Monika Ridinger

A genome-wide association study of alcohol dependence

Excessive alcohol consumption is one of the leading causes of preventable death in the United States. Approximately 14% of those who use alcohol meet criteria during their lifetime for alcohol dependence, which is characterized by tolerance, withdrawal, inability to stop drinking, and continued drinking despite serious psychological or physiological problems. We explored genetic influences on alcohol dependence among 1,897 European-American and African-American subjects with alcohol dependence compared with 1,932 unrelated, alcohol-exposed, nondependent controls. Constitutional DNA of each subject was genotyped using the Illumina 1M beadchip. Fifteen SNPs yielded P < 10−5, but in two independent replication series, no SNP passed a replication threshold of P < 0.05. Candidate gene GABRA2, which encodes the GABA receptor α2 subunit, was evaluated independently. Five SNPs at GABRA2 yielded nominal (uncorrected) P < 0.05, with odds ratios between 1.11 and 1.16. Further dissection of the alcoholism phenotype, to disentangle the influence of comorbid substance-use disorders, will be a next step in identifying genetic variants associated with alcohol dependence.

Genome-wide Association Study of Alcohol Dependence

CONTEXT Alcohol dependence is a serious and common public health problem. It is well established that genetic factors play a major role in the development of this disorder. Identification of genes that contribute to alcohol dependence will improve our understanding of the mechanisms that underlie this disorder. OBJECTIVE To identify susceptibility genes for alcohol dependence through a genome-wide association study (GWAS) and a follow-up study in a population of German male inpatients with an early age at onset. DESIGN The GWAS tested 524,396 single-nucleotide polymorphisms (SNPs). All SNPs with P < 10(-4) were subjected to the follow-up study. In addition, nominally significant SNPs from genes that had also shown expression changes in rat brains after long-term alcohol consumption were selected for the follow-up step. SETTING Five university hospitals in southern and central Germany. PARTICIPANTS The GWAS included 487 male inpatients with alcohol dependence as defined by the DSM-IV and an age at onset younger than 28 years and 1358 population-based control individuals. The follow-up study included 1024 male inpatients and 996 age-matched male controls. All the participants were of German descent. MAIN OUTCOME MEASURES Significant association findings in the GWAS and follow-up study with the same alleles. RESULTS The GWAS produced 121 SNPs with nominal P < 10(-4). These, together with 19 additional SNPs from homologues of rat genes showing differential expression, were genotyped in the follow-up sample. Fifteen SNPs showed significant association with the same allele as in the GWAS. In the combined analysis, 2 closely linked intergenic SNPs met genome-wide significance (rs7590720, P = 9.72 x 10(-9); rs1344694, P = 1.69 x 10(-8)). They are located on chromosome region 2q35, which has been implicated in linkage studies for alcohol phenotypes. Nine SNPs were located in genes, including the CDH13 and ADH1C genes, that have been reported to be associated with alcohol dependence. CONCLUSIONS This is the first GWAS and follow-up study to identify a genome-wide significant association in alcohol dependence. Further independent studies are required to confirm these findings.

Genetic variation in the PNPLA3 gene is associated with alcoholic liver injury in caucasians.

A recent genome‐wide study revealed an association between variation in the PNPLA3 gene and liver fat content. In addition, the PNPLA3 single‐nucleotide polymorphism rs738409 (M148I) was reported to be associated with advanced alcoholic liver disease in alcohol‐dependent individuals of Mestizo descent. We therefore evaluated the impact of rs738409 on the manifestation of alcoholic liver disease in two independent German cohorts. Genotype and allele frequencies of rs738409 (M148I) were determined in 1,043 alcoholic patients with or without alcoholic liver injury and in 376 at‐risk drinkers from a population‐based cohort. Relative to alcoholic patients without liver damage (n = 439), rs738409 genotype GG was strongly overrepresented in patients with alcoholic liver cirrhosis (n = 210; OR 2.79; Pgenotype = 1.2 × 10−5; Pallelic = 1.6 × 10−6) and in alcoholic patients without cirrhosis but with elevated alanine aminotransferase levels (n = 219; OR 2.33; Pgenotype = 0.0085; Pallelic = 0.0042). The latter, biochemically defined association was confirmed in an independent population‐based cohort of at‐risk drinkers with a median alcohol intake of 300 g/week (OR 4.75; Pgenotype = 0.040; Pallelic = 0.022), and for aspartate aminotransferase (AST) levels. Frequencies of allele PNPLA3 rs738409(G) in individuals with steatosis and normal alanine aminotransferase (ALT) and AST levels were lower than in alcoholics without steatosis and normal ALT/AST (Pcombined = 0.03). The population attributable risk of cirrhosis in alcoholic carriers of allele PNPLA3 rs738409(G) was estimated at 26.6%. Conclusion: Genotype PNPLA3 rs738409(GG) is associated with alcoholic liver cirrhosis and elevated aminotransferase levels in alcoholic Caucasians. (HEPATOLOGY 2011)

Genome-wide association study of alcohol dependence: significant findings in African- and European-Americans including novel risk loci

We report a GWAS of alcohol dependence (AD) in European-American (EA) and African-American (AA) populations, with replication in independent samples of EAs, AAs and Germans. Our sample for discovery and replication was 16 087 subjects, the largest sample for AD GWAS to date. Numerous genome-wide significant (GWS) associations were identified, many novel. Most associations were population specific, but in several cases were GWS in EAs and AAs for different SNPs at the same locus,showing biological convergence across populations. We confirmed well-known risk loci mapped to alcohol-metabolizing enzyme genes, notably ADH1B (EAs: Arg48His, P=1.17 × 10−31; AAs: Arg369Cys, P=6.33 × 10−17) and ADH1C in AAs (Thr151Thr, P=4.94 × 10−10), and identified novel risk loci mapping to the ADH gene cluster on chromosome 4 and extending centromerically beyond it to include GWS associations at LOC100507053 in AAs (P=2.63 × 10−11), PDLIM5 in EAs (P=2.01 × 10−8), and METAP in AAs (P=3.35 × 10−8). We also identified a novel GWS association (1.17 × 10−10) mapped to chromosome 2 at rs1437396, between MTIF2 and CCDC88A, across all of the EA and AA cohorts, with supportive gene expression evidence, and population-specific GWS for markers on chromosomes 5, 9 and 19. Several of the novel associations implicate direct involvement of, or interaction with, genes previously identified as schizophrenia risk loci. Confirmation of known AD risk loci supports the overall validity of the study; the novel loci are worthy of genetic and biological follow-up. The findings support a convergence of risk genes (but not necessarily risk alleles) between populations, and, to a lesser extent, between psychiatric traits.

A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis

Alcohol misuse is the leading cause of cirrhosis and the second most common indication for liver transplantation in the Western world. We performed a genome-wide association study for alcohol-related cirrhosis in individuals of European descent (712 cases and 1,426 controls) with subsequent validation in two independent European cohorts (1,148 cases and 922 controls). We identified variants in the MBOAT7 (P = 1.03 × 10−9) and TM6SF2 (P = 7.89 × 10−10) genes as new risk loci and confirmed rs738409 in PNPLA3 as an important risk locus for alcohol-related cirrhosis (P = 1.54 × 10−48) at a genome-wide level of significance. These three loci have a role in lipid processing, suggesting that lipid turnover is important in the pathogenesis of alcohol-related cirrhosis.

Effects of the Circadian Rhythm Gene Period 1 (Per1) on Psychosocial Stress-Induced Alcohol Drinking

OBJECTIVE Circadian and stress-response systems mediate environmental changes that affect alcohol drinking. Psychosocial stress is an environmental risk factor for alcohol abuse. Circadian rhythm gene period 1 (Per1) is targeted by stress hormones and is transcriptionally activated in corticotropin releasing factor-expressing cells. The authors hypothesized that Per1 is involved in integrating stress response and circadian rhythmicity and explored its relevance to alcohol drinking. METHOD In mice, the effects of stress on ethanol intake in mPer1-mutant and wild-type mice were assessed. In humans, single nucleotide polymorphisms (SNPs) in hPer1 were tested for association with alcohol drinking behavior in 273 adolescents and an adult case-control sample of 1,006 alcohol-dependent patients and 1,178 comparison subjects. In vitro experiments were conducted to measure genotype-specific expression and transcription factor binding to hPer1. RESULTS The mPer1-mutant mice showed enhanced alcohol consumption in response to social defeat stress relative to their wild-type littermates. An association with the frequency of heavy drinking in adolescents with the hPer1 promoter SNP rs3027172 and with psychosocial adversity was found. There was significant interaction between the rs3027172 genotype and psychosocial adversity on this drinking measure. In a confirmatory analysis, association of hPer1 rs3027172 with alcohol dependence was shown. Cortisol-induced transcriptional activation of hPer1 was reduced in human B-lymphoblastoid cells carrying the risk genotype of rs3027172. Binding affinity of the transcription factor Snail1 to the risk allele of the hPer1 SNP rs3027172 was also reduced. CONCLUSIONS The findings indicate that the hPer1 gene regulates alcohol drinking behavior during stressful conditions and provide evidence for underlying neurobiological mechanisms.

KCNJ6 is associated with adult alcohol dependence and involved in gene × early life stress interactions in adolescent alcohol drinking.

Alcohol abuse and dependence have proven to be complex genetic traits that are influenced by environmental factors. Primate and human studies have shown that early life stress increases the propensity for alcohol abuse in later life. The reinforcing properties of alcohol are mediated by dopaminergic signaling; however, there is little evidence to indicate how stress alters alcohol reinforcement. KCNJ6 (the gene encoding G-protein-coupled inwardly rectifying potassium channel 2 (GIRK2)) is a brain expressed potassium channel with inhibitory effects on dopaminergic tone. The properties of GIRK2 have been shown to be enhanced by the stress peptide corticotrophin-releasing hormone. Therefore, we sought to examine the role of KCNJ6 polymorphisms in adult alcohol dependence and stress-related alcohol abuse in adolescents. We selected 11 SNPs in the promoter region of KCNJ6, which were genotyped in 1152 adult alcohol dependents and 1203 controls. One SNP, rs2836016, was found to be associated with alcohol dependence (p=0.01, false discovery rate). We then assessed rs2836016 in an adolescent sample of 261 subjects, which were characterized for early life stress and adolescent hazardous drinking, defined using the Alcohol Use Disorders Identification Test (AUDIT), to examine gene–environment interactions. In the adolescent sample, the risk genotype of rs2836016 was significantly associated with increased AUDIT scores, but only in those individuals exposed to high levels of psychosocial stress in early life (p=0.01). Our findings show that KCNJ6 is associated with alcohol dependence and may moderate the effect of early psychosocial stress on risky alcohol drinking in adolescents. We have identified a candidate gene for future studies investigating a possible functional link between the response to stress and alcohol reinforcement.

Association of ADH4 genetic variants with alcohol dependence risk and related phenotypes: results from a larger multicenter association study

Genetic variants of the alcohol‐metabolizing enzyme ADH4, located on chromosome 4q22–4q23, have been related to alcohol dependence (AD) risk in previous research. The aim of this association study in a large multicenter sample of alcohol‐dependent individuals and controls is to confirm ADH4 single nucleotide polymorphism (SNP) and haplotype association with AD and relevant related phenotypes. One thousand, six hundred and twenty‐two (1622) inpatient subjects and 1469 control subjects with DSM‐IV. AD from four addiction treatment centres were included. Characteristics of AD and related phenotypes including alcohol withdrawal, Cloningers type I and II and first ages of drinking, regular drinking and AD onset were obtained using standardized structured interviews. After subjects were genotyped for 2 ADH4 polymorphisms, single SNP case‐control and haplotype analyses were conducted. Both variants—rs1800759 and rs1042364—and the A‐A and C‐G haplotypes were significantly related to AD across samples. Furthermore, associations with AD‐related phenotypes and subtypes revealed a potential protective influence of this haplotype. This study confirms the significant relationship of ADH4 variants with AD and related phenotypes. While the rs1800759 and rs1042364 A‐A haplotype had a potential protective influence on the risk for several AD‐related phenotypes, this effect is rather small compared to functional variants of other alcohol or acetaldehyde‐metabolizing enzymes like ALDH2*2 or ADH1B*2.

Sex-Specific Role for Adenylyl Cyclase Type 7 in Alcohol Dependence

Background Alcohol has been shown to critically modulate cyclic adenosine-3′,5′ monophosphate (cAMP) signaling. A number of downstream effectors that respond to the cAMP signals (e.g., protein kinase A, cAMP response element binding protein) have, in turn, been examined in relation to alcohol consumption. These studies did not, however, delineate the point at which the actions of alcohol on the cAMP cascade might translate into differences in drinking behavior. To further understand the role of cAMP synthesis in alcohol drinking and dependence, we investigated a specific adenylyl cyclase isoform, adenylyl cyclase (AC) Type 7, whose activity is selectively enhanced by ethanol. Methods We measured alcohol consumption and preference in mice in which one copy of the Adcy7 gene was disrupted (Adcy7+/−). To demonstrate relevance of this gene for alcohol dependence in humans, we tested the association of polymorphisms in the ADCY7 gene with alcohol dependence in a sample of 1703 alcohol-dependent individuals and 1347 control subjects. Results We show that Adcy7+/− female mice have higher preference for alcohol than wild-type mice, whereas there is little difference in alcohol consumption or preference between Adcy7+/− male mice and wild-type control subjects. In the human sample, we found that single nucleotide polymorphisms in ADCY7 associate with alcohol dependence in women, and these markers are also associated with ADCY7 expression (messenger RNA) levels. Conclusions These findings implicate adenylyl cyclase Type 7 as a critical component of the molecular pathways contributing to alcohol drinking and the development of alcohol dependence.

Analysis of carbohydrate deficient transferrin serum levels during abstinence.

An alcohol-associated change in the serum transferrin glycoform pattern, carbohydrate-deficient transferrin (CDT), is used as a biomarker of chronic moderate to heavy alcohol consumption. Furthermore, CDT is employed as a marker of abstinence. Here, we analyzed CDT in patients with chronic excessive alcohol abuse at the beginning and during abstinence. Twenty-nine alcohol dependent patients were recruited from an in-patient abstention program. Reported drinking levels were at least 100 g/d (range up to 450 g/d; mean: 248.9±94.7 g/d) within the last month before study entry. Blood samples were drawn at the beginning and during the abstention program and the relative concentration (%CDT) of CDT was determined using ion exchange followed by immunodetermination of CDT. At study entry, 25/29 patients had a %CDT level above the established cutoff. Although CDT levels declined during abstinence in most patients, in ten patients with %CDT levels just above the cutoff at the start of the program, the CDT values remained elevated 6 weeks after cessation of drinking. Our data indicate that %CDT levels below the cutoff cannot even rule out long lasting excessive alcohol abuse. Further, measurement of %CDT should be interpreted with special care when used as a marker of alcohol abstinence.