Genetic Markers Related to Alcohol Use and Abuse

Chapter 4

Genetic Markers Related to Alcohol Use and Abuse Joshua A. Bornhorst1 and Gwendolyn McMillin1,2 1

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States, 2ARUP Laboratory and Department of Pathology,

University of Utah, Salt Lake City, UT, United States

INTRODUCTION Ethanol (alcohol) use has been an important aspect of many human societies since the beginning of recorded history. However, the excessive use of alcohol is associated with many negative consequences, contributing to increased medical costs, birth defects, lost productivity, and violent crime. In the United States approximately 150 billion dollars per year is spent in alcohol-related accidents and illness [1,2]. There is an evidence that the contribution of alcohol abuse to the global disease burden is larger than that of any other controlled or illicit substances, with the notable exclusion of tobacco [3,4]. It is believed that 3.2%
5.9% of global deaths and as much as 4%
5% of overall global disease can be attributed to alcohol consumption [3,5,6]. A great deal of work has gone into elucidating genetic markers for susceptibility of an individual for alcohol abuse. Although trait markers are generally in research and developmental stages, there are many biochemical markers of alcohol abuse that have been well established [7
9]. Increasingly, there is increasing interest in clinically useful genetic markers [10,11]. This chapter explores genetic markers that could be employed to identify susceptibility to alcohol dependence and to improve therapeutic efforts (see Table 4.1). Alcohol consumption is not always associated with the negative health and welfare impacts, and it should be noted that often consumption may not result in adverse consequences. However, consumption patterns can be linked to disease especially for chronic disease states [6,14]. The definition of “excessive use” of alcohol varies. Greater than 40 g a day of ethanol for men and 20 g a day for women is considered “hazardous” or “harmful” [15]. An acute intake of more than 5
7 drinks in males and 3
5 drinks (assuming 10 g of ethanol per drink) is also considered harmful [16]. Differences in alcohol abuse prevalence are observed for different ages, racial backgrounds, gender, and socioeconomic classes. Alcohol abuse prevalence is generally highest among young white males and unmarried individuals [17,18]. Per capita consumption steadily increased from 1970 to 2007 [19]. Furthermore, between 2006 and 2014 the number of alcohol-related emergency department visits increased 62% [20]. The risk of other adverse health effects is well known to increase with heavy and regular alcohol consumption [16]. Alcohol abuse is known to be directly associated with a number of different medical conditions (Table 4.1), including hepatic, cardiovascular, psychiatric, and neurological disorders [12,15,21,22]. It also should be noted that some studies have shown that drinkers of ,30 g of alcohol per day may have a lower mortality rate than those who abstain from drinking altogether [16,23,24]. The World Health Organization estimates that alcohol abuse affects 76.3 million people globally, and has identified reducing alcohol utilization as a priority area in international public health [14]. Alcohol abuse is not, by definition, alcohol dependency or alcoholism. Many environmental, clinical, and genetic factors contribute to the development of alcoholism (see Fig. 4.1). Clinical alcohol dependency is considered as a chronic disease for which diagnosis is defined by Diagnostic and Statistical Manual of Mental Disorders (DSM), Cloninger type I
II, and Feighner I
II criteria. Using the diagnostic criteria, as specified in DSM-IV, 19.3 million Americans required treatment for alcohol abuse or dependence in 2007 [25]. Briefly, the DSM-IV differentiates abuse from dependence on the frequency of events resulting in impairment or distress within specific domains of everyday

Critical Issues in Alcohol and Drugs of Abuse Testing. DOI: https://doi.org/10.1016/B978-0-12-815607-0.00004-6 Copyright © 2019 Elsevier Inc. All rights reserved.

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Critical Issues in Alcohol and Drugs of Abuse Testing

TABLE 4.1 Selected Health Risks and the Estimated Percent of Worldwide Disease Burden Related to Alcohol Consumption Disorder

Percent of Disease Burden Related to Alcohol (%)

Cirrhosis of the liver

32

Cancer of mouth and oropharynx

19

Esophageal cancer

29

Liver carcinoma

25

Breast cancer

7

Depression

2

Epilepsy Ischemic heart disease Hemorrhagic stroke

12 2 10

Source: Adapted from Room R, Babor T, Rehm J. Alcohol and public health. Lancet 2005;365 (9458):519
530; Rehm J, Room R, Graham K, Monteiro M, et al. The relationship of average volume of alcohol consumption and patterns of drinking to burden of disease: an overview. Addiction 2003;98 (9):1209
1228 and “WHO World Health Report 2002: reducing risks, promoting healthy life,” Geneva, World Health Organization.

FIGURE 4.1 Factors contributing to alcoholism.

life and the DSM-V presented additional recommendations [26]. Diagnosis of dependence requires three or more events within a 12-month period and evidence of tolerance, withdrawal, or relapse [25,27]. In the United States the prevalence of alcohol dependence is 5.4% in males and 2.3% in females. By way of comparison, only 0.6% of US adults exhibit drug dependence on at a given time, although their lifetime drug dependence prevalence is 10.3% [28,29], yet alcoholism frequently coexists with other addictions, including illicit substance abuse and nicotine dependence more often than be expected by chance [30]. It is estimated that as many as 18% of all adults

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exhibit alcohol abuse over the course of their lifetime and 12.5% will be dependent on alcohol at some point in their lives. However, alcohol dependency is under-diagnosed, and it is estimated that only about one-tenth of all individuals who exhibit alcohol dependence receive treatment [18,31]. Interestingly, considerable overlap has been recognized between the incidence of alcohol dependency and mental illness. For example, susceptibility to alcoholism may be linked to antisocial personality disorder, conduct disorder, and personality characteristics such as impulsivity, aggressiveness, thrill-seeking, and rebelliousness. Efforts continue to further elucidate the biological changes in specific brain regions associated with the dependence or addiction process [32]. While deemed the most reliable observational criteria for diagnosis of alcohol abuse or dependence, the DSM criteria are often highly subjective.

ROLE OF LABORATORY TESTING There is a clear need for laboratory testing to identify alcoholism, alcohol abuse, and susceptibility to alcoholism. In addition to biochemical changes or markers associated with alcohol use [33], genetic testing could potentially be used to identify individuals who are predisposed to developing alcoholism or who have clinically relevant abnormalities of ethanol metabolism. A number of the candidate genes and polymorphisms have been identified based on their involvement in alcohol metabolism or the neuronal response to alcohol. Evaluation of variants of these genes and the development of genetic testing may also be useful from a pharmacogenetic perspective, in guiding drug and dose selection for detoxification and abstinence therapy. Alcohol dependence is a complex psychiatric disorder affected by both genetic and environmental influences. Although alcoholism does not show a clear pattern of direct Mendelian inheritance, studies have shown that the genetic contribution to alcohol use disorder etiology is considerable [34
40]. Controversially, while some studies indicate that the prevalence of alcohol-associated disorders varies among ethnic minorities, other studies indicate no difference in total alcohol overall consumption [41
43]. A number of policy groups have recommended the implementation of alcohol screening and brief intervention strategies in routine health care settings in which it can reduce health care costs, costs associated with emergency room visits and result in a lower overall disease burden [41,43
46]. Thus it appears beneficial to have genetic testing to identify individuals who are predisposed to or otherwise at elevated risk for developing alcoholism. Candidate genes are identified based on their involvement in alcohol metabolism (see Figs. 4.1 and 4.2) such as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), as well as those involved in neuronal response to alcohol such as gammaaminobutyric acid (GABA) receptor subunits, and serotonin (5-hydroxytryptamine, 5-HT) transporters. These and other genetic tests may also be useful from a pharmacogenetic perspective, in guiding drug and dose selection for detoxification and abstinence therapy. This overview of the genetic markers that may be employed to identify susceptibility to alcohol dependence, to select therapeutic options to treat abuse, and to monitor therapeutic compliance.

FIGURE 4.2 Schematic of alcohol metabolism.

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Critical Issues in Alcohol and Drugs of Abuse Testing

GENETIC MARKERS OF ALCOHOL DEPENDENCE As indicated previously, alcohol predisposition and dependence is due to a complex blend and interaction of environmental
behavioral factors and genetic determinants (Fig. 4.1). Social, cultural, and biological factors all contribute to the differences in alcohol use and abuse. An important means for identification of individuals who are at risk for alcoholism may include genetic testing [47]. Based on analysis of large, well-characterized cohorts of twins (nearly 10,000 twin pairs), alcoholism is a moderately to highly heritable psychiatric disease, with heritability of more than 0.5, and about equally across genders [35,48]. Worldwide studies based on diverse technologies, linkage of human polymorphic markers, genetic association studies, and investigation of candidate genes, are accumulating to identify specific genes involved in the development of this disorder. Advances in molecular genetic profiling technology are changing from single-locus methodologies designed to detect single nucleotide polymorphisms (SNPs) to methods designed to detect many SNPs, multigene testing, and even genome-wide analysis. Therefore it is important to identify genes that predispose individuals from diverse populations to alcoholism, genes that alter treatment response, and genes that interact with other environmental factors. In this chapter an overview of the best characterized genetic markers is provided. Original research articles and more comprehensive reviews of specific details describing genetics of alcoholism should be consulted for further information [10,11,49
56]. Potential genetic indicators of alcohol abuse and susceptibility are listed in Table 4.2.

Alcohol and Aldehyde Dehydrogenases Metabolism of ethanol is a two-stage process of elimination: ADH oxidizes ethanol to acetaldehyde, a toxic intermediate, which is in turn converted to acetate, primarily by the mitochondrial form of ALDH2. A simplified schematic of this pathway is shown in Fig. 4.2. Acetaldehyde is a toxic metabolite which may be involved in the addiction process [57]. ADH exists as a polygene family on chromosome 4, divided into class I and class II genes. Approximately 70% of alcohol metabolism is mediated by isozymes expressed in the liver by class I genes: ADH1A, ADH1B (previously known as ADH2), and ADH1C. Clinically significant polymorphisms with altered affinity for ethanol and altered metabolic activity are described, the best studied of which include the ADH1B*2 (His47Arg) and the ADH1B*3 (Arg369Cys). These alleles are associated with low affinity for alcohol, but high activity ( . 30-fold higher than normal) and rapid conversion of ethanol to acetaldehyde. The ADH1B*2 is common in Asian populations (allele frequency of approximately 0.9) and is associated with facial flushing due to the accumulation of acetaldehyde. This allele is also associated with resistance to alcoholism; the allele frequency was found to be 0.73 in nonalcoholics and 0.48 in TABLE 4.2 Potential Genetic Indicators of Alcohol Abuse and Susceptibility Observational Markers DSM-IV and V diagnostic criteria Observation of the use of other substances Some General Genetic Markers of Alcohol Abuse Susceptibility Alcohol and aldehyde dehydrogenases Neurotransmitter systems Dopamine (DA) associated markers GABA ACh Glutamate Serotonin NPY ACN9 Opioids Potential Methods for Identification of Markers and evaluation of Alcohol Abuse Susceptibility Transcriptomics Genomics Metabolomics Proteomics Epigenomics Next generation whole-genome sequencing

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alcoholics. The ADH1B*3 allele is most common in Native American and African populations [58
63]. Several of the genes that encode ADH and ALDH enzymes exhibit functional polymorphisms that result in interindividual variation in ethanol metabolism capacity. Genome-wide association studies have linked the number of variants to alcohol phenotypes including relative risk of alcoholism [64
67]. At least 19 putative genes and several pseudogenes are included in the ALDH gene superfamily, yet just ALDH1 (chromosome 9, cytosolic) and ALDH2 (chromosome 12, mitochondrial) are associated specifically with acetaldehyde oxidation. The ALDH2*2 (Glu487Lys), common to several ethnic populations, leads to reduced activity and like the ADH1B*2, it is associated with accumulation of acetaldehyde following alcohol intake. Promoter variants in ALDH1 (ALDH1A1*2, a 17 bp deletion, and ALDH1A1*3, a 3 bp insertion) are associated with reduced expression of ALDH. All of these ALDH alleles are associated with resistance to alcoholism. People who inherit the combination of a rapid metabolizing ADH allele combined with the impaired metabolizing ALDH alleles had particularly reduced risk for alcoholism. Also of interest is the drug disulfiram which is used in prevention of alcoholism relapse. This drug acts to mimic this phenotype through inhibition of ALDH activity [68,69]. Kuo et al. [59] tested polymorphisms in seven ADH genes, along with the ALDH1A1 and ALDH2 genes, for association with alcohol dependence. In this study, numerous SNPs in the ADH gene were associated with alcohol dependence. Another large case-control study found associations between AD and ADH5 genotypes; also, diplotypes of ADH1A, ADH1B, ADH7, and ALDH2 were linked to AD in European- or African-Americans [59]. The ALDH2*2 (Glu487Lys) polymorphism located in the ADLH2 variant enzyme is prevalent in many Asian ethnic groups and produces acetaldehyde with an associated alcohol-flushing response reaction similar to that observed in the ADH1B*2 allele (also see http://www.aldh.org) [70
72]. The clinical significance of these and additional ALDH genes relative to alcohol use, intolerance, and abuse remains to be defined.

Neurotransmitter Systems The role of neurotransmitters in the brain is to either stimulate or inhibit the flow of impulse between neurons. Many neurotransmitter systems are involved in the positive, reinforcing, and aversive effects of alcohol. Here the potential involvement of genes involved in dopamine (DA), GABA, cholinergic, glutamate, serotonin, neuropeptide Y (NPY), ACN9, and opioid-mediated neurotransmission is described. Most emphasis is placed on the dopaminergic system, as it has been most extensively studied relative to alcoholism till date. Note that for each system, genes associated with the production, transport, storage, and destruction of neurotransmitters may be relevant, in addition to the genes coding for receptors and signal transduction mechanisms associated with actual neurotransmission. The cumulative and/or compensatory effect(s) of individual genetic variants is not well understood, but here association and potential consequences of affected genes are briefly discussed for selected aspects of each major neurotransmitter system [73].

Dopamine The DA system is one of the major contributors to the development of anxiety and depression. This neurotransmitter system is widely associated with rewarding, euphoric response to many drugs of abuse, including alcohol, and therefore may contribute to alcoholism predisposition and vulnerability. DA receptors fall within a class of metabotropic G protein
coupled receptors. There are five subtypes of DA receptors: D1, D2, D3, D4, and D5. The D2 regulates DA synthesis and releases in the presynaptic region, and has been the most studied in relation to alcohol-related phenotypes. This association is based on the thought that the rewarding effects of alcohol are mediated through the mesolimbic DA system [74], called also dopaminergic reward system [75]. In particular, the TaqI-A1 polymorphism in the DRD2 gene has been the subject of numerous association studies to explore the relationship between the A1 allele of DRD2 with alcohol consumption and dependence, with somewhat conflicting results. Some of the studies report a significantly higher frequency of the A1 allele in alcoholic compared with nonalcoholic populations, suggesting an increased susceptibility to alcohol dependence with this allele [76
79]. A large meta-analysis (44 studies with 5273 cases and 3995 controls) considering genotyping data of the Taq1 polymorphism showed a small but significant association of alcohol dependency [80] in individuals with homozygote or heterozygote for the A1 allele. Other studies have been less supportive of this association [81
83] leaving the role of this polymorphism in susceptibility for alcohol dependence inconclusive. However, these inconsistent results may be explained by the recent discovery that the TaqI-A1 polymorphism is in fact located 10 kb downstream from the DRD2 gene and causes an amino acid substitution in one of the ankyrin repeats in kinase domain 1 of the ANKK1 gene [84]. This data suggests that the TaqI-A1 polymorphism, in combination with polymorphisms of the ANKK1 and/or DRD2 could be

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Critical Issues in Alcohol and Drugs of Abuse Testing

epistatically associated with psychopathic traits in alcohol-dependent patients [85,86]. Further study will be required to understand this relationship. The dopamine D4 receptor (DRD4) is a G protein
coupled receptor with a D2-like activity, in addition to an inhibitory activity of the adenyl cyclase [87]. A 48-base (DRD4 E8 48-bp) of variable number of tandem repeats (VNTR) on exon 3, ranging from 2 to 11 repeats [88], is the most studied polymorphism; 2
5 repeats define the short, and 6
10 repeats define the long allele [89]. The 7-repeat variant of these polymorphisms reduces the ability of the DA to inhibit the cyclic AMP. The “long allele” has been associated by some studies with a “novelty seeking” [90] trait that is often present in the alcoholic phenotype. The dopamine transporter (SLC6A3) is a membrane-spanning protein that binds to DA terminating the DA signal by removing the neurotransmitter from the synapses into a neuron. A VNTR polymorphism in the 30 untranslated region of the DA transporter gene (DAT1) has been shown to affect the expression of the transporter [91]. Several case-control studies and family-based association studies have reported conflicting associations with the DAT1 VNTR polymorphism and alcohol-related phenotypes; however, most of these studies had very small sample sizes [92
98]. Elevated frequency of the 7-repeat and a decreased frequency of the 9-allele were found in Japanese alcoholics compared to control subjects [93]. Catechol-O-methyltransferase (COMT), dopamine-β-hydroxylase (DβH) and monoamide oxidase are the major enzymes catalyzing metabolism of DA, norepinephrine, and other catecholamines. Genetic polymorphisms in these genes have been described and used as markers for these loci sites. The role of the corresponding genes in alcohol use and dependence is still controversial [99
103]. The COMT gene is found on chromosome 21q11 and contains the functional polymorphism Val158Met in soluble COMT (S-COMT) or Val108Met in membrane-bound COMT (MB-COMT) [104
106]. The Val158/108 enzyme has more activity than the Met158/108 form and is linked with decreased amounts of DA [35]. The Met158/108 allele is associated with higher consumption of alcohol in men [107] and higher anxiety levels in women [108]. In the COMBINE Alcoholism Treatment Study, relationships between candidate loci and drug metabolism or alcohol addiction were explored [109,110]. In the COMT gene, the Val158Met polymorphism was associated with brain endogenous opioid function, response to stress and anxiety, and differences in cognitive and emotional processes.

Dopamine Metabolizing Enzymes DβH catalyzes the conversion of DA to norepinephrine. The DβH *444 G . A polymorphism, was associated with alcoholism [111]. Two studies did not establish association between a DβH-1021 polymorphism and alcoholism [112,113].

GABA GABA is the major inhibitory neurotransmitter in the central nervous system. GABAB receptors are a family of G protein
coupled receptors that stimulate the opening of potassium channels. The GABAergic system is thought responsible for producing sedation, anxiolytic, and muscular relaxant effects of ethanol as well as for exhibiting signs of withdrawal. The effects of GABA are mediated through two primary receptor types: GABAA and GABAB. The GABAA receptor is a ligand-gated ion channel (modulates chloride conductance into neurons), and is composed of six different subunits, designed as α, β, γ, δ, ρ, and ε; GABAB receptors are coupled to G proteins. Precise composition of individual receptors affects response to GABA-agonists such as ethanol. Most of the GABAA genes are organized into clusters located on chromosomes 4, 5, and 15. Linkage and association studies consistently support the association between alcohol dependence and GABAA α2, GABA β1, and GABA β3 genes [53,114,115]. Fewer studies have identified a role of GABAB receptors and alcohol dependence [116,117]. GABAC receptors, which differ in complexity of structure, abundance, distribution, and function from GABAA and GABAB receptors, can be found in retinal, hippocampus, spinal cord, and pituitary tissues [118,119]. The GABA system interacts with the DA system to reinforce the effects of alcohol. Pharmacological treatments that target the inhibitory and excitatory modulators of the CMDS may prevent relapse or reduce heavy drinking have been proposed [118,120]. Clinical trials on the anticonvulsant drug topiramate, which facilitating GABA function through interaction with the GABAA receptor, have significantly reduced drinking in alcohol-dependent subjects [118,120].

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Acetylcholine Acetylcholine (ACh) is a neurotransmitter found in the brain and autonomic nervous system; it interacts with muscarinic and nicotinic receptors. A region on chromosome 7, including 11 SNPs, within and flanking the gene encoding for the muscarine ACh receptor subtype M2 (CHRM2) was significantly associated by linkage with alcohol dependence [121
123]. Several genetic studies have implied that these genes may contribute to alcohol and nicotine coaddition [124
127]. Chatterjee et al. suggest that FDA-approved ACh receptor ligands, such as varenicline and mecamylamine, may be useful in the treatment of alcohol abuse as well as smoking cessation [128].

Glutamate Glutamate is the primary excitatory neurotransmitter in the central nervous system and ethanol potently and selectively inhibits glutamate receptors. One receptor, a calcium-conducting ligand-gated glutamate receptor known as the Nmethyl-D-aspartate (NMDA) receptor, is thought to be particularly involved in development of alcoholism. The inhibitory effect of ethanol is overcome with prolonged ethanol exposure, by way of compensatory “upregulation” of NMDA receptor expression. These changes are believed to underlie the development of ethanol tolerance and dependence as well as acute and delayed signs of withdrawal, particularly agitation and seizures. Allelic variants of the NMDAR1 and the NMDAR2B receptor genes were associated with alcoholism and related traits [129], but not consistently in other studies [130]. Withdrawal from chronic alcohol usage lead to increased activity in the NMDA receptor and increased influx of calcium may be attributed to neurotoxicity and neuronal cell death. Topiramate, which can decrease the release of DA, has shown effectiveness in the management of alcohol dependence [131].

Serotonin Genes involved in the regulation of the serotonin (5-HT) system provide plausible candidate genes for involvement in alcohol consumption and abuse and dependence [132]. The serotonin transporter is responsible for serotonin reuptake and is a key regulator of serotonin availability in the synaptic cleft. A variant in the gene that codes for the transporter (5HTT, on chromosome 17), the 5-HTTLPR polymorphism, is associated with alcohol consumption. The serotonin transporter (5-HTT) is postulated as a marker for alcohol dependency [133]. Chronic alcohol intake may exert neurotoxic effects upon neurotransmitter systems such as the serotonergic system [134]. The short
long alleles of 5HTTLPR result in either 14 (short, S) or 16 (long, L) copies of a 20
23 base pair repeated sequence and affect expression of the gene. The S variant is associated with reduced expression of the transporter protein and was correlated with an increased risk of alcohol dependency [69,70] and may also affect the risk of relapse in abstinent patients, but these associations are somewhat controversial [135,136].

Neuropeptide Y Several lines of evidence in both human and animal studies suggest that variation in NPY or its receptor genes (NPY1R, NPY2R, and NPY5R) is associated with alcohol dependence as well as alcohol withdrawal symptoms. Reduced activity of the NPY system may be associated with increased alcohol intake [137]. Sequence variations in NPY receptor genes are associated with alcohol dependence, particularly a severe subtype of alcohol dependence characterized by withdrawal symptoms, comorbid alcohol, and cocaine dependence [138].

ACN9 The ACN9 protein is a mitochondrial protein that is involved in gluconeogenesis and metabolism of ethanol or acetate into carbohydrate [77]. Chromosome 7 that encodes ACN9 protein has shown consistent evidence of linkage with a variety of phenotypes related to alcohol dependence in the collaborative study on the genetics of alcoholism (COGA) project. Four of the eight most significant SNPs were located in or very near the ACN9 gene which encodes for the ACN9 protein, a novel mitochondrial protein involved in gluconeogenesis and the assimilation of ethanol or acetate into carbohydrate [77].

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Critical Issues in Alcohol and Drugs of Abuse Testing

Opioids The opioid system has been implicated in association with alcoholism, particularly because opioid antagonists are used successfully to manage alcohol dependence. While no significant association has been made between the mu and delta opioid receptor genes and alcohol dependence, the kappa receptor gene (OPRK1) and a gene encoding for an opioid ligand, prodynorphin (PDYN), were strongly associated with a risk of alcohol dependence [54].

PHARMACOGENETICS OF ALCOHOL DEPENDENCE TREATMENT Therapeutic approaches to alcohol abuse disorders are based in large part on acute detoxification, followed by longterm measures to prevent relapse. Indeed, addiction, risk of relapse, and remission are commonly compared in parallel to diagnosis and management of chronic disease. Several classes of drugs have been utilized for alleviation of alcohol withdrawal symptoms and dependency treatment [139]. The genetic components associated with alcohol abuse disorders, while not always defined specifically, remain strong, based on familial and twin studies as well as data described in the previous section. It is therefore logical that both the genetic factors responsible for disease and the genetic factors that may predispose therapeutic success or failure should be considered in selection of treatment modalities and management of rehabilitation. Complimentary to traditional genetics, pharmacogenetics associates genetics with interindividual variability in the two major processes responsible for drug action: pharmacokinetics and pharmacodynamics. Thus, from the perspective of pharmacogenetics, variants in genes associated with either metabolism (pharmacokinetics) or response (pharmacodynamics) of any drug used to treat alcohol dependency could impact efficacy of the drug and optimal dose requirements. Variants in genes associated with pharmacokinetics such as the genes that code for cytochrome P450 enzymes, will impact the time required for drug clearance and time to achieve steady-state concentrations. As a consequence, drug dose and interval of dosing may influence efficacy. Genes associated with pharmacodynamics correlate with the sensitivity or resistance to a particular drug action and hence, drug and drug dose requirements. Pharmacogenetic markers may therefore become important in directing addiction treatment by personalizing both drug and dose selection prior to drug administration [140
143]. There are several classes of drugs applied to the treatment of alcohol abuse and dependence. Here, potential pharmacogenetic markers relative to alcohol detoxification (diazepam) and dependency treatment (disulfiram, naltrexone, acamprosate, ondansetron, topiramate, finasteride, dexmedetomidine, and bromocriptine) are discussed. Mechanism of action of disulfiram is shown in Fig. 4.2. Examples of drugs used for alcohol dependence therapy are listed in Table 4.3.

Alcohol Detoxification Benzodiazepines are the most commonly used class of drugs to treat withdrawal symptoms associated with ethanol detoxification. While dosing may be titrated to clinical need, it may be important to consider pharmacogenetic testing that could avoid unintentional overdose and fail to respond. Mechanisms of metabolism for individual benzodiazepines should be investigated because some pathways are complex and active metabolites are common. In addition, the kinetics for elimination of each drug and active metabolite vary substantially. Using diazepam as an example, several active metabolites are formed, including nordiazepam, oxazepam, and temazepam with half-lives of approximately 20
40 h for diazepam and nordiazepam, and half-lives of approximately 4
12 h for oxazepam and temazepam. These metabolic reactions are catalyzed by cytochrome P450 (CYP) isozymes, particularly CYP2C19 and CYP3A4. These compounds are further metabolized by formation of glucuronide and other conjugates. Substantial variation in CYP and glucuronyltransferase genes has been associated with altered kinetics and risk of toxicity. Although the safety margin for benzodiazepines is good for acute administration, clinical impact of genetic variation in benzodiazepine metabolism could inadvertently contribute to toxicity if the drugs are administered frequently or for a long period of time [143]. The mechanism of action of benzodiazepines is primarily explained by affinity for the central GABAA receptor. As such, it is possible that impaired GABAergic function would prevent or impair the clinical sensitivity to benzodiazepine therapy. In the case of benzodiazepine resistance, treatment through a non-GABAergic mechanism may be required. Indeed, dexmedetomidine, a central alpha2-receptor agonist, has also been used successfully to treat alcohol withdrawal [144]. Negative modulators of NMDA glutamate receptors (e.g., acamprosate) may be useful agents for the treating withdrawal signs and symptoms [145,146].

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TABLE 4.3 Pharmacogenetics of Treatment for Alcoholism Generic Name

Trade Name

Indication for Use

Candidate Gene Related to Pharmacokinetics

Candidate Gene Related to Pharmacodynamics

Diazepam

Valium

Prevent withdrawal

CYP2C19

GABRA

CYP3A4 Many CYPs

ADH

Disulfiram

Antabuse

Maintain abstinence

Naltrexone

Depade

Maintain abstinence

OPRM1

Acamprosate

Campral

Maintain abstinence

GABRA NMDA

Ondansetron

Zofran

Experimental

Topiramate

Topamax

Experimental

CYP3A4

HTR3A GABRA GLUR

Finasteride

Propecia

Experimental

CYP3A4

GABRA

Dexmedetomidine

Precedex

Experimental

CYP2A6

ADRA2A

Bromocriptine

Parlodel

Experimental

CYP3A4

DRD2

Alcohol Dependence Treatment Many drugs, acting through different or unknown mechanisms have been successfully used to treat alcohol dependence by minimizing cravings, tapering drinking behavior, and preventing recurrence of dependency on alcohol and other substances. Long-term treatment for months to years is often required, so it is important that safety and efficacy of the drugs and dose selected are considered. Pharmacogenetic testing may assist in this selection process, by predicting drug sensitivity or resistance, and optimizing dose and dosing intervals. Early intervention may benefit from administration of disulfiram, which inhibits ALDH and promotes accumulation of acetaldehyde, leading to an unpleasant reaction (Fig. 4.2). This negative reinforcement approach, while it does not itself prevent craving, may reduce drinking behavior. Genetic variations in the ADH and ALDH genes can affect response to the alcohol dependence treatment drug disulfiram. Perhaps not surprisingly, patient compliance with disulfiram administration is reported to be poor [147,148]. Pharmacogenetic testing for ADH or ALDH variants may be considered prior to administration of disulfiram, because persons with impaired ALDH may not respond to the drug [149]. Naltrexone is another well-accepted therapy for long-term prevention of alcoholism [150]. Naltrexone is an opioid receptor antagonist and has been shown most effective for persons who possess the OPRM1 variants, particularly the Asn40 allele [151]. Craving and anxiety are treated effectively with bromocriptine, a DA antagonist, when the TaqI-A1 allele was present with DRD2. Acamprosate, which has effects at both opioid and glutamate receptors have also been used successfully to treat alcohol dependence, as might be expected based on speculation of these neurotransmitter system’s involvements in the pathogenesis of alcoholism [152]. Finally, antiepileptic drugs, long known to inhibit excessive excitation in the central nervous system, have proven successful applied to dependency treatment, particularly in reducing impulsive behavior [151,153]. Examples of antiepileptic drugs used successfully to treat alcoholism include topiramate, gabapentin, and levetiracetam [154
156].

CONCLUSIONS Advances in molecular profiling technologies within the disciplines of genomics, transcriptomics, proteomics, and metabolomics will likely play an essential role in the development of prevention strategies and personalized treatments of alcoholism, as well as the identification of new therapeutic targets. In addition to available biochemical markers, an increasing number of gene variant markers have been identified. Continued evolution in array and sequencing

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Critical Issues in Alcohol and Drugs of Abuse Testing

technologies, along with the continued establishment of large data and sample repositories are important to achieve progress in a complex disorder like alcoholism. The identification of genes associated with alcohol dependence can be accomplished by several methods including candidate gene studies, whole-genome association, and linkage studies. In whole-genome studies, broad chromosomal regions are linked to a target phenotypic expression factor by means of a panel of polymorphisms [7,53]. One example of a family-based data set is the COGA that is targeted toward the identification of genes contributing to alcoholism [157,158]. A number of genome-wide association studies have been carried out which have supported many of the genotype
phenotype associations described earlier in this work [55]. Investigations will increasingly need to account for both genomic and epigenomic data [159]. Epigenetic studies often demonstrate substance-specific changes to gene expression that is linked to substance intake patterns [10]. These patterns are based on changes in chemical modifications of DNA and chromatin which alters expression of genetic material. Epigenetic changes can regulate gene expression without changing the DNA code itself and have been shown to be linked to the development of many diseases processes [160
162]. Alcohol intake is now well demonstrated to modify chromatin and appears to be involved in the maintenance of alcohol addiction [163]. This is an emerging and exciting frontier in our understanding and potential treatment of alcohol dependence. Alcohol abuse and dependency is a tremendous social problem, contributing to disease, crime, and inappropriate behavior. Chemical biomarkers such as mean corpuscular volume, gamma-glutamyltransferase, carbohydrate deficient transferring, ethyl glucuronide, fatty acid ethyl esters, and other chemical biomarkers may provide evidence of alcohol use, abuse, and compliance with abstinence. However, genetic testing may provide additional tools for management of alcohol abuse by detecting individuals at risk for alcoholism. It is well recognized that the risk of developing alcoholism is closely related to both environmental and genetic factors. In fact, it is estimated that 50%
60% of the factors associated with alcoholism are genetic in origin. There are currently 10 tests available for alcohol dependency according to the national genetic test registry [164]. Genetic testing to identify individuals at risk for alcohol abuse may prevent alcohol use disorders, allow for early intervention, or direct therapy. Therapy of alcoholism may include medications that curb craving and anxiety, and may be optimized through the use of pharmacogenetic testing. Indeed, testing genes related to the metabolism of ethanol, the pathogenesis of alcoholism, and the genes linked to successful therapy may be employed to identify susceptibility to alcohol use disorders [13].

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