Genetic markers of alcohol abuse

ELSEVIER

Clinica Chimica Acta 257 (1997) 199 250

Genetic markers of alcohol abuse Ralph A. Ferguson, David M. Goldberg* Department o)C Clinical Biochemistry, Banting Institute, University o[ Toronto, 100 Banting Street. Toronto, Ontario, Canada M5G IL5

Received 29 November 1995: revised 3 April 1996; accepted 4 April 1996

Abstract

In this paper, we review the current status of genetic markers for the development of alcohol abuse. Family, twin, half-sibling and adoption studies of alcoholic subjects suggest that the heritability of liability to alcoholism is at least 50%. These findings have fuelled intensive investigation in the fields of neurology, biochemistry, genetics and molecular biology aimed at the identification of markers for the risk of alcoholism. The most promising of these are discussed in detail. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) polymorphisms, specifically the ADH3*I, ADH2*2, and ALDH2*2 genotypes appear to confer a protective effect against alcoholism, most notably in Oriental subjects. Caucasian alcohol abusers and their first-degree relatives exhibit depressed platelet monoamine oxidase activity, the degree of which is greater in Type II than Type I alcoholics. Electrophysiological characteristics of alcoholics and those at risk for developing alcoholism have also been identified, including the reduced amplitude of the event-related brain potential and, after ethanal ingestion, characteristic EEG a-wave activity. Lower platelet adenylate cyclase activity is seen in alcoholics compared to controls, presumably as a result of over-expression of an inhibitory G-protein. Markers related to other signal transduction pathways of the central nervous system including the serotoninergic, muscarinic and dopaminergic systems are also discussed. In this group of markers, the putative association between the inheritance of the A1 allele of the D2 dopamine receptor and the susceptibility to alcoholism provides the most dramatic illustration of the challenges presently existing in this field of scientific investigation. Current limitations in the definition, diagnosis and classification of alcoholism, the confounding influences of race and gender on association studies, as well as the statistical approach of linkage studies are discussed as they relate to the endeavor to uncover valid genetic markers for the risk of alcoholism. Copyright © 1997 Elsevier Science B.V. * Corresponding author. Fax: + 1 416 9785650. 0009-8981/97/$17.00 Copyright © 1997 Elsevier Science B.V. All rights reserved PII S0009-8981 (96)06444-3

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Keywords: Alcoholism; Alcohol; Drug dependence; Genetics; Inheritance; Alcohol dehydrogenase; Aldehyde dehydrogenase; Monoamine oxidase; Dopamine receptor; Gamma aminobutyric acid; Adenylate cyclase; Serotonin; Event-related brain potential; Electroencephalography

1. Introduction

Much enthusiasm has been generated in recent years regarding the beneficial effects of moderate alcohol consumption on human health. These effects include, but are not limited to, the apparent cardioprotective effect of moderate alcohol consumption [1-4]. In contrast, the toxic effects of alcohol have been appreciated for a much longer period of time. The medical consequences of chronic alcohol abuse, both direct and indirect, may be observed in any of the major body systems [5]. Furthermore, alcohol addiction is known to be a causative factor of many harmful psychiatric and sociologic sequelae and it is well established as a major risk factor for suicide [6]. In the United States, 80% of murders, 80% of domestic violence and 50% of fatal automobile accidents involve a person who is intoxicated [7]. The destructive consequences of alcoholism at the individual, familial and societal levels are well appreciated. In light of this, scientific inquiry has endeavored to describe the etiology of this disease in order that a better understanding of alcoholism may provide useful therapeutic and/or preventative strategies. When one speaks of markers of alcoholism, the distinction between markers of state and markers of trait must be made. The former are usually the consequence of organic disease and/or toxicity associated with alcohol consumption. While some of these biochemical markers are useful in the detection of alcohol abuse prior to the onset of irreversible organ and tissue damage, their utility in this respect remains limited [8]. Markers of trait, on the other hand, are by definition risk markers for the development of alcoholism. Thus, these markers should be useful in identifying the individual at risk - - whether or not a drop of alcohol has ever passed his lips. In this review, we address the current status of the markers of risk for the development of alcoholism. Before we begin, however, we will briefly review some pertinent aspects related to this subject. These include the metabolism of alcohol and the definition(s) and diagnosis of alcoholism. I. 1. Pharmacokinetics, actions and metabolism of alcohol Since only a small percentage of ingested alcohol is absorbed through the stomach, the emptying time of the stomach dictates how long the alcohol is

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retained before absorption. Thus, food, drugs, and any other factors which retard gastric emptying will likewise retard the appearance of alcohol in the blood. Due to the phenomenon of acute tolerance, the CNS actions of alcohol are more pronounced during the absorptive phase than during the elimination phase. Thus, the faster and higher the levels of blood alcohol are reached, the more pronounced is the intoxication. The concentration of alcohol which is ingested is also a determinant of the kinetics of the absorptive phase. Absolute (100%) alcohol irritates and inflames the gastric and duodenal mucosa, thereby limiting its absorption. On the other hand, the rate of absorption is also limited when alcohol is present in dilute form ( ~ 20°/°). It is interesting to note that the 'ideal' concentration of alcohol (i.e. that which is associated with the fastest absorption) is ~ 40% [7] - - corresponding to the concentration of alcohol in most commercially distilled spirits. Ethanol is a small polar molecule which distributes widely throughout the body following its absorption. It is metabolized predominantly by liver alcohol dehydrogenase (ADH) to acetaldehyde - - a noxious molecule that will be discussed in greater detail in the section dedicated to ADH and aldehyde dehydrogenase (ALDH) isoforms. A L D H oxidizes acetaldehyde to acetic acid which may then be used in various metabolic pathways associated with fat, carbohydrate and protein metabolism. The elimination of ethanol from the blood generally approximates a zero-order process, although this rate is variable. For example, males have generally lower elimination rates than females (mean values of 0.15 and 0.18 g/l/h, respectively, [9]). At either high ( > 3 g/t) or low ( < 0.2 g/l) blood concentrations, the elimination becomes nearly first order and is accelerated at the higher concentration ( ~ 0.22 g/l/h, [10,11]). Furthermore, drinking behavior is associated with altered disposition of blood alcohol. Winek and Murphy [12] demonstrated that alcoholics have higher than normal elimination rates, averaging approximately 0.3 g/l/h. The acute effects of alcohol intoxication arise from its effects on the CNS. These are described by Porter and Moyer [13] as causing no apparent influence at low blood alcohol levels (0.1-0.5 g/l) to euphoria and decreased inhibitions ( < 1.2 g/l), decreased orientation and coordination (1.0-3.0 g/l) and progression to coma (3.5-5.0 g/l) or death from respiratory arrest at very high levels ( > 4.5 g/l). It was once felt that the CNS effects of ethanol arise primarily from its effects on neuronal membrane permeability and fluidity. Indeed, the concept that ethanol is a lipid solvent which alters the general physiology of neuronal membranes by altering the lipids is still found in contemporary publications [7]. But, as pointed out by Koob and Bloom [14], such a non-specific mechanism is incompatible with the neuropsychopharmacological profile of ethanol in light of its known effects on motor coordination, arousal,

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cognition, and its euphoric and anxiolytic effects. Instead, ethanol acts in selective areas of the brain by specific, albeit poorly understood, modification of neuronal discharge patterns and neurotransmitter metabolism [14].

1.2. Definitions of alcoholism Recent scientific inquiries into the biochemical and genetic bases of alcoholism have done much to illuminate a phenomenon which has been recognized since antiquity. Indeed, the dangers of excessive alcohol consumption are described dramatically in Biblical writings [15,16]. The recent advances in our understanding of alcoholism may be rightfully attributed to progress in such areas as laboratory technology, genetic epidemiology and various other methodological refinements which have brought alcoholism to its present position as a valid subject of medical and scientific investigation. In 1967, alcoholism was designated as a disease by the American Medical Association. Unfortunately, this declaration did not extinguish the philosophical debate over whether or not alcoholism is a disease, a personality disorder or simply a moral weakness [17-20]. Nor should it have done so. As Erickson [21] points out, declaring a disorder a disease does not make it so. However, we now have a large body of scientific evidence which firmly establishes the disease concept of alcoholism [21-23]. Furthermore, the original AMA designation of alcoholism as a disease has been supported by a detailed and scientifically validated report [24]. While it is true that the debate over whether or not alcoholism is a disease has been put to rest, the definition of 'alcoholism' remains a point of considerable contention. As pointed out below in the section on dopamine receptors, ambiguity over selection criteria for alcoholic subjects can confound studies of genetic markers of alcoholism. The DSM-III-R [25] criteria for substance-dependence disorders represents a popular diagnostic tool which is considered to be reliable and valid [26,27]. A largely behavioral concept of alcoholism is employed by the DSM-III-R to differentiate between 'alcohol abuse' and 'alcohol dependence'. This scheme was designed to allow a differential diagnosis of milder and recent-onset cases of alcohol abuse vs. alcohol dependence accompanied by physiological states of tolerance and dependence. However, criticism of this distinction as being artificial has arisen. For instance, Schuckit et al. [28] applied the DSM-III [29] criteria for alcohol abuse and dependence to 403 male primary alcoholic patients. The two diagnostic groups differed in the amount of alcohol consumed per day (being greater in the 'alcohol-dependents') but not in the frequency of alcohol consumption, drug-use profiles, psychiatric histories, family histories of psychiatric disorders or in the demographic descriptors. A comprehensive critical evaluation

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of the DSM-III-R is provided by Miller [7]. Many such recommendations have been adopted in the recently released DSM-IV (see Section 8). 1.3. Questionnaires as diagnostic tools Despite its limitations, the DSM has enjoyed prominence as the gold standard for the selection of alcoholic and control populations by those investigating the association between putative genetic markers and alcoholism. Many questionnaires are available to facilitate the diagnosis of alcoholism on the basis of self-reported behavior and attitudes. Some of these, such as the four CAGE questions [30] were developed to serve as screening tools for the physician in general clinical practice. Others are more elaborate, such as the twenty-five-question Michigan Alcoholism Screening Test (MAST) [31]. These share with the DSM the great disadvantages associated with denial by the suspected alcoholic under investigation. The 'masking' of alcohol questionnaires within larger ones concerning other lifestyle and health issues has been undertaken in order to minimize any embarrassment that the person under interview may experience in talking about alcohol-related questions. In hospital settings at least, this strategy does not appear to be useful [32]. In addition to denial, the utility of the questionnaire in the structured interview is further compromised by biases related to such factors as race [33,34], gender [34,35], and pregnancy [36]. In a recent review of 20 studies of the MAST, Storgaard et al. [37] report sensitivities and specificities ranging from 0.36-1.00 and 0.36-0.96, respectively. Reported positive and negative predictive values ranged from 0.24-0.96 and 0.78-1.00, respectively. The authors identified the estimated population prevalence of alcoholism and the diagnostic method against which the MAST was compared as being amongst the largest influences on the widely discrepant validity measures obtained. This is not surprising, as data from self-reported alcohol-related behavior and/or attitudes are a major component of these estimates. Compounding this unfortunate situation is the utilization of ill-defined 'in-house' or otherwise modified forms of established questionnaires. As Miller [7] points out, in light of the limitations of current diagnostic tools and the consequent limitations in our estimates of incidence and prevalence, the magnitude and severity of drug and alcohol abuse cannot be accurately assessed. 1.4. Sub-types gf alcoholism Clinical and epidemiological research over the past three decades has demonstrated that there is significant heterogeneity amongst those who are

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diagnosed as being alcoholics. For example, any two such persons may differ with respect to a wide variety of characteristics including genetic predisposition, personality characteristics, age at which alcohol abuse begins, and the severity of alcohol-related sequelae [38]. Methodological approaches which have been used in the typology of alcoholism have included clinical and statistical description, genetic epidemiology, studies of high risk groups and treatment matching. Thus, alcoholics are now described by a number of theoretically appropriate measures such as psychopathology, personality, drinking patterns, family history, and alcohol-related consequences [38,39]. Alcoholic typologies which incorporate multiple defining characteristics include Cloninger's neurobiological learning model [40], Morey and Skinner's hybrid model [41], Zucker's developmental model [42] and the vulnerability-severity classification of Babor et al. [43]. The array of terminology and definitions arising from these different typological approaches may strike many as confusing. On closer examination, however, these multidimensional typologies do describe somewhat homogeneous clusters of alcoholics. Two such classification systems will serve to emphasize this point. The first is the genetic-epidemiological approach of Cloninger and the second is the approach of Babor et al. which is based on descriptive clinical assessment and prospective prognostic evaluation. Cloninger [40] classified clinical sub-types of alcoholism by evaluating data from adoption and twin studies and proposed the neurobiological learning model of alcoholism which identifies two genetic sub-types. Type 1 (milieulimited) alcoholics have characteristics which include a later onset of alcohol-related problems, the development of psychological rather than physical dependence and guilt related to their alcohol use. Type II (male-limited) alcoholics, on the other hand, manifest alcohol problems at an earlier age, show spontaneous or compulsive alcohol-seeking behavior and are socially disruptive when indulging. Babor and colleagues [43] have applied empirical clustering techniques, prognostic evaluation and comprehensive descriptive assessment to alcoholism systematics. They have identified two groups, termed Type A and Type B. The former group is characterized by later onset, fewer childhood risk factors and less alcohol-related problems. Type B alcoholics, on the other hand, possess childhood risk factors, a family history of alcoholism, early onset of alcohol-related problems, greater severity of dependence, polydrug use, a longer treatment history, greater psychopathology and more life stress. Furthermore, Type A and Type B alcoholics also differ with respect to outcomes of alcohol treatment. The similarities between the classification systems above are also seen in the other aforementioned classification systems. Thus it is possible that the various typology theories now found in the literature are describing the

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same phenomenon. In other words, there appears to exist two basic 'types' of alcoholic. The first is characterized by later onset, slower course, fewer complications, less psychological impairment and a better prognosis. The second is characterized by genetic precursors, early onset, a more rapid course, more severe symptoms, greater psychological impairment and a poorer prognosis. A recent volume of the Annals of the New York Academy of Sciences is dedicated to the subject of alcoholic typology [39,44-48]. It is evident that 'alcoholism' cannot be described by a unidimensional measure. Thus, a single marker for the risk of alcoholism will, in itself, have a modest predictive value. The search for the basis of individual variability in addictive liability has to date been conducted by individual research groups which tend to 'specialize' in a specific putative marker or family of markers for the risk of alcoholism. As the approach to identifying such markers matures, so will the approach to evaluating their utility, not in isolation but as part of a constellation of markers.

2. Family studies of alcoholism It is widely believed that alcoholism arises from an interplay of environmental and genetic factors. Physiological, neurological, biochemical and personality studies of alcoholics and their children provide evidence that heritable factors do exist which predispose to the development of alcoholism [49-54]. The heritability of alcoholism is suggested by the fact that children of alcoholic parents are at increased risk for developing alcoholism [55-57]. Cotton [55] reviewed 39 family studies which were conducted over 40 years and involved 6251 alcoholic and 4083 non-alcoholic probands. Her analysis led her to estimate that one of three alcoholics has at least one alcoholic parent. In addition, her survey indicates that the incidence of alcoholism is significantly lower among relatives of non-alcoholics than among relatives of alcoholics. Historically, many family studies have been retrospective. Nevertheless, prospective studies do exist, and these support the role of family history in the etiology of alcoholism. One such study was undertaken with 1380 New Jersey subjects. Data gathered on the youths at 12, 15 and 18 years of age and twice thereafter at 3-year intervals have demonstrated that the number of self-reported alcohol and/or drug-related problems of family history positive subjects was about twice that of family history negative subjects [58]. Furthermore, a 30-year follow-up of 161 sons of alcoholic Danish fathers showed that these individuals are twice as likely as matched controls to develop alcohol or drug dependence [48,59]. The observation that alcoholism runs in families is, in itself, not proof positive that there exists a genetic component for this disease. It is possible

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that children learn behavior from alcoholic parents that increases their vulnerability to the disease. This phenomenon has been termed vertical cultural transmission [60]. Nevertheless, study designs may be tailored to distinguish such environmental influences from genetic mechanisms of inheritance. Examples of these design strategies are twin, half-sibling and adoption studies. Twin studies have investigated the heritability of alcoholism by comparing the concordance rates between monozygotic (MZ) and dizygotic (DZ) twins. Since MZ twins are genetically equivalent, whereas DZ twins share only half their genes, traits that are under genetic influences will be reflected by a greater concordance among the former than the latter group. As shown in Table 1, there exist several large studies that demonstrate a greater concordance among MZ than DZ twins for alcoholism. However, not all investigators have been able to demonstrate this phenomenon. Murray et al. [63] failed to observe a significant concordance. Likewise, neither Pickens et al. [64] nor McGue et al. [65] were able to demonstrate a significant difference in MZ/DZ concordance for alcoholism in female same-sex twin pairs. It is possible that the relatively small sample size of these studies accounts for their negative findings. By contrast, a large investigation of 1030 female-female twin pairs in Virginia found a significant genetic component for alcoholism [66]. This report evaluated the concordance for

Table 1 Twin studies of the inheritability of alcoholism No. twin pairs Characteristics

Concordance (%)

Ref.

MZT ~ DZT b M Z T / D Z T 174 15 924 56 86 (male) 44 (female) l l4 (male) 55 (female) 181 (male) 87 (female) 1030 (female)

Alcoholism Alcoholism Alcoholism Alcohol dependence Alcohol dependence Alcohol abuse and/or dependence Alcohol abuse and/or dependence Alcoholism Alcoholism Alcoholismc Narrow Intermediate Broad

71 26 21 59 25 76 36 77 39

32 14 25 36 5 61 25 54 42

2.2 2.0 0.8 1.6 5.0 1.2 1.4 1.4 0.92

26 32 47

12 24 32

2.2 1.3 1.5

[61] [62] [63] [64]

[65] [66]

~Monozygotic twins; bDizygotic twins; cSee text for details of categories.

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alcoholism defined: (1) 'narrowly' as alcoholism with tolerance or dependence; (2) 'intermediately' as alcoholism with or without tolerancedependence corresponding to the DSM-III-R definition of alcohol dependence; or (3) 'broadly' as alcoholism with or without tolerance-dependence or problem drinking. The probandwise concordance was higher in MZ than in DZ twins for all the aforementioned definitions of the disease (Table 1). Furthermore, these subjects exhibited an inherited liability to alcoholism of 51 59°/,, [66,67], similar to the degree of heritability determined by large studies of male twins [68]. Half-sibling investigations also point to a genetic component of alcoholism. Schuckit et al. [69] reported that 65°/,, of alcoholic half-siblings had an alcoholic biological parent compared with only 20°/,, of non-alcoholic half-siblings. Finally, adoption studies have been utilized to discriminate genetic from environmental factors in the etiology of alcoholism. A Danish investigation of 133 male adoptees, separated from their parents by 6 weeks of age, found that 18% of those with a positive biological paternal history developed alcoholism compared with only 5% of the adoptees who did not have a positive biological family history for alcoholism [70]. Furthermore, the sons of alcoholic parents who were adopted away had the same increased risk (about three- to fourfold over controls) of becoming alcoholics as their biological brothers who were raised by their alcoholic parents [71]. This latter observation, that the sons of alcoholics have an increased susceptibility to developing alcoholism whether or not they are raised by their alcoholic biological parents, indicates an appreciable genetic component in the etiology of this disease. Other adoption studies have evaluated alcoholism exclusively in women. Of 913 adopted Swedish women, alcohol abuse was threefold more frequent in adopted-out daughters of alcoholic women than in daughters of non-alcoholic parents. Interestingly, there was no such difference observed in the adopted-out daughters when the fathers and not the mothers were identified as alcohol abusers [72,73]. In contrast, Kendler et al. [67] found that genetic vulnerability to alcoholism is transmitted equally from mothers and fathers to their daughters (n = 1030 femalefemale twin pairs). The evidence for a genetic basis of alcoholism is summarized in Table 2. It is clear from this overview that the mode of inheritance of alcoholism is far from established. This is a disease which has both a complex mode of transmission and susceptibility to environmental factors. Nonetheless, the genetic-epidemiological evidence points to many possibilities. Among these are that genetic 'trait' markers to determine risk for the development of alcoholism may someday replace 'state' markers of alcohol abuse. The progression to this goal requires the definition of the genes that control

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Table 2 Evidence for genetic basis of alcoholism Family studies Twin studies Half-sibling studies Adoption studies

33% incidence of at least one alcoholic parent Incidence much lower among relatives of non-alcoholics Much greater concordance for alcoholism among monozygotic than dizygotic twins 65% of alcoholic half-siblings have alcoholic biological parent compared with 20% among non-alcoholics 18% of those with positive paternal history develop alcoholism compared with 5% of those without Adopted out sons of alcoholics had same increased risk of alcoholism (three- to fourfold) as brothers raised by biological parents

these traits. Significant research has been undertaken to investigate several candidate genes, gene products and genetic markers for alcoholism. In the following sections, we review evidence linking the risk for alcoholism with several putative genetic markers including the A D H and A L D H genotypes, monoamine oxidase and adenyl cyclase expression, and dopaminergic receptor polymorphisms.

3. Alcohol and aldehyde dehydrogenases

3.1. Alcohol dehydrogenase There are at least six genes for human alcohol dehydrogenase (ADH; EC 1.1.1.1.). The products of all six genes are expressed in liver, but some are found in other tissues including lung, stomach, cornea, and oesophagus. They are divided into four different classes (Table 3). The Class I isoenzymes are homodimers and heterodimers composed of ~, fl, and 7 subunits. They are the most important enzymes in ethanol metabolism because of their low Km for substrate. Their structural genes are located on the long arm of chromosome 4 [75]. The Class I enzymes are also notable in demonstrating genetic polymorphism at two loci, ADH2 and ADH3. Three different subunits (ill, f12, f13) are encoded by the ADH~, ADH~, and A D H 3 genes, respectively. The properties of these polymorphic forms are summarized in Table 4. The fl protein subunits at the ADH2 locus differ by one amino acid from each other. The fll isozyme has a very low Km for ethanol and a relatively low Vmax. It is found most commonly in Caucasians and is frequently designated ADH2*I. The f12f12 form also has a low Km but its Vmax is 40-fold higher than that of fl~fll. It is common among Asians and is designated ADH2*2. The properties of fl~fl2 heterodimers fall between those

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Table 3 Gene loci and protein products of alcohol and aldehyde dehydrogenases [74] Class

Alcohol I I I II III -IV

Gene locus

dehydrogenase~' ADH1 ADH2 ADH3 ADH4 ADH5 ADH6 ADH7

Protein product

Properties

fl 7 7r Z -a

Low K b hepatic enzyme High Very High High

Aldehyde dehydrogenas¢ -ALDH 1 ALDH 1 .-ALDH 2 ALDH 2

Km hepatic enzyme high Km enzyme K m gastric and hepatic enzyme K m gastric and esophageal enzyme

High K m cytosolic enzyme Low K m mitochondrial enzyme

"All A D H s are cytosolic and dimeric. Class 1 subunits can form heterodimers. bMichaelis-Menten constant for ethanol. CThe A L D H s are homotetramers.

of the homodimeric isoenzymes. The third isoenzyme, f13f13 (designated ADH2*3) has a high Km for ethanol and high Vm,x and has been identified only among Africans. Polymorphism at the A D H 3 locus is due to two different subunits (71, 72) corresponding to the ADH~ and A D H 2 alleles, respectively. In an alternative nomenclature ADH3*I encodes the 7~ subunit, while ADH3*2 encodes the 72 subunit. These differ by two amino acids, and the homodimer 7171 has a twofold greater Vm,x than the 7272 homodimeric form. This polymorphism is of lesser consequence than that at ADH2, since the differences in kinetic Table 4 Polymorphisms of alcohol and aldehyde dehydrogenases [74] Gene locus

Alleles

Protein product

Enzyme properties

ADH2

ADH2*I ADH2*2 ADH2*3

//1 f12 f13

Low K~, low Vnbl a x Low K m, high V,.... High K m, high Vm,~X

ADH3

ADH3*I ADH3*2

~)1 )'2

Low Km, higher Vn.... Low Kin, lower V,.....

ALDH2

ALDH2* l ALDH2*2

ALDH-E ALDH-K

Active Inactive

~Km refers to the Michaelis Menten constant for ethanol. ~'Comparisons of Vm~,x of the isoenzymes indicate differences within fl or i' groups.

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properties are much smaller, and the heterogeneity at this locus is very much less; for example, the ADH3*I allele frequency in Chinese is 0.91. The remaining classes of A D H isoenzymes are not thought to be of major significance in ethanol metabolism and have received very little attention in population studies. Using the polymerase chain reaction (PCR) with samples of whole blood to amplify genomic DNA sequences of EXON 3 and EXON 9 from the ADH2 gene and of EXON 8 from the ADH3 gene, Couzigou et al. [76] compared the polymorphism at these loci among 46 alcoholic cirrhotic patients and 39 controls. The distributions were similar in the two populations. None of the alcoholic liver disease subjects or controls were homozygous //2, and the /13 allele was not found in any of these French subjects. This study confirmed the earlier findings of a Japanese group reporting no differences between patients with alcoholic liver disease and a control group at the ADH2 locus [77]. The French authors expanded their observations in a subsequent paper [78], and noted that ADH3 polymorphism did not affect the rate of ethanol elimination in healthy volunteers. Using identical PCR techniques on whole blood samples from Chinese men living in Taiwan, Thomasson et al. [79,80] reported that alcoholics had significantly lower frequencies of both the ADH2*2 and ADH3*I alleles than non-alcoholics from the same population. This difference was independent of ALDH2 genotype (see below). Among alcoholics homozygous for ADH2*I, the ADH3*2 allele frequency was significantly higher than in the non-alcoholic population (p < 0.001). Thus there appears to be a linkage between the two alleles in the alcoholic subjects. In a previous report, ADH2 genotypes were compared in non-alcoholic Japanese and patients with alcoholic liver disease, no difference in allele frequency being demonstrated [77]. However, only 10-16% of alcoholics develop liver disease, so that those that do so may not be genotypically representative of the alcoholic population in general. The functional significance of these findings is that individuals possessing ADH2*2 and ADH3*I alleles generate acetaldehyde much more rapidly after ethanol consumption than do individuals with the alternate alleles and are thus less tolerant to ethanol. Chinese subjects lacking these alleles therefore appear to be at higher risk for the development of alcohol abuse. By contrast with these findings in Orientals, Gilder et al. [81] were unable to detect any differences in ADH2 and ADH3 genotypes in D N A extracted from whole blood of 82 Caucasians receiving treatment for alcohol-related problems, and 84 controls. Sherman and colleagues [82-84], in a series of papers, have described a restriction fragment length polymorphism (RFLP) in the ADH2 gene in leukocyte D N A digested with the restriction enzyme PvuII. The two-allele polymorphism was designated A and B (Table 5). In

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Table 5 ADH2 RFLP genotypes and allele frequencies in controls and alcohol misusers [82] Group

Controls Alcoholics

No. in group

23 45

ADH2 genotype

Allele frequency (%)

AA

AB

BB

A

B

18 7

3 19

2 19

85 37

15 63

control subjects the allele frequencies were 85% for A and 15% for B compared with 37% and 63%, respectively, in alcohol abusers (p < 0.001). Furthermore, the B allele was significantly associated with severe liver damage (p < 0.05) as well as alcohol dependency and a family history of alcohol abuse. An analysis of the sequence of ADH2 indicated that the additional restriction site is probably in a non-coding region, suggesting that this base alteration does not affect enzymatic function. It is intriguing that this polymorphism was detected using a probe containing a coding sequence specific for the f12 allele which is very uncommon (2% or less) in the white European population studied [76,85]. The authors therefore suggested that the RFLP is in linkage disequilibrium with either a polymorphism in an adjacent regulatory sequence, resulting in a change in ADH2 expression, or with a coding region of a neighbouring gene. They also examined the ADH3 genotypes of 26 patients with alcoholic cirrhosis and 16 controls. The allele frequencies for ADH3*I and ADH3*2 were 39% and 62% in the cirrhotics compared with 63% and 38% respectively in the controls (p = 0.05). These frequencies are in agreement with those reported earlier by Couzigou et al. [76] and Day et al. [851.

3.2. Aldehyde dehydrogenase Aldehyde dehydrogenase (ALDH, EC 1.2.1.3.) which converts acetaldehyde to acetate exists in four forms in human liver identifiable by starch gel electrophoresis [86]. The mitochondrial enzyme, ALDH2, has a low Km and is believed to be responsible for the majority of acetaldehyde oxidation. The enzyme is a homotetramer, and its gene is located on chromosome 12 [87]. The normal allele is designated ALDH2* 1, but a point mutation in the gene produces a mutant allele with deficient activity, designated ALDH2*2, which is dominant over the normal allele. Thus, subjects who are both homozygous and heterozygous for ALDH2*2 lack detectable ALDH2 activity in liver. ALDH2 deficiency is relatively common among Asians, being associated with facial flushing and other unpleasant symptoms when alcohol is con-

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sumed, presumably due to elevated levels of blood acetaldehyde [88-90]. The ALDH2*2 phenotype is much less common in Japanese alcoholics than in the general population [91]. Thomasson et al. [79] examined the genonomic D N A of Chinese males living in Taiwan. This was extracted from peripheral leukocytes and EXON 12 of the ALDH2 gene was amplified using PCR. Of 100 subjects divided equally between alcohol abusers and non-alcoholics, 48% of the latter and 12% of the former had at least one ALDH2*2 allele and were therefore deficient in ALDH2 activity. It is presumed that the slow removal of acetaldehyde after alcohol consumption in ALDH2*2 subjects causes flushing and other symptoms which act to protect these individuals against the risk of alcohol abuse. Many reports have confirmed this association among Orientals, but it seems to play no role in other racial groups. Investigating 160 Caucasian alcohol abusers and controls, Gilder et al. [81] failed to detect the ALDH2*2 allele in any of the subjects. On the other hand, Sherman et al. [83,84] examined the relationship of alcohol-induced flushing in Caucasian subjects and their erythrocyte A L D H 1 activity. The role of this enzyme is unknown, and its high Km for acetaldehyde (30/zmol/1 as opposed to 1 /lmol/1 for mitochondrial ALDH2) would preclude a principal role in aldehyde metabolism. Fifty percent of females and 8% of males reported flushing after a small amount of alcohol. At least two-thirds of affected individuals reported a similar reaction in other family members. All of the flushers had reduced erythrocyte ALDH1 activity and this relationship was confirmed in pedigree studies of two families with affected members. Surprisingly, there was no difference between the stated average weekly alcohol intake between 'flushers' and those who did not manifest this response. Neither differences in the blood ethanol or acetaldehyde concentrations, nor in the elimination rates for these two constituents could be detected. Laser Doppler measurements of cutaneous blood flow after oral ethanol demonstrated that flushing in the A L D H 1-deficient Caucasians was very much less than that experienced by ALDH2-deficient Orientals. The absence of unpleasant symptoms, apart from flushing, in A L D H 1-deficient subjects appears to provide no protection against alcohol abuse. Indeed, in a large evaluation of twin pairs (n = 5831), Slutske et al. [92] concluded that self-reported alcohol-related flushing is not a protective factor for alcoholism in Australian Caucasians. Sherman et al. have also described polymorphism in the ALDH5 gene, and identified three separate mutations in both Caucasian and Oriental populations [93]. So far, these mutations have not been linked to either alcohol abuse or alcoholic liver disease. Higuchi et al. [94] evaluated the ALDH2 phenotype by isoelectric focusing of hair root lysates taken from 282 Japanese men and women. Men with inactive ALDH2 drank alcohol less often and in lesser amounts than those

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with active ALDH2 (p < 0.001). A lower monthly alcohol consumption was also apparent among ALDH2-deficient females (p < 0.005). Approximately 86% of those who reported always experiencing facial flushing after drinking had inactive ALDH2, whereas 86% of those who sometimes experienced flushing and 96% of those who never flushed had active ALDH2. Moreover, among flushers with active ALDH2, nearly all reported that they could continue to drink whereas less than half those flushers with inactive ALDH2 were able to do so (p < 0.001). Interestingly, 30% of the fathers of subjects with inactive ALDH2 were unable to drink, compared with 14.7% of fathers of those with ALDH2*I phenotype (p < 0.01). The effect of the ALDH2*2 allele as a genetic deterrent of heavy alcohol drinking among Asians was examined in a total of 1300 Japanese alcoholics admitted in the years 1979, 1986 and 1992 (Table 6). Over this 13-year period, alcohol consumption and alcohol-related problems had both shown a major per capita increase in Japan. Genotyping was performed on DNA from lymphocytes using PCR amplification. There was no change in the incidence of the homozygous ALDH2*2 phenotype over this period, but heterozygosity increased among the alcohol abusers from 2.5% in 1979 to 8% and 13% in 1986 and 1992, respectively. Homozygosity for ALDH2*2 appears to completely suppress the development of alcoholism whereas the suppressive effect of the heterozygous status is incomplete, and seems to be influenced by social and cultural factors [94,95]. In an analysis of 264 Japanese males [96], those who were heterozygous for ALDH2*2 had a range of symptoms after alcohol consumption including facial flushing, warmth, drowsiness, palpitation and nausea much more frequently than those with the ALDH2*I phenotype, but less frequently than those who were homozygous ALDH2*2. It was speculated that a small proportion of the enzyme in heterozygotes may consist of tetramers comprising only the normal sub-unit, thus conferring some enzyme activity. This is in line with a suggestion by Enomoto et al. [97]. Of significant interest is a recent report on the relative importance of the ALDH2*2 allele and North American acculturation on alcohol consumpTable 6 Percentage distribution p a t t e r n s of A L D H 2 in Japanese alcoholics in three different years

[95] A L D H 2 genotypes

1979 n = 400

1986 n = 400

1992 n = 500

3-year total n = 1300

ALDH2*2/2*2 A L D H 2 * 1/2*2 A L D H 2 * 1/2* 1

0.0 2.5 97.5

0.0 8.0 92.0

0.0 13.0 87.0

0.0 8.2 91.8

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tion by Oriental males born in the United States and Canada [98]. Results of this study show that subjects carrying the inactive ALDH2 allele drink two-thirds less alcohol, exhibit one-third the prevalence of binge drinking and are three times more likely to be abstainers than subjects carrying the gene for the active enzyme. In addition, binge drinking and abstinence rates for Orientals with the active ALDH2 are not significantly different than the corresponding rates for Caucasian North American males. The authors estimate that the A L D H mutation predicts two-thirds of the alcohol consumption and excessive alcohol use by Oriental males born in North America whereas acculturation in North American society accounts for no more than 11% of the variance in overall alcohol consumption. In summary, the ALDH2*2 phenotype, which is found only in Orientals, confers protection against alcohol abuse especially when present in the homozygous form by virtue of delaying the clearance of acetaldehyde. Individuals possessing the ADH2*2 and ADH3*I alleles will generate and accumulate acetaldehyde much more rapidly than those with other alleles. However, among Caucasians, where the ALDH2*2 allele is not represented, this additional acetaldehyde can be cleared without major effect, and therefore these genes do not predispose to alcohol abuse. On the other hand, when they occur in Orientals with deficient ALDH2 activity, they will increase acetaldehyde concentrations following alcohol consumption to particularly high levels and will therefore confer additional genetic protection against the risk of alcohol abuse. This fact is further supported by the results of a study of 1116 Japanese subjects which demonstrated higher odds ratios for alcoholism in those with active ALDH2 than those with inactive ALDH2. The odds ratios of both these groups increased from homozygous ADH2*2 to heterozygous ADH2*2 to homozygous ADH2* 1 [99]. Polymorphisms of other A L D H genes are not informative for alcohol abuse. The main conclusions to be drawn from this extensive literature is that there is no inherited defect of alcohol metabolism among Caucasians that can separate those prone to alcohol abuse from those who are resistant; among Orientals, A D H and A L D H phenotypes are more useful in predicting who is resistant to alcoholism rather than those who are susceptible, a distinction that flushing in response to alcohol is also capable of suggesting, but with less certainty. In other words, the more common variants of these enzymes are 'permissive' rather than 'predisposing' to alcohol abuse. In Oriental subjects, the uncommon variants make drinking an unpleasant experience due to acetaldehyde toxicity.

4. Monoamine oxidase

Monoamine oxidase (MAO, E.C. 1.4.3.4) is a mitochondrial flavoenzyme that catalyzes the oxidative deamination of primary amines, including the

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naturally occurring catecholamines and indolamines. Its principal location is in the outer mitochondrial membrane. The enzyme exists in two forms designated A and B. The two forms are distinguished by their sensitivity to different inhibitors: the A form to clorgyline, and the B form to L-deprenil [100,101]. Whereas both forms of the enzyme are present in the human brain, only the B form is present in human platelets [102,103]. Both high and low platelet MAO activities have been reported in subjects with affective disorders. The structural genes for both forms of human MAO have been assigned to the X chromosome. However, regulation of the catalytically active forms of the enzyme is exercised at the level of transcription; by post-translational modifications; and by environmental factors probably modulating the previous two steps [104]. The notion that platelets share similar biochemical processes (and possibly a common embryological origin) with neurons has led to their use as a surrogate for cerebral tissue in studies designed to elucidate enzyme abnormalities in neurological and psychiatric disorders. In 1975, Gottfries et al. reported that brain MAO activity was decreased in alcoholics [105]. This observation was confirmed by Oreland et al. [106] several years later. In 1977, Wiberg et al. described low activities of platelet MAO in human alcoholics compared with matched controls [107]. This observation was very rapidly confirmed and extended by many different groups of investigators [108-111]. An observation that initially caused some confusion was a sharp increase in platelet MAO activity over the first few days of withdrawal of alcohol, with a gradual return to the initial lower levels associated with abusive drinking over the next 2-3 weeks [112,113] as demonstrated in Fig. 1. Among the explanations offered for the initially low levels was that they reflected inhibition by acetaldehyde, iron deficiency or a lack of Vitamin B6. Putative inhibitors of MAO were also reported in the urine of alcoholics. However, these possibilities have been convincingly excluded. The increase upon withdrawal was explained as a result of stress, increased catecholamine excretion, or to thrombocytosis which often occurs on cessation of drinking to rectify the thrombocytopenia which is a common consequence of alcoholism. Newly produced platelets are larger and contain more MAO activity, so that an increase occurs in the percentage of newer platelets with a resulting increase in MAO activity when expressed per platelet [113]. The outcome of these observations is that for meaningful results, platelet MAO activities should be determined after a period of withdrawal from alcohol. Alexopoulos et al. [114] recommended that samples should not be tested within 6 weeks of abstinence in comparisons with reference populations. When this is done, the mean values in alcoholics have been consistently lower than those of matched controls.

R.A. Ferguson, D.M. Goldberg / Clinica Chimica Acta 257 (1997) 199-250

216

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Fig. 1. Percentage changes (mean + SE) in platelet MAO activity stimulated by cessation of drinking by male alcoholics. Reproduced, with permission, from [113]. These observations took on more significance when it was demonstrated, although not very convincingly, that first-degree relatives of alcoholics tended to have lower platelet M A O activities than control subjects lacking a family history of alcoholism [110,115]. Further, when alcoholic subjects were classified into the Cloninger Types I and II, it was observed that the platelet M A O activity was distinctly low in the latter group but essentially normal in the former [116]. Because this sub-classification appears to be relevant to the etiology, epidemiology and treatment of alcoholism, it seemed important to determine whether platelet MAO activity was indeed a marker for Cloninger Type II alcoholism and, if so, how accurate it was in discriminating between the two types. In 1988, Pandey et al. [117] measured platelet M A O activity in 75 patients with alcoholism and 123 normal controls. In the alcoholics, platelet M A O values were consistent with a mixture of two normal distributions. Those in the lower distribution were younger, developed alcoholism at an earlier age, and had a higher frequency of relatives who were alcoholics (Table 7). The distribution of the data indicated that 91.4% of the male alcoholic patients had decreased M A O activities relative to normal controls. Using a statistically derived cut-off point of 26.2 units, 61 of their male subjects fell into

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217

the low M A O category and six into the high activity (normal values) category. A problem with this report is that if one accepts the validity of this model, the incidence of Type II alcoholics in this group of in-patients admitted for detoxification exceeded that of Type I by a factor of 10, a much higher proportion than in similar populations (see below). In a subsequent study also utilizing alcoholics who had been admitted as in-patients, Sullivan et al. [118] took the opposite approach: they first classified the patients into Types I and II on the basis of a structured clinical interview accompanied by the administration of various questionnaires and tests. Twenty-five subjects were diagnosed as Type I and 31 as Type II. The platelet M A O activities in these two groups and in control subjects (healthy hospital staff) are shown in Fig. 2. The Type I subjects had a lower mean value than the controls, and the mean for the Type II group was significantly lower than that of both controls and Type I subjects. An analysis of distribution for normality suggested that the best fit for the data was a mixture of three normal distributions corresponding to controls, Type I and Type II alcoholics, although the authors pointed out that a larger sample size would be required to attain statistical significance. Von Knorring et al. [119] studied a total of 99 subjects out of 107 consecutive males treated for alcoholism as in-patients. Classification of the subjects into Type I and Type II based upon criteria previously published by these authors revealed 37 Type I and 62 Type II alcoholics with the characteristics shown in Table 8. The distribution of platelet M A O activities in the alcoholics and in 36 healthy controls is presented in Fig. 3. Although the data show major overlap, the Chi-square test for activities above and below 1 SD established significant differences between Type II and controls, Table 7 Clinical variables in alcoholics stratified by low and high platelet MAO activity [117] Variable

Low MAO (n = 64) High MAO (n = 11)

Sex

61 M 3F 39.0 _+8.4 15.0 ___5.3 26.8 _+8.4 9 31 42 21

Age (yearsF MAO activityb Age of onset of alcoholism (years)a History of major depression Parental history of major depression 0 1 alcoholic family members >_2 alcoholic family members

6M 5F 47.2 _+8.6 32.7 +_4.5 33.2 _+9.2 3 5 11 0

aMean ± SD. bMean _+SD, nmol [~4C]tyraminemetabolized per mg platelet protein per hour at 37°C.

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Fig. 2. Platelet MAO activity (mean__+SE indicated) observed for male type I and type II alcoholics following at least 4 weeks of abstinence compared to non-alcoholic controls. Reproduced, with permission, from [118]. and between Type II and Type I, but the difference between Type I and controls was not significant. The authors speculated that Type I! alcoholics would be characterized by a serotonergic deficit, a hypothesis supported by the high incidence of psychiatric disorders marked by aggressive and suicidal behaviour as well as depression in this group. They further suggested that serotonin uptake blockers should prove useful in Type II but not Type I subjects. However, they also admitted that the clinical classification was often at variance with the platelet MAO activity, and it was uncertain whether the latter could truly serve as a marker for Type II patients. The nature of the proposed serotonergic deficit among chronic alcoholics and its relationship to MAO has been the subject of hypothesis and speculation [120-122]. In alcoholism, depression, and other conditions associated with low platelet MAO activity, low concentrations of serotonin metabolites were found in CSF [123], suggesting impaired removal of serotonin and therefore prolonged action of this neurotransmitter. The reduced

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219

metabolism of serotonin may also be accompanied by enhancement of its uptake as documented for platelets (see below). The beneficial effect of serotonin-uptake blockers in these conditions is consistent with this notion. The most comprehensive study of these issues has been published by Devor et al. [124]. Their data on inheritance of MAO activity were consistent with a partially recessive single major locus with modest multifactorial background effects. By comparing catalytic and mass concentration assays using a monoclonal antibody to MAO-B, they demonstrated that the reduction in MAO was due to diminished catalytic activity of the individual MAO-B molecules rather than to a decline in their number per platelet. Analysis of the co-segregation of enzyme activity with alcoholism gave a value of r = - 0.29 to - 0.34 depending upon the substrate concentration used for the MAO assay. When family members of alcoholics were divided into those with high and low platelet MAO activities, the incidence of alcoholism in the latter was 54.3% compared with 34.3% in the former, and the incidences of other psychiatric illnesses were 57.1% and 38.1% in the two respective groups. They perceptively pointed out that the increased family history of alcoholism and psychiatric disease among Type II alcoholics is a self-fulfilling prophecy in the sense that these criteria are themselves used to assign subjects to this classification. Thus, platelet MAO may be a biological or genetic marker of an underlying pathophysiological process leading to alcoholism and other psychiatric illness rather than a marker of alcoholism itself. Moreover, Type II alcoholics share personality profiles similar to other psychiatric patients with low platelet MAO, including aggressive and suicidal behaviour and impulsive personality traits [119]. Table 8 Characteristics of type 1 and type 2 male alcoholics [119] Characteristic

Type 1 (n = 37)

Type 2 (n = 62)

Age of first alcohol consumption (years) Age of first subjective alcohol problems (years) Age of first treatment contact (years) Glue misuse Cannabis misuse Amphetamine misuse Illegal use of minor tranquillizers Aggressive after alcohol consumption Absent from work due to alcoholism Job loss due to alcoholism Arrested when drunk Drunk while driving Other forms of criminality

18.0 _+4.0 36.3 ± 7.7 41.9 _+9.7 1 (3%) 0 0 0 7 (19%) 28 (76%) 7 (19%) 24 (65%) 18 (49%) 2 (5%)

16.3 _+ 3.3 23.8 _+ 4.7 27.8 _+ 7.1 17 (27%) 29 (47%) 12 (19%) 13 (21%) 34 (55%) 57 (92%) 29 (47%) 55 (89%) 42 (68%) 32 (52"/,,)

220

R.A. Ferguson, D.M. Goldberg

Clinica Chimica Acta 257 (1997) 199-250

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Reviewing the literature on platelet MAO activity, one is impressed by the relatively small numbers of subjects forming the basis for each individual report. Until investigations involving several thousand of subjects (alcoholics, their relatives, and normal controls) have been performed, it seems unlikely that these dilemmas will be resolved, and even if they are, it appears certain that the results will be more helpful in understanding the biological factors contributing to alcoholism rather than as a tool to assign risk within a normal healthy population.

5. Dopamine receptors The dopaminergic system is an important component of the CNS neurotransmission network. In fact, over one half of the catecholamine content of the brain is made up of dopamine. Immuno- and histochemical studies have indicated that dopaminergic neurons play a central role in the neuro-biochemistry of the basal ganglia (including those of the caudate nucleus), the

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221

nucleus accumbens, the olfactory tubule, the central nucleus of the amygdala, the median eminence and the frontal cortex. Dopaminergic neurons play a role in a host of behavorial and physiological control mechanisms such as those involved in hormone secretion, voluntary movement and emotion. It is not surprising therefore, that many clinically important psychoactive drugs are known to have high affinity for dopamine receptors [125]. Indeed, drugs that target dopamine receptors are used in the medical treatment of such disorders as schizophrenia and Parkinson Disease. Furthermore, the dopaminergic system is strongly implicated in maladaptive seeking behaviour of substances such as alcohol and cocaine. Such behaviour appears to be driven in part by dopamine-mediated reinforcement in the mesocorticolimbic 'reward' pathway [14]. Alcohol stimulates the release of dopamine and its metabolites from the rat brain both in vitro [126] and in vivo [127,128]. These observations, in addition to those described above, have been interpreted as strongly implicating the dopaminergic system of the CNS (particularly the mesolimbic and mesocortical pathways) in the biochemistry of alcoholic and other forms of addictive behaviour. Nevertheless, it has not been until the development of molecular biological techniques such as PCR and RFEP analysis that hypotheses concerning dopamine neurobiology could be tested visa vis inheritable risks for alcoholism. Dopamine receptor polymorphism has come to be an important area of investigation in this regard. 5. I. Genetics and structure of the dopamine receptor

Historically, the dopaminergic receptors have been identified and characterized exclusively by biochemical and pharmacological criteria. Radioligand binding and cell physiological techniques were successful in establishing the presence of two dopamine receptor sub-types, D1 and D2, with the former being 15 times more sensitive to dopamine than the latter. The D1 neurotransmitter receptor was found to exert its neuronal effects via Gs mediated stimulation of adenyl cyclase [129], whereas agonist stimulation of the D2 receptor effected adenyl cyclase inhibition via Gi [130]. Bunzow et al. [131] were the first to clone a dopamine receptor gene. The rat D2 receptor gene was isolated by using a hamster f12 adrenoceptor gene hybridization probe under low stringency conditions. The authors characterized the receptor on the basis of amino-acid sequence, tissue distribution of mRNA, and the pharmacological properties of the gene product as expressed in mouse fibroblast cells. This pioneering work of Bunzow and colleagues was quickly followed by the cloning, sequencing and chromosomal mapping of the human D2 receptor gene [132,133]. The gene is located on chromosome 11 at q22 q23

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(Fig. 4). This is of particular interest given that chromosome 11 had previously been linked to alcoholism [134-137]. The D2 gene consists of two alleles, A1 and A2, as defined by 2hD2G1 probe hybridization to Taql-digests [132,133]. This clone contains the eighth exon and 16.5 kbase pairs of the 3'-flanking sequence of the D2 dopamine receptor gene. Grandy et al. [133] also demonstrated that the coding region of the D2 receptor gene is composed of six introns and seven exons from which the structure of the gene product can be deduced (Fig. 5). The D2 receptor is not only structurally but also pharmacologically related to two of three more re-

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Fig. 4. Ideogram of chromosome 11 as determined by southern blot analysis of TaqI-digested DNA prepared from five cell lines and hybridized with 2hD2GI. Each of the five cell line regions indicated above possessed different rearrangements of human chromosome 11. The approximate location of DRD2 at 1lq22-23, is also illustrated. Reproduced, with permission, from [133].

R.A. Ferguson, D.M. Goldberg /Clinica Chimica Acta 257 (1997) 199 250

[el

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Fig. 5. The D2 receptor shares structural similarity with the D3 and D4 receptors as illustrated above. Each contain ~ 420 amino acids of which the homologous peptides are indicated by solid circles. Reproduced, with permission, from [138].

cently identified dopamine receptor sub-types. Dopamine receptors D3, D4, and D2 are approximately 420 amino acids in size and share an overall homology of about 30% [139,140]. A fifth receptor D5 is more closely related to the D1 receptor than any one of the aforementioned receptors [138]. 5.2. Association studies of the D2 receptor and alcoholism

An important milestone in the investigation of the relationship between the dopaminergic system of the CNS and alcoholism occurred in 1990 with the publication of a study by Blum, Noble and colleagues which demonstrated an association of the A1 allele of the human dopamine D2 receptor gene and alcoholism. These investigators assessed gene polymorphisms of brain frontal grey cortex tissue in the cadavers of 35 alcoholics and 35 non-alcoholics. Four restriction endonucleases were employed to search for possible polymorphic associations with alcoholism by nine DNA probes (including those for the genes of alcohol dehydrogenase, monoamine oxidase, tyrosine hydroxylase, and the D2 receptor). Only D2 receptor polymorphisms, determined by the Taq 1 2hD2G1 probe, were associated with

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alcoholism. Specifically, the presence of the A1 allele was found to correctly identify 77% of the alcoholic subjects whereas its absence correctly identified 72% of the non-alcoholic subjects. Given the fact that this gene is located on a somatic chromosome (chromosome 11), it is not surprising that no significant sex difference is observed for the A1 allelic distribution in alcoholic and non-alcoholic subjects [141]. Blum's report generated a torrent of enthusiasm that was fuelled by the possibility that a previously elusive genetic linkage to the very serious and widespread disease of alcoholism had finally been identified. The excitement continued to escalate as the proposed association was further supported. One of these studies, by Blum et al. [142], involved 159 subjects classified as non-alcoholics (n = 43), less severe alcoholics (n = 44), severe alcoholics (n = 52) and children of alcoholics (n = 20). Both the combined alcoholic group, as well as children of alcoholics, were found to have a significantly greater association with the A1 allele than non-alcoholics as revealed by molecular biological investigation of peripheral lymphocytes. The strongest allelic relationship was demonstrable when severe alcoholics were compared with non-alcoholics. This study was supported by another by Comings et al. [143], who found a significant difference in the frequency of the A1 allele in 104 alcoholics (42%) as compared with 108 controls who were not evaluated for alcoholism (22%). A physiological consequence of dopamine receptor polymorphism was demonstrated in vitro by Noble et al. [144]. Sixty-six samples of caudate nucleus tissue (available from the original 1990 study) were characterized by tritiated spironolactone ligand studies. While no significant difference was observed in the binding affinity of caudate nucleus tissue of alcoholics and non-alcoholics, tissues from the former group showed a significantly lower binding capacity. Moreover, a progressive reduction in the number of binding sites was found in subjects with A2/A2, A2/A1, and A1/A1 alleles. Further evidence linking dopamine receptor polymorphisms to changes in neurophysiology was found in an evaluation of three groups of Caucasian boys (32 sons of active alcoholic fathers, 36 sons of recovering alcoholic fathers and 30 sons of social drinkers). The A1 allele frequency in these boys was 0.313, 0.139 and 0.133, respectively, and was associated with longer P3 latency [145]. Indeed, P3 latency (see later section) had been advocated previously as a marker of risk for alcoholism [146] and the association between this and the dopamine receptor AI allele, as shown by Noble, provided physiological-based support for the growing genetic evidence linking alcoholism with this allele. Furthermore, studies of cognitive function have uncovered an association between reduced visuospatial performance (commonly observed in alcoholics) and the presence of the A1 allele in alcohol- and drug-naive boys [147].

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5.2. I. Dissenting studies As the story of the investigation of alcoholism and dopamine receptor polymorphism evolved, there appeared a growing and appreciable body of evidence against an association of the A1 allele and the risk of alcoholism. Indeed, within the year of Blum's original publication, Bolos et al. [148] reported that they had failed to find an association between the presence of the A1 allele and alcoholism in 40 white alcoholics and 127 matched controls. Another study of 32 white alcoholics (10 of whom were categorized as severe alcoholics) and 25 white controls demonstrated A1 frequencies of 41% and 12%, respectively [149]. Association of alcoholism with the A I allele was significant when controls were compared with the severe alcoholic subset (A1 frequency = 60% in the latter group) but not when they were compared with the less severe cases. Furthermore, no evidence of linkage or cosegregation was observed after multigenerational pedigree analysis of 17 nuclear families [149]. Attempts to replicate a positive association between the A1 allele and alcoholism by Gelernter et al. [150] also failed. This latter paper relates similar frequencies of the A1 and A2 allele in 44 white alcoholics (0.23 and 0.77) and 68 controls (0.20 and 0.80) respectively. Subtyping the alcoholic group by family history, antisocial personality disorder, physical withdrawal symptoms or severity of alcoholism was not successful in uncovering a significant difference in allele frequency. Similar findings, i.e. lack of support for Blum and Noble's hypothesis, have been recorded by Cook et al. [151] in a study of 20 alcoholics and 20 controls, as well as by Gejman et al. [152] in an investigation of predominantly white patients with schizophrenia (n = 106) and alcoholism (n = 113) as compared with controls (n = 34). 5.3. Factors contributing to conflicting results 5.3.1. Classification The discrepancies in the studies which either support or do not support the association between alcoholism and the A1 dopaminergic receptor allele have been the subject of much debate. For instance, Noble and Paredes [153] point out that many of the latter studies, such as those by Bolos et al. [148] and Gelernter et al. [150], are biased by the fact that they neither screened for alcoholism in their population-based control groups nor included severely ill alcoholics (e.g. those with cirrhosis) in the experimental group. Noble and Paredes emphasized that when the 'healthy' alcoholic group is stratified on the basis of ethanol consumption over the 2 months preceding the study (i.e. > 300 drinks vs. < 300 drinks), a significantly greater prevalence of the AI allele in the former group is revealed. This criticism would not appear to apply to the study of Turner et al. [154] who,

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upon examining 47 Caucasian males with severe alcoholism as indicated by DSM-III-R criteria, could not demonstrate a significant difference in A1 allele frequency in this group (19%) as compared with control frequencies as reported by either Blum et al. [141] or Bolos et al. [148]. These investigators also failed to show a significant effect for alcoholics with severe medical complications. The large majority of the alcoholics classified as severe by Turner et al. were under treatment because of drinking and driving problems. Noble and Paredes, therefore, counter that these patients were suffering from acute rather than chronic alcoholism, and would be more appropriately classified as less-severe alcoholics. Such arguments might at first glance appear as superfluous debates based upon mere semantics. Yet it is not known how to classify the subjects of these studies in a truly objective and relevant manner. Indeed there is no consensus at all on the definition of 'severe' alcoholism.

5.3.2. Definition drift It is evident that differences in the definition and classification of alcoholism (e.g. as severe or less severe) are a considerable source of heterogeneity amongst the aforementioned studies. Kidd [155] calls attention to the 'definition drift' that has occurred in the investigation of the putative association of the D2 dopamine receptor gene and alcoholism. He points out that as the numerous studies attempt to replicate Blum and Noble's original work, each has slightly modified the original definition of the disorder. This is a c o m m o n p h e n o m e n o n in replication studies and appears to arise from the temptation of investigators to revise the definition of the disorder based on the strongest association found in the new data. Thus, according to Kidd [155], the association of the A1 allele (defined by TaqIA digestion and R F L P analysis) with alcoholism was initially thought to be direct, but has evolved to a relationship with severe alcoholism, and beyond that to an indication of susceptibility to addictive behavior [156,157]. G o o d m a n [158] states that even elementary concepts of alcoholism need to be more sharply described to avoid confusion. He defines alcoholism broadly as either one of two types. One form, which he classifies as 'alcoholism 1', is a chronic disease associated with one or more of hepatitis, hepatic cirrhosis, pancreatitis, gastritis, cardiomyopathy, peripheral neuropathy, and encephalopathy. This is distinct from, but often confused with, what he calls 'alcoholism 2' - - a behavior pattern of excessive alcohol consumption. On the basis of the contradictory findings described above, he feels that the evidence points towards a possible role of the A1 allele in the involvement of alcohol toxicity and not drinking behavior per se.

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5.3.3. Ethnic variation Another criticism of many of the available reports involves the subject of racial heterogeneity. The studies emanating from the United States are primarily, but by no means exclusively, studies upon whites. This is also true for investigations of other nationals including Canadians [159], Finns [160] and French [161]. There is evidence from Japan [162] that the A1 allele is associated with alcoholism in that race and is positively correlated with the severity of alcoholism. The need to adequately control for racial and ethnic factors in these studies would not exist if dopamine polymorphisms did not differ appreciably amongst different populations. This is evidently not the case. Barr and Kidd [163] showed that the frequency of the A1 allele varies greatly (0.09-0.75) according to ethnicity. Furthermore, within the same race, great variation in A1 allele frequency may be observed as shown by Goldman et al. [164]. This latter study demonstrated a fourfold variation in A1 incidence in the Caucasian subjects assessed up to that time. In addition he found significant variation in the dopamine Taql A1 allele frequency between Caucasians (0.18-0.20), American Blacks (0.38), Jemez Pueblo Indians (0.63) and Cheyenne Indians (0.80). This fact underscores the necessity to appropriately control for ethnicity when evaluating the association of the A1 polymorphism with alcoholism. An interesting approach to this problem has been suggested by Lander [165] who points out that the shortcomings of many genetic association studies could be overcome by using the parents of the affected individuals as controls. By comparing the genes of interest on the chromosomes of alcoholics with those that are not passed on by their parents, Lander suggests that a large part of the statistical bias associated with population stratification and sampling error could be averted. 5.3.4. Statistical considerations Linkage studies, while rarer than studies of association, have failed to uncover an association between the A1 allele and alcoholism [148,149]. Because of its simplicity compared with linkage studies, which require genetic analysis of extended kindreds, association study design has enjoyed much greater popularity. One must remember, however, that the power of association studies depends on the weight of a priori evidence in favor of etiologically relevant variation of one candidate gene [150]. We have previously outlined the evidence for the involvement of the dopaminergic system in psychiatric diseases in general, and alcoholism in particular [14,129,135]. Despite this evidence, Crowe [166] argues that we are not yet sufficiently knowledgeable about the biology of psychiatric diseases to be able to identify candidate genes with confidence. Furthermore, the prior likelihood for any one 'candidate' gene in this area of investigation is very low, given the tens of thousands of genes that are expressed in the brain [167].

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Arguing that any single candidate gene for alcoholism has a low prior probability, Crowe [166] asserts that a significance level o f p < 0.00001 is a minimal requirement to support a 'significant' association, as this is necessary to limit the rate of false-positives to 5%. Blum and Noble's original paper [141] fails to meet this criterion. The statistical pitfalls of association studies have led many to accept that consistent replication of a study's findings is the best evidence for a true association. Nevertheless, the statistical hurdles of such studies merit more scrutiny, and replication of a positive study is in itself not enough to prove association [155,166]. Consistent with this history of contradictory findings, meta-analyses of studies which investigate the putative association of the A1 allele and alcoholism have arrived at different conclusions [150,156,168]. Meta-analyses by Pato et al. [168] and Noble [156] find evidence for an increased risk of alcoholism in subjects carrying the A1 allele. On the other hand, Gelernter's [150] meta-analysis is not supportive of an association. Furthermore, Gelernter concludes that heterogeneity among studies (related to ethnic differences and sampling error) is a more likely explanation for differences observed in the prevalence of the A1 allele than is an association with alcoholism. 5.3.5. What is the A1 allele7 We must offer one final caveat to the reader regarding the majority of dopamine receptor association studies conducted to date. This is the fact that the A1 allele is simply a marker and does not reflect the existence of an actual variant of the D2 receptor gene. Indeed, the A1 allele has been estimated to be situated i0000 base pairs from the actual gene that codes for the final protein product [169]. Furthermore, it appears that no structural (i.e. coding) mutation exists in the dopamine D2 receptor gene in alcoholics, as determined by denaturing gradient gel electrophoresis, even though a substantial number of the subjects were known to have Taql 2hD2G1 probe-defined allelic variation [152]. Criticisms relating to these facts have led investigators to identify other, more relevant, alleles of the D2, and other, dopamine receptor genes. An addition to the 2hD2G1 probe was provided through the work of Hauge et al. [170]. The 2hD2G2 cloned probe contains the exons 2 - 7 of the D2 dopamine receptor gene and is therefore much more closely associated with the gene than is 2hD2G1 (Fig. 6). The newer probe detects two allelic Taql R F L P variants called B1 (4.6 kbasepairs) and B2 (4.1 kbasepairs) which appear to be in linkage disequilibrium with TaqlA RFLPs [170]. Using the 2hD2G2 probe and (Caucasian) samples from one of their previous studies, Blum et al. [171] found the B1 allele to be present in 13.3% of the non-alcoholics (n = 30), 16.7% of the less severe alcoholics (n = 36)

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and 46.9% of the severe alcoholics (n = 49). The prevalence of the B1 allele in the severe alcoholic group was significantly greater than in either the non-alcoholic group or in the less severe alcoholic group (p < 0.01 for both comparisons). Nevertheless, other studies that have utilized R F L P s that span coding regions of the D2 receptor gene have failed to illustrate an association of the D2 region with alcoholism [172]. Furthermore, attempts to uncover associations between alcoholism and other dopamine receptor polymorphisms (e.g. D4) have been unsuccessful [173]. Thus it would appear that the controversy is fated to continue, for the time being. 5.3.6. Current status

In the preceding discussion, we have attempted to provide information that is valuable in the interpretation of the disparate findings regarding the putative association of the D2 dopamine receptor system and the etiology of alcoholism. We hope to have given the reader not only a narrative of the history of this particular chapter in the study of alcoholism, but also an awareness of the pitfalls that may be encountered when attempting to interpret the data which are presently available. Indeed, no side of this polarized debate can claim to be supported exclusively by the data of flawless studies. Recommendations such as those made by Crowe [166] for the minimization of false-positive results and the standardization and optimization of methodological approach, if heeded, should result in a clearer understanding of the possible relationship of the genes of the dopaminergic system (and, indeed, any other genes) and alcoholism.

p 4.6 Kb

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3

4

5

6

7

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Fig. 6. Map of the D2 receptor gene. Polymorphic and non-polymorphic TaqI sites are indicated by T* and T, respectively.A polymorphic microsatelliteis indicated by TG. Exons are shown by numbered boxes. Horizontal lines represent introns and flanking regions of the gene. Also indicated are the regions corresponding to genomic phage 2hD2G1 and 2hD2G2 probes. Reproduced, with permission, from [156].

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6. Other markers related to neurotransmission

6.1. Gamma aminobutyric acid Gamma-aminobutyric acid (GABA) is an amino acid which is not found in proteins, but has a major role as a neurotransmitter in the central nervous system. In a receptor-binding study of post mortem brains of alcoholics compared with matched controls, Tran et al. [174] found that the binding of the GABA agonist muscimol was significantly enhanced. Subsequently, plasma GABA concentrations were reported to be significantly lower in abstinent alcoholic men compared with matched controls [175]. More recently, Moss et al. [176] described an investigation in which serial plasma GABA determinations were performed after administering a placebo beverage or ethanol to sons of alcoholic fathers (high risk) and matched controls from families without alcoholism (low risk). The study was meticulously performed in a randomized double-blind fashion with triplicate assays at each time point over a 3-h period. The authors used sophisticated statistical techniques to assert that alcohol increased the plasma GABA concentrations in the high risk group whereas these were lowered in the low risk group. They also claimed that high risk subjects had significantly less plasma GABA during the placebo stimulation. Their data (Fig. 7) display very high variance, since the error bars represent SEM. Although the mean values at zero time differed in the two groups by about 6 ng/ml, this persisted throughout the 3-h period of observation. In itself, this difference was not statistically significant at any time point. It is possible that, purely by chance, the 10 high-risk patients happened to have lower values than the 10 low-risk patients independent of their risk of alcoholism, and since the values were not affected by placebo, the difference persisted. It is interesting that there was very little difference between the 0-time mean values for alcohol stimulation, and the overlap continued throughout the 3-h observation. The authors hypothesized that plasma GABA is a marker linked to genetic vulnerability to alcoholism, or a biochemical correlate of behaviour which predisposes to subsequent alcoholism. We find their evidence unconvincing.

6.2. Adenylate cyclase Tabakoff et al. [177] demonstrated that platelet adenylate cyclase (AC) activity, when stimulated by a number of agents including cesium fluoride and prostaglandin El, was significantly lower among alcoholic subjects compared with controls. This difference persisted after cessation of alcohol consumption over several years. No difference in unstimulated AC activity

R.A. Ferguson, D.M. Gohtberg / Clinica E7~imica Acta 257 (1997) 199 250

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Fig. 7. GABA-like activity in plasma in high risk (HR, x) and low risk (LR, 3) males following oral challenge with (A) placebo and (B) 0.8 g/kg ethanol. Data are presented as the mean _+SE. Reproduced, with permission, from [176]. was noted between the two groups. They went on to conduct family and genetic studies on both basal and stimulated AC activities in an effort to understand their mode of inheritance and possible co-segregation with risk for alcohol abuse [178,179]. In a detailed examination of 115 members of 14 families, significant evidence of familial transmission for basal AC activity was observed, but no major gene effect was indicated, and the multifactorial background effect, while present, was not large. With stimulated AC activity, there was evidence for familial transmission with a substantial major gene effect and a modest multifactorial background; this major gene was transmitted as a Mendelian co-dominant. In discussing these findings, the authors refer to the uncertain nature of the basal (unstimulated) AC

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activity. On the other hand, the stimulated AC activity has been well delineated, with the receptor being coupled to the membrane-bound enzyme through a G-protein system. They speculated that the major gene effect indicated by their segregation analysis specifies the G-protein rather than the enzyme. They were unable to correlate stimulated AC activity with status for alcoholism in their subjects and interpreted this as meaning either no relationship between this phenomenon and risk of alcoholism, or that the families were too heterogeneous to allow co-segregation to be detected. Low receptor-activated AC activity had earlier been demonstrated in the lymphocytes of alcoholics by Diamond et al. [180]. More recently, Waltman et al. [181] reported that lymphocyte membranes from abstinent alcoholics had decreased basal AC and stimulated AC compared with both controls and actively drinking alcoholics. Immunoblot analysis revealed a threefold increase in the level of Gi2~ protein and its mRNA in the lymphocytes of abstinent alcoholics compared with controls and actively drinking alcoholics. They postulated that the enhanced expression of this inhibitory G-protein accounts for the reduced AC activity in abstinent alcoholics. 6.3. Platelet serotonin uptake

The interference by alcohol with neurotransmission processes, together with the pathogenic role of serotonin in psychiatric diseases have elicited interest in the effects of alcohol upon serotonin uptake by platelets. Increased uptake was demonstrated in alcoholic patients, even after prolonged abstinence [182]. A dissociation was shown for the binding of serotonin and imipramine [183]. The latter binding site showed no difference between alcoholic and non-alcoholic subjects whereas the Vmaxof serotonin uptake was increased in alcoholics without change in Km. Subsequently, these authors examined platelet serotonin uptake in former alcoholics and their offspring [184]. The Vmaxfor serotonin uptake was higher in offspring of alcoholics (both adult and children) relative to their respective control groups, while former alcoholics had very much higher Vn,,~ values (Fig. 8). The authors intend to extend this work and to follow-up the family members with a view to determining whether indeed high Vm~ values for platelet serotonin uptake are associated with a high risk of alcohol dependence.

7. Other markers

It is not surprising that sensitivity to alcohol may play a role in determining susceptibility to develop alcohol dependence (Table 9), in line with the

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protection afforded by A D H and A L D H genotypes predisposing to acetaldehyde accumulation. What is more surprising is that this sensitivity, or rather resistance to the inebriating effects of alcohol, may have a genetic basis [185]. It is not illogical, however, that the more one is able to drink, the more one will drink, thus risking the harmful effects of alcohol, including dependence. The interest of neurophysiologists in alcohol abuse has generated a sizeable literature on electrophysiological abnormalities associated with this condition and their possible inheritance. One of the most widely investigated parameters is the P3 component (Factor 2) of the Event-Related Brain Potential (ERBP). Two features of this component have been examined: its amplitude, and its latency in response to the applied stimulus (visual or auditory). There is overwhelming evidence for a reduction in P3 amplitude in alcoholics and their offspring [146,186,187]. The latency in response appears to be affected in a more subtle way. In keeping with its sedative effects, alcohol ingestion increases the P3 latency period, but recovery is faster among males whose fathers are alcoholics than among the sons of non-alcoholics [188]. Results of investigations using resting encephalographic (EEG) measures have been confusing. These have focused primarily upon alpha-wave activity which can be divided into slow-frequency and fast-frequency components. Ehlers and Schuckit [191] demonstrated that individuals at high risk for alcoholism had significantly more energy in their baseline fast alpha frequency than did controls. This is in contrast to other investigations which

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P3 ERBP

Electrophysiological

Endocrinologi- HPA-axis cal

fl-activity

-activity

Subjective criteria

Alcohol sensitivity

50

Marker

Category

Acute elevation of ACTH and cortisol in abstinenct alcoholics may be related to relapse risk Cortisol elevation following acute alcohol ingestion is higher in daughters of male alcoholics than controls

Reduced amplitude may be co-transmitted with alcoholism Increased latency in boys is associated with presence of A1 allele of D2 receptor More energy in fast frequency range is seen in sons of alcoholic fathers than in controls Sons of alcoholic fathers demonstrate greater decreases in mean frequency after ethanol than do controls Ethanol-induced changes in slow frequency range are related to the observed BAC in sons of alcoholic fathers Elderly and middle-aged non-alcoholics with alcoholic biological relatives show elevated levels in comparison with controls Increased risk for alcoholism is associated with greater activity in fast frequency range ACTH response to C R H is decreased in abstinent alcoholics

Sons of alcoholic fathers show faster return to baseline latency following ethanol consumption than do controls

[185]

Lower sensitivity at 20 years is associated with fourfold greater likelihood of developing alcoholism by 30 years Amplitude reduced in alcoholics and sons of alcoholic fathers

[199]

[196]

[196-198]

[1951

[194]

[193]

[192]

[189,190] [145] [191]

[188]

[146,186,187]

[185]

Ref.

Decreased sensitivity in sons of alcoholic fathers

Characteristic

Table 9 Selected markers of increased risk for alcohol abuse

r

t~ ,,q

5

Muscarinic

GABA'ergic

Adrenergic

CSF M H P G correlates negatively with family history of alcoholism and reported history of alcohol consumption Plasma GABA-like activity is lower in males at risk for alcoholism following ethanol ingestion Serotonin transporter ligand binding is diminished in brains of alcoholics Genetic sensitivity to the motor-impairing effect of moderate ethanol doses has a likely biological basis in GABAA receptor point mutation Subjects with positive family history for alcoholism display decreased cerebellar baseline glucose metabolism as well as blunted cerebellar metabolic response to lorazepam compared with controls Thalamic M1 and M2 receptor subtypes diminished in older (58-84 years) but not younger (19 57 years) chronic alcoholics compared with controls

Lower prolactin levels following acute ingestion of alcohol by daughters of male alcoholics than for controls Stimulated GH secretion is less in alcoholics who relapse compared to controls and alcoholics who abstain Gs alpha subunit quantity of temporal cortex is reduced in alcoholics

Prolactin

GH

Characteristic

Marker

[207]

[206]

[204] [205]

[2031

[202]

[201]

[200]

[199]

Ref.

ACTH adrenocorticotropic hormone; BAC, blood alcohol curve: CRH, corticotrophic hormone; CSF, cerebrospinal fluid; ERBP, event-related brain potential: GABA, gamma aminobutyric acid; GH, growth hormone; HPA, hypothalamo-pituitary-adrenal; MHPG, 3-methoxy-4-hydroxy-phenylglycol.

Signal transduction

Category

Table 9 (continued)

t3~

I kxa

t~ ,.q

2~

236

R.A. Ferguson, D.M. Goldberg / Clinica Chimica Acta 257 (1997) 199 250

have failed to demonstrate significant group differences in a variety of baseline EEG frequency band activities [192,193]. Nevertheless, high risk males demonstrate greater increases in slow alpha activity on the ascending curve (acute sensitization) and significantly faster recovery to baseline slow alpha activity during the descending phase (acute tolerance) of the blood alcohol curve compared to controls [193]. Many investigators have examined the effects of alcohol exposure and withdrawal upon components of the endocrine system. Hormonal responses to alcohol as well as changes in neurotransmitter behavior have been evaluated in the relatives of alcoholics as possible genetic markers of predisposition. These and other selected markers are described in Table 9. Finally, an RFLP of the human collagen type I A2 gene has been proposed as a means of predicting the susceptibility to liver damage in alcoholics, as well as suggesting an inherited basis for the latter [208].

8. Concluding remarks The preceding overview of genetic determinants of alcoholism and their molecular biochemistry betrays the immaturity of our present understanding in this area of scientific inquiry. The promise of the A1 allele of the D2 receptor gene as a direct marker for alcoholism and the euphoria with which this claim was initially greeted serve as a paradigm in this respect. Thus, the discovery of the 'alcoholism gene' with all its attendant therapeutic and preventative possibilities remains, for the time being, elusive. Nonetheless, important advances have been made in our understanding of the molecular basis of this disease, and undoubtedly they will continue. In the meantime, much can be done. First of all, a framework for the rigorous and standardized evaluation of potential genetic markers must be constructed. The diagnostic criteria of alcohol abuse and dependence and the measures of the severity of alcohol dependence need to be clarified. These problems are being addressed with strategies exemplified by the formulation of the DSM-IV. Extensive literature review, re-analysis of existing databases and multi-center field trials (stratified by sex, ethnicity and age) have been employed in order to establish empirical criteria [209] which can be applied by practitioners and research scientists alike for the diagnosis of alcoholism. The DSM-IV may not be the solution for all the problems in testing the genetic markers of alcoholism that have been identified in this review. Nevertheless, it is hoped that this new system will be an improvement over the old. Secondly, how should we use those markers that prove to yield respectable diagnostic efficiencies? Past experience has shown that universal

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primary prevention (i.e. targeting all individuals of the community) is largely ineffective and should be abandoned for, or at least complemented by, strategies which focus upon the sub-groups at risk within that community [210,211]. In this context, good genetic markers are expected to be invaluable tools for the identification of families and individuals who stand to benefit from preventative and/or interventional treatment. Thirdly, recognition of the genetic components of alcoholism is now firmly established and has led to expanded criteria for chemical dependency and an improved understanding of its causes. This is evidenced by the incorporation of genetic etiological criteria with environmental etiological criteria advocated by those of the psychosocial tradition to form a new theory of chemical dependency known as the biopsychosocial model [212]. Fourthly, genetic analysis of individual subjects may allow improved outcome in the therapy of alcoholism. There are many pharmacotherapeutic agents presently used in the treatment of alcoholism. These include dopamine receptor antagonists (e.g. tiapride) which are effective in reducing common abstinence symptoms such as anxiety, depression and insomnia [213]. The dopamine receptor agonist bromocriptine reduces the desire to drink, the frequency of drinking and the psychosocial dysfunction of chronic alcoholics [214]. Propranolol is an effective anxiolytic for alcoholics [215]. In addition, serotonin uptake inhibitors have been shown to be effective in, for example, reducing alcohol consumption and increasing the period of abstinence in heavy drinkers and alcoholics seeking treatment [216-220]. To date, the aforementioned pharmacotherapeutic agents have been restricted largely to research activity. Indeed, only two pharmacological agents are presently approved by the FDA for use in the treatment of alcohol dependence; these being disulfiram and naltrexone [221]. Disulfiram acts to inhibit ALDH activity, thereby causing the unpleasant alcoholrelated flush reaction described in Section 3. Its utility in the treatment of alcoholism, however, has been disappointing for many reasons, not the least of which is poor patient compliance [221,222]. In contrast to disulfiram, the opiate antagonist naltrexone has recently received much attention in the North American press, presumably because of its more recent FDA approval for use in the treatment of alcohol dependence. While the media hype surrounding naltrexone may be over-optimistic, it does appear to be efficacious in the treatment of alcohol dependence as shown by two independent double-blind, 12-week, placebo-controlled clinical trials [223,224]. Volpicelli et al. [223] have determined that naltrexone-treated alcoholic subjects have lower relapse rates, fewer drinking days and less craving for alcohol than do placebo-treated subjects. O'Malley and colleagues [224,225] have demonstrated that naltrexone treatment of alcoholics

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is more effective than placebo in reducing drinking and resolving alcoholrelated problems. Among subjects who sampled alcohol during the study, those who received naltexone and therapeutic counselling were least likely to relapse to heavy drinking [224]. The molecular mechanisms by which naltrexone exerts its effects are not fully known. Presumably, opioid receptor antagonism suppresses alcoholinduced dopamine release in the nucleus accumbens thereby blocking the reinforcing properties of alcohol [226]. Indeed, in retrospective analysis of subjects who lapsed from abstinence during their earlier study, Volpicelli et al. found that a greater proportion of the naltrexone-treated group, as opposed to the placebo-treated group, reported that the 'high' produced by alcohol ingestion was significantly less than usual [227]. The authors suggest that the diminution of pleasure associated with alcohol ingestion in the former group was a significant factor in their lower relapse rate [227,228]. These pharmacotherapeutic agents cover a wide spectrum of neurotransmission targets. Theoretically, they might be tailored to the specific genotype of the patient (e.g. adrenergic, dopaminergic and/or serotonergic system defects) in order to enhance their therapeutic outcomes. Support for this 'pharmacogenetic' approach is provided by a 6-week double blind placebo-controlled study of 83 alcoholics in which bromocriptine treatment outcome was evaluated in respect to D2 dopamine receptor genotype. The greatest alleviation of craving and anxiety was observed in the bromocriptine-treated alcoholics possessing the A1 allele whereas attrition was highest in the placebo-treated A1 alcoholics [229]. Certainly, more studies of this nature are warranted. Finally, the economic returns on programs that are successful in identifying and preventing alcoholism or improving its treatment are potentially enormous. Needless to say, however, both molecular biological testing and screening are expensive undertakings. Selected screening may be more cost effective than general population screening for alcoholism, but this remains to be established. Exacerbating these costs is the relatively lengthy period of time required to see the return on the investment of alcoholism treatment. For example, 4 years are required following the initiation of alcoholism treatment to see the health care costs of treated alcoholic cohorts fall to those levels observed for non-alcoholics [230]. Furthermore, the US spends $1.6 billion annually on alcohol and drug treatment, prevention and research. The bulk of this enormous sum (73%) is spent on treatment programs [231]. Yet this pales in comparison to the $177 billion which is lost from the US economy annually through the consequences of abusive alcohol consumption [232]. The potential savings should serve as a stimulus to continue research into the genetic components of alcohol abuse. If the present achievements are less than spectacular, they must not be permitted

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to discourage further effort and new investigative approaches. Cautious optimism should eventually prevail.

Acknowledgements We wish to thank Mrs Pat Machado for her assistance in preparing this manuscript. RAF was supported by an Ontario Ministry of Health Postdoctoral Fellowship in Clinical Biochemistry.

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