Diana G. Patterson

Genomewide linkage study in the Irish affected sib pair study of alcohol dependence: evidence for a susceptibility region for symptoms of alcohol dependence on chromosome 4.

Alcoholism is a relatively common, chronic, disabling and often treatment-resistant disorder. Evidence from twin and adoption studies indicates a substantial genetic influence, with heritability estimates of 50–60%. We conducted a genome scan in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD). Most probands were ascertained through alcoholism treatment settings and were severely affected. Probands, affected siblings and parents were evaluated by structured interview. A 4 cM genome scan was conducted using 474 families of which most (96%) were comprised by affected sib pairs. Nonparametric and quantitative linkage analyses were conducted using DSM-IV alcohol dependence (AD) and number of DSM-IV AD symptoms (ADSX). Quantitative results indicate strong linkage for number of AD criteria to a broad region of chromosome 4, ranging from 4q22 to 4q32 (peak multipoint LOD=4.59, P=2.1 × 10−6, at D4S1611). Follow-up analyses suggest that the linkage may be due to variation in the symptoms of tolerance and out of control drinking. There was evidence of weak linkage (LODs of 1.0–2.0) to several other regions, including 1q44, 13q31, and 22q11 for AD along with 2q37, 9q21, 9q34 and 18p11 for ADSX. The location of the chromosome 4 peak is consistent with results from prior linkage studies and includes the alcohol dehydrogenase gene cluster. The results of this study suggest the importance of genetic variation in chromosome 4 in the etiology and severity of alcoholism in Caucasian populations.

The Irish Affected Sib Pair Study of Alcohol Dependence: study methodology and validation of diagnosis by interview and family history.

BACKGROUND This article is the first report of the Irish Affected Sib Pair Study of Alcohol Dependence, whose goal is to detect the genomic location of susceptibility loci for alcohol dependence (AD). This article describes phenotypic characteristics of the probands, siblings, and parents included in the sample and examines agreement among different sources of diagnostic information, including the validity of family history (FH) assessment. METHODS Structured diagnostic interviews were conducted with 1414 individuals from 591 families ascertained in Ireland. AD was assessed among 1201 probands and affected siblings with use of the Semi-Structured Assessment for the Genetics of Alcoholism and among 213 parents with use of a modified version of the Structured Clinical Interview for DSM. Probands and siblings were also assessed for drinking history, comorbid disorders, and other clinical characteristics. FH reports based on FH-Research Diagnostic Criteria were obtained for 1113 of these individuals as well as for 3652 first-degree relatives who were not interviewed. RESULTS Sample characteristics confirm the severity of AD among the affected individuals. Agreement between FH ratings and diagnoses based on direct interviews was high for both parent-offspring and sibling-sibling comparisons (e.g., positive and negative predictive values > 80% for a range of cutoffs). Agreement among individuals about their family members was also high for a single item (1 month or more of drinking problems, tetrachoric r = 0.86-0.98), the total number of DSM-IV AD symptoms (polychoric r = 0.86-0.96), and classifications based on a range of cutoffs (kappa = 0.75-0.80). Use of multiple informants improved classification accuracy only slightly (6-10%). CONCLUSIONS The authors successfully collected data for a large sample of affected sibling pairs for molecular genetic analysis of AD. Individuals with AD were able to provide accurate evaluations of alcoholism symptoms in their parents and adult siblings. A single screening item performed nearly as well as the full scale. Collecting information from multiple informants may not be cost effective for the gain in predictive accuracy. FH information collected from affected informants can be a valuable source of diagnostic information for family studies of alcoholism.

A genome-wide linkage analysis for the personality trait neuroticism in the Irish affected sib pair study of alcohol dependence

Neuroticism is a personality trait which reflects individual differences in emotional stability and vulnerability to stress and anxiety. Consistent evidence shows substantial genetic influences on variation in this trait. The present study seeks to identify regions containing susceptibility loci for neuroticism using a selected sib‐pair sample from Ireland. Using Merlin regress, we conducted a 4 cM whole‐genome linkage analysis on 714 sib‐pairs. Evidence for linkage to neuroticism was found on chromosomes 11p, 12q, and 15q. The highest linkage peak was on 12q at marker D12S1638 with a Lod score of 2.13 (−log p = 2.76, empirical P‐value <0.001). Our data also support gender specific loci for neuroticism, with male specific linkage regions on chromosomes 1, 4, 11, 12, 15, 16, and 22, and female specific linkage regions on chromosomes 2, 4, 9, 12, 13, and 18. Some genome regions reported in the present study replicate findings from previous linkage studies of neuroticism. These results, together with prior studies, indicate several potential regions for quantitative trait loci for neuroticism that warrant further study.

Neurotransmitter and neuromodulator genes associated with a history of depressive symptoms in individuals with alcohol dependence.

BACKGROUND Depressive symptoms are common among individuals with alcohol use disorders and impact treatment outcome. Substantial overlap exists among the neurobiological systems proposed in the pathophysiology of depressive and alcohol use disorders; however, specific genetic effects contributing to risk for depressive comorbidity remain poorly understood. METHODS This study examines the association of depressive symptom scores for lifetime depression (the sum of DSM-IV major depression co-endorsed criteria for lifetime depression) with markers in 120 candidate genes in 554 alcohol-dependent individuals. The candidate genes code for molecules involved in dopamine, serotonin, glutamate, gamma-aminobutyric acid (GABA), and opioid neurotransmission, cell signaling, pharmacokinetics, stress biology, and behavioral control. Analyses were conducted at the single marker level with experimentwise permutation to control for multiple testing. RESULTS Results revealed nominal associations for markers in 20 genes. Following experimentwise permutation, markers in the corticotropin-releasing hormone-binding protein (CRHBP) the μ-opioid receptor (OPRM1) and the β1 subunit of GABA A (GABA(A)) receptors (GABRB1) met or exceeded the significance threshold. None of the markers associated with depressive symptom scores were significantly associated with alcohol dependence symptom scores. CONCLUSION These findings suggest potential risk genes for depressive symptoms in alcohol-dependent individuals.

Associations of glutamate decarboxylase genes with initial sensitivity and age-at-onset of alcohol dependence in the Irish Affected Sib Pair Study of Alcohol Dependence

BACKGROUND The relation of gamma-aminobutyric acid (GABA) to alcohol dependence (AD) has been widely studied. Several previous studies suggest that GABA may be involved in alcohol withdrawal, tolerance, and the symptoms that form an AD diagnosis. The genes coding for glutamate decarboxylase (GAD), the rate-limiting enzyme in GABA synthesis, are of potential interest for their association to ethanol consumption and AD. There are two isoforms of GAD, GAD1 and GAD2, which were reported to be associated with AD in males of Han Taiwanese (GAD1) and Russian (GAD2) ancestry. The present study examined the association of the two GAD isoforms with AD and relevant alcohol-related traits in the Irish Affected Sib Pair Study of Alcohol Dependence [Prescott, C.A., Sullivan, P.F., Myers, J.M., Patterson, D.G., Devitt, M., Halberstadt, L.J., Walsh, D., Kendler, K.S., 2005. The Irish Affected Sib Pair Study of Alcohol Dependence: study methodology and validation of diagnosis by interview and family history. Alcohol.-Clin. Exp. Res. 29 (3) 417-429]. METHODS Participants were recruited in Ireland, including 575 independent cases who met DSM-IV AD criteria and 530 controls, screened for heavy drinking. We first conducted case-control analyses of the GAD genes with AD and, within the cases, examined associations with age at onset of AD, withdrawal symptoms, and two quantitative measures: initial sensitivity and tolerance (based on scales from the Self-Rating of the Effects of Ethanol) [Schuckit, M.A., Smith, T.L., Tipp, J.E., 1997. The self-rating of the effects of alcohol (SRE) form as a retrospective measure of the risk for alcoholism. Addiction 92, 979-988]. A total of 29 SNPs were genotyped for GAD1 and GAD2 using the Illumina GoldenGate protocols. Statistical procedures were implemented to control for false discovery rates (FDR). RESULTS Nine of 29 markers with minor allele frequencies less than 0.01 were removed from standard analysis; the remaining 20 markers were all in Hardy-Weinberg equilibrium. Three markers in the intronic regions of GAD1 were associated with initial sensitivity to alcohol (P=0.002); the associations remained significant after a FDR based correction for multiple testing. In addition, one marker located 3kb upstream of GAD1 exhibited association with age at onset of AD (P=0.0001). Gender specific effects were observed in results of both single marker and haplotype analyses. CONCLUSION We found no evidence for the association of GAD genes with AD but significant association of GAD1 with initial sensitivity and age at onset of AD. Our findings suggest that the underlying pathophysiology regulated by genes like GAD1 may be more directly related to the component processes that form AD than to the clinical disorder.

Limited Associations of Dopamine System Genes With Alcohol Dependence and Related Traits in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD)

BACKGROUND Over 50 years of evidence from research has established that the central dopaminergic reward pathway is likely involved in alcohol dependence (AD). Additional evidence supports a role for dopamine (DA) in other disinhibitory psychopathology, which is often comorbid with AD. Family and twin studies demonstrate that a common genetic component accounts for most of the genetic variance in these traits. Thus, DA-related genes represent putative candidates for the genetic risk that underlies not only AD but also behavioral disinhibition. Many linkage and association studies have examined these relationships with inconsistent results, possibly because of low power, poor marker coverage, and/or an inappropriate correction for multiple testing. METHODS We conducted an association study on the products encoded by 10 DA-related genes (DRD1-D5, SLC18A2, SLC6A3, DDC, TH, COMT) using a large, ethnically homogeneous sample with severe AD (n = 545) and screened controls (n = 509). We collected genotypes from linkage disequilibrium (LD)-tagging single nucleotide polymorphisms (SNPs) and employed a gene-based method of correction. We tested for association with AD diagnosis in cases and controls and with a variety of alcohol-related traits (including age-at-onset, initial sensitivity, tolerance, maximum daily drinks, and a withdrawal factor score), disinhibitory symptoms, and a disinhibitory factor score in cases only. A total of 135 SNPs were genotyped using the Illumina GoldenGate and Taqman Assays-on-Demand protocols. RESULTS Of the 101 SNPs entered into standard analysis, 6 independent SNPs from 5 DA genes were associated with AD or a quantitative alcohol-related trait. Two SNPs across 2 genes were associated with a disinhibitory symptom count, while 1 SNP in DRD5 was positive for association with the general disinhibitory factor score. CONCLUSIONS Our study provides evidence of modest associations between a small number of DA-related genes and AD as well as a range of alcohol-related traits and measures of behavioral disinhibition. While we did conduct gene-based correction for multiple testing, we did not correct for multiple traits because the traits are correlated. However, false-positive findings remain possible, so our results must be interpreted with caution.

Genome-wide linkage scans for major depression in individuals with alcohol dependence

Major depression is more prevalent among individuals with alcoholism than in the general population. Twin studies have found a moderate degree of genetic correlation for alcohol dependence (AD) and major depression (MD), suggesting the existence of loci that confer susceptibility to both disorders. The aim of the present study was to conduct genome-wide linkage analyses to identify loci and to replicate prior evidence for linkage to MD, and to search for linkage regions that may confer risk to the co-occurrence of depression and alcoholism in a sample of sib-pairs affected with AD. A set of 1020 microsatellite markers (average marker spacing of 4cM) were genotyped in 1289 subjects, which consisted of 473 informative families for analysis of depressive traits and 626 sibling pairs for analysis of symptoms of MD and AD. For univariate linkage results for depression, there were six regions (1q, 2p, 4q, 12q, 13q, and 22q) with multipoint LOD scores in excess of 1.00; the highest peak was on chromosome 4q32.3 near marker D4S2952 (LOD=2.17, p=0.0008) for symptoms of MD. Bivariate linkage analysis of symptoms of MD and AD identified only one region at 22q11.21 with LOD>1, which overlapped with the region for symptoms of MD. Several of these regions replicate previously reported linkage results for major depression and emotion-related traits and events, such as neuroticism and suicide attempts. These identified genomic locations, together with results from prior studies, indicate potential regions of interests that may contain susceptibility loci to the risk of depression among individuals with alcohol dependence.

Alcohol dependence is associated with the ZNF699 gene, a human locus related to Drosophila hangover, in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD) sample.

Because tolerance is an important aspect of alcohol dependence (AD) in humans, recent evidence showing that the Drosophila gene hang is critically involved in the development of alcohol tolerance in the fly suggests that variation in related human loci might be important in the etiology of alcohol-related disorders. The orthology of hang in mammals is complex, but a number of human gene products (including ZNF699) with similar levels of amino-acid identity (18–26%) and similarity (30–41%), are consistently identified as the best matches with the translated hang sequence. We tested for association between the dichotomous clinical phenotype of alcohol dependence and seven single nucleotide polymorphisms (SNPs) in ZNF699 in our sample of 565 genetically independent cases and 496 siblings diagnosed with AD, and 609 controls. In analyses of genetically independent cases and controls, four of the seven single markers show strong evidence for association with AD (0.00003<Fishers exact P<0.001), and the most significant single marker, rs7254880, tags an associated haplotype with frequency 0.071 in cases compared to 0.034 in controls (χ2 15.563, P<0.00008, 5000 permutation P<0.001, OR 2.17); inclusion of affected siblings gives similar results. Expression analyses conducted in independent postmortem brain samples show that expression of ZNF699 mRNA is significantly reduced in the dorsolateral prefrontal cortex of individuals carrying this haplotype compared with other observed haplotype combinations.

A systematic gene-based screen of chr4q22–q32 identifies association of a novel susceptibility gene, DKK2, with the quantitative trait of alcohol dependence symptom counts

Studies of alcohol dependence (AD) have consistently found evidence of linkage on chromosome 4q21-q32. A genome-wide linkage scan in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD) sample also provided its strongest evidence of linkage on chromosome 4q22-q32 using an index of AD severity based on the count of DSM-IV AD symptoms (ADSX; LOD = 4.59). We conducted a systematic, gene-centric association study using 518 LD-tagging single nucleotide polymorphisms (SNPs) in the 65 known and predicted genes within the 1-LOD interval surrounding the linkage peak. Case-only regression analysis with the quantitative variable of ADSX was performed in the 562 genetically independent cases; nominal support for association was demonstrated by 32 tagging SNPs in 14 genes. We did not observe study-wide significance, but gene-wise correction for multiple testing with the Nyholt procedure yielded empirical evidence of association with two genes, DKK2 (dickkopf homolog 2) (P = 0.007) and EGF (epidermal growth factor) (P = 0.025) in the IASPSAD sample. Three SNPs in DKK2 (rs427983; rs419558; rs399087) demonstrated empirical significance. Assessment of possible replication in 847 cases of European descent from a large independent sample, the Collaborative Study of the Genetics of Alcoholism, yielded replication for DKK2 but not EGF. We observed genotypic and phenotypic replication for DKK2 with the three SNPs yielding significant association with ADSX in the IASPSAD sample. Haplotype-specific expression measurements in post-mortem tissue samples suggested a functional role for DKK2. This evidence notwithstanding, replication is needed before confidence can be placed in these findings.

Empirically Defined Subtypes of Alcohol Dependence in an Irish Family Sample

Alcohol dependence (AD) is clinically and etiologically heterogeneous. The goal of this study was to explore AD subtypes among a sample of 1221 participants in the Irish Affected Sib Pair Study of Alcohol Dependence, all of whom met DSM-IV criteria for AD. Variables used to identify the subtypes included major depressive disorder, antisocial personality disorder, illicit drug dependence (cannabis, sedatives, stimulants, cocaine, opioids, and hallucinogens), nicotine dependence, the personality traits of neuroticism and novelty seeking, and early alcohol use. Using latent class analysis, a 3-class solution was identified as the most parsimonious description of the data. Individuals in a Mild class were least likely to have comorbid psychopathology, whereas a severe class had highest probabilities of all comorbid psychopathology. The third class was characterized by high probabilities of major depression and higher neuroticism scores, but lower likelihood of other comorbid disorders than seen in the severe class. Overall, sibling pair resemblance for class was stronger within than between classes, and was greatest for siblings within the severe class, suggesting a stronger familial etiology for this class. These findings are consistent with the affective regulation and behavioral disinhibition subtypes of alcoholism, and are in line with prior work suggesting familial influences on subtype etiology.