The clinical overlap between monogenic Familial Hemiplegic Migraine (FHM) and common migraine subtypes, and the fact that all three FHM genes are involved in the transport of ions, suggest that ion transport genes may underlie susceptibility to common forms of migraine. To test this leading hypothesis, we examined common variation in 155 ion transport genes using 5257 single nucleotide polymorphisms (SNPs) in a Finnish sample of 841 unrelated migraine with aura cases and 884 unrelated non-migraine controls. The top signals were then tested for replication in four independent migraine case-control samples from the Netherlands, Germany and Australia, totalling 2835 unrelated migraine cases and 2740 unrelated controls. SNPs within 12 genes (KCNB2, KCNQ3, CLIC5, ATP2C2, CACNA1E, CACNB2, KCNE2, KCNK12, KCNK2, KCNS3, SCN5A and SCN9A) with promising nominal association (0.00041 < P < 0.005) in the Finnish sample were selected for replication. Although no variant remained significant after adjusting for multiple testing nor produced consistent evidence for association across all cohorts, a significant epistatic interaction between KCNB2 SNP rs1431656 (chromosome 8q13.3) and CACNB2 SNP rs7076100 (chromosome 10p12.33) (pointwise P = 0.00002; global P = 0.02) was observed in the Finnish case-control sample. We conclude that common variants of moderate effect size in ion transport genes do not play a major role in susceptibility to common migraine within these European populations, although there is some evidence for epistatic interaction between potassium and calcium channel genes, KCNB2 and CACNB2. Multiple rare variants or trans-regulatory elements of these genes are not ruled out.

Catechol‐O‐methyltransferase (COMT) catalyzes the breakdown of catechol neurotransmitters, including dopamine, which plays a prominent role in drug reward. A common single nucleotide polymorphism (SNP), G472A, codes for a Val158Met substitution and results in a fourfold down regulation of enzyme activity. We sequenced exon IV of COMT gene in search for novel polymorphisms and then genotyped four out of five identified by direct sequencing, using TaqMan assay on 266 opioid‐dependent and 173 control subjects. Genotype frequencies of the G472A SNP varied significantly (P = 0.029) among the three main ethnic/cultural groups (Caucasians, Hispanics, and African Americans). Using a genotype test, we found a trend to point‐wise association (P = 0.053) of the G472A SNP in Hispanic subjects with opiate addiction. Further analysis of G472A genotypes in Hispanic subjects with data stratified by gender identified a point‐wise significant (P = 0.049) association of G/A and A/A genotypes with opiate addiction in women, but not men. These point‐wise significant results are not significant experiment‐wise (at P < 0.05) after correction for multiple testing. No significant association was found with haplotypes of the three most common SNPs. Linkage disequilibrium patterns were similar for the three ethnic/cultural groups. © 2008 Wiley‐Liss, Inc.

There is strong evidence for a genetic contribution to individual differences in vulnerability to drug addictions. Studies have shown that the 68-base pair repeat polymorphism in the promoter region of the human prodynorphin gene contains a putative AP-1 binding site, and that three or four repeat copies result in greater transcriptional activation. Here, we report on a separate cohort of 302 subjects ascertained and characterized extensively by Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition and Addiction Severity Index criteria as: (1) a control group of 127 subjects with no history of alcohol or drug abuse or dependence; (3) a case group of 82 with cocaine dependence only; and (3) a case group of 93 with cocaine and alcohol codependence. The promoter region of the prodynorphin gene containing the repeat was amplified from genomic DNA by polymerase chain reaction and analyzed via gel electrophoresis. Statistical tests were performed with data stratified by the three major ethnic groups studied: African American, Caucasian and Hispanic. For analyses, genotypes were grouped into short (1,1; 1,2; 2,2), short/long (1,3; 2,3; 1,4; 2,4) and long (3,3; 3,4; 4,4) repeats. Deviation from Hardy-Weinberg Equilibrium in the African American control group necessitated testing for association using grouped genotypes rather than grouped alleles. In controls, a significant difference was found in grouped genotype distribution among ethnicities. We found a point-wise, but not experiment-wise across-ethnicities, significant difference in grouped genotype frequency between the cocaine/alcohol-codependent group and the controls in African Americans, with genotypes containing longer alleles found at higher frequency in the codependent group.

There is strong evidence for a genetic contribution to individual differences in vulnerability to drug addictions. Studies have shown that the 68-base pair repeat polymorphism in the promoter region of the human prodynorphin gene contains a putative AP-1 binding site, and that three or four repeat copies result in greater transcriptional activation. Here, we report on a separate cohort of 302 subjects ascertained and characterized extensively by Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition and Addiction Severity Index criteria as: (1) a control group of 127 subjects with no history of alcohol or drug abuse or dependence; (3) a case group of 82 with cocaine dependence only; and (3) a case group of 93 with cocaine and alcohol codependence. The promoter region of the prodynorphin gene containing the repeat was amplified from genomic DNA by polymerase chain reaction and analyzed via gel electrophoresis. Statistical tests were performed with data stratified by the three major ethnic groups studied: African American, Caucasian and Hispanic. For analyses, genotypes were grouped into short (1,1; 1,2; 2,2), short/long (1,3; 2,3; 1,4; 2,4) and long (3,3; 3,4; 4,4) repeats. Deviation from Hardy–Weinberg Equilibrium in the African American control group necessitated testing for association using grouped genotypes rather than grouped alleles. In controls, a significant difference was found in grouped genotype distribution among ethnicities. We found a point-wise, but not experiment-wise across-ethnicities, significant difference in grouped genotype frequency between the cocaine/alcohol-codependent group and the controls in African Americans, with genotypes containing longer alleles found at higher frequency in the codependent group.

The most common single nucleotide polymorphism in the coding region of the human mu opioid receptor gene is the A118G variant, an adenine to guanine transition at nucleotide position 118 of the coding sequence of the gene. This polymorphism codes for an asparagine to aspartic acid substitution at amino acid 40 in the amino‐terminus, thereby removing a potential extracellular glycosylation site. Using in vitro cellular expression assays, this variant has been reported to change binding of the endogenous agonist beta‐endorphin and signaling of the receptor following binding of beta‐endorphin. Three clinical studies report that A118G genotype affects opioid antagonist‐mediated increases in cortisol levels. These studies demonstrate a functional role of this variant in responses to endogenous and exogenous opioids. To further characterize function, we expressed the prototype and variant receptors in two types of cells (human 293 embryonic kidney cells and Syrian hamster adenovirus‐12‐induced tumor cells). Stable expression of variant and prototype receptors was characterized by differences in levels of cell surface binding capacity ( B max ), forskolin‐induced cAMP accumulation, as well as agonist‐induced accumulation of cAMP (EC 50 ) for several agonists, but not for beta‐endorphin. In contrast, transiently expressed variant receptors showed only a minor difference in cell surface binding capacity compared to the prototype, and no differences in cAMP EC 50 values.

Discovery and characterization of the functional A118G mu-(mu)-opioid receptor variant led to hypotheses, now in part proven, about its role in alterations of endogenous human physiology and in responses to opioid antagonist administration. Differences in cellular expression levels, ligand binding, and signal transduction for variant receptors have been documented in vitro. Human genetic studies also indicate that individuals carrying one or two copies of the 118G allele may have increased risk for opiate and alcohol addictions and that this polymorphism may also explain some of the variability in success of opioid antagonist treatment for alcoholism. Future research will further define the role of the A118G variant in addictive diseases and their treatment, in pain perception and opioid analgesia, and for a myriad of other responses mediated by the mu-opioid receptor.

Throughout the long history of opiold drug use by humans, it has been known that opioids are powerful analgesics, but they can cause addiction. It has also been observed, and is now substantiated by multiple reports and studies, that during opioid treatment of severe and short-term pain, addiction arises only rarely. However, when opioids are extended to patients with chronic pain, and therapeutic opioid use is not confined to patients with severe and short-lived pain, compulsive opiold seeking and addiction arising directly from opioid treatment of pain become more visible. Although the epidemiological evidence base currently available is rudimentary, it appears that problematic opioid use arises in some fraction of opioid-treated chronic pain patients, and that problematic behaviors and addiction are problems that need to be addressed. Since the potentially devastating effects of addiction can substantially offset the benefits of opioid pain relief, it seems timely to reexamine addiction mechanisms and their relevance to the practice of long-term opioid treatment for pain. This article reviews the neurobiological and genetic basis of addiction, its terminology and diagnosis, the evidence on addiction rates during opioid treatment of chronic pain and the implications of biological mechanisms in formulating rational opioid treatment regimes.

The Publisher regrets that in the published version of the above article, the Abstract was inadvertently omitted. Please find the Abstract below. Abstract Throughout the long history of opioid drug use by humans, it has been known that opioids are powerful analgesics, but they can cause addiction. It has also been observed, and is now substantiated by multiple reports and studies, that during opioid treatment of severe and short-term pain, addiction arises only rarely. However, when opioids are extended to patients with chronic pain, and therapeutic opioid use is not confined to patients with severe and short-lived pain, compulsive opioid seeking and addiction arising directly from opioid treatment of pain become more visible. Although the epidemiological evidence base currently available is rudimentary, it appears that problematic opioid use arises in some fraction of opioid-treated chronic pain patients, and that problematic behaviors and addiction are problems that need to be addressed. Since the potentially devastating effects of addiction can substantially offset the benefits of opioid pain relief, it seems timely to reexamine addiction mechanisms and their relevance to the practice of long-term opioid treatment for pain. This article reviews the neurobiological and genetic basis of addiction, its terminology and diagnosis, the evidence on addiction rates during opioid treatment of chronic pain and the implications of biological mechanisms in formulating rational opioid treatment regimes.

Objectives 5-hydroxytryptamine (serotonin)-1B receptors (HTR1B) may play an important role in psychiatric disorders and drug and alcohol dependence. In this study we report on genotype, molecular haplotype and statistically estimated haplotype analyses of previously identified polymorphisms in positions - 261T > G, -161A > T, 129C > T, 861G > C and 1180A > G of the HTR1B gene in ethnically diverse populations (African-Americans, Caucasians, Hispanics and Asians) including 235 former heroin addicts and 161 control subjects from New York City, The objectives were to test for an association of molecular and statistically estimated haplotypes and genotypes in HTR1B gene with heroin addiction and to compare results provided by molecular and statistically estimated haplotyping methods.Methods Genotype analysis was performed using a standard TaqMan protocol. Molecular haplotype analysis of the subset of polymorphisms consisting of -261T > G, -161A > T and 129C > T was performed using a protocol specially designed by our group, using fluorescent PCR. This is based on use of allele-specific primers complementary to flanking polymorphisms and a fluorescently labeled sequence-specific TaqMan probe set complementary to an internal polymorphism of the haplotype region. Every individual's statistically inferred haplotype pair agreed with the individual's haplotype pair determined by molecular haplotyping.Results and conclusion A point-wise significant association of haplotype pairs containing allele G at position 1180 with protective effect from heroin addiction in Caucasians was found. A point-wise nominally significant association of allele 1180G with a protective effect from heroin addiction was found in Caucasians. Statistically significant differences across four ethnic groups in control subjects for allelic frequencies of -261T > G and -161A > T were found.

The mu opioid receptor is centrally involved in the development of the addictive diseases. It also modulates the stress responsive hypothalamic–pituitary–adrenal axis. Receptors encoded by the variant 118G polymorphism in exon 1 of the mu opioid receptor gene have a threefold increase in beta-endorphin binding and beta-endorphin is three times more potent in receptor-mediated activation of G protein-coupled inwardly rectifying potassium channels. Humans with this variant have increased stress response following opioid antagonism. Here, we study basal levels of adrenocorticotropic hormone and cortisol in subjects with this variant. In all, 59 healthy adults were genotyped and had morning levels of adrenocorticotropic hormone and cortisol measured following intravenous administration of saline placebo. Subjects with a 118G allele had significantly greater levels of cortisol than subjects with the prototype gene. Groups did not differ in levels of adrenocorticotropic hormone. A planned comparison revealed significantly greater cortisol in females with at least one copy of the 118G allele compared to females with the prototype gene. There was no significant effect of gender alone, nor was there a significant interaction between gender and genotype, on ACTH or cortisol. Subjects with at least one copy of the 118G allele have increased basal levels of cortisol, which may influence the susceptibility to and treatment of the stress responsive dyscrasia.

Genetic variation may partially underlie complex personality and physiological traits—such as impulsivity, risk taking and stress responsivity—as well as a substantial proportion of vulnerability to addictive diseases. Furthermore, personality and physiological traits themselves may differentially affect the various stages of addiction, defined chronologically as initiation of drug use, regular drug use, addiction/dependence and potentially relapse. Here we focus on recent approaches to the study of genetic variation in these personality and physiological traits, and their influence on and interaction with addictive diseases.

The μ-opioid receptor (MOR), through its effects on reward and stress-responsivity, modulates alcohol intake in both animal and human laboratory studies. We have previously demonstrated that the frequently occurring A118G single-nucleotide polymorphism (SNP) in exon 1 of the MORgene (OPRM1), which encodes an amino-acid substitution, is functional and receptors encoded by the variant 118G allele bind the endogenous opioid peptide β-endorphin with three-fold greater affinity than prototype receptors. Other groups subsequently reported that this variant alters stress-responsivity in normal volunteers and also increases the therapeutic response to naltrexone (a μ-preferring opioid antagonist) in the treatment of alcohol dependence. We compared frequencies of genotypes containing an 118G allele in 389 alcohol-dependent individuals and 170 population-based controls without drug or alcohol abuse or dependence. The A118G SNP was present in the Hardy–Weinberg equilibrium with an overall frequency of the 118G allele of 10.9%. There was a significant overall association between genotypes with an 118G allele and alcohol dependence (p=0.0074). The attributable risk for alcohol dependence in subjects with an 118G allele was 11.1%. There was no difference in A118G genotype between type 1 and type 2 alcoholics. In central Sweden, the functional variant 118G allele in exon 1 of OPRM1 is associated with an increased attributable risk for alcohol dependence.

Opiate and cocaine addictions are major social and medical problems that impose a significant burden on society. Despite the size and scope of these problems, there are few effective treatments for these addictions. Methadone maintenance is an effective and most widely used treatment for opiate addiction, allowing normalization of many physiological abnormalities caused by chronic use of short-acting opiates. There are no pharmacological treatments for cocaine addiction. Epidemiological, linkage, and association studies have demonstrated a significant contribution of genetic factors to the addictive diseases. This article reviews the molecular genetics and pharmacogenetics of opiate and cocaine addictions, focusing primarily on genes of the opioid and monoaminergic systems that have been associated with or have evidence for linkage to opiate or cocaine addiction. This evidence has been marshaled either through identification of variant alleles that lead to functional alterations of gene products, altered gene expression, or findings of linkage or association studies. Studies of polymorphisms in the μ opioid receptor gene, which encodes the receptor target of some endogenous opioids, heroin, morphine, and synthetic opioids, have contributed substantially to knowledge of genetic influences on opiate and cocaine addiction. Other genes of the endogenous opioid and monoaminergic systems, particularly genes encoding dopamine β-hydroxylase, and the dopamine, serotonin, and norepinephrine transporters have also been implicated. Variants in genes encoding proteins involved in metabolism or biotransformation of drugs of abuse and also of treatment agents are reviewed.

Preprodynorphin and preproenkephalin are protein precursors from which are derived two classes of opioid neurotransmitter peptides. Dynorphin A(1–17) is produced by proteolytic processing of prodynorphin, and processing of proenkephalin yields the enkephalin peptides. We report here on the isolation and sequencing of multiple clones for these two mRNAs from a cDNA library. Two cDNA clones of preprodynorphin contained the full-length sequence (2.35kb) with the primary structure predicted from the guinea pig gene sequence. In contrast, one clone encoded the full-length sequence but also an additional 192nt at the 5′ end. This sequence has high homology to the 5′ flanking region of the human preprodynorphin gene, and RNase protection assays demonstrated that in addition to a primary initiation site, transcription of this mRNA is initiated at several sites 160–190nt 5′ with respect to the primary site. This difference may alter translational efficiency or mRNA stability. The sequence of preproenkephalin cDNA clones confirmed the structure predicted from the gene sequence. One clone, however, contained sequences encoded by exons 2 and 3, and initiated within the first intron (intron A) of the gene. We used RNase protection mapping to assess the abundance in the brain and pituitary of preproenkephalin transcripts that initiate within intron A. These studies confirmed that the primary transcription start site is 28 nucleotides downstream from the TATAA site, and that intron A sequences are not present in significant amounts in these tissues.

The roots of the Laboratory of the Biology of the Addictive Diseases are in the development of methadone maintenance for the treatment of opiate addiction. Methadone maintenance therapy continues to be one of the major effective forms of addiction pharmacotherapy and underscores the importance of biological factors in the physiology and treatment of the addictive diseases. Recent work in the Laboratory has focused on the neurobiological, neurochemical, neuroendocrine and behavioral aspects of addictive diseases (principally cocaine and the opiate addictions), using an interdisciplinary approach. The models we have focused on range from in vitro molecular biology and neuroscience, to in vivo animal models, to experiments in normal human populations and patients with specific addictive diseases, and most recently to the human molecular genetics of different addictive diseases. Two long-term corollary hypotheses have guided the Laboratory's work: (1) That the endogenous opioid peptide/receptor systems play a central role in the addictive states and therefore in their treatment. (2) That atypical responsivity to stressors (e.g., in the hypothalamic-pituitary-adrenal axis) plays a role in vulnerability and relapse to specific addictive diseases. This atypical responsivity may be drug-induced, environmentally acquired, and/or due to genetic variation.

The kappa opioid receptor (KOR) plays a role in stress responsivity, opiate withdrawal and responses to cocaine. KOR activation by its endogenous ligand dynorphin A(1-17) decreases basal and drug-induced striatal levels of dopamine. The complete structure of the human KOR gene (hOPRK1) has not been previously determined. This study: (i) characterized the genomic structure of the hOPRK1 gene; (ii) identified single nucleotide polymorphisms (SNPs) in the hOPRK1 gene; and (iii) investigated possible associations of these variants with vulnerability to develop heroin addiction. Analysis of 5'-RACE cDNA clones revealed the presence of a novel exon 1 ranging in length from 167 to 251 nucleotides in the 5' 5'-untranslated region of the hOPRK1 mRNA. We found that the hOPRK1 gene has four major exons and three introns, similar to rodent OPRK1 genes. Direct sequencing of amplified DNA containing all four exons and intron 1 of the hOPRK1 gene were evaluated for polymorphisms in 291 subjects (145 former heroin addicts and 146 controls). Twelve SNPs were identified, nine novel variants and three previously reported SNPs. Using logistic regression with opioid dependence as the dependent variable, the 36G>T SNP exhibited a point-wise significant association (P = 0.016) with disease status. The number of haplotypes seen in the three ethnic groups were nine, six and five for African-Americans, Caucasians, and Hispanics, respectively, with corresponding significance levels for differences in haplotype frequencies between cases and controls of P = 0.0742, 0.1015 and 0.0041. Combining ethnicities by Fisher's method yields an empirical significance level of P = 0.0020.

Drug addiction is a complex disorder that has a large spectrum of causes. Vulnerability to addiction has been shown in twin studies to have a robust genetic component. This genetic basis for addiction has general and specific components for each drug abused. Although many genes have been implicated in drug addiction, only a handful have either been replicated to have an association or to have an identified functional mechanism related to specific effects of abused drugs. A few selected genetic variants that currently look promising for the study of alcohol, opiate, and cocaine addiction are discussed in this article.