Epigenetic Effects in HPA Axis Genes Associated with Cortical Thickness, ERP Components and SUD Outcome.

Association between familial loading for alcohol use disorders (AUD) and event-related potentials (ERPs) suggests a genetic basis for these oscillations though much less is known about epigenetic pathways influenced by environmental variation. Early life adversity (ELA) influences negative outcomes much later in life. The stress-activated neuropeptide corticotropin-releasing hormone (CRH) contributes to the deleterious effects of ELA on brain structure and function in animals. Accordingly, we hypothesized that ELA would be related to cortical thickness and electrophysiological characteristics through an epigenetic effect on CRH receptor type-1 (CRHR1) methylation. A total of 217 adolescent and young adult participants from either multiplex alcohol dependence or control families were scanned using magnetic resonance imaging (MRI) at 3T and cortical thickness was determined. Longitudinal follow-up across childhood, adolescence, and young adulthood provided developmental ERP data and measures of adversity. Blood samples for genetic and epigenetic analyses were obtained in childhood. Cortical thickness and visual ERP components were analyzed for their association and tested for familial risk group differences. Visual P300 amplitude at Pz and cortical thickness of the left lateral orbitofrontal region (LOFC), were significantly related to risk group status. LOFC cortical thickness showed a negative correlation with CRHR1 methylation status and with childhood total stress scores from the Life Stressors and Social Resources Inventory (LISRES). Stress scores were also significantly related to P300 amplitude recorded in childhood. The present results suggest that early life adversity reflected in greater total LISRES stress scores in childhood can impact the methylation of the CRHR1 gene with implications for brain development as seen in cortical thickness and electrophysiological signals emanating from particular brain regions.

Cross-generational effects of alcohol dependence in humans on HRAS and TP53 methylation in offspring.

AIM: We hypothesized that cross-generational effects of alcohol exposure could alter DNA methylation and expression of the HRAS oncogene and TP53 tumor suppressor gene that drive cancer development. METHODS: DNA methylation of the HRAS and TP53 genes was tested in samples from young participants (Mean age of 13.4 years). RESULTS: Controlling for both personal use and maternal use of substances during pregnancy, familial alcohol dependence was associated with hypomethylation of CpG sites in the HRAS promoter region and hypermethylation of the TP53 gene. CONCLUSION: The results suggest that ancestral exposure to alcohol can have enduring effects that impact epigenetic processes such as DNA methylation that controls expression of genes that drive cancer development such as HRAS and TP53.

Longitudinal predictors of cannabis use and dependence in offspring from families at ultra high risk for alcohol dependence and in control families.

Cannabis use is common among adolescents. Identification of the factors associated with continued heavy use into young adulthood and development of cannabis abuse and dependence is of considerable importance. The role of familial risk for addiction and an associated endophenotype, P300 amplitude, has not previously been related to cannabis use and dependence. A prospective longitudinal study spanning childhood and young adulthood provided the opportunity for exploring these factors, along with genetic variation, in the cannabis use behaviors of 338 young adult offspring from high and low familial risk for alcohol dependence families (ages 19-30). P300 data were collected multiple times in childhood. The association between young adult patterns of cannabis use or cannabis abuse/dependence was tested with genetic variation in the cannabinoid gene, CNR1, the ANKK1-DRD2 gene, and childhood developmental trajectories of P300. Young adult patterns of cannabis use was characterized by three patterns: (i) no use throughout; (ii) declining use from adolescence through young adulthood; and (iii) frequent use throughout. Following the low P300 trajectory in childhood predicted cannabis abuse and dependence by young adulthood. A four SNP ANKK1-DRD2 haplotype (G-G-G-C) was found to be significantly associated with the frequency of use patterns (P = 0.0008). Although CNR1 variation overall was not significantly associated with these patterns, among individuals with cannabis abuse/dependence the presence of one or both copies of the rs806368 A > G minor allele conferred a 5.4-fold increase (P = 0.003) in the likelihood that they would be in the frequent and persistent use group rather than the declining use group.

ACN9 and alcohol dependence: family-based association analysis in multiplex alcohol dependence families.

A previous genome-wide linkage study of alcohol dependence (AD) in the Pittsburgh-based multiplex family study found suggestive evidence for linkage on Chromosome 7q, a region in which the ACN9 gene is located. Using the same two generation Pittsburgh family data in which linkage was found, data for a third generation was added. The expanded sample included 133 pedigrees with 995 individuals. Finer mapping was undertaken using six SNPs extending from rs1917939 to rs13475 with minor allele frequency (MAF) ≥0.15 and pair-wise linkage disequilibrium (LD) of r(2) <0.8 using the HapMap CEU population. Binary affection status, visual, and auditory P300 data were tested for family-based association. Family-based analyses found all six SNPs associated with affected status. Three SNPs are located upstream of the gene, two SNPs are within intron 1 and one is in Exon 4. FBAT P-values for the six SNPs ranged between 0.05 and 0.0005. Haplotype analysis revealed one four-SNP block formed by rs10499934, rs7794886, rs12056091, and rs13475 with an overall significant association at P = 0.0008. Analysis of visual P300 amplitude data, a known endophenotype of alcohol dependence risk, revealed a significant association for SNPs within intron 1 and exon 4 under a dominant model of transmission. Family-based association analysis shows the ACN9 gene significantly associated with alcohol dependence and P300 amplitude variation. The potential importance of the ACN9 gene for AD risk may be related to its role in gluconeogenesis which may be involved in the regulation of alcohol metabolism.

Familial risk for alcohol dependence and developmental changes in BMI: the moderating influence of addiction and obesity genes.

AIM: Familial loading for alcohol dependence (AD) and variation in genes reported to be associated with AD or BMI were tested in a longitudinal study. MATERIALS & METHODS: Growth curve analyses of BMI data collected at approximately yearly intervals and obesity status (BMI > 30) were examined. RESULTS: High-risk males were found to have higher BMI than low-risk males, beginning at age 15 years (2.0 kg/m(2) difference; p = 0.046), persisting through age 19 years (3.3 kg/m(2) difference; p = 0.005). CHRM2 genotypic variance predicted longitudinal BMI and obesity status. Interactions with risk status and sex were also observed for DRD2 and FTO gene variation. CONCLUSION: Variation at loci implicated in addiction may be influential in determining susceptibility to increased BMI in childhood and adolescence.

Cholinergic receptor gene (CHRM2) variation and familial loading for alcohol dependence predict childhood developmental trajectories of P300.

P300 amplitude in childhood predicts substance use disorders by young adulthood. Trajectories of visual P300 amplitude show an association between low amplitude P300 and familial risk for alcohol dependence (AD). Variation in the cholinergic muscarinic receptor gene (CHRM2) has previously been associated with P300 amplitude and AD. The present study used group based trajectory modeling of auditory P300 data collected longitudinally from offspring in families with and without familial loading for AD to determine if specific trajectories would be associated with familial risk and CHRM2 variation. Trajectory modeling confirms previous reports of an association between the low visual P300 trajectory with high familial risk in male offspring. This association was detected in offspring in the 8-12 age range, but not in 13-18 or 19-29 year olds or in high-risk female offspring. CHRM2 association analysis with P300 finds 8-12 year olds who are homozygous for the T allele of rs1824024 are 2.6 times more likely to follow a P300 trajectory characterized by lower and slower change regardless of familial loading. Combining the odds for being male and having a TT genotype results in odds of 6.5 that individuals will follow the low P300 trajectory.

Family-based association analysis of alcohol dependence implicates KIAA0040 on Chromosome 1q in multiplex alcohol dependence families.

BACKGROUND: A previous genome-wide linkage study of alcohol dependence in multiplex families found a suggestive linkage result for a region on Chromosome 1 near microsatellite markers D1S196 and D1S2878. The KIAA0040 gene has been mapped to this region (1q24 - q25). A recent genome-wide association study using SAGE (the Study of Addiction: Genetics and Environment) and COGA (Collaborative Study on the Genetics of Alcoholism) found five SNPs within the KIAA0040 gene significantly associated with alcohol dependence. A meta-analysis using data from these sources also found the KIAA0040 gene significantly associated with alcohol dependence. METHODS: Using family data consisting of 1000 individuals with phenotypic data (762 with both phenotype and DNA), finer mapping of a 0.3 cM region that included the KIAA0040 gene and a flanking gene was undertaken using SNPs with minor allele frequency (MAF) ≥ 0.15 and pair-wise linkage disequilibrium (LD) of r2 < 0.8 using the HapMap CEU population. RESULTS: Significant FBAT p-values were observed for six SNPs, four within the KIAA0040 gene (rs2269650, rs2861158, rs1008459, rs2272785) and two adjacent to KIAA0040 (rs10912899 and rs3753555). Five haplotype blocks of varying size were identified using HAPLOVIEW. Analysis using the haplotype-based test function of FBAT revealed one two-SNP block (rs1008459-rs2272785) associated with alcohol dependence. This block showed a pattern of transmission in which one haplotype, CT, with a frequency of 0.577 was found to be over-transmitted to affected offspring (p = 0.017) while another haplotype, AG, with a frequency of 0.238 was found to be under-transmitted to affected offspring (p = 0.006). A three-SNP block (rs1008459-rs2272785-rs375355) showed an overall significant association (p = 0.011) with alcohol dependence with the haplotype ACT over-transmitted to affected offspring (p = 0.016) and the haplotype GAG under-transmitted (p = 0.002). CONCLUSIONS: Family-based association analysis shows the KIAA0040 gene significantly associated with alcohol dependence. The potential importance of the KIAA0040 gene for AD risk is currently unknown. However, the present results support earlier findings from a genome-wide association study.

Amygdala Volume in Offspring from Multiplex for Alcohol Dependence Families: The Moderating Influence of Childhood Environment and 5-HTTLPR Variation.

BACKGROUND: The increased susceptibility for developing alcohol dependence seen in offspring from families with alcohol dependence may be related to structural and functional differences in brain circuits that influence emotional processing. Early childhood environment, genetic variation in the serotonin transporter-linked polymorphic region (5-HTTLPR) of the SLCA4 gene and allelic variation in the Brain Derived Neurotrophic Factor (BDNF) gene have each been reported to be related to volumetric differences in the temporal lobe especially the amygdala. METHODS: Magnetic resonance imaging was used to obtain amygdala volumes for 129 adolescent/young adult individuals who were either High-Risk (HR) offspring from families with multiple cases of alcohol dependence (N=71) or Low-Risk (LR) controls (N=58). Childhood family environment was measured prospectively using age-appropriate versions of the Family Environment Scale during a longitudinal follow-up study. The subjects were genotyped for Brain-Derived Neurotrophic Factor (BDNF) Val66Met and the serotonin transporter polymorphism (5-HTTLPR). Two family environment scale scores (Cohesion and Conflict), genotypic variation, and their interaction were tested for their association with amygdala volumes. Personal and prenatal exposure to alcohol and drugs were considered in statistical analyses in order to more accurately determine the effects of familial risk group differences. RESULTS: Amygdala volume was reduced in offspring from families with multiple alcohol dependent members in comparison to offspring from control families. High-Risk offspring who were carriers of the S variant of the 5-HTTLPR polymorphism had reduced amygdala volume in comparison to those with an LL genotype. Larger amygdala volume was associated with greater family cohesion but only in Low-Risk control offspring. CONCLUSIONS: Familial risk for alcohol dependence is an important predictor of amygdala volume even when removing cases with significant personal exposure and covarying for prenatal exposure effects. The present study provides new evidence that amygdala volume is modified by 5-HTTLPR variation in High-Risk families.

ASTN1 and alcohol dependence: family-based association analysis in multiplex alcohol dependence families.

A previous genome-wide linkage study of alcohol dependence (AD) in multiplex families found a suggestive linkage result for a region on Chromosome 1 near microsatellite markers D1S196 and D1S2878. The ASTN1 gene is in this region, a gene previously reported to be associated with substance abuse, bipolar disorder and schizophrenia. Using the same family data consisting of 330 individuals with phenotypic data and DNA, finer mapping of a 26 cM region centered on D1S196 was undertaken using SNPs with minor allele frequency (MAF) ≥ 0.15 and pair-wise linkage disequilibrium (LD) of r(2) < 0.8 using the HapMap CEU population. Significant FBAT P-values for SNPs within the ASTN1 gene were observed for four SNPs (rs465066, rs228008, rs6668092, and rs172917), the most significant, rs228008, within intron 8 had a P-value of 0.001. Using MQLS, which allows for inclusion of all families, we find three of these SNPs with MQLS P-values < 0.003. In addition, two additional neighboring SNPs (rs10798496 and rs6667588) showed significance at P = 0.002 and 0.03, respectively. Haplotype analysis was performed using the haplotype-based test function of FBAT for a block that included rs228008, rs6668092, and rs172917. This analysis found one block (GCG) over-transmitted and another (ATA) under-transmitted to affected offspring. Linkage analysis identified a region consistent with the association results. Family-based association analysis shows the ASTN1 gene significantly associated with alcohol dependence. The potential importance of the ASTN1 gene for AD risk may be related its role in glial-guided neuronal migration.

Cerebellum volume in high-risk offspring from multiplex alcohol dependence families: association with allelic variation in GABRA2 and BDNF.

Offspring from families with multiple cases of alcohol dependence have a greater likelihood of developing alcohol dependence (AD) and related substance use disorders. Greater susceptibility for developing these disorders may be related to structural differences in brain circuits that influence the salience of rewards or modify the efficiency of information processing and AD susceptibility. We examined the cerebellum of 71 adolescent/young adult high-risk (HR) offspring from families with multiple cases of alcohol dependence (multiplex families), and 60 low-risk (LR) controls with no family history of alcohol or drug dependence who were matched for age, gender, socioeconomic status and IQ, with attention given to possible effects of personal use of substances and maternal use during pregnancy. Magnetic resonance images were acquired on a General Electric 1.5-Tesla scanner and manually traced (BRAINS2) blind to clinical information. GABRA2 and BDNF variation were tested for their association with cerebellar volumes. High-risk offspring from multiplex AD families showed greater total volume of the cerebellum and total gray matter (GM), in comparison with LR controls. An interaction between allelic variation in GABRA2 and BDNF genes was associated with GM volumes, suggesting that inherited variation in these genes may promote early developmental differences in neuronal proliferation of the cerebellum.

Disruption of orbitofrontal cortex laterality in offspring from multiplex alcohol dependence families.

BACKGROUND: Increased susceptibility for developing alcohol dependence (AD) might be related to structural differences in brain circuits that influence the salience of rewards and/or modify the efficiency of information processing. The role of the orbitofrontal cortex (OFC) in regulating emotional processing is increasingly being recognized along with its association with impulsive behavior. METHODS: Magnetic resonance imaging was used to measure the OFC in 107 high- and low-risk offspring (mean age 17.6 +/- 4.69 years) from either multiplex AD families or control families. Region of interest measures including segmented values were obtained by reliable raters using BRAINS2 software. Statistical analyses were adjusted for intracranial volume, age, socioeconomic status (SES), IQ, and handedness. The Multidimensional Personality Questionnaire (MPQ) was administered to determine scale scores for Control. Genotyping was performed for the serotonin transporter (5-HTT) gene and the brain-derived neurotrophic factor (BDNF) gene. RESULTS: High-risk offspring from multiplex for AD families showed decreased right/left OFC volumes in comparison with control subjects. Smaller volume in the right hemisphere was significantly associated with variation in the 5-HTT and BDNF genes. White matter (WM) ratios showed a positive correlation with MPQ Control scale scores, indicating that reduced OFC WM is related to greater impulsivity. CONCLUSIONS: Offspring from multiplex families for AD manifest genetic susceptibility by exhibiting disruption in the laterality of the OFC volume that is related to greater impulsivity (lower Control scale scores). This disruption in OFC laterality is related to variation in genes associated with neuronal growth.

Dopaminergic mutations: within-family association and linkage in multiplex alcohol dependence families.

Animal and human studies of addiction indicate that the D2 dopamine receptor (DRD2) plays a critical role in the mechanism of drug reward. D2 receptor density in the brains of alcoholics has been shown to be reduced relative to controls. Previous studies of DRD2 in association with alcohol dependence using variation in the TaqI A locus were highly controversial. Recently, a synonymous mutation, C957T, in the coding region of the human DRD2 gene has been identified which appears to have functional effects including alteration in receptor availability. In order to determine if susceptibility to alcohol dependence (AD) within multiplex alcohol dependence families would be altered by the C957T in the coding region of the D2 gene, within-family association was studied in members of Caucasian multiplex alcohol dependence families. Members of control families with no personal alcohol or substance dependence history were included for case/control comparisons. Analyses performed to detect within-family association showed evidence favoring an association for the C957T polymorphism (P = 0.038). Linkage analyses of polymorphisms in this region showed that only the C957T locus remained of interest (P = 0.015). Evidence for the C957T T allele having a role in AD susceptibility at the population level using a case/control comparison was statistically marginal (P = 0.062), but was consistent with the family data results. These results support a role for DRD2 as a susceptibility gene for alcohol dependence within multiplex families at high risk for developing alcohol dependence.

The role of the GABRA2 polymorphism in multiplex alcohol dependence families with minimal comorbidity: within-family association and linkage analyses.

OBJECTIVE: The genes encoding the gamma-aminobutyric acid(A) (GABA(A)) receptor have been the focus of several recent studies investigating the genetic etiology of alcohol dependence. Analyses of multiplex families found a particular gene, GABRA2, to be highly associated with alcohol dependence, using within-family association tests and other methods. Results were confirmed in three case-control studies. The objective of this study was to investigate the GABRA2 gene in another collection of multiplex families. METHOD: Analyses were based on phenotypic and genotypic data available for 330 individuals from 65 bigenerational pedigrees with a total of 232 alcohol-dependent subjects. A proband pair of same-sex siblings meeting Diagnostic and Statistical Manual of Mental Disorders, Third Edition, criteria for alcohol dependence was required for entry of a family into the study. One member of the proband pair was identified while in treatment for alcohol dependence. Linkage and association of GABRA2 and alcohol dependence were evaluated using SIBPAL (a nonparametric linkage package) and both the Pedigree Disequilibrium Test and the Family-Based Association Test, respectively. RESULTS: We find no evidence of a relationship between GABRA2 and alcohol dependence. Linkage analyses exhibited no linkage using affected/affected, affected/unaffected, and unaffected/unaffected sib pairs (all p's < .13). There was no evidence of a within-family association (all p's > .39). CONCLUSIONS: Comorbidity may explain why our results differ from those in the literature. The presence of primary drug dependence and/or other psychiatric disorders is minimal in our pedigrees, although several of the other previously published multiplex family analyses exhibit a greater degree of comorbidity.

A genome wide search for alcoholism susceptibility genes.

Alcoholism is currently one of the most serious public health problems in the US. Lifetime prevalence rates are relatively high with one in five men and one in 12 women meeting criteria for this condition. Identification of genetic loci conferring an increased susceptibility to developing alcohol dependence could strengthen prevention efforts by informing individuals of their risk before abusive drinking ensues. Families identified through a double proband methodology have provided an exceptional opportunity for gene-finding because of the increased recurrence risks seen in these sibships. A total of 360 markers for 22 autosomes were spaced at an average distance of 9.4 cM and genotyping performed for 330 members of these multiplex families. Extensive clinical data, personality variation, and event-related potential characteristics were available for reducing heterogeneity and detecting robust linkage signals. Multipoint linkage analysis using different analytic strategies give strong support for loci on chromosomes 1, 2, 6, 7, 10, 12, 14, 16, and 17.