- Email: [email protected]
INFLUENCE OF NICOTINIC RECEPTOR GENE – CHRNA5 – ON CRACK COCAINE DEPENDENCE AND SEVERITY
S1016
Abstracts
current data, 97% of target was coved at 10x. Post-sequencing calling and recalibration were performed per the Genome Analysis Toolkit (GATK) best practices. Markers were limited to those with a minimum genotype quality score of 20 and a minimum depth of 10, in addition to having passed GATK variant quality score recalibration (VQSR). Results: In the data from the first 133 cases, we detected 688949 variants. After filtering for variants having a minor allele frequency (MAF) 4 0.05 in European data from ExAC, UK10K, or 1000 Genomes, 299191 remained, 72277 (24.2%) of which were novel. We identified 1372 loss-of-function (LOF), 29247 missense, and 19365 synonymous changes. We focused preliminary analyses on alcohol metabolizing genes. We detected 6 variants present at higher MAF than found in UK10K control data: ADH5 (rs28730652, 2 = 8.62, p= 0.003; rs201786928 2 =8.31, p = 0.004), ADH6 (rs189318592, 2 = 36.63, p = 1.43E-9; rs146084391, 2 = 25.57, p = 4.27E-7), and ADH1B (rs375691484, 2 = 18.31, p= 1.87E-5; rs41275699 2= 10.42, p= 0.001). Some of these changes may have relevant functional consequence. Further, these markers are currently being genotyped in our full case-control sample. Discussion: Since known LOF mutations in ADH1B and ALDH2 reduce risk for AUD, we are currently performing analyses of these 2 genes as well as the complete set of alcohol and aldehyde metabolizing genes (ADH1-6, ALDH118, CAT and CYP2E1, N = 27 loci) to test the hypothesis that numbers of high-impact variants in these genes are significantly different than matched gene sets in the AUD cases. We matched for 1) coding basepair length, 2) genomic interval length, 3) N of exons, N of 4) LOF, 5) missense, and 6) synonymous mutations, and the 7) probability of LOF intolerance (pLI) from ExAC data to create comparison gene sets for each gene of interest (GOI) to determine if the GOI were different from their matched clusters for LOF, missense, and synonymous mutations. Here we have identified 6 possibly functional rare variants in key alcohol dehydrogenase genes. In addition, we are applying a novel method to investigate whether patterns of variation in these target genes are different than would be expected based on matched case genes determined by current reference data.
Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.euroneuro.2017.08.418
M112. A STUDY ON THE ASSOCIATION OF POLYMORPHISMS AND METHYLATION STATUS OF DOPAMINE PATHWAY GENES IN ALCOHOL DEPENDENCE
polymorphisms and methylation changes affecting transcription of the genes involved in various dopamine neurotransmitter gene pathways. The aim of this study was to identify polymorphisms and methylation changes in the SNCA and DAT genes of dopamine pathway in subjects with AD. Methods: A total of 145 AD subjects from the outpatient department of Psychiatry and 110 healthy volunteers were recruited for the study. All subjects were interviewed with a semi-structured and WHO assist questionnaire. Blood samples drawn were subjected to DNA extraction, followed by sodium bisulphite modification and methylation specific PCR of the SNCA and DAT genes. DNA was also used to screen six polymorphisms of the dopamine pathway namely DRD4 120 bp duplication, DRD3 Ser9Gly, DRD2 Taq1α, COMT rs4680, COMT-287A4G and DRD4 -521C/T by PCR/RFLP. Genotype frequencies were calculated and their significance assessed using chi-square test and correlated with duration of alcohol use, age at onset of alcohol use, quantity of alcohol consumed (gms/day) and liver function. Statistical analysis was done through SPSS software V21.0. Results: Of all the polymorphisms studied COMT Val158Met (rs4680) showed significant association with AD (p-0.03) compared to the others. SNCA gene methylation levels were significantly different between AD subjects and controls (p o 0.0001), while the difference between methylation levels of the DAT gene were similar between subjects and controls (p = 0.839). Analysis of methylation levels of the DAT gene with duration of alcohol use showed an increase in methylation from one allele to both alleles being methylated with increase in duration of use ranging from 7.8071.31 to 15.1270.864 years respectively (p = 0.003). Similar increase in methylation levels were also observed with age of the subject which ranged from 30.072.25 to 37.2371.01 years (p = 0.043). Discussion: In the present study, significant difference between COMT rs4680 was observed between cases and controls. More heterozygosity was observed in cases which imply presence of low activity allele of COMT gene which affects the activity of enzyme and makes the individual more vulnerable to alcohol dependence. However, increased methylation at promoter region of DAT gene with both age and duration of alcohol use may lead to less reuptake of dopamine from the synapse. Whereas, decreased methylation at promoter region of SNCA gene may help in the formation of synapse and hence increased dopamine neurotransmission. Therefore, this preliminary report suggests association of COMT rs4680 and methylation changes of SNCA and DAT genes with alcohol dependence.
Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.euroneuro.2017.08.419
n
Ranjan Gupta , Renu Singh, Atul Ambekar, Tripti Grover, Arundhati Sharma AIIMS
Background: Alcohol Dependence (AD), a chronic relapsing disorder, is a serious social and public health concern globally. Risk of AD is reportedly increased due to
M113. INFLUENCE OF NICOTINIC RECEPTOR GENE –
CHRNA5 – ON CRACK COCAINE DEPENDENCE AND SEVERITY n
Jaqueline B. Schuch ,1, Angelita P. Aroche1, Diego L. Rovaris1, Anderson Stolf2, Breno Sanvicente Vieira3,
Abstracts Eduardo S. Vitola1, Eugênio H. Grevet1, Flavio Pechanski2, Felix Henrique Paim Kessler2, Lisia von Diemen2, Rodrigo Grassi-Oliveira3, Claiton Bau1
S1017 Josep Antoni Ramos-Quiroga5, Marta Ribases6, n Miguel Casas7, Bru Cormand1, Noèlia Fernàndez-Castillo ,1 1
1
Universidade Federal do Rio Grande do Sul 2 Center for Drug and Alcohol Research/HCPA 3 Pontifical Catholic University of Rio Grande do Sul
Background: Crack cocaine is a highly addictive substance and this dependence has a significant prevalence worldwide, especially in Brazil. Several factors are involved on crack cocaine dependence, and heritability estimates have highlighted the influence of genetic variants in the dependence susceptibility. Previous studies have shown that genetic variants in nicotine receptor genes are not only associated with nicotine abuse, but also with cocaine dependence. Our aim was to investigate the effects of polymorphisms in the nicotinic receptor alpha 5 gene CHRNA5 - on crack cocaine dependence. Methods: Three hundred crack cocaine users and six hundred and thirty-three healthy controls were enrolled in this study. The sample was composed of white Brazilians with European descent. Diagnoses and clinical assessments were performed according to DSM-IV criteria. The Addiction Severity Index - 6 (ASI-6) was used to assess the severity of dependence. Three polymorphisms were analyzed in the CHRNA5 gene (rs588765, rs16969968, and rs514743). Logistic regression models and general linear models were used to evaluated the effect of SNPs on crack cocaine addiction and severity of dependence, respectively. Results: Significant effects were found for two polymorphisms. Genotypes TT-rs588765 and AA-rs16969968 were associated with reduced risk to crack cocaine dependence (OR= 0.581, P= 0.037; OR = 0.532, P= 0.009, respectively). However, only a trend towards association for rs514743 was detected (TT, OR = 0.571, P =0.065). Analyses regarding severity of dependence did not reveal any significant association between SNPs and scores generated by ASI-6. Discussion: We replicated previous findings regarding the influence of CHRNA5 gene on cocaine addiction. Notwithstanding, studies have demonstrated that rs16969968 apparently presents opposite effects in nicotine and crack/ cocaine addictions. Our perspectives include increasing sample size and further explore other aspects possibly involved with addiction, such as age of first use and psychiatric comorbidities. Potential implications of these genetic factors should be more explored in future studies.
Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.euroneuro.2017.08.420
M114. EXPLORING ALLELE SPECIFIC METHYLATION (ASM) IN DRUG DEPENDENCE SUSCEPTIBILITY Laura Pineda-Cirera1, Judit Cabana-Domínguez1, Carlos Roncero2, Laia Rodriguez-Cintas3, Lara Grau-López2, R. Felipe Palma-Álvarez3, Constanza Daigre4,
University of Barcelona, Centro Nacional de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III. Institut de Biomedicina de la Universitat de Barcelona (IBUB), Institut de Recerca Sant Joan de Déu (IR-SJD), Esplugues de Llobregat 2 Addiction and Dual Diagnosis Unit, Psychiatric Services, Hospital Universitari Vall d’Hebron-ASPB, Biomedical Network Research Center on Mental Health (CIBERSAM), Instituto de Salud Carlos III, Hospital Universitari Vall d’Hebron, Barcelona Universitat Autònoma de Barcelona 3 Addiction and Dual Diagnosis Unit, Psychiatric Services, Hospital Universitari Vall d’Hebron-ASPB, Universitat Autònoma de Barcelona 4 Addiction and Dual Diagnosis Unit, Psychiatric Services, Hospital Universitari Vall d’Hebron-ASPB, Biomedical Network Research Center on Mental Health (CIBERSAM), Instituto de Salud Carlos III 5 Hospital Universitari Vall d’Hebron, Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Instituto de Salud Carlos III, Psychiatric Genetics Unit, Group of Psychiatry, Mental Health and Addiction, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Universitat Autònoma de Barcelona 6 Psychiatric Genetics Unit, Institut Recerca Hospital Universitari Vall Hebron (VHIR), Biomedical Network Research Center on Mental Health (CIBERSAM), Instituto de Salud Carlos III, Hospital Universitari Vall d’Hebron 7 Hospital Universitari Vall d’Hebron, Biomedical Network Research Center on Mental Health (CIBERSAM), Instituto de Salud Carlos III, Universitat Autònoma de Barcelona
Background: Drug addiction is a neuropsychiatric disorder where long-lasting changes in gene expression within particular regions of the brain play an important role. Work over the past decade has demonstrated a crucial role for epigenetic mechanisms in driving stable changes in gene expression in several tissues, including the brain. Allelespecific methylation (ASM) is a common epigenetic mechanism consisting on SNPs that correlate with differential levels of methylation at CpG sites. The aim of our study is to assess the possible contribution of ASM in different brain regions to drug dependence susceptibility. Methods: We performed a SNP selection based on two previous studies that reported thousands of ASM SNPs in different brain regions of post-mortem human samples. By combining both studies, we obtained a total of 43,132 SNPs CpG pairs that include 33,944 SNPs and 5,036 CpG islands. From those, we obtained a sub-list of 184 SNPs using the following selection criteria: cis associations between SNPs and CpG sites, correlation (R2) of DNA methylation with gene expression Z0.5 and selection of only one SNP per CpG site, the one showing the best correlation (R2). Subsequently, we evaluated the possible contribution of these SNPs to drug dependence predisposition through a case-control association study in a sample of 577 drugdependent patients and 655 sex-matched controls from Spain, and then we followed-up the significant associations
Abstracts
current data, 97% of target was coved at 10x. Post-sequencing calling and recalibration were performed per the Genome Analysis Toolkit (GATK) best practices. Markers were limited to those with a minimum genotype quality score of 20 and a minimum depth of 10, in addition to having passed GATK variant quality score recalibration (VQSR). Results: In the data from the first 133 cases, we detected 688949 variants. After filtering for variants having a minor allele frequency (MAF) 4 0.05 in European data from ExAC, UK10K, or 1000 Genomes, 299191 remained, 72277 (24.2%) of which were novel. We identified 1372 loss-of-function (LOF), 29247 missense, and 19365 synonymous changes. We focused preliminary analyses on alcohol metabolizing genes. We detected 6 variants present at higher MAF than found in UK10K control data: ADH5 (rs28730652, 2 = 8.62, p= 0.003; rs201786928 2 =8.31, p = 0.004), ADH6 (rs189318592, 2 = 36.63, p = 1.43E-9; rs146084391, 2 = 25.57, p = 4.27E-7), and ADH1B (rs375691484, 2 = 18.31, p= 1.87E-5; rs41275699 2= 10.42, p= 0.001). Some of these changes may have relevant functional consequence. Further, these markers are currently being genotyped in our full case-control sample. Discussion: Since known LOF mutations in ADH1B and ALDH2 reduce risk for AUD, we are currently performing analyses of these 2 genes as well as the complete set of alcohol and aldehyde metabolizing genes (ADH1-6, ALDH118, CAT and CYP2E1, N = 27 loci) to test the hypothesis that numbers of high-impact variants in these genes are significantly different than matched gene sets in the AUD cases. We matched for 1) coding basepair length, 2) genomic interval length, 3) N of exons, N of 4) LOF, 5) missense, and 6) synonymous mutations, and the 7) probability of LOF intolerance (pLI) from ExAC data to create comparison gene sets for each gene of interest (GOI) to determine if the GOI were different from their matched clusters for LOF, missense, and synonymous mutations. Here we have identified 6 possibly functional rare variants in key alcohol dehydrogenase genes. In addition, we are applying a novel method to investigate whether patterns of variation in these target genes are different than would be expected based on matched case genes determined by current reference data.
Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.euroneuro.2017.08.418
M112. A STUDY ON THE ASSOCIATION OF POLYMORPHISMS AND METHYLATION STATUS OF DOPAMINE PATHWAY GENES IN ALCOHOL DEPENDENCE
polymorphisms and methylation changes affecting transcription of the genes involved in various dopamine neurotransmitter gene pathways. The aim of this study was to identify polymorphisms and methylation changes in the SNCA and DAT genes of dopamine pathway in subjects with AD. Methods: A total of 145 AD subjects from the outpatient department of Psychiatry and 110 healthy volunteers were recruited for the study. All subjects were interviewed with a semi-structured and WHO assist questionnaire. Blood samples drawn were subjected to DNA extraction, followed by sodium bisulphite modification and methylation specific PCR of the SNCA and DAT genes. DNA was also used to screen six polymorphisms of the dopamine pathway namely DRD4 120 bp duplication, DRD3 Ser9Gly, DRD2 Taq1α, COMT rs4680, COMT-287A4G and DRD4 -521C/T by PCR/RFLP. Genotype frequencies were calculated and their significance assessed using chi-square test and correlated with duration of alcohol use, age at onset of alcohol use, quantity of alcohol consumed (gms/day) and liver function. Statistical analysis was done through SPSS software V21.0. Results: Of all the polymorphisms studied COMT Val158Met (rs4680) showed significant association with AD (p-0.03) compared to the others. SNCA gene methylation levels were significantly different between AD subjects and controls (p o 0.0001), while the difference between methylation levels of the DAT gene were similar between subjects and controls (p = 0.839). Analysis of methylation levels of the DAT gene with duration of alcohol use showed an increase in methylation from one allele to both alleles being methylated with increase in duration of use ranging from 7.8071.31 to 15.1270.864 years respectively (p = 0.003). Similar increase in methylation levels were also observed with age of the subject which ranged from 30.072.25 to 37.2371.01 years (p = 0.043). Discussion: In the present study, significant difference between COMT rs4680 was observed between cases and controls. More heterozygosity was observed in cases which imply presence of low activity allele of COMT gene which affects the activity of enzyme and makes the individual more vulnerable to alcohol dependence. However, increased methylation at promoter region of DAT gene with both age and duration of alcohol use may lead to less reuptake of dopamine from the synapse. Whereas, decreased methylation at promoter region of SNCA gene may help in the formation of synapse and hence increased dopamine neurotransmission. Therefore, this preliminary report suggests association of COMT rs4680 and methylation changes of SNCA and DAT genes with alcohol dependence.
Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.euroneuro.2017.08.419
n
Ranjan Gupta , Renu Singh, Atul Ambekar, Tripti Grover, Arundhati Sharma AIIMS
Background: Alcohol Dependence (AD), a chronic relapsing disorder, is a serious social and public health concern globally. Risk of AD is reportedly increased due to
M113. INFLUENCE OF NICOTINIC RECEPTOR GENE –
CHRNA5 – ON CRACK COCAINE DEPENDENCE AND SEVERITY n
Jaqueline B. Schuch ,1, Angelita P. Aroche1, Diego L. Rovaris1, Anderson Stolf2, Breno Sanvicente Vieira3,
Abstracts Eduardo S. Vitola1, Eugênio H. Grevet1, Flavio Pechanski2, Felix Henrique Paim Kessler2, Lisia von Diemen2, Rodrigo Grassi-Oliveira3, Claiton Bau1
S1017 Josep Antoni Ramos-Quiroga5, Marta Ribases6, n Miguel Casas7, Bru Cormand1, Noèlia Fernàndez-Castillo ,1 1
1
Universidade Federal do Rio Grande do Sul 2 Center for Drug and Alcohol Research/HCPA 3 Pontifical Catholic University of Rio Grande do Sul
Background: Crack cocaine is a highly addictive substance and this dependence has a significant prevalence worldwide, especially in Brazil. Several factors are involved on crack cocaine dependence, and heritability estimates have highlighted the influence of genetic variants in the dependence susceptibility. Previous studies have shown that genetic variants in nicotine receptor genes are not only associated with nicotine abuse, but also with cocaine dependence. Our aim was to investigate the effects of polymorphisms in the nicotinic receptor alpha 5 gene CHRNA5 - on crack cocaine dependence. Methods: Three hundred crack cocaine users and six hundred and thirty-three healthy controls were enrolled in this study. The sample was composed of white Brazilians with European descent. Diagnoses and clinical assessments were performed according to DSM-IV criteria. The Addiction Severity Index - 6 (ASI-6) was used to assess the severity of dependence. Three polymorphisms were analyzed in the CHRNA5 gene (rs588765, rs16969968, and rs514743). Logistic regression models and general linear models were used to evaluated the effect of SNPs on crack cocaine addiction and severity of dependence, respectively. Results: Significant effects were found for two polymorphisms. Genotypes TT-rs588765 and AA-rs16969968 were associated with reduced risk to crack cocaine dependence (OR= 0.581, P= 0.037; OR = 0.532, P= 0.009, respectively). However, only a trend towards association for rs514743 was detected (TT, OR = 0.571, P =0.065). Analyses regarding severity of dependence did not reveal any significant association between SNPs and scores generated by ASI-6. Discussion: We replicated previous findings regarding the influence of CHRNA5 gene on cocaine addiction. Notwithstanding, studies have demonstrated that rs16969968 apparently presents opposite effects in nicotine and crack/ cocaine addictions. Our perspectives include increasing sample size and further explore other aspects possibly involved with addiction, such as age of first use and psychiatric comorbidities. Potential implications of these genetic factors should be more explored in future studies.
Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.euroneuro.2017.08.420
M114. EXPLORING ALLELE SPECIFIC METHYLATION (ASM) IN DRUG DEPENDENCE SUSCEPTIBILITY Laura Pineda-Cirera1, Judit Cabana-Domínguez1, Carlos Roncero2, Laia Rodriguez-Cintas3, Lara Grau-López2, R. Felipe Palma-Álvarez3, Constanza Daigre4,
University of Barcelona, Centro Nacional de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III. Institut de Biomedicina de la Universitat de Barcelona (IBUB), Institut de Recerca Sant Joan de Déu (IR-SJD), Esplugues de Llobregat 2 Addiction and Dual Diagnosis Unit, Psychiatric Services, Hospital Universitari Vall d’Hebron-ASPB, Biomedical Network Research Center on Mental Health (CIBERSAM), Instituto de Salud Carlos III, Hospital Universitari Vall d’Hebron, Barcelona Universitat Autònoma de Barcelona 3 Addiction and Dual Diagnosis Unit, Psychiatric Services, Hospital Universitari Vall d’Hebron-ASPB, Universitat Autònoma de Barcelona 4 Addiction and Dual Diagnosis Unit, Psychiatric Services, Hospital Universitari Vall d’Hebron-ASPB, Biomedical Network Research Center on Mental Health (CIBERSAM), Instituto de Salud Carlos III 5 Hospital Universitari Vall d’Hebron, Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Instituto de Salud Carlos III, Psychiatric Genetics Unit, Group of Psychiatry, Mental Health and Addiction, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Universitat Autònoma de Barcelona 6 Psychiatric Genetics Unit, Institut Recerca Hospital Universitari Vall Hebron (VHIR), Biomedical Network Research Center on Mental Health (CIBERSAM), Instituto de Salud Carlos III, Hospital Universitari Vall d’Hebron 7 Hospital Universitari Vall d’Hebron, Biomedical Network Research Center on Mental Health (CIBERSAM), Instituto de Salud Carlos III, Universitat Autònoma de Barcelona
Background: Drug addiction is a neuropsychiatric disorder where long-lasting changes in gene expression within particular regions of the brain play an important role. Work over the past decade has demonstrated a crucial role for epigenetic mechanisms in driving stable changes in gene expression in several tissues, including the brain. Allelespecific methylation (ASM) is a common epigenetic mechanism consisting on SNPs that correlate with differential levels of methylation at CpG sites. The aim of our study is to assess the possible contribution of ASM in different brain regions to drug dependence susceptibility. Methods: We performed a SNP selection based on two previous studies that reported thousands of ASM SNPs in different brain regions of post-mortem human samples. By combining both studies, we obtained a total of 43,132 SNPs CpG pairs that include 33,944 SNPs and 5,036 CpG islands. From those, we obtained a sub-list of 184 SNPs using the following selection criteria: cis associations between SNPs and CpG sites, correlation (R2) of DNA methylation with gene expression Z0.5 and selection of only one SNP per CpG site, the one showing the best correlation (R2). Subsequently, we evaluated the possible contribution of these SNPs to drug dependence predisposition through a case-control association study in a sample of 577 drugdependent patients and 655 sex-matched controls from Spain, and then we followed-up the significant associations