Association of GABRA2 with Drug Dependence in the Collaborative Study of the Genetics of Alcoholism Sample

Results from twin studies suggest that overlapping genetic factors influence alcohol dependence and illicit drug dependence. Using data from the Collaborative Study on the Genetics of Alcoholism (COGA), we examined the association between 69 SNPs in the GABA_A receptor gene cluster on chromosome 4 and marijuana and illicit drug dependence, individually, and as co-occurring phenotypes with alcohol dependence. Results suggested association between marijuana dependence and illicit drug dependence with SNPs in the GABRA2 gene. Interestingly, the evidence for association previously observed with alcohol dependence came only from individuals with comorbid illicit drug dependence. There was no association with other genes in the GABA_A cluster on chromosome 4 with illicit drug dependence.     

Novel exonic mu-opioid receptor gene (OPRM1) polymorphisms not associated with opioid dependence.

The mu-opioid receptor (MOR) mediates reward and dependence associated with opioids and other commonly abused substances. Variability in the MOR gene, OPRM1, may influence risk for opioid dependence. In this study, associations between two single nucleotide polymorphisms (SNPs), dbSNP rs540825 and dbSNP rs562859, and opioid dependence were investigated. The two SNPs are located in the protein coding region of the novel exon X of an alternative splice variant of OPRM1, and can be detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. Genotyping at the two SNPs was performed for 170 severe opioid dependent individuals and 128 carefully screened controls. Although no differences were found between cases and controls, there were significant prevalence differences between African-American (AA) subjects and European-American (EA) subjects for SNP 540825 allele and genotype frequencies. The 540825 and 562859 polymorphisms were found to be in complete linkage disequilibrium (LD) for both ethnic groups, and LD existed between the 562859 SNP and the A(-1320)G SNP in the promoter region of OPRM1 in AAs, based on genotyping data previously carried out on the same subjects. LD between these two markers, separated by 55 kb, links the entire distance studied in this project. The results indicate that polymorphisms in the novel splice variant are not associated with opioid dependence, but are in LD with other polymorphisms in OPRM1.     

Allelic and haplotypic association of GABRA2 with alcohol dependence.

Alcohol dependence is a highly prevalent disorder that is associated with serious morbidity and mortality. Because the GABAA neurotransmitter receptor is an important mediator for several behavioral effects of alcohol, genes encoding GABA-related proteins are functional candidates to influence risk of alcohol dependence. Two genome-wide scans showed linkage of alcohol dependence to a region on chromosome 4p, which contains a cluster of genes encoding GABAA receptor subunits. A recent effort to fine map that region showed a haplotypic association of alcohol dependence to the gene encoding the GABAA receptor alpha-2 subunit (GABRA2). We examined 10 single nucleotide polymorphisms (SNPs) spanning the coding region of this gene in samples of European American subjects with alcohol dependence (n = 446), and controls (n = 334) screened to exclude substance use disorders. There was evidence of association to alcohol dependence for seven adjacent markers spanning 98,000 bp in the middle and 3'-portion of the GABRA2 gene (range of P-values = 0.008-0.03). When the subset of the alcohol-dependent subjects excluding those with a diagnosis of cocaine or opioid dependence or major depressive episode (n = 198) was examined, the strength of the association was increased across these 7 SNPs (range of P-values = 0.002-0.007). Two common haplotypes in this region accounted for 90.8% of chromosomes. The more common haplotype was present in 55.6% of control group chromosomes versus 48.2% of alcohol-dependent subjects (P = 0.007) and 45.8% of subjects with alcohol dependence but no co-morbid drug dependence or depression (P = 0.003). These findings replicate and extend recently reported findings, which together underscore the potential contribution of polymorphic variation at the GABRA2 locus to the risk for alcohol dependence.     

Association between two mu-opioid receptor gene (OPRM1) haplotype blocks and drug or alcohol dependence

We examined 13 single nucleotide polymorphisms (SNPs) spanning the coding region of the mu-opioid receptor gene (OPRM1), among 382 European Americans (EAs) affected with substance dependence [alcohol dependence (AD) and/or drug dependence (DD)] and 338 EA healthy controls. These SNPs delineated two haplotype blocks. Genotype distributions for all SNPs were in Hardy-Weinberg equilibrium (HWE) in controls, but in cases, four SNPs in Block I and three SNPs in Block II showed deviation from HWE. Significant differences were found between cases and controls in allele and/or genotype frequencies for six SNPs in Block I and two SNPs in Block II. Association of SNP4 in Block I with DD (allele: P=0.004), SNP5 in Block I with AD and DD (allele: P< or =0.005 for both) and two SNPs in Block II with AD (SNP11 genotype: P=0.002; SNP12 genotype: P=0.001) were significant after correction for multiple testing. Frequency distributions of haplotypes (constructed by five tag SNPs) differed significantly for cases and controls (P<0.001 for both AD and DD). Logistic regression analyses confirmed the association between OPRM1 variants and substance dependence, when sex and age of subjects and alleles, genotypes, haplotypes or diplotypes of five tag SNPs were considered. Population structure analyses excluded population stratification artifact. Additional supporting evidence for association between OPRM1 and AD was obtained in a smaller Russian sample (247 cases and 100 controls). These findings suggest that OPRM1 intronic variants play a role in susceptibility to AD and DD in populations of European ancestry.     

Association analysis of delta-opioid receptor gene polymorphisms in methamphetamine dependence/psychosis.

The role of the delta-opioid receptor (OPRD1) in methamphetamine (MAP) addiction was investigated using association analysis between OPRD1 gene polymorphisms and MAP dependence/psychosis. DNA samples from Japanese patients with MAP dependence/psychosis were analyzed to find polymorphisms in OPRD1 gene exons and exon-intron boundaries. One novel single nucleotide polymorphism (SNP) in intron 1 and two SNPs in exon 3 were identified. The two SNPs in exon 3 were in linkage disequilibrium. No significant difference was observed in either genotypic or allelic frequencies of these SNPs between controls (n = 260) and MAP dependent/psychotic patients (n = 170). Global analyses using the three SNPs and subcategory analyses on clinical parameters also showed no significant differences. These results suggest that the OPRD1 gene variants may not be a factor in vulnerability to MAP dependence/psychosis.     

Nonreplication of association between mu-opioid-receptor gene (OPRM1) A118G polymorphism and substance dependence.

In the present investigation we hypothesized the A118G (Asn40Asp) polymorphism of the mu-opioid receptor gene (OPRM1) as a particular vulnerability factor for heroin and alcohol dependence. Therefore, we tested this hypothesis in two independent large samples by two different methods: a case-control sample (comprising n = 287 heroin and n = 221 alcohol study subjects versus n = 365 nondependent controls) and a family-controlled sample of 111 parent-offspring trios of heroin-dependent study subjects and 75 parent-offspring trios of alcohol-dependent study subjects to avoid stratification artifacts. In both patient samples and by both methods we were unable to corroborate the hypothesis of OPRM1 A118G polymorphism as a particular risk factor for any kind of substance dependence including opioid addiction. In addition, there was no significant association between the endophenotype of the individuals under study (e.g., comorbidity, severity of illness) and a particular genotype of OPRM1.     

Haplotype block structure of the genomic region of the mu opioid receptor gene

The opioid system is involved in the action of opiate drugs, opioid addiction, pain experience and analgesia. Individual differences in opioid effect may be attributed in part to genetic variations. Long-range cis regulatory elements and intronic variants are potential sources of functional diversity. Recently, we have detected association of two intronic OPRM1 variants with heroin addiction in European Americans. In this study, we analyzed the genetic variations in the OPRM1 100?kb 5'-flanking region and intron 1 in the HapMap Caucasian population. Four major linkage disequilibrium blocks were identified, consisting of 28, 22, 15 and 42 single-nucleotide polymorphisms (SNPs), respectively. The locations of these blocks are (-100 to -90), (-90 to -67), (-20 to -1) and (+1 to +44)?kb, respectively. The two intronic variants, indicated in our recent study, are part of a distinct haplogroup that includes SNPs from intron 1, and the proximal 5' region. The 118G (rs1799971) allele is part of a different haplogroup that includes several variants in the distal 5' region that may have a regulatory potential. These findings were corroborated by genotyping eight SNPs in a sample of European Americans and suggest an extended OPRM1 locus with potential new regulatory regions.     

Association of μ-opioid receptor gene (OPRM1) haplotypes with postoperative nausea and vomiting

Genetic variants, such as single-nucleotide polymorphisms (SNPs), of the μ-opioid receptor gene (OPRM1) might be associated with individual differences in opioid sensitivity, as well as with the incidence and severity of postoperative nausea and vomiting (PONV). The goal of the present study was to determine, in a cohort of Japanese surgical patients, genotypes and haplotypes of several SNPs in the OPRM1 gene, and their association with PONV during the early (first 24 h) postoperative period. We examined the incidence and severity of PONV, during the first 24 h after surgery, in 85 Japanese patients receiving intravenous patient-controlled analgesia fentanyl analgesia for postoperative pain control. Eight tag SNPs of the OPRM1 gene (rs1799971, A/G; rs510769, G/A; rs4870266, G/A; rs3798683, G/A; rs1323042, A/C; rs609623, C/T; rs9397685, A/G; and rs644261, C/G) were selected based on their minor allele frequency (>10 %) and linkage disequilibrium strength (<80 %), and genotyped for haplotype analysis and determination of associations with PONV. Only one out of eight investigated SNPs, rs9397685, in the intronic part of the OPRM1 gene was associated with differences in the occurrence and severity of PONV. We also found four common haplotypes with a frequency of >10 % in the investigated patients, including GGGAACAC (33 %), AGGGACAC (19 %), GGGAACGC (12 %), and AGAGACAC (10 %). The severity of PONV in carriers of the GGGAACGC haplotype was significantly lower than in the carriers of the other haplotypes (P < 0.05). One intronic SNP, rs9397685, and haplotypes constructed from eight SNPs within the OPRM1 gene locus might be involved in the severity of PONV associated with general anesthesia and opioid administration. This novel finding, if validated and verified in larger and additional ethnic cohorts, might contribute to better knowledge of the contribution of the OPRM1 gene to PONV.     

Association between single nucleotide polymorphisms in the mu opioid receptor gene (<Emphasis Type="Italic">OPRM1</Emphasis>) and self-reported responses to alcohol in American Indians

Background  

Variation in response to the hedonic and adverse effects of a substance is in part an inherited factor that may influence its use, abuse and dependence. The mu opioid receptor is the primary site of action for opiates and individuals with polymorphisms in this receptor appear to have variation in the CNS effects of opiates. Several studies have suggested that this receptor may also mediate some of the effects of non-opioid drugs, such as alcohol. The purpose of this study was to investigate associations between 13 single nucleotide polymorphisms in the mu opioid receptor gene (OPRM1) with self-reported responses to alcohol, an endophenotype associated with the development of alcohol dependence, in American Indians living on eight contiguous reservations.     

Sequence variability and candidate gene analysis in complex disease: association of mu opioid receptor gene variation with substance dependence

To analyze candidate genes and establish complex genotype-phenotype relationships against a background of high natural genome sequence variability, we have developed approaches to (i) compare candidate gene sequence information in multiple individuals; (ii) predict haplotypes from numerous variants; and (iii) classify haplotypes and identify specific sequence variants, or combinations of variants (pattern), associated with the phenotype. Using the human mu opioid receptor gene (OPRM1) as a model system, we have combined these approaches to test a potential role of OPRM1 in substance (heroin/cocaine) dependence. All known functionally relevant regions of this prime candidate gene were analyzed by multiplex sequence comparison in 250 cases and controls; 43 variants were identified and 52 different haplotypes predicted in the subgroup of 172 African-Americans. These haplotypes were classified by similarity clustering into two functionally related categories, one of which was significantly more frequent in substance-dependent individuals. Common to this category was a characteristic pattern of sequence variants [-1793T-->A, -1699Tins, -1320A-->G, -111C-->T, +17C-->T (A6V)], which was associated with substance dependence. This study provides an example of approaches that have been successfully applied to the establishment of complex genotype-phenotype relationships in the presence of abundant DNA sequence variation.     

A Genome-Wide Association Study of Behavioral Disinhibition

We report results from a genome wide association study (GWAS) of five quantitative indicators of behavioral disinhibition: nicotine, alcohol consumption, alcohol dependence, illicit drugs, and non-substance related behavioral disinhibition. The sample, consisting of 7,188 Caucasian individuals clustered in 2,300 nuclear families, was genotyped on over 520,000 SNP markers from Illumina’s Human 660W-Quad Array. Analysis of individual SNP associations revealed only one marker-component phenotype association, between rs1868152 and illicit drugs, with a p value below the standard genome-wide threshold of 5 × 10^?8. Because we had analyzed five separate phenotypes, we do not consider this single association to be significant. However, we report 13 SNPs that were associated at p < 10^?5 for one phenotype and p < 10^?3 for at least two other phenotypes, which are potential candidates for future investigations of variants associated with general behavioral disinhibition. Biometric analysis of the twin and family data yielded estimates of additive heritability for the component phenotypes ranging from 49 to 70 %, GCTA estimates of heritability for the same phenotypes ranged from 8 to 37 %. Consequently, even though the common variants genotyped on the GWAS array appear in aggregate to account for a sizable proportion of heritable effects in multiple indicators of behavioral disinhibition, our data suggest that most of the additive heritability remains “missing”.     

Genetic association study of GABRA2 single nucleotide polymorphisms and electroencephalography in alcohol dependence

The gamma aminobutyric acid (GABA) system has been implicated in the susceptibility to develop alcohol dependence and in determining electroencephalogram (EEG) beta activity. The role of the GABA receptor alpha-2 gene (GABRA2) in human alcohol dependence was determined in a genetic and electrophysiological study. The study population comprised 586 white UK individuals with alcohol dependence but a very low prevalence of co-morbid drug dependence, and 603 ancestrally matched healthy controls. Genotyping for seven GABRA2 single nucleotide polymorphisms (SNPs), identified from the literature as positively associated with alcohol dependence, was performed with success rates of 90% or greater. EEGs were available in 32 selected patients who had been abstinent from alcohol for a minimum of 24 months and in 138 ancestrally matched healthy controls. None of the SNPs showed allelic or haplotypic association with alcohol dependence. All markers were in Hardy Weinberg equilibrium (HWE) in the controls. HWE for marker rs279841 in the alcohol dependent sample was p=0.0199 and combined p=0.0166. Linkage disequilibrium patterns appear to be very similar to that observed in the HapMap CEU data. A significantly higher prevalence of excess EEG fast activity was found in the patients (31 vs. 14%, p=0.018). A significant relationship was found between the presence of excess EEG fast activity and GABRA2 SNPs rs548583, rs279871 and rs279841. This allelic association study provides no evidence for an association between GABRA2 polymorphisms and alcohol dependence. However, a significant relationship was identified between GABRA2 and excess EEG fast activity. This dissociation of effect may reflect the fact that the EEG is a more direct marker of phenotypic GABRA2 expression than the more heterogeneous alcohol dependence phenotype.     

Sequence variations in the mu-opioid receptor gene (OPRM1) associated with human addiction to heroin

Human mu-opioid receptor (OPRM1) is the major site for the analgesic action of most opioid drugs such as morphine, methadone and heroin. It was previously reported that a single nucleotide polymorphism (SNP) in exon1 (c.118A-->G) of OPRM1 might modestly alter the affinity in beta-endorphin-Mu interaction. Using denaturing high performance liquid chromatography (DHPLC) the complete coding region of the OPRM1 gene was screened for SNPs in Han-Chinese heroin addicts and normal control. Three novel SNPs were detected, one in exon3, one in intron3 and one in the 3' untranslated region. The SNP c.118A-->G reportedly altered the interaction of Mu receptor with opioid had no statistically significant correlation with heroin addition in Han Chinese. However, addicted subjects with the SNP in intron2 (IVS2 +31G-->A) tended to show much higher heroin intake dosages than those without this SNP. We also observed that individuals carrying both SNP c.118A-->G and IVS2 +31G-->A consumed relatively more drugs compared to other addicts. Thus our study further highlights the importance of studing the various regions of the mu opioid receptor gene, coding as well as non-coding, for genetic markers that may be linked to, or directly contribute to opioid drug-seeking behavior.     

Nonreplication of an association of SGIP1 SNPs with alcohol dependence and resting theta EEG power

A recent study in a sample of Plains Indians showed association between eight single nucleotide polymorphisms (SNPs) located in the SGIP1 gene and resting θ electroencephalogram (EEG) power. This association appeared to generalize to alcohol use disorders, for which EEG power is a potential endophenotype. We analyzed a large, diverse sample for replication of the association of these implicated SGIP1 SNPs (genotyped on the Illumina 1M platform) with alcohol dependence (N=3988) and θ EEG power (N=1066). We found no evidence of association of the earlier implicated SGIP1 SNPs with either alcohol dependence or θ EEG power (all P>0.15) in this sample. The earlier implicated SNPs located in SGIP1 gene showed no association with alcohol dependence or θ EEG power in this sample of individuals with European and/or African ancestry. This failure to replicate may be the result of differences in ancestry between this sample and the original sample.     

Association between DRD2/ANKK1 TaqIA polymorphism and common illicit drug dependence: Evidence from a meta-analysis

BackgroundGrowing evidence indicated conflicting results about the dopamine receptor D2 (DRD2)/kinase domain containing 1 gene (ANKK1) TaqIA single nucleotide polymorphism (rs1800497) and common illicit drug dependence risk including stimulants, opioid and marijuana. We conducted a meta-analysis to evaluate the association between the polymorphism and common illicit drug dependence risk.MethodA total of 25 available studies (26 subgroups) testing the association between the polymorphism and common illicit drug dependence were examined through Oct 2013. Pooled odds ratios (ORs) and 95% confidence intervals (CI) were estimated using fixed- and random-effects models when appropriate. Heterogeneity and publication bias were evaluated.ResultsWe found the DRD2/ANKK1 TaqIA polymorphism was significantly associated with increased risk of opioid dependence under homozygote, dominant, and recessive genetic model, respectively (homozygote: OR = 1.546, 95%CI = 1.279–1.87; dominant: OR = 1.265, 95%CI = 1.055–1.516; recessive: OR = 1.409, 95%CI = 1.182–1.680). Subgroup analyses were similar to the results of the total population by ethnicity and quality score. Besides, we also found that Caucasian and low-quality studies were major sources of heterogeneity for opioid dependence. We failed to find any significant association between the polymorphism and stimulants or marijuana neither in total population nor subgroup analyses under any genetic model.ConclusionsThe current meta-analysis suggested that DRD2/ANKK1 TaqIA polymorphism might be associated with opioid dependence risk, but not associated with stimulants or marijuana dependence.     

Genomic screen for loci associated with alcohol dependence in Mission Indians.

Alcohol dependence is a leading cause of morbidity and mortality in Native Americans, yet biological factors underlying the disorder in this ethnic group remain elusive. This study's aims were to map susceptibility loci for DSM-III-R alcohol dependence and two narrower alcohol-related phenotypes in Mission Indian families. Each participant gave a blood sample and completed an interview using the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) that was used to make alcohol dependence diagnoses and the narrower phenotypes of withdrawal, and drinking severity. Genotypes were determined for a panel 791 microsatellite polymorphisms. Analyses of multipoint variance component LOD scores for the dichotomous DSM-III-R phenotype revealed no peak LOD scores that exceeded 2.0 at any chromosome location. Two chromosomes, 4 and 12, had peak LOD scores that exceeded 2 for the alcohol use severity phenotype and three chromosomes 6, 15, 16 were found to have peaks with LOD scores that exceeded 2 for the withdrawal phenotype. Evidence for linkage to chromosomes 4 and 15, and 16 have been reported previously for alcohol related phenotypes whereas no evidence has as yet been reported for chromosomes 6 and 12. Combined linkage and association analysis suggest that alcohol dehydrogenase 1B gene polymorphisms are partially responsible for the linkage result on chromosome 4 in this population. These results corroborate the importance of several chromosomal regions highlighted in prior segregation studies in alcoholism and further identify new regions of the genome that may be unique to either the restricted phenotypes evaluated or this population of Mission Indians.     

No association of the GABAA receptor genes on chromosome 5 with alcoholism in the collaborative study on the genetics of alcoholism sample.

A substantial body of literature suggests that gamma-aminobutyric acid (GABA) may be involved in the neurochemical pathways contributing to alcohol use and related disorders. Chromosome 5 contains a cluster of GABA(A) receptor genes, GABRA1, GABRA6, GABRB2, and GABRG2, which have been among the most extensively studied in relation to alcohol use. These studies have yielded mixed results. Using data from large, multiplex alcoholic families collected as part of the Collaborative Study on the Genetics of Alcoholism (COGA), we sought to provide more conclusive evidence regarding the role of the GABA(A) receptor genes on chromosome 5. Multiple single nucleotide polymorphisms (SNPs) were tested in each of the four chromosome 5q GABA(A) receptor genes, and we conducted both classic trio-based association analyzes and extended pedigree analyzes. We found no consistent evidence of association with alcohol dependence or alcohol dependence comorbid with antisocial personality disorder (ASPD) for any of the regions tested in the chromosome 5 GABA(A) receptor genes. These analyses suggest that the GABA(A) receptor genes on chromosome 5 do not play a strong role in alcohol dependence. Future studies are planned to test whether these genes are more important in influencing behavioral endophenotypes related to the risk of alcohol dependence.     

No evidence of association between genetic variants of the PDCD1 ligands and SLE

PDCD1, an immunoreceptor involved in peripheral tolerance has previously been shown to be genetically associated with systemic lupus erythematosus (SLE). PDCD1 has two ligands whose genes are located in close proximity on chromosome 9p24. Our attention was drawn to these ligands after finding suggestive linkage to a marker (gata62f03, Z=2.27) located close to their genes in a genome scan of Icelandic families multiplex for SLE. Here, we analyse Swedish trios (N=149) for 23 single nucleotide polymorphisms (SNPs) within the genes of the PDCD1 ligands. Initially, indication of association to eight SNPs was observed, and these SNPs were therefore also analysed in Mexican trios (N=90), as well as independent sets of patients and controls from Sweden (152 patients, 448 controls) and Argentina (288 patients, 288 controls). We do not find support for genetic association to SLE. This is the first genetic study of SLE and the PDCD1 ligands and the lack of association in several cohorts implies that these genes are not major risk factors for SLE.