DRD4 exon III VNTR polymorphism-susceptibility factor for heroin dependence? Results of a case-control and a family-based association approach

Dopaminergic abnormalities are implicated in the pathogenesis of substance abuse.1 Recently, two reports have been published suggesting an association between opioid dependence and presence of long alleles of the dopamine D4 receptor (DRD4) gene exon III VNTR.2, 3 We have attempted to replicate this finding using a two-tiered strategy employing independent case-control and family-based association samples. Our study was possibly the largest candidate gene association study to date on opioid dependence in a sample of 815 subjects, 396 of whom were patients. We found long alleles of the DRD4 exon III VNTR in similar frequency among 285 heroin addicts and 197 controls. Furthermore, no preferential transmission of long alleles to affected offspring was observed in a sample of 111 patients and their parents. Our results, therefore, do not support the hypothesis that alleles of the DRD4 exon III VNTR are susceptibility factors for opioid dependence in man. Molecular Psychiatry(2000) 5, 101-104.     

Minor genetic variants of the dopamine D4 receptor (DRD4) polymorphism are associated with novelty seeking in healthy Japanese subjects

Although an association between the dopamine receptor D4 (DRD4) gene and personality traits had been previously investigated, results from previous studies were not conclusive. This may be due to the method of grouping used, which categorized the gene population into two groups based on the length of the variable number of tandem repeats (VNTR) in exon 3. In the present study, we categorized 616 healthy Japanese subjects into more than two groups by further subdividing the DRD4 48-bp VNTR polymorphism, and compared Temperament and Character Inventory (TCI) scores among the groups. A significant difference was found between the DRD4 48-bp VNTR polymorphism and novelty seeking (p = 0.016). The novelty-seeking scores in the subjects carrying the 5/5 genotype were significantly higher than in those carrying the 2/2 genotype (p = 0.002) or the 4/4 genotype (p = 0.005). However, when the conventional method of grouping was used (i.e., short alleles vs. long alleles), there were no significant associations between the DRD4 VNTR polymorphism and any TCI scores. Our results suggest that minor 5-repeat allele is associated with high novelty-seeking scores in healthy Japanese subjects.     

多巴胺D4受体第3外显子48 bp 可变重复序列多态性与抽动障碍的关联分析

目的研究多巴胺D4受体第3外显子48 bp可变重复序列（DRD4 exonⅢ48 bpVNTR）多态性是否与抽动障碍相关联。方法根据临床诊断及是否合并注意缺陷多动障碍（ADHD），将178例抽动障碍患者（患者组）分为抽动障碍组［包括Tourette 综合征（TS）和慢性抽动障碍（CT），共91例］及合并组［合并ADHD 的TS或CT，共87例］两个亚组，采用病例-对照关联分析方法，以及聚合酶链反应、VNTR多态性分析等技术，进行抽动障碍与DRD4 exonⅢ48 bpVNTR多态性的关联分析，将其等位基因按是否≥5个48 bp重复片段分为2组进行比较，并与209名正常人进行对照。结果患者组、抽动障碍组及合并组的DRD4 exonⅢ 48 bpVNTR等位基因频率与对照组比较，差异无显著性（χ2值分别为5.81,3.76，6.78；P值分别为0.21,0.44,0.15），仅合并组与DRD4 exonⅢ48 bpVNTR多态性的≥5个48 bp的长重复等位基因存在关联（χ2=5.55,P=0.02）。结论 DRD4 exonⅢ48 bpVNTR多态性长重复等位基因与合并ADHD的抽动障碍存在关联，该等位基因可能是中国人群合并ADHD的抽动障碍的遗传危险因素。     

Association between VNTR polymorphism in promoter region of prodynorphin (PDYN) gene and heroin dependence

Within the core promoter region of prodynorphin (PDYN), a 68-bp sequence was found to occur as a polymorphism element, either singular or as tandemly repeated two, three or four times. We report the sequence of a novel allele (5-repeats). Our study revealed the existence of an ancestral nucleotide (A) at 29th position of the VNTR in human. In total, 442 heroin addicts and 799 controls were included in this study. The present findings revealed a male-limited association between VNTR polymorphism and heroin dependence risk.     

Epistatic effects between variants of kappa-opioid receptor gene and A118G of mu-opioid receptor gene increase susceptibility to addiction in Indian population

Objective

Unequivocal evidence suggests contribution of κ-opioid receptor (KOR) in addiction to drugs of abuse. A study was undertaken to identify the single nucleotide polymorphisms (SNP) at selective areas of kappa opioid receptor 1 (OPRK1) gene in heroin as well as in alcohol addicts and to compare them with that in control population. The potential interaction of the identified KOR SNPs with A118G of μ opioid receptor was also investigated.

Methods

Two hundred control subjects, one hundred thirty heroin and one hundred ten alcohol addicts, all male and residing in Kolkata, a city in eastern India, volunteered for the study. Exons 3 and 4 of OPRK1 and the SNP, A118G of mu opioid receptor 1 (OPRM1) in the DNA samples were genotyped by sequencing and restriction fragment length polymorphism respectively. The SNPs identified in the population were analyzed by odds ratio and its corresponding 95% confidence interval was estimated using logistic regression models. SNP–SNP interactions were also investigated.

Results

Three SNPs of OPRK1, rs16918875, rs702764 and rs963549, were identified in the population, none of which showed significant association with addiction. On the other hand, significant association was observed for A118G with heroin addiction (χ² = 7.268, P = 0.0264) as well as with alcoholic addition (χ² = 6.626, P = 0.0364). A potential SNP–SNP interaction showed that the odds of being addicted was 2.51 fold in heroin subjects [CI (95%) = 1.1524 to 5.4947, P = 0.0206] and 2.31 fold in alcoholics [CI (95%) = 1.025 to 5.24, P = 0.0433] with the OPRK1 (rs16918875) and A118G risk alleles than without either. A significant interaction was also identified between GG/AG of A118G and GG of rs702764 [O.R (95%) = 2.04 (1.279 to 3.287), P = 0.0029] in case of opioid population.

Conclusion

Our study suggests that set associations of polymorphisms may be important in determining the risk profile for complex diseases such as addiction.     

Analysis of monoamine oxidase A (MAO-A) promoter polymorphism in male heroin-dependent subjects: behavioural and personality correlates

Summary. The promoter of the monoamine oxidase A (MAO-A) gene was analysed to test whether length variation of the repeat polymorphism contributes to variation in individual vulnerability to aggressive-criminal behaviour, and liability to heroin dependence. The repeat number of the MAO-A polymorphism was assessed in 199 male subjects of Italian descent, a sample comprising 95 healthy subjects and 104 heroin-dependent subjects including 52 addicted individuals with violent behaviour and antisocial personality disorder. The frequency of the low-activity 3-repeat allele was significantly higher in violent offenders among heroin addicts, compared to addicted individuals without antisocial behaviour (34.6 vs. 15.4%; p<0.03) and controls (18.9%; p<0.05). No significant difference was evidenced in the frequencies of the MAO-A alleles between heroin-dependent subjects in general and control subjects. High activity 4-repeat allele frequency was significantly higher in addicted individuals without antisocial behavior compared to antisocial-aggressive heroin-dependent subjects (76.9 vs. 55.8%; p<0.02). Buss Durkee Hostility Inventory (BDHI) mean total scores were significantly higher in heroin addicts than in controls (p<0.001), and in antisocial-violent heroin addicts in comparison with addicted individuals without antisocial behaviour (p<0.005). Among heroin addicts BDHI irritability, suspiciousness and resentment subscales scores were found significantly higher in low activity 3-repeat allele subjects than in high activity alleles subjects (p<0.001; p<0.05; p<0.05, respectively). No association was found between MAO-A polymorphism and suicide history. Our findings suggest that the low-activity 3-repeat allele of the MAO-A promoter polymorphism confers increased susceptibility to antisocial-violent behavior and aggressiveness, rather than drug dependence per se, in heroin-dependent males.     

Role of gene-gene/gene-environment interaction in the etiology of eastern Indian ADHD probands

Associations between attention deficit hyperactivity disorder (ADHD) and genetic polymorphisms in the dopamine receptors, transporter and metabolizing enzymes have been reported in different ethnic groups. Gene variants may affect disease outcome by acting synergistically or antagonistically and thus their combined effect becomes an important aspect to study in the disease etiology. In the present investigation, interaction between ten functional polymorphisms in DRD4, DAT1, MAOA, COMT, and DBH genes were explored in the Indo-Caucasoid population. ADHD cases were recruited based on DSM-IV criteria. Peripheral blood samples were collected from ADHD probands (N = 126), their parents (N = 233) and controls (N = 96) after obtaining informed written consent for participation. Genomic DNA was subjected to PCR based analysis of single nucleotide polymorphisms and variable number of tandem repeats (VNTRs). Data obtained was examined for population as well as family-based association analyses. While case-control analysis revealed higher occurrence of DAT1 intron 8 VNTR 5R allele (P = 0.02) in cases, significant preferential transmission of the 7R-T (DRD4 exon3 VNTR-rs1800955) and 3R-T (MAOA-u VNTR-rs6323) haplotypes were noticed from parents to probands (P = 0.02 and 0.002 respectively). Gene-gene interaction analysis revealed significant additive effect of DBH rs1108580 and DRD4 rs1800955 with significant main effects of DRD4 exon3 VNTR, DAT1 3′UTR and intron 8 VNTR, MAOA u-VNTR, rs6323, COMT rs4680, rs362204, DBH rs1611115 and rs1108580 thereby pointing towards a strong association of these markers with ADHD. Correlation between gene variants, high ADHD score and low DBH enzymatic activity was also noticed, especially in male probands. From these observations, an impact of the studied sites on the disease etiology could be speculated in this ethnic group.     

Family-based association study between monoamine oxidase A (MAOA) gene promoter VNTR polymorphism and Tourette’s syndrome in Chinese Han population

To clarify the association of monoamine oxidase A– variable number of tandem repeat (MAOA-pVNTR) with susceptibility to Tourette’s syndrome (TS) in Chinese Han population we discuss the genetic contribution of MAOA–VNTR in 141 TS patients including all their parents in Chinese Han population using transmission disequilibrium test (TDT) design. Our results revealed that no significant association was found in the MAOA gene promoter VNTR polymorphism and TS in Chinese Han population (TDT = 1.515, df = 1, p > 0.05). The negative result may be mainly due to the small sample size, but we don’t deny the role of gene coding serotonergic or monoaminergic structures in the etiology of TS.     

Effects of source of DNA on genotyping success rates and allele percentages in the Preschoolers with Attention-Deficit/Hyperactivity Disorder Treatment Study (PATS)

OBJECTIVE: In children diagnosed with attention-deficit/hyperactivity disorder (ADHD) and their parents, who were participants of the Preschool ADHD Treatment Study (PATS), we assessed the effect of source of DNA (from buccal or blood cells) on the genotyping success rate and allele percentages for the five polymorphisms in three candidate genes (DAT1, DRD4, and SNAP 25) investigated in the PATS pharmacogenetic study of response to stimulant medication. METHOD: At baseline assessment, 241 individuals (113 probands and 128 parents) consented to participate; 144 individuals (52 probands and 92 parents) provided blood samples from venipuncture, and 97 individuals (61 probands and 36 parents) provided buccal samples from cheek swab as specimens for isolation of DNA. Three types of polymorphisms-variable number of tandem repeat (VNTR) polymorphism, tandem duplication polymorphism (TDP), and single nucleotide polymorphism (SNP)-were evaluated, including the DRD4 gene 48-bp VNTR in exon III, the DAT1 gene 40-bp VNTR in 3'-untranslated region, the DRD4 gene TDP 120-bp duplication in the promoter region, the SNAP-25 gene TC-1069 SNP, and the SNAP-25 gene TG-1065 SNP. Standard procedures were used to genotype individuals for each of these five polymorphisms. RESULTS: Using the methods available in 2004, the genotyping success rate was on the average much greater for DNA from blood cells than buccal cells (e.g., 91% vs. 54% in probands). For some polymorphisms (DRD4-VNTR, DRD4-TDP, and SNAP25-TC SNP), allele proportion also varied by blood versus buccal source of DNA (e.g., 26.5% vs. 18.6% for the 7-repeat allele of the DRD4 gene). CONCLUSIONS: The much lower success rate for genotyping based on DNA from buccal than blood cells is likely due to the quality of DNA derived from these two sources. The observed source differences in allele proportion may be due to self-selection related to choice of how specimens were collected (from cheek swab or venipuncture), or to a selective detection of some alleles based on differences in DNA quality.     

多巴胺2受体基因TaqIA多态性与美沙酮维持治疗海洛因依赖患者心理症状相关性研究

目的研究昆明市美沙酮维持治疗A门诊接受美沙酮维持治疗的汉族海洛因依赖者多巴胺D2受体基因TaqIA多态性与接受美沙酮维持治疗海洛因依赖患者心理症状的相关性,探讨影响病人治疗效果的遗传因素。方法对81例接受美沙酮维持治疗的海洛因依赖者实施SCL-90测试,应用聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）技术,检测多巴胺D2受体基因TaqIA基因多态,比较不同基因型与SCL-90各因子的关系。结果 A1＋组（A1/A1,A1/A2）与A1-组（A2/A2）SCL-90各因子分无显著性差异。结论D2受体基因TaqIA多态性与美沙酮维持治疗海洛因依赖患者心理症状可能不存在相关性。     

Molecular genetics and attention in ADHD

A research program at UC Irvine has investigated the molecular genetic basis of ADHD by focusing on one candidate gene (DRD4) and the highly variable 48 bp VNTR polymorphism in exon 3. Initial studies revealed that the 7R variant is over-represented in ADHD samples, and a subsequent study suggested that the 7R allele is associated with clear excesses in behavior but not with some cognitive deficits thought to be core feature of the disorder. The next phase of this research program showed that (1) the common 7R allele was the product of positive selection, (2) other variation in and around the DRD4 gene is in tight linkage disequilibrium with the 7R allele but not the 4R allele, and (3) more rare 7R variants in the ADHD clinical sample than expected. Based on this program of research, we suggest that the 7R VNTR variant is responsible for the observed association of the DRD4 gene with ADHD, and that a challenge for the future is understanding what other genetic and/or environmental factors influence this association and affect clinical outcome of the disorder.     

Associations of serotonin transporter gene promoter polymorphisms and monoamine oxidase A gene polymorphisms with oppositional defiant disorder in a Chinese Han population

Background

Oppositional defiant disorder (ODD) is a behavioral disorder that mainly refers to a recurrent pattern of disobedient, defiant, negativistic and hostile behaviors toward authority figures. Previous studies have showed associations of serotonin transporter (5-HTT) and monoamine oxidase A (MAOA) with behavioral and psychiatric disorders. The purposes of this study were to investigate the potential association of 5-HTT gene promoter polymorphism (5-HTTLPR) and MAOA gene polymorphism with susceptibility to ODD in a Han Chinese school population.

Methods

The 5-HTTLPR gene polymorphism and the MAOA gene polymorphism were genotyped in a case–control study of 257 Han Chinese children (123 ODD and 134 healthy controls).

Results

There was significant difference in the allele distribution of 5-HTTLPR (χ²?=?7.849, P?=?0.005) between the ODD and control groups. Further, there were significant differences in genotype (χ²?=?5.168, P?=?0.023) and allele distributions (χ²?=?10.336, P?=?0.001) of the MAOA gene polymorphism that is variable-number tandem repeat (MAOA-uVNTR) between two groups. Moreover, there were significant differences in genotype (χ²?=?4.624, P?=?0.032) and allele distributions (χ²?=?9.248, P?=?0.002) of MAOA-uVNTR only in the male ODD and healthy groups.

Conclusions

Our results suggest that 5-HTTLPR and MAOA-uVNTR gene variants may contribute to susceptibility to ODD. Further, MAOA-uVNTR gene polymorphism may play a role in susceptibility to ODD only in male children.

     

Interaction between serotonin 5-HT2A receptor gene and dopamine transporter (DAT1) gene polymorphisms influences personality trait of persistence in Austrian Caucasians

We examined 89 normal volunteers using Cloninger's Temperament and Character Inventory (TCI). Genotyping the 102T/C polymorphism of the serotonin 5HT2A receptor gene and the ser9gly polymorphism in exon 1 of the dopamine D3 receptor (DRD3) gene was performed using PCR-RFLP, whereas the dopamine transporter (DAT1) gene variable number of tandem repeats (VNTR) polymorphism was investigated using PCR amplification followed by electrophoresis in an 8% acrylamide gel with a set of size markers. We found a nominally significant association between gender and harm avoidance (P=0.017; women showing higher scores). There was no association of either DAT1, DRD3 or 5HT2A alleles or genotypes with any dimension of the TCI applying Kruskal–Wallis rank-sum tests. Comparing homozygote and heterozygote DAT1 genotypes, we found higher novelty seeking scores in homozygotes (P=0.054). We further found a nominally significant interaction between DAT1 and 5HT2A homo-/heterozygous gene variants (P=0.0071; DAT1 and 5HT2A genotypes P value of 0.05), performing multivariate analysis of variance (MANOVA). Examining the temperamental TCI subscales, this interaction was associated with persistence (genotypes: P=0.004; homo-/heterozygous gene variants: P=0.0004). We conclude that an interaction between DAT1 and 5HT2A genes might influence the temperamental personality trait persistence.     

Monoamine oxidase A regulates antisocial personality in whites with no history of physical abuse

Objective

Preclinical and human family studies clearly link monoamine oxidase A (MAOA) to aggression and antisocial personality (ASP). The 30–base pair variable number tandem repeat in the MAOA promoter regulates MAOA levels, but its effects on ASP in humans are unclear.

Methods

We evaluated the association of the variable number tandem repeat of the MAOA promoter with Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, ASP disorder (ASPD) traits in a community sample of 435 participants from the Hopkins Epidemiology of Personality Disorders Study.

Results

We did not find an association between the activity of the MAOA allele and ASPD traits; however, among whites, when subjects with a history of childhood physical abuse were excluded, the remaining subjects with low-activity alleles had ASPD trait counts that were 41% greater than those with high-activity alleles (P < .05).

Conclusion

The high-activity MAOA allele is protective against ASP among whites with no history of physical abuse, lending support to a link between MAOA expression and antisocial behavior.     

Repeat variation in the human PER2 gene as a new genetic marker associated with cocaine addiction and brain dopamine D2 receptor availability

Low dopamine D2 receptor (D2R) levels in the striatum are consistently reported in cocaine abusers; inter-individual variations in the degree of the decrease suggest a modulating effect of genetic makeup on vulnerability to addiction. The PER2 (Period 2) gene belongs to the clock genes family of circadian regulators; circadian oscillations of PER2 expression in the striatum was modulated by dopamine through D2Rs. Aberrant periodicity of PER2 contributes to the incidence and severity of various brain disorders, including drug addiction. Here we report a newly identified variable number tandem repeat (VNTR) polymorphism in the human PER2 gene (VNTR in the third intron). We found significant differences in the VNTR alleles prevalence across ethnic groups so that the major allele (4 repeats (4R)) is over-represented in non-African population (4R homozygosity is 88%), but not in African Americans (homozygosity 51%). We also detected a biased PER2 genotype distribution among healthy controls and cocaine-addicted individuals. In African Americans, the proportion of 4R/three repeat (3R) carriers in healthy controls is much lower than that in cocaine abusers (23% vs 39%, P=0.004), whereas among non-Africans most 3R/4R heterozygotes are healthy controls (10.5% vs 2.5%, P=0.04). Analysis of striatal D2R availability measured with positron emission tomography and [¹¹C]raclopride revealed higher levels of D2R in carriers of 4R/4R genotype (P<0.01). Taken together, these results provide preliminary evidence for the role of the PER2 gene in regulating striatal D2R availability in the human brain and in vulnerability for cocaine addiction.     

Dopamine D4 receptor (DRD4) polymorphism and adaptability trait during infancy: a longitudinal study in 1- to 5-month-old neonates

We have examined the relationship between the common dopamine D4 receptor (DRD4) exon III repeat polymorphism and infants' behavior measured with the Italian version of the Early and Revised Infancy Temperament Questionnaires (EITQ/RITQ) in 122 Italian neonates at 1 and 5 months of life, when the genetic contribution to the behavior can be more clearly assessed. Two-way (genotype × age) analysis of variance revealed a significant correlation with the temperamental subscale of adaptability [F(1, 120)=5.26, P<0.02]. At 1 month of life (early assessment), infants with long (L) DRD4 alleles presented significantly low scores (L 2.61±0.073; S 2.84+0.79; Newman-Keuls P=0.03) in comparison with the high scores of infants with short (S) alleles (L 2.4±0.059; S 2.25±0.57). These differences were not detected at 5 months of life (late assessment), denoting a strong environmental effect at this age on the genetic background. These results confirm and extend the genetic influence of the DRD4 gene in human temperament at birth. Electronic Publication     

Association of dopamine D4 receptor (<Emphasis Type="Italic">DRD4</Emphasis>) exon III repeat polymorphism with temperament in 3-year-old infants

The long forms of the dopamine D4 receptor (DRD4) exon III repeat polymorphism (L-DRD4) have been linked in some studies to the adult personality trait of novelty seeking (NS), as well as to infant personality traits related to interest and activity. The current investigation extends the results of our previous longitudinal study on 1- to 5-month-old neonates assessed by the Early and Revised Infancy Temperament Questionnaire (EITQ/RITQ), in which we found a significant correlation between the DRD4 polymorphism and the adaptability trait at 1 month of age. In this study, we examined the relationship between children′s behavior at 3 years of age, measured with the Toddler Temperament Scale (TTS), and DRD4 exon III repeat polymorphism. We found a significant association between the behavioral dimension of intensity of reaction and DRD4 genotypes. Current data failed to confirm the association with the adaptability trait. None of the extraversion and/or exploratory behavior measures was related to the L-DRD4 allele, as expected. In contrast, children with 4/7 genotypes showed worse response to new stimuli compared with 4/4 genotypes. This study corroborates only in part previous results on the link between the DRD4 gene and human temperament.