G  415  0.20  0.36  0.717  0.870  rs804280  C  415  0.46  0.87  0.384  0.828  rs3729856  G  415  0.17  0.10  0.924  0.940  rs867858  C  415  −0.10  −0.15  0.877  0.940  rs1062219  A  412  −1.15  −1.78  0.076  0.418  rs804290  A  415  −1.60  −1.40  0.161  0.548  rs11785481  A  415  0.33  0.22  0.825  0.935  rs12458  T  413  −0.32  −0.61  0.541  0.828  rs3203358  C  415  −1.57  −1.43  0.153  0.548  SNP  A1a  N  BETA  STAT  P-value  FDR  rs6990313  A  414  1.14  2.09  0.037  0.418  rs10105409  G  415  1.55  2.43  0.015  0.262  rs6601604  A  415  0.24  0.47  0.642  0.845  rs10112596  A  415  −2.24  −3.33  0.001*  0.032*  rs12550668  G  415  −0.76  −0.95  0.344  0.828  rs2898292  G  415  0.94  1.72  0.086  0.418  rs4840579  A  415  0.30  0.58  0.560  0.828  rs11250159  A  415  1.07  1.73  0.085  0.418  rs17153694  A  412  1.03  1.17  0.241  0.746  rs17153698  A  415  0.83  1.58  0.115  0.489  rs6983129  C  414  0.34  0.65  0.516  0.828  rs2898295  A  415  0.22  0.43  0.671  0.845  rs11250163  C  415  −0.27  −0.32  0.753  0.883  rs13275657  A  411  −1.33  −0.94  0.346  0.828  rs2029969  G  415  −0.38  −0.63  0.532  0.828  rs2173117  A  415  −0.44  −0.74  0.461  0.828  rs3779664  A  415  −0.44  −0.43  0.665  0.845  rs3735814  A  415  0.92  1.86  0.064  0.418  rs2740434  A  415  0.42  0.77  0.440  0.828  rs2645399  A  414  0.42  0.83  0.405  0.828  rs11784693  A  414  0.33  0.53  0.598  0.845  rs804283  G  415  0.06  0.08  0.940  0.940  rs17153747  G  415  −0.72  −0.61  0.545  0.828  rs804282  C  414  −0.04  −0.09  0.927  0.940  rs13264774  A  415  0.42  0.67  0.506  0.828  rs13273672  G  415  0.20  0.36  0.717  0.870  rs804280  C  415  0.46  0.87  0.384  0.828  rs3729856  G  415  0.17  0.10  0.924  0.940  rs867858  C  415  −0.10  −0.15  0.877  0.940  rs1062219  A  412  −1.15  −1.78  0.076  0.418  rs804290  A  415  −1.60  −1.40  0.161  0.548  rs11785481  A  415  0.33  0.22  0.825  0.935  rs12458  T  413  −0.32  −0.61  0.541  0.828  rs3203358  C  415  −1.57  −1.43  0.153  0.548  aAllele 1 refers to minor allele. bAnalysis controls for age, gender, African ancestry informative markers and European ancestry informative markers. *P < 0.05. There are several explanations for these discrepant results. First, as our study consisted of a relatively small sample size, we may have lacked adequate power to detect small effects, which is a limitation of the current study. This limitation might be particularly relevant given the number of SNPs that did not have a MAF > 5%. While Karpyak et al. (2014) used a sample of over 800 AD cases, we were limited to 534 and 510 AUD cases in the EA and AA subgroups, respectively. Clinical heterogeneity, such as differences in anxiety or participant status, may also account for our inability to replicate past studies. Edenberg et al. (2010) and Treutlein et al. (2009) used a sample of participants receiving treatment for their alcohol use, while our cohort included both treatment-seeking and non-treatment-seeking individuals. Although all AUD patients in the present study received a diagnosis based on the DSM-IV, it is possible that the two cohorts represent different phenotypes, which may have confounded the analysis. Furthermore, it is likely that multiple genes are involved in AUD, with only their interaction accumulating to account for a significant proportion of the variance. Therefore, additional studies of genetic variation are needed to elucidate whether polymorphisms of the GATA4 gene interact with other genes to contribute to the genetic risk for AUD and other alcohol-related phenotypes. Given that one SNP in the EA group (rs6601604) and three SNPs in the AA group (rs6990313, rs11250159 and rs17153694) did not survive correction for multiple comparisons, our data indicate a need for further replication studies with larger sample sizes. Karpyak et al. (2014) used gene-level testing to identify an association between AUD diagnoses and GATA4 variation at the gene-level. Future studies should use this additional analysis to replicate these findings and identify any gene-level association between GATA4 variants and alcohol-related clinical phenotypes. Confirming a gene-level association between GATA4 and AUD would provide a target for identifying and treating maladaptive alcohol use. Our finding of a relationship between SNP rs10112596 and an anxiety measure in the AA group with AUD is novel, as this marker has not yet been associated with any alcohol-related phenotype. ANP levels might underlie this correlation, as decreased ANP levels have been associated with increased anxiety among individuals with AUD during detoxification (Kiefer et al., 2002). This result also supports GATA4 as a potential new target for research on comorbid AUD and anxiety, particularly among those of African ancestry. Consistent with a prior study that found a moderating effect of ethnicity on the association between alcohol abuse and an anxiety-related neuroendocrine biomarker, we found this association between GATA4 and anxiety in only the AA group with AUD (Ransome et al. 2017). Our finding suggests that the minor allele in rs10112596 might provide a protective effect in lowering anxiety susceptibility among African Americans with AUD. However, given our sample size, this association should be further examined in future studies. We did not find any association that survived correction for multiple testing between GATA4 SNPs and several other alcohol-related phenotypes. Further studies should utilize gene-level testing to determine whether there is a gene-level association between GATA4 and comorbid AUD and alcohol-related phenotypes. Our study endeavored to extend the knowledge of the genetic components relating to AUD. We did not replicate previous studies that found an association between GATA4 variants and risk for AUD; however, further studies with larger samples and gene-level testing techniques are needed. We did find an association between rs10112596 and anxiety in the AA group, suggesting that this SNP may contribute to risk for AUD and anxiety in individuals of AA but not EA. This finding also implicates GATA4 in the relationship between AUD and anxiety, indicating a possible protective effect of the rs10112596 A minor allele. This investigation contributes meaningfully to the field because it extends the discovery of certain genotypes that may be associated with a higher risk of developing and maintaining AUD, as well as those genotypes that constitute part of a genetic ‘protective’ factor. As medicine and treatment plans are becoming more personalized and patient-specific, it becomes crucial to elucidate the mechanisms behind the genetic contribution to complex disorders. Ideally, genetic information will provide us with the tools to better diagnose and prevent psychiatric disorders, including AUD. Genetics can also provide meaningful information regarding the underlying biological basis of a disease when designing treatment strategies. An understanding of genetic susceptibility to AUD may inform the development of individualized pharmacological interventions that may provide patient-specific drug efficacy. SUPPLEMENTARY MATERIAL Supplementary data are available at Alcohol And Alcoholism online. Conflict of Interest Statement The authors declare no conflict of interest. Funding This work was supported by the National Institutes of Health (NIH) intramural funding [ZIA-AA000242; Section on Clinical Genomics and Experimental Therapeutics; to F.W.L.; Division of Intramural Clinical and Biological Research of the National Institute on Alcohol Abuse and Alcoholism (NIAAA)]. REFERENCES American Psychiatric Association. 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Association Analysis Between Genetic Variation in GATA Binding Protein 4 (GATA4) and Alcohol Use Disorder

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Oxford University Press
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Medical Council on Alcohol and Oxford University Press 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US.
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0735-0414
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1464-3502
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10.1093/alcalc/agx120
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Abstract

Abstract Aims Previous genetic association studies have shown that variation in the GATA4 gene encoding the GATA binding protein 4, a binding protein that binds to the ANA sequence GATA, increase susceptibility for alcohol use disorder (AUD). In this study, we aimed to replicate those findings in an independent sample and analyze their association with anxiety. Methods Overall, 1044 individuals with AUD [534 European American (EA), 510 African Americans (AA)] and 645 controls [413 EA, 232 AA] were genotyped using 34 markers. Genotype and allele frequencies were compared between cases and controls using chi-square analysis. Other phenotype data were analyzed for possible associations with GATA4 single-nucleotide polymorphisms (SNPs) in individuals with AUD. Results Rs6601604 was nominally significantly associated with AUD in EA, and 3 SNPs (rs6990313, rs11250159 and rs17153694) showed trend-level significance (P < 0.10) in AA. However, none of the SNPs were significant after correcting for multiple testing. Haplotype analysis of the 34 SNPs did not find a significant association between haplotype blocks and AUD diagnosis after correcting for multiple testing. From the phenotype analysis, anxiety was associated with GATA4 SNP rs10112596 among the AA group with AUD after a correction for multiple testing. Conclusions Although previous studies have shown a relationship between variants of the GATA4 gene and a diagnosis of AUD, we did not replicate these findings in our sample. Additional studies of variation in this gene are needed to elucidate whether polymorphisms of the GATA4 gene are associated with AUD and other alcohol-related phenotypes. Short Summary GATA4 variants were not associated with AUD in either the European ancestry or African ancestry groups after correcting for multiple comparisons. Rs10112596 demonstrated a significant relationship with an anxiety measure among the African ancestry group with AUD. INTRODUCTION The prevalence, individual health risks and societal costs of excessive alcohol use demonstrate the importance of identifying underlying causes of pathological alcohol consumption and developing novel treatment approaches (Bouchery et al., 2011; Stahre et al., 2014; Grant et al., 2015). Genetic factors account for ~40–60% of the variance in risk of developing alcohol use disorder (AUD) (Rietschel and Treutlein, 2013; Tawa et al., 2016); however, AUD is a complex disorder, with many factors contributing to its onset and maintenance. Identification of the underlying genetic risk variants will further our understanding of the disorder’s neurobiology and may direct the development of novel individualized (i.e. pharmacogenetic) treatment options for AUD. Recent case-control genome-wide association studies (GWAS) implicate GATA binding protein 4 (GATA4), a gene located on chromosome 8, in associations with vulnerability to AUD diagnosis (Treutlein et al., 2009; Edenberg et al., 2010). While a previous candidate gene study found a significant association between GATA4 and AUD using gene-level testing (Karpyak et al., 2014), a more recent study by Degenhardt et al. (2016) failed to show an association between rare GATA4 variants and AUD. However, it should be noted that Degenhardt et al. (2016) attempted to identify only rare risk-associated variants, which precluded them from identifying likely protective variants in GATA4. The GATA4 gene encodes transcription factor GATA4, which regulates the expression of the atrial natriuretic peptide (ANP), among other proteins (McBride and Nemer, 2001). Importantly, GATA4 protein is expressed throughout cells in the central nervous system (CNS). Reduced ANP expression in the CNS is associated with the dysregulation of stress and anxiety mechanisms in the brain, suggesting a possible link between ANP and AUD (Jorde et al., 2014). ANP also influences hypothalamic–pituitary–adrenal (HPA) axis functioning, as well as amygdala activation, further supporting the relationship between ANP and AUD-related phenotypes (McBride and Nemer, 2001). Clinically, post-detoxification patients with AUD and decreased ANP plasma levels report increased craving and anxiety levels compared to both detoxified patients with AUD and higher ANP plasma levels, as well as controls (Kiefer et al., 2002). GATA4 also acts as a transcription factor for brain natriuretic peptide (BNP), a peptide involved in the regulation of the cardiovascular system. Interestingly, BNP is also involved in stress responses when found in the CNS (Amir et al., 2010). One study demonstrated a relationship between GATA4 binding site methylation and BNP expression among alcohol-dependent patients experiencing alcohol withdrawal (Glahn et al., 2016). Previous GWAS highlight an association between the specific intronic single-nucleotide polymorphism (SNP) rs13273672 in the GATA4 gene and AUD-related phenotypes (Kiefer et al., 2011; Jorde et al., 2014). In a randomized, double-blind, placebo-controlled study, Kiefer et al. (2011) showed that alcohol-dependent individuals with the rs13273672 G allele had a decreased time to relapse following Acamprosate treatment compared to A allele carriers with AUD. Furthermore, the G allele of this SNP was predictive of a significant decrease in variance in ANP plasma concentration compared to the A allele. Acamprosate is an FDA-approved pharmacological intervention for AUD that decreases cravings to reduce relapse risk; it is thought to primarily work through the glutamatergic system, although the exact mechanism of action remains unclear (Kiefer et al., 2011). Further research identified the AA genotype as associated with stronger alcohol cue-induced amygdala activation, and this association was predictive of a lower relapse risk (Jorde et al., 2014). Zois et al. (2016) expanded on this work by identifying an interaction between GATA4 genotype and gray matter volume on relapse risk, such that the AA genotype group showed an association between greater gray matter and a reduced relapse risk. This provides further support for the possible protective nature of the AA genotype. Furthermore, AUD has been shown to be associated with mood and anxiety disorders, neuroticism and alcohol withdrawal (Regier et al., 1990; Malouff et al., 2007; Becker and Mulholland, 2014), all of which may play a role in genetic predisposition to AUD (Tawa et al., 2016). In particular, much research has determined the significant co-occurrence of AUD and anxiety disorders, and it is likely that genetic variation influences this comorbidity (Poikolainen, 2000; Smith and Randall, 2012). According to the common factor model proposed by Smith , a third variable (e.g. genetic susceptibility) explains the presence of both AUD and anxiety disorders. In line with this model, Merikangas et al. (1996) found that relatives of patients with anxiety disorders had an increased risk for alcohol dependence. This result could be partly explained by shared genetic factors influencing susceptibility to AUD and anxiety disorders. Moreover, a review by Kenna et al. (2012) highlights more recent research that has found an association between 5-HTTLPR, a 5-HT transporter polymorphism, and both alcohol dependence and anxiety symptoms. Given the high degree of heritability of AUD and frequent comorbid occurrence of anxiety symptoms, identifying genetic risk factors that contribute to their shared pathophysiology may improve our understanding of comorbid AUD and anxiety, as well as inform the development of pharmacological treatments. Therefore, the present study aimed to explore associations between genetic variation in GATA4 and anxiety, as assessed by the Brief Scale for Anxiety. In summary, previous studies indicate a possible association between variation in the GATA4 gene and AUD. However, the underlying mechanisms of this relationship are still relatively unknown and poorly understood. Therefore, additional studies of SNPs within GATA4 and their association with AUD-related phenotypes are needed. In this case-control study, we sought to (a) replicate findings associating variants in GATA4 with increased susceptibility to AUD and (b) determine associations between GATA4 variants and alcohol-related clinical phenotypes, specifically anxiety, as assessed by the Brief Scale for Anxiety. Identifying genetic variants associated with AUD and related clinical phenotypes could be used to identify individuals at risk of developing AUD. Ultimately, this could inform the development of more targeted pharmacological prevention and treatment approaches for AUD. MATERIALS AND METHODS Participants This study was approved by the Institutional Review Board at the National Institutes of Health (NIH). All participants provided written informed consent and permission to use collected samples. Out of 1778 individuals with collected samples, 1044 individuals with AUD [534 European Americans (EA), 510 African Americans (AA)] and 645 controls [413 EA and 232 AA] took part in this study. The 89 missing participants were excluded because they did not have a completed SCID diagnosis. Study participants were recruited between 2005 and 2016 from the inpatient unit and outpatient clinic of the Laboratory of Clinical and Translational Studies at the National Institute on Alcohol Abuse and Alcoholism (NIAAA), NIH (Bethesda, MD). Participants were recruited from three screening protocols, all of which excluded those under 18 years of age. Two of the screening protocols included only those in good health without major medical problems, and excluded individuals that were under court-mandated or involuntary treatment. The third protocol excluded prisoners, as well as pregnant women. Alcohol-dependent subjects were diagnosed with alcohol dependence according to the Diagnostic and Statistical Manuel for Mental Disorders, 4th edn, Text-revised (DSM-IV-TR) (American Psychiatric Association, 2000). Participants were diagnosed using the Structured Clinical Interview (SCID-I) for DSM-IV-TR (First, et al., 2002). Given the overlap between the DSM-IV alcohol dependence criteria and the Diagnostic and Statistical Manual of Mental Disorders, 5th edn (DSM-5) (American Psychiatric Association, 2013) AUD criteria, all participants also met criteria for AUD; however, a separate clinical interview was not conducted. Informed consent was obtained from all subjects who participated in accordance with the Declaration of Helsinki. Genotyping and SNP selection Large-scale genotyping was performed at the NIAAA Laboratory of Neurogenetics using the Illumina OmniExpress BeadChip (Illumina, San Diego, CA). Data for all SNPs located within the GATA4 gene that were genotyped on the array were extracted using PLINK version 1.07 (Purcell et al., 2007) (http://pngu.mgh.harvard.edu/purcell/plink/), based on start and end base pair positions for the gene located on chromosome 8 (11561716, 1161750; GRCh37/hg19 assembly). This procedure resulted in genotype data for 34 SNPs. Ancestry informative markers (AIMs; n = 2500) were also extracted from the Illumina array to calculate ancestral proportions for all study participants. Using methods described previously for an AIM panel including 186 markers (Hodgkinson et al., 2008), which were not available for the current data set, the ancestry assessment identified six ethnic factors (Africa, Europe, Asia, Far East Asia, Oceania and Americas). An analysis of the 34 SNPs among the full sample (n = 1778) found that all were in Hardy–Weinberg equilibrium (HWE) except rs12550668 (P < 0.005) and rs3729856 (P < 0.033) in the EA group. In the AA group, all SNPs were in HWE except rs6601604 (P < 0.03), rs804280 (P < 0.004) and rs867858 (P < 0.003).The same analysis found that rs10105409 in the EA group and rs13275657, rs17153747, rs3729856, rs804290 and rs11785481 in the AA group all had minor allele frequencies (MAF) < 5%. All other SNPs had a MAF > 5%. Analysis Allele frequencies for each SNP were determined using PLINK for the sample as a whole, and then separately for subjects of European and African ancestry (based on self-report). Due to multiple differences in allele frequency across the 34 SNPs, subsequent analyses were conducted separately in each group. Single marker association analyses were conducted using frequency comparison by chi-square test, which is the standard case-control method in PLINK, with adjustment for multiple comparisons performed using the Benjamini–Hochberg method for false discovery rate (FDR) (Benjamini and Hochberg, 1995). The threshold for FDR was set at q = 0.05. Haplotype blocks were determined using HaploView software (Barrett et al., 2005), with haplotype blocks defined using the default D’/LOD method. Haplotype association tests using these defined blocks were conducted in PLINK, and were corrected for multiple comparisons using permutation tests (5000 permutations). Participants also completed a variety of clinical assessments, including the Alcohol Dependence Scale (ADS; Skinner and Allen, 1982), Montgomery Asberg Depression Rating Scale (MADRS; Montgomery and Asberg, 1979), Brief Scale for Anxiety (BSA; Tyrer et al., 1984), State-Trait Anxiety Inventory (STAI; Spielberger et al., 1970), Clinical Institute Withdrawal Assessment for Alcohol (CIWA; Sullivan et al., 1989) and NEO-PI-R (Costa and McCrae, 2002). Sample sizes for these assessments are inconsistent due to missing data, particularly among the control group who were not administered these assessments until later in the study’s recruitment. Single marker association and haplotype analyses were conducted for these continuous outcomes using linear regression models in PLINK. These analyses controlled for age, gender, and African and European ancestry via the AIMS scores for Africa and Europe, based on research showing age, gender and ethnicity differences in alcohol consumption and its related consequences (Delker et al. 2016). RESULTS Table 1 shows the demographic information of participants, as well as differences in the clinical assessments between groups. As expected, AUD participants had significantly greater scores in all alcohol-related phenotype measures when compared to controls in both the EA and AA group. To analyze the association between the 34 GATA4 SNPs and AUD diagnosis, single marker association analyses were conducted using frequency comparison by chi-square test with adjustment for multiple comparisons using the Benjamini–Hochberg method for FDR. Results revealed that one SNP (rs6601604) was nominally significantly associated with AUD in the EA group (P = 0.036). However, this SNP was not significant after adjusting for multiple comparisons (Table 2). Three SNPs (rs6990313, rs11250159 and rs17153694) trended towards a significant association with AUD in the AA group (Ps ≤ 0.065) (Table 3), but these SNPs failed to reach trend-level significance after adjusting for multiple comparisons. The a priori SNP of interest, rs13273672, was not significantly associated with AUD for either ancestry group in our sample. Table 1. Demographic and clinical assessment information   European ancestry  African ancestry  AUD (n = 534)  Controls (n = 413)  P-valuea  AUD (n = 510)  Controls (n = 232)  P-valuea  Gender count (female)  166 (31.1%)  175 (42.4%)  0.0003  140 (27.5%)  107 (46.1%)  <0.0001  Mean age (SD)  42.5 (11.4)  32.0 (12.1)  <0.0001  43.2 (10.1)  35.6 (11.0)  <0.0001  Mean ADS score (SD)  21.8 (8.3) [n = 451]  2.1 (4.1) [n = 95]  <0.0001  17.7 (8.6) [n = 345]  1.3 (3.9) [n = 94]  <0.0001  Mean MADRS score (SD)  15.3 (9.7) [n = 444]  1.5 (3.5) [n = 166]  <0.0001  11.2 (9.4) [n = 441]  1.2 (3.1) [n = 155]  <0.0001  Mean BSA score (SD)  11.1 (7.0) [n = 446]  1.3 (2.6) [n = 166]  <0.0001  8.7 (7.2) [n = 441]  1.1 (2.6) [n = 155]  <0.0001  Mean STAI score (SD)  43.2 (13.6) [n = 199]  33.6 (11.5) [n = 185]  <0.0001  41.1 (12.4) [n = 235]  32.4 (11.9) [n = 145]  <0.0001  Mean neuroticism score (SD)  56.3 (11.5) [n = 483]  44.8 (10.0) [n = 377]  <0.0001  54.6 (9.7) [n = 444]  44.9 (8.4) [n = 202]  <0.0001    European ancestry  African ancestry  AUD (n = 534)  Controls (n = 413)  P-valuea  AUD (n = 510)  Controls (n = 232)  P-valuea  Gender count (female)  166 (31.1%)  175 (42.4%)  0.0003  140 (27.5%)  107 (46.1%)  <0.0001  Mean age (SD)  42.5 (11.4)  32.0 (12.1)  <0.0001  43.2 (10.1)  35.6 (11.0)  <0.0001  Mean ADS score (SD)  21.8 (8.3) [n = 451]  2.1 (4.1) [n = 95]  <0.0001  17.7 (8.6) [n = 345]  1.3 (3.9) [n = 94]  <0.0001  Mean MADRS score (SD)  15.3 (9.7) [n = 444]  1.5 (3.5) [n = 166]  <0.0001  11.2 (9.4) [n = 441]  1.2 (3.1) [n = 155]  <0.0001  Mean BSA score (SD)  11.1 (7.0) [n = 446]  1.3 (2.6) [n = 166]  <0.0001  8.7 (7.2) [n = 441]  1.1 (2.6) [n = 155]  <0.0001  Mean STAI score (SD)  43.2 (13.6) [n = 199]  33.6 (11.5) [n = 185]  <0.0001  41.1 (12.4) [n = 235]  32.4 (11.9) [n = 145]  <0.0001  Mean neuroticism score (SD)  56.3 (11.5) [n = 483]  44.8 (10.0) [n = 377]  <0.0001  54.6 (9.7) [n = 444]  44.9 (8.4) [n = 202]  <0.0001  aChi-square test for gender; t-test for all remaining continuous variables. Table 1. Demographic and clinical assessment information   European ancestry  African ancestry  AUD (n = 534)  Controls (n = 413)  P-valuea  AUD (n = 510)  Controls (n = 232)  P-valuea  Gender count (female)  166 (31.1%)  175 (42.4%)  0.0003  140 (27.5%)  107 (46.1%)  <0.0001  Mean age (SD)  42.5 (11.4)  32.0 (12.1)  <0.0001  43.2 (10.1)  35.6 (11.0)  <0.0001  Mean ADS score (SD)  21.8 (8.3) [n = 451]  2.1 (4.1) [n = 95]  <0.0001  17.7 (8.6) [n = 345]  1.3 (3.9) [n = 94]  <0.0001  Mean MADRS score (SD)  15.3 (9.7) [n = 444]  1.5 (3.5) [n = 166]  <0.0001  11.2 (9.4) [n = 441]  1.2 (3.1) [n = 155]  <0.0001  Mean BSA score (SD)  11.1 (7.0) [n = 446]  1.3 (2.6) [n = 166]  <0.0001  8.7 (7.2) [n = 441]  1.1 (2.6) [n = 155]  <0.0001  Mean STAI score (SD)  43.2 (13.6) [n = 199]  33.6 (11.5) [n = 185]  <0.0001  41.1 (12.4) [n = 235]  32.4 (11.9) [n = 145]  <0.0001  Mean neuroticism score (SD)  56.3 (11.5) [n = 483]  44.8 (10.0) [n = 377]  <0.0001  54.6 (9.7) [n = 444]  44.9 (8.4) [n = 202]  <0.0001    European ancestry  African ancestry  AUD (n = 534)  Controls (n = 413)  P-valuea  AUD (n = 510)  Controls (n = 232)  P-valuea  Gender count (female)  166 (31.1%)  175 (42.4%)  0.0003  140 (27.5%)  107 (46.1%)  <0.0001  Mean age (SD)  42.5 (11.4)  32.0 (12.1)  <0.0001  43.2 (10.1)  35.6 (11.0)  <0.0001  Mean ADS score (SD)  21.8 (8.3) [n = 451]  2.1 (4.1) [n = 95]  <0.0001  17.7 (8.6) [n = 345]  1.3 (3.9) [n = 94]  <0.0001  Mean MADRS score (SD)  15.3 (9.7) [n = 444]  1.5 (3.5) [n = 166]  <0.0001  11.2 (9.4) [n = 441]  1.2 (3.1) [n = 155]  <0.0001  Mean BSA score (SD)  11.1 (7.0) [n = 446]  1.3 (2.6) [n = 166]  <0.0001  8.7 (7.2) [n = 441]  1.1 (2.6) [n = 155]  <0.0001  Mean STAI score (SD)  43.2 (13.6) [n = 199]  33.6 (11.5) [n = 185]  <0.0001  41.1 (12.4) [n = 235]  32.4 (11.9) [n = 145]  <0.0001  Mean neuroticism score (SD)  56.3 (11.5) [n = 483]  44.8 (10.0) [n = 377]  <0.0001  54.6 (9.7) [n = 444]  44.9 (8.4) [n = 202]  <0.0001  aChi-square test for gender; t-test for all remaining continuous variables. Table 2. Associations between GATA4 gene SNPs and AUD in EA samplea SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.10  0.09  1.37  1.21  0.243  0.836  rs10105409  G/A  0.01  0.00  0.05  1.16  0.818  0.904  rs6601604  A/G  0.29  0.34  4.42  0.81  0.036*  0.836  rs10112596  A/G  0.17  0.19  1.33  0.87  0.248  0.836  rs12550668  A/G  0.40  0.43  1.74  0.88  0.188  0.836  rs2898292  G/A  0.10  0.10  0.00  1.00  0.995  0.995  rs4840579  G/A  0.39  0.40  0.28  0.95  0.597  0.836  rs11250159  A/C  0.08  0.09  0.65  0.87  0.421  0.836  rs17153694  A/G  0.07  0.08  0.18  0.93  0.670  0.836  rs17153698  A/G  0.16  0.15  0.24  1.06  0.625  0.836  rs6983129  C/A  0.47  0.48  0.59  0.93  0.444  0.836  rs2898295  A/G  0.50  0.47  1.06  1.10  0.304  0.836  rs11250163  C/A  0.47  0.45  0.69  1.08  0.407  0.836  rs13275657  A/G  0.19  0.20  0.64  0.91  0.423  0.836  rs2029969  G/A  0.38  0.36  0.53  1.07  0.465  0.836  rs2173117  A/C  0.33  0.30  1.78  1.14  0.182  0.836  rs3779664  A/G  0.14  0.16  1.08  0.87  0.299  0.836  rs3735814  A/G  0.48  0.49  0.18  0.96  0.671  0.836  rs2740434  A/G  0.33  0.34  0.13  0.97  0.720  0.844  rs2645399  A/G  0.34  0.36  0.49  0.93  0.486  0.836  rs11784693  A/G  0.29  0.32  1.44  0.89  0.231  0.836  rs804283  G/A  0.29  0.31  1.24  0.89  0.265  0.836  rs17153747  G/A  0.13  0.12  0.75  1.13  0.386  0.836  rs804282  C/A  0.45  0.46  0.40  0.94  0.529  0.836  rs13264774  A/G  0.15  0.15  0.02  1.02  0.880  0.935  rs13273672  G/A  0.30  0.29  0.26  1.05  0.610  0.836  rs804280  C/A  0.43  0.44  0.32  0.95  0.574  0.836  rs3729856  G/A  0.14  0.14  0.16  1.06  0.689  0.836  rs867858  C/A  0.31  0.31  0.05  1.02  0.824  0.904  rs1062219  A/G  0.45  0.46  0.20  0.96  0.655  0.836  rs804290  A/G  0.24  0.22  1.31  1.13  0.253  0.836  rs11785481  A/G  0.14  0.15  0.26  0.93  0.607  0.836  rs12458  T/A  0.33  0.31  0.36  1.06  0.548  0.836  rs3203358  C/G  0.33  0.32  0.01  1.01  0.917  0.944  SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.10  0.09  1.37  1.21  0.243  0.836  rs10105409  G/A  0.01  0.00  0.05  1.16  0.818  0.904  rs6601604  A/G  0.29  0.34  4.42  0.81  0.036*  0.836  rs10112596  A/G  0.17  0.19  1.33  0.87  0.248  0.836  rs12550668  A/G  0.40  0.43  1.74  0.88  0.188  0.836  rs2898292  G/A  0.10  0.10  0.00  1.00  0.995  0.995  rs4840579  G/A  0.39  0.40  0.28  0.95  0.597  0.836  rs11250159  A/C  0.08  0.09  0.65  0.87  0.421  0.836  rs17153694  A/G  0.07  0.08  0.18  0.93  0.670  0.836  rs17153698  A/G  0.16  0.15  0.24  1.06  0.625  0.836  rs6983129  C/A  0.47  0.48  0.59  0.93  0.444  0.836  rs2898295  A/G  0.50  0.47  1.06  1.10  0.304  0.836  rs11250163  C/A  0.47  0.45  0.69  1.08  0.407  0.836  rs13275657  A/G  0.19  0.20  0.64  0.91  0.423  0.836  rs2029969  G/A  0.38  0.36  0.53  1.07  0.465  0.836  rs2173117  A/C  0.33  0.30  1.78  1.14  0.182  0.836  rs3779664  A/G  0.14  0.16  1.08  0.87  0.299  0.836  rs3735814  A/G  0.48  0.49  0.18  0.96  0.671  0.836  rs2740434  A/G  0.33  0.34  0.13  0.97  0.720  0.844  rs2645399  A/G  0.34  0.36  0.49  0.93  0.486  0.836  rs11784693  A/G  0.29  0.32  1.44  0.89  0.231  0.836  rs804283  G/A  0.29  0.31  1.24  0.89  0.265  0.836  rs17153747  G/A  0.13  0.12  0.75  1.13  0.386  0.836  rs804282  C/A  0.45  0.46  0.40  0.94  0.529  0.836  rs13264774  A/G  0.15  0.15  0.02  1.02  0.880  0.935  rs13273672  G/A  0.30  0.29  0.26  1.05  0.610  0.836  rs804280  C/A  0.43  0.44  0.32  0.95  0.574  0.836  rs3729856  G/A  0.14  0.14  0.16  1.06  0.689  0.836  rs867858  C/A  0.31  0.31  0.05  1.02  0.824  0.904  rs1062219  A/G  0.45  0.46  0.20  0.96  0.655  0.836  rs804290  A/G  0.24  0.22  1.31  1.13  0.253  0.836  rs11785481  A/G  0.14  0.15  0.26  0.93  0.607  0.836  rs12458  T/A  0.33  0.31  0.36  1.06  0.548  0.836  rs3203358  C/G  0.33  0.32  0.01  1.01  0.917  0.944  aN = 534 cases, 413 controls. bAlleles 1 and 2 refer to minor and major allele, respectively. cMAF = minor allele frequency. *P < 0.05. Table 2. Associations between GATA4 gene SNPs and AUD in EA samplea SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.10  0.09  1.37  1.21  0.243  0.836  rs10105409  G/A  0.01  0.00  0.05  1.16  0.818  0.904  rs6601604  A/G  0.29  0.34  4.42  0.81  0.036*  0.836  rs10112596  A/G  0.17  0.19  1.33  0.87  0.248  0.836  rs12550668  A/G  0.40  0.43  1.74  0.88  0.188  0.836  rs2898292  G/A  0.10  0.10  0.00  1.00  0.995  0.995  rs4840579  G/A  0.39  0.40  0.28  0.95  0.597  0.836  rs11250159  A/C  0.08  0.09  0.65  0.87  0.421  0.836  rs17153694  A/G  0.07  0.08  0.18  0.93  0.670  0.836  rs17153698  A/G  0.16  0.15  0.24  1.06  0.625  0.836  rs6983129  C/A  0.47  0.48  0.59  0.93  0.444  0.836  rs2898295  A/G  0.50  0.47  1.06  1.10  0.304  0.836  rs11250163  C/A  0.47  0.45  0.69  1.08  0.407  0.836  rs13275657  A/G  0.19  0.20  0.64  0.91  0.423  0.836  rs2029969  G/A  0.38  0.36  0.53  1.07  0.465  0.836  rs2173117  A/C  0.33  0.30  1.78  1.14  0.182  0.836  rs3779664  A/G  0.14  0.16  1.08  0.87  0.299  0.836  rs3735814  A/G  0.48  0.49  0.18  0.96  0.671  0.836  rs2740434  A/G  0.33  0.34  0.13  0.97  0.720  0.844  rs2645399  A/G  0.34  0.36  0.49  0.93  0.486  0.836  rs11784693  A/G  0.29  0.32  1.44  0.89  0.231  0.836  rs804283  G/A  0.29  0.31  1.24  0.89  0.265  0.836  rs17153747  G/A  0.13  0.12  0.75  1.13  0.386  0.836  rs804282  C/A  0.45  0.46  0.40  0.94  0.529  0.836  rs13264774  A/G  0.15  0.15  0.02  1.02  0.880  0.935  rs13273672  G/A  0.30  0.29  0.26  1.05  0.610  0.836  rs804280  C/A  0.43  0.44  0.32  0.95  0.574  0.836  rs3729856  G/A  0.14  0.14  0.16  1.06  0.689  0.836  rs867858  C/A  0.31  0.31  0.05  1.02  0.824  0.904  rs1062219  A/G  0.45  0.46  0.20  0.96  0.655  0.836  rs804290  A/G  0.24  0.22  1.31  1.13  0.253  0.836  rs11785481  A/G  0.14  0.15  0.26  0.93  0.607  0.836  rs12458  T/A  0.33  0.31  0.36  1.06  0.548  0.836  rs3203358  C/G  0.33  0.32  0.01  1.01  0.917  0.944  SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.10  0.09  1.37  1.21  0.243  0.836  rs10105409  G/A  0.01  0.00  0.05  1.16  0.818  0.904  rs6601604  A/G  0.29  0.34  4.42  0.81  0.036*  0.836  rs10112596  A/G  0.17  0.19  1.33  0.87  0.248  0.836  rs12550668  A/G  0.40  0.43  1.74  0.88  0.188  0.836  rs2898292  G/A  0.10  0.10  0.00  1.00  0.995  0.995  rs4840579  G/A  0.39  0.40  0.28  0.95  0.597  0.836  rs11250159  A/C  0.08  0.09  0.65  0.87  0.421  0.836  rs17153694  A/G  0.07  0.08  0.18  0.93  0.670  0.836  rs17153698  A/G  0.16  0.15  0.24  1.06  0.625  0.836  rs6983129  C/A  0.47  0.48  0.59  0.93  0.444  0.836  rs2898295  A/G  0.50  0.47  1.06  1.10  0.304  0.836  rs11250163  C/A  0.47  0.45  0.69  1.08  0.407  0.836  rs13275657  A/G  0.19  0.20  0.64  0.91  0.423  0.836  rs2029969  G/A  0.38  0.36  0.53  1.07  0.465  0.836  rs2173117  A/C  0.33  0.30  1.78  1.14  0.182  0.836  rs3779664  A/G  0.14  0.16  1.08  0.87  0.299  0.836  rs3735814  A/G  0.48  0.49  0.18  0.96  0.671  0.836  rs2740434  A/G  0.33  0.34  0.13  0.97  0.720  0.844  rs2645399  A/G  0.34  0.36  0.49  0.93  0.486  0.836  rs11784693  A/G  0.29  0.32  1.44  0.89  0.231  0.836  rs804283  G/A  0.29  0.31  1.24  0.89  0.265  0.836  rs17153747  G/A  0.13  0.12  0.75  1.13  0.386  0.836  rs804282  C/A  0.45  0.46  0.40  0.94  0.529  0.836  rs13264774  A/G  0.15  0.15  0.02  1.02  0.880  0.935  rs13273672  G/A  0.30  0.29  0.26  1.05  0.610  0.836  rs804280  C/A  0.43  0.44  0.32  0.95  0.574  0.836  rs3729856  G/A  0.14  0.14  0.16  1.06  0.689  0.836  rs867858  C/A  0.31  0.31  0.05  1.02  0.824  0.904  rs1062219  A/G  0.45  0.46  0.20  0.96  0.655  0.836  rs804290  A/G  0.24  0.22  1.31  1.13  0.253  0.836  rs11785481  A/G  0.14  0.15  0.26  0.93  0.607  0.836  rs12458  T/A  0.33  0.31  0.36  1.06  0.548  0.836  rs3203358  C/G  0.33  0.32  0.01  1.01  0.917  0.944  aN = 534 cases, 413 controls. bAlleles 1 and 2 refer to minor and major allele, respectively. cMAF = minor allele frequency. *P < 0.05. In addition to tests of single SNP associations, we ran haplotype analyses of the 34 SNPs. Interestingly, the haplotype structure differed in the EA and AA populations. There were nine haplotype blocks in the EA group (Supplementary Fig. S1), and seven haplotype bocks in the AA group (Supplementary Fig. S2). Two haplotype blocks (Blocks 1 and 5) were nominally significantly related to AUD in the EA group (P = 0.037, P = 0.015, respectively). Block 1 included rs6601604 (Supplementary Table S1), which was nominally significantly associated with AUD in the single SNP association (Table 2). One block (Block 9) reached trend-level significance in this group (P = 0.087) (Supplementary Table S1). Two haplotype blocks (Block 2 and Block 3) trended towards a significant relationship with AUD in the AA group (Ps ≤ 0.068). Block 3 contained both rs11250159 and rs17153694 (Supplementary Table S2), both of which trended towards significance in the single SNP association (Table 3). There was no significant association between haplotype blocks and AUD diagnosis after correction for multiple testing. Further analyses of continuous phenotype outcomes co-varied for age, gender, and African and European ancestry. These analyses showed no significant relationships that survived multiple comparisons between the GATA4 SNPs and scores on the ADS, MADRS, STAI, CIWA or NEO Neuroticism (data not shown). When analyzing only those with current AUD, one phenotype, anxiety, as measured by the BSA was significantly associated with GATA4 SNP rs10112596 when adjusting for multiple comparisons (P = 0.032) in the AA ancestry only (Table 4). This SNP was not in a haplotype block. Table 3. Associations between GATA4 gene SNPs and AUD in AA samplea SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.28  0.32  3.39  0.80  0.065  0.742  rs10105409  G/A  0.17  0.19  0.96  0.87  0.327  0.930  rs6601604  A/G  0.43  0.41  0.62  1.09  0.431  0.930  rs10112596  A/G  0.16  0.14  0.86  1.16  0.353  0.930  rs12550668  G/A  0.11  0.10  0.66  1.16  0.417  0.930  rs2898292  G/A  0.30  0.27  2.06  1.20  0.151  0.921  rs4840579  A/G  0.47  0.46  0.40  1.07  0.527  0.930  rs11250159  A/C  0.21  0.17  3.41  1.31  0.065  0.742  rs17153694  A/G  0.09  0.06  3.79  1.54  0.051  0.742  rs17153698  A/G  0.32  0.31  0.29  1.07  0.588  0.930  rs6983129  C/A  0.38  0.35  1.46  1.15  0.226  0.930  rs2898295  A/G  0.39  0.38  0.12  1.04  0.728  0.930  rs11250163  C/A  0.10  0.10  0.02  0.98  0.898  0.930  rs13275657  A/G  0.04  0.03  0.64  1.27  0.425  0.930  rs2029969  G/A  0.24  0.24  0.01  0.99  0.927  0.930  rs2173117  A/C  0.24  0.23  0.34  1.08  0.559  0.930  rs3779664  A/G  0.06  0.06  0.13  1.09  0.721  0.930  rs3735814  A/G  0.49  0.49  0.06  1.03  0.802  0.930  rs2740434  A/G  0.31  0.31  0.01  1.01  0.930  0.930  rs2645399  A/G  0.48  0.51  0.69  0.91  0.408  0.930  rs11784693  A/G  0.22  0.21  0.12  1.05  0.726  0.930  rs804283  G/A  0.13  0.11  1.72  1.26  0.190  0.921  rs17153747  G/A  0.05  0.04  0.27  1.15  0.605  0.930  rs804282  C/A  0.46  0.48  0.18  0.95  0.671  0.930  rs13264774  A/G  0.22  0.26  1.82  0.84  0.177  0.921  rs13273672  G/A  0.37  0.38  0.20  0.95  0.657  0.930  rs804280  C/A  0.42  0.41  0.20  1.05  0.655  0.930  rs3729856  G/A  0.02  0.02  0.63  0.74  0.428  0.930  rs867858  C/A  0.23  0.23  0.06  0.97  0.810  0.930  rs1062219  A/G  0.17  0.17  0.03  1.03  0.867  0.930  rs804290  A/G  0.05  0.03  2.20  1.54  0.138  0.921  rs11785481  A/G  0.03  0.03  0.03  0.94  0.852  0.930  rs12458  T/A  0.39  0.39  0.02  0.98  0.892  0.930  rs3203358  C/G  0.06  0.06  0.19  1.11  0.667  0.930  SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.28  0.32  3.39  0.80  0.065  0.742  rs10105409  G/A  0.17  0.19  0.96  0.87  0.327  0.930  rs6601604  A/G  0.43  0.41  0.62  1.09  0.431  0.930  rs10112596  A/G  0.16  0.14  0.86  1.16  0.353  0.930  rs12550668  G/A  0.11  0.10  0.66  1.16  0.417  0.930  rs2898292  G/A  0.30  0.27  2.06  1.20  0.151  0.921  rs4840579  A/G  0.47  0.46  0.40  1.07  0.527  0.930  rs11250159  A/C  0.21  0.17  3.41  1.31  0.065  0.742  rs17153694  A/G  0.09  0.06  3.79  1.54  0.051  0.742  rs17153698  A/G  0.32  0.31  0.29  1.07  0.588  0.930  rs6983129  C/A  0.38  0.35  1.46  1.15  0.226  0.930  rs2898295  A/G  0.39  0.38  0.12  1.04  0.728  0.930  rs11250163  C/A  0.10  0.10  0.02  0.98  0.898  0.930  rs13275657  A/G  0.04  0.03  0.64  1.27  0.425  0.930  rs2029969  G/A  0.24  0.24  0.01  0.99  0.927  0.930  rs2173117  A/C  0.24  0.23  0.34  1.08  0.559  0.930  rs3779664  A/G  0.06  0.06  0.13  1.09  0.721  0.930  rs3735814  A/G  0.49  0.49  0.06  1.03  0.802  0.930  rs2740434  A/G  0.31  0.31  0.01  1.01  0.930  0.930  rs2645399  A/G  0.48  0.51  0.69  0.91  0.408  0.930  rs11784693  A/G  0.22  0.21  0.12  1.05  0.726  0.930  rs804283  G/A  0.13  0.11  1.72  1.26  0.190  0.921  rs17153747  G/A  0.05  0.04  0.27  1.15  0.605  0.930  rs804282  C/A  0.46  0.48  0.18  0.95  0.671  0.930  rs13264774  A/G  0.22  0.26  1.82  0.84  0.177  0.921  rs13273672  G/A  0.37  0.38  0.20  0.95  0.657  0.930  rs804280  C/A  0.42  0.41  0.20  1.05  0.655  0.930  rs3729856  G/A  0.02  0.02  0.63  0.74  0.428  0.930  rs867858  C/A  0.23  0.23  0.06  0.97  0.810  0.930  rs1062219  A/G  0.17  0.17  0.03  1.03  0.867  0.930  rs804290  A/G  0.05  0.03  2.20  1.54  0.138  0.921  rs11785481  A/G  0.03  0.03  0.03  0.94  0.852  0.930  rs12458  T/A  0.39  0.39  0.02  0.98  0.892  0.930  rs3203358  C/G  0.06  0.06  0.19  1.11  0.667  0.930  aN = 510 cases, 232 controls. bAlleles 1 and 2 refer to minor and major allele, respectively. cMAF = minor allele frequency. Table 3. Associations between GATA4 gene SNPs and AUD in AA samplea SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.28  0.32  3.39  0.80  0.065  0.742  rs10105409  G/A  0.17  0.19  0.96  0.87  0.327  0.930  rs6601604  A/G  0.43  0.41  0.62  1.09  0.431  0.930  rs10112596  A/G  0.16  0.14  0.86  1.16  0.353  0.930  rs12550668  G/A  0.11  0.10  0.66  1.16  0.417  0.930  rs2898292  G/A  0.30  0.27  2.06  1.20  0.151  0.921  rs4840579  A/G  0.47  0.46  0.40  1.07  0.527  0.930  rs11250159  A/C  0.21  0.17  3.41  1.31  0.065  0.742  rs17153694  A/G  0.09  0.06  3.79  1.54  0.051  0.742  rs17153698  A/G  0.32  0.31  0.29  1.07  0.588  0.930  rs6983129  C/A  0.38  0.35  1.46  1.15  0.226  0.930  rs2898295  A/G  0.39  0.38  0.12  1.04  0.728  0.930  rs11250163  C/A  0.10  0.10  0.02  0.98  0.898  0.930  rs13275657  A/G  0.04  0.03  0.64  1.27  0.425  0.930  rs2029969  G/A  0.24  0.24  0.01  0.99  0.927  0.930  rs2173117  A/C  0.24  0.23  0.34  1.08  0.559  0.930  rs3779664  A/G  0.06  0.06  0.13  1.09  0.721  0.930  rs3735814  A/G  0.49  0.49  0.06  1.03  0.802  0.930  rs2740434  A/G  0.31  0.31  0.01  1.01  0.930  0.930  rs2645399  A/G  0.48  0.51  0.69  0.91  0.408  0.930  rs11784693  A/G  0.22  0.21  0.12  1.05  0.726  0.930  rs804283  G/A  0.13  0.11  1.72  1.26  0.190  0.921  rs17153747  G/A  0.05  0.04  0.27  1.15  0.605  0.930  rs804282  C/A  0.46  0.48  0.18  0.95  0.671  0.930  rs13264774  A/G  0.22  0.26  1.82  0.84  0.177  0.921  rs13273672  G/A  0.37  0.38  0.20  0.95  0.657  0.930  rs804280  C/A  0.42  0.41  0.20  1.05  0.655  0.930  rs3729856  G/A  0.02  0.02  0.63  0.74  0.428  0.930  rs867858  C/A  0.23  0.23  0.06  0.97  0.810  0.930  rs1062219  A/G  0.17  0.17  0.03  1.03  0.867  0.930  rs804290  A/G  0.05  0.03  2.20  1.54  0.138  0.921  rs11785481  A/G  0.03  0.03  0.03  0.94  0.852  0.930  rs12458  T/A  0.39  0.39  0.02  0.98  0.892  0.930  rs3203358  C/G  0.06  0.06  0.19  1.11  0.667  0.930  SNP  A1/A2b  MAF casesc  MAF controls  Chi square  Odds ratio  P-value  FDR  rs6990313  A/C  0.28  0.32  3.39  0.80  0.065  0.742  rs10105409  G/A  0.17  0.19  0.96  0.87  0.327  0.930  rs6601604  A/G  0.43  0.41  0.62  1.09  0.431  0.930  rs10112596  A/G  0.16  0.14  0.86  1.16  0.353  0.930  rs12550668  G/A  0.11  0.10  0.66  1.16  0.417  0.930  rs2898292  G/A  0.30  0.27  2.06  1.20  0.151  0.921  rs4840579  A/G  0.47  0.46  0.40  1.07  0.527  0.930  rs11250159  A/C  0.21  0.17  3.41  1.31  0.065  0.742  rs17153694  A/G  0.09  0.06  3.79  1.54  0.051  0.742  rs17153698  A/G  0.32  0.31  0.29  1.07  0.588  0.930  rs6983129  C/A  0.38  0.35  1.46  1.15  0.226  0.930  rs2898295  A/G  0.39  0.38  0.12  1.04  0.728  0.930  rs11250163  C/A  0.10  0.10  0.02  0.98  0.898  0.930  rs13275657  A/G  0.04  0.03  0.64  1.27  0.425  0.930  rs2029969  G/A  0.24  0.24  0.01  0.99  0.927  0.930  rs2173117  A/C  0.24  0.23  0.34  1.08  0.559  0.930  rs3779664  A/G  0.06  0.06  0.13  1.09  0.721  0.930  rs3735814  A/G  0.49  0.49  0.06  1.03  0.802  0.930  rs2740434  A/G  0.31  0.31  0.01  1.01  0.930  0.930  rs2645399  A/G  0.48  0.51  0.69  0.91  0.408  0.930  rs11784693  A/G  0.22  0.21  0.12  1.05  0.726  0.930  rs804283  G/A  0.13  0.11  1.72  1.26  0.190  0.921  rs17153747  G/A  0.05  0.04  0.27  1.15  0.605  0.930  rs804282  C/A  0.46  0.48  0.18  0.95  0.671  0.930  rs13264774  A/G  0.22  0.26  1.82  0.84  0.177  0.921  rs13273672  G/A  0.37  0.38  0.20  0.95  0.657  0.930  rs804280  C/A  0.42  0.41  0.20  1.05  0.655  0.930  rs3729856  G/A  0.02  0.02  0.63  0.74  0.428  0.930  rs867858  C/A  0.23  0.23  0.06  0.97  0.810  0.930  rs1062219  A/G  0.17  0.17  0.03  1.03  0.867  0.930  rs804290  A/G  0.05  0.03  2.20  1.54  0.138  0.921  rs11785481  A/G  0.03  0.03  0.03  0.94  0.852  0.930  rs12458  T/A  0.39  0.39  0.02  0.98  0.892  0.930  rs3203358  C/G  0.06  0.06  0.19  1.11  0.667  0.930  aN = 510 cases, 232 controls. bAlleles 1 and 2 refer to minor and major allele, respectively. cMAF = minor allele frequency. DISCUSSION Previous studies have found evidence that implicates the GATA4 gene in susceptibility to alcohol dependence (Treutlein et al., 2009; Edenberg et al., 2010; Karpyak et al., 2014). In particular, the SNP rs13273672 has been found to be related to variance in ANP expression, alcohol-induced cue reactivity and relapse risk (Kiefer et al., 2011; Jorde et al., 2014; Zois et al., 2016). This case-control study aimed to replicate previous GWAS and candidate gene studies relating GATA4 and SNP rs13273672 with AUD and alcohol-related phenotypes. Although previous studies have shown a relationship between variants of this gene and a diagnosis of AUD, we did not replicate these findings in our sample. Table 4. Associations between GATA4 gene SNPs and Brief Scale for Anxiety (BSA) scores in AA sample with AUD SNP  A1a  N  BETA  STAT  P-value  FDR  rs6990313  A  414  1.14  2.09  0.037  0.418  rs10105409  G  415  1.55  2.43  0.015  0.262  rs6601604  A  415  0.24  0.47  0.642  0.845  rs10112596  A  415  −2.24  −3.33  0.001*  0.032*  rs12550668  G  415  −0.76  −0.95  0.344  0.828  rs2898292  G  415  0.94  1.72  0.086  0.418  rs4840579  A  415  0.30  0.58  0.560  0.828  rs11250159  A  415  1.07  1.73  0.085  0.418  rs17153694  A  412  1.03  1.17  0.241  0.746  rs17153698  A  415  0.83  1.58  0.115  0.489  rs6983129  C  414  0.34  0.65  0.516  0.828  rs2898295  A  415  0.22  0.43  0.671  0.845  rs11250163  C  415  −0.27  −0.32  0.753  0.883  rs13275657  A  411  −1.33  −0.94  0.346  0.828  rs2029969  G  415  −0.38  −0.63  0.532  0.828  rs2173117  A  415  −0.44  −0.74  0.461  0.828  rs3779664  A  415  −0.44  −0.43  0.665  0.845  rs3735814  A  415  0.92  1.86  0.064  0.418  rs2740434  A  415  0.42  0.77  0.440  0.828  rs2645399  A  414  0.42  0.83  0.405  0.828  rs11784693  A  414  0.33  0.53  0.598  0.845  rs804283  G  415  0.06  0.08  0.940  0.940  rs17153747  G  415  −0.72  −0.61  0.545  0.828  rs804282  C  414  −0.04  −0.09  0.927  0.940  rs13264774  A  415  0.42  0.67  0.506  0.828  rs13273672  G  415  0.20  0.36  0.717  0.870  rs804280  C  415  0.46  0.87  0.384  0.828  rs3729856  G  415  0.17  0.10  0.924  0.940  rs867858  C  415  −0.10  −0.15  0.877  0.940  rs1062219  A  412  −1.15  −1.78  0.076  0.418  rs804290  A  415  −1.60  −1.40  0.161  0.548  rs11785481  A  415  0.33  0.22  0.825  0.935  rs12458  T  413  −0.32  −0.61  0.541  0.828  rs3203358  C  415  −1.57  −1.43  0.153  0.548  SNP  A1a  N  BETA  STAT  P-value  FDR  rs6990313  A  414  1.14  2.09  0.037  0.418  rs10105409  G  415  1.55  2.43  0.015  0.262  rs6601604  A  415  0.24  0.47  0.642  0.845  rs10112596  A  415  −2.24  −3.33  0.001*  0.032*  rs12550668  G  415  −0.76  −0.95  0.344  0.828  rs2898292  G  415  0.94  1.72  0.086  0.418  rs4840579  A  415  0.30  0.58  0.560  0.828  rs11250159  A  415  1.07  1.73  0.085  0.418  rs17153694  A  412  1.03  1.17  0.241  0.746  rs17153698  A  415  0.83  1.58  0.115  0.489  rs6983129  C  414  0.34  0.65  0.516  0.828  rs2898295  A  415  0.22  0.43  0.671  0.845  rs11250163  C  415  −0.27  −0.32  0.753  0.883  rs13275657  A  411  −1.33  −0.94  0.346  0.828  rs2029969  G  415  −0.38  −0.63  0.532  0.828  rs2173117  A  415  −0.44  −0.74  0.461  0.828  rs3779664  A  415  −0.44  −0.43  0.665  0.845  rs3735814  A  415  0.92  1.86  0.064  0.418  rs2740434  A  415  0.42  0.77  0.440  0.828  rs2645399  A  414  0.42  0.83  0.405  0.828  rs11784693  A  414  0.33  0.53  0.598  0.845  rs804283  G  415  0.06  0.08  0.940  0.940  rs17153747  G  415  −0.72  −0.61  0.545  0.828  rs804282  C  414  −0.04  −0.09  0.927  0.940  rs13264774  A  415  0.42  0.67  0.506  0.828  rs13273672  G  415  0.20  0.36  0.717  0.870  rs804280  C  415  0.46  0.87  0.384  0.828  rs3729856  G  415  0.17  0.10  0.924  0.940  rs867858  C  415  −0.10  −0.15  0.877  0.940  rs1062219  A  412  −1.15  −1.78  0.076  0.418  rs804290  A  415  −1.60  −1.40  0.161  0.548  rs11785481  A  415  0.33  0.22  0.825  0.935  rs12458  T  413  −0.32  −0.61  0.541  0.828  rs3203358  C  415  −1.57  −1.43  0.153  0.548  aAllele 1 refers to minor allele. bAnalysis controls for age, gender, African ancestry informative markers and European ancestry informative markers. *P < 0.05. Table 4. Associations between GATA4 gene SNPs and Brief Scale for Anxiety (BSA) scores in AA sample with AUD SNP  A1a  N  BETA  STAT  P-value  FDR  rs6990313  A  414  1.14  2.09  0.037  0.418  rs10105409  G  415  1.55  2.43  0.015  0.262  rs6601604  A  415  0.24  0.47  0.642  0.845  rs10112596  A  415  −2.24  −3.33  0.001*  0.032*  rs12550668  G  415  −0.76  −0.95  0.344  0.828  rs2898292  G  415  0.94  1.72  0.086  0.418  rs4840579  A  415  0.30  0.58  0.560  0.828  rs11250159  A  415  1.07  1.73  0.085  0.418  rs17153694  A  412  1.03  1.17  0.241  0.746  rs17153698  A  415  0.83  1.58  0.115  0.489  rs6983129  C  414  0.34  0.65  0.516  0.828  rs2898295  A  415  0.22  0.43  0.671  0.845  rs11250163  C  415  −0.27  −0.32  0.753  0.883  rs13275657  A  411  −1.33  −0.94  0.346  0.828  rs2029969  G  415  −0.38  −0.63  0.532  0.828  rs2173117  A  415  −0.44  −0.74  0.461  0.828  rs3779664  A  415  −0.44  −0.43  0.665  0.845  rs3735814  A  415  0.92  1.86  0.064  0.418  rs2740434  A  415  0.42  0.77  0.440  0.828  rs2645399  A  414  0.42  0.83  0.405  0.828  rs11784693  A  414  0.33  0.53  0.598  0.845  rs804283  G  415  0.06  0.08  0.940  0.940  rs17153747  G  415  −0.72  −0.61  0.545  0.828  rs804282  C  414  −0.04  −0.09  0.927  0.940  rs13264774  A  415  0.42  0.67  0.506  0.828  rs13273672  G  415  0.20  0.36  0.717  0.870  rs804280  C  415  0.46  0.87  0.384  0.828  rs3729856  G  415  0.17  0.10  0.924  0.940  rs867858  C  415  −0.10  −0.15  0.877  0.940  rs1062219  A  412  −1.15  −1.78  0.076  0.418  rs804290  A  415  −1.60  −1.40  0.161  0.548  rs11785481  A  415  0.33  0.22  0.825  0.935  rs12458  T  413  −0.32  −0.61  0.541  0.828  rs3203358  C  415  −1.57  −1.43  0.153  0.548  SNP  A1a  N  BETA  STAT  P-value  FDR  rs6990313  A  414  1.14  2.09  0.037  0.418  rs10105409  G  415  1.55  2.43  0.015  0.262  rs6601604  A  415  0.24  0.47  0.642  0.845  rs10112596  A  415  −2.24  −3.33  0.001*  0.032*  rs12550668  G  415  −0.76  −0.95  0.344  0.828  rs2898292  G  415  0.94  1.72  0.086  0.418  rs4840579  A  415  0.30  0.58  0.560  0.828  rs11250159  A  415  1.07  1.73  0.085  0.418  rs17153694  A  412  1.03  1.17  0.241  0.746  rs17153698  A  415  0.83  1.58  0.115  0.489  rs6983129  C  414  0.34  0.65  0.516  0.828  rs2898295  A  415  0.22  0.43  0.671  0.845  rs11250163  C  415  −0.27  −0.32  0.753  0.883  rs13275657  A  411  −1.33  −0.94  0.346  0.828  rs2029969  G  415  −0.38  −0.63  0.532  0.828  rs2173117  A  415  −0.44  −0.74  0.461  0.828  rs3779664  A  415  −0.44  −0.43  0.665  0.845  rs3735814  A  415  0.92  1.86  0.064  0.418  rs2740434  A  415  0.42  0.77  0.440  0.828  rs2645399  A  414  0.42  0.83  0.405  0.828  rs11784693  A  414  0.33  0.53  0.598  0.845  rs804283  G  415  0.06  0.08  0.940  0.940  rs17153747  G  415  −0.72  −0.61  0.545  0.828  rs804282  C  414  −0.04  −0.09  0.927  0.940  rs13264774  A  415  0.42  0.67  0.506  0.828  rs13273672  G  415  0.20  0.36  0.717  0.870  rs804280  C  415  0.46  0.87  0.384  0.828  rs3729856  G  415  0.17  0.10  0.924  0.940  rs867858  C  415  −0.10  −0.15  0.877  0.940  rs1062219  A  412  −1.15  −1.78  0.076  0.418  rs804290  A  415  −1.60  −1.40  0.161  0.548  rs11785481  A  415  0.33  0.22  0.825  0.935  rs12458  T  413  −0.32  −0.61  0.541  0.828  rs3203358  C  415  −1.57  −1.43  0.153  0.548  aAllele 1 refers to minor allele. bAnalysis controls for age, gender, African ancestry informative markers and European ancestry informative markers. *P < 0.05. There are several explanations for these discrepant results. First, as our study consisted of a relatively small sample size, we may have lacked adequate power to detect small effects, which is a limitation of the current study. This limitation might be particularly relevant given the number of SNPs that did not have a MAF > 5%. While Karpyak et al. (2014) used a sample of over 800 AD cases, we were limited to 534 and 510 AUD cases in the EA and AA subgroups, respectively. Clinical heterogeneity, such as differences in anxiety or participant status, may also account for our inability to replicate past studies. Edenberg et al. (2010) and Treutlein et al. (2009) used a sample of participants receiving treatment for their alcohol use, while our cohort included both treatment-seeking and non-treatment-seeking individuals. Although all AUD patients in the present study received a diagnosis based on the DSM-IV, it is possible that the two cohorts represent different phenotypes, which may have confounded the analysis. Furthermore, it is likely that multiple genes are involved in AUD, with only their interaction accumulating to account for a significant proportion of the variance. Therefore, additional studies of genetic variation are needed to elucidate whether polymorphisms of the GATA4 gene interact with other genes to contribute to the genetic risk for AUD and other alcohol-related phenotypes. Given that one SNP in the EA group (rs6601604) and three SNPs in the AA group (rs6990313, rs11250159 and rs17153694) did not survive correction for multiple comparisons, our data indicate a need for further replication studies with larger sample sizes. Karpyak et al. (2014) used gene-level testing to identify an association between AUD diagnoses and GATA4 variation at the gene-level. Future studies should use this additional analysis to replicate these findings and identify any gene-level association between GATA4 variants and alcohol-related clinical phenotypes. Confirming a gene-level association between GATA4 and AUD would provide a target for identifying and treating maladaptive alcohol use. Our finding of a relationship between SNP rs10112596 and an anxiety measure in the AA group with AUD is novel, as this marker has not yet been associated with any alcohol-related phenotype. ANP levels might underlie this correlation, as decreased ANP levels have been associated with increased anxiety among individuals with AUD during detoxification (Kiefer et al., 2002). This result also supports GATA4 as a potential new target for research on comorbid AUD and anxiety, particularly among those of African ancestry. Consistent with a prior study that found a moderating effect of ethnicity on the association between alcohol abuse and an anxiety-related neuroendocrine biomarker, we found this association between GATA4 and anxiety in only the AA group with AUD (Ransome et al. 2017). Our finding suggests that the minor allele in rs10112596 might provide a protective effect in lowering anxiety susceptibility among African Americans with AUD. However, given our sample size, this association should be further examined in future studies. We did not find any association that survived correction for multiple testing between GATA4 SNPs and several other alcohol-related phenotypes. Further studies should utilize gene-level testing to determine whether there is a gene-level association between GATA4 and comorbid AUD and alcohol-related phenotypes. Our study endeavored to extend the knowledge of the genetic components relating to AUD. We did not replicate previous studies that found an association between GATA4 variants and risk for AUD; however, further studies with larger samples and gene-level testing techniques are needed. We did find an association between rs10112596 and anxiety in the AA group, suggesting that this SNP may contribute to risk for AUD and anxiety in individuals of AA but not EA. This finding also implicates GATA4 in the relationship between AUD and anxiety, indicating a possible protective effect of the rs10112596 A minor allele. This investigation contributes meaningfully to the field because it extends the discovery of certain genotypes that may be associated with a higher risk of developing and maintaining AUD, as well as those genotypes that constitute part of a genetic ‘protective’ factor. As medicine and treatment plans are becoming more personalized and patient-specific, it becomes crucial to elucidate the mechanisms behind the genetic contribution to complex disorders. 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Alcohol and AlcoholismOxford University Press

Published: Feb 5, 2018

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