November 3-4, 1997
Rockville, Maryland

U.S. Department of Health and Human Services
Public Health Service
National Institutes of Health

ALCOHOL-RELATED GENETICS IN ANIMALS

REFERENCES

APPENDICES

 A summary of FY97 genetics awards is detailed below.

Genetics

Percentage of Genetics to Total

The National Institute on Alcohol Abuse and Alcoholism's (NIAAA) Subcommittee for the Review of the Extramural Research Portfolio for Genetics met on 3-4 November 1997. The charge to the Subcommittee was to examine the appropriateness of the breadth, coverage, and balance of the genetics research portfolio, identifying research areas that are well covered and others which are either under-investigated or which otherwise warrant significantly increased attention. The Subcommittee was asked also to provide specific external advice and guidance on the scope and direction of the Institute's extramural research activities in the genetics area.

The Subcommittee for the Review of the Extramural Research Portfolio for Genetics consisted of two NIAAA Advisory Council members (one of whom served as a co-chair), an expert in genetics served as the other co-chair, and an advisory group of six individuals. Three of these individuals have demonstrated expertise in alcohol-related areas and three individuals have demonstrated expertise in non-alcohol-related areas (see Appendix A).

The review process was initiated by having experts (see Appendix B) in genetics prepare written assessments of the state of knowledge, gaps in knowledge, and research opportunities. NIAAA program staff (see Appendix C) presented the current extramural portfolio, categorized into the areas of individuals at high risk, twin and adoption studies, linkage and association studies, and alcohol-related genetics in animals (see below). All information was shared with experts, selected NIAAA staff, and the co-chairs and advisory group before the meeting.

On 3-4 November 1997, experts and NIAAA program staff made abbreviated presentations of their material followed by discussion among all of the participants, including representatives from other NIH Institutes and guests (see Appendix D). After completing this process, the co-chairs and advisory group, with input from the experts, delineated the following list of research priorities. 

• Significantly increase the overall investment in the genetics portfolio and specifically increase support for young investigators to encourage them to enter into the field of the genetics of alcoholism.
• Increase the emphasis on the study of the development of alcohol use and misuse and the associated risk factors in genetically informative human populations. Such studies should consider issues of gene-environment interactions and correlation and the role of comorbidity.
• Increase emphasis on gene expression studies in animal models with the hope that these will develop to the point that they become viable in human populations.
• Increase the emphasis on the gene-finding techniques of linkage and association of alcoholism in human populations. This will require both the support of data collection efforts and the development of statistical and analytical techniques to optimally analyze such data.
• Develop centralized technology resources for animal models, including space for maintenance of transgenics, knockouts, knockins, and advanced intercrossed lines; cryopreservation; informatics; and development of web site.

 Other areas for consideration include: 

Sponsoring a conference on human-animal phenotypes, with goal of consensus on what new animal phenotypes should be developed.

• Development of inducible, tissue-specific genes in animals.
• Encourage/seduce animal geneticists studying learning and memory to include alcohol-related behavior.

 Psychosocial Phenotypes

 Genetic Basis for Alcoholism in Humans

 Twin and Adoption Studies

 Quantitative Trait Loci (QTL)

It has been acknowledged for some time that alcoholism aggregates in families (Cotton, 1979) and appears to be genetically influenced (Goodwin et al., 1973). These observations have resulted in a number of studies designed to compare the offspring of alcoholic parent(s) with those of nonalcoholic parents in order to determine biological and psychosocial characteristics that might predispose an individual to consume excessive amounts of alcoholic beverages when exposed to opportune environmental circumstances. Because these investigations of populations at high-risk for alcoholism study individuals prior to the development of alcohol dependence/ abuse, they may be more likely to reveal causative factors. High-risk methods can also be used to examine gene-environment interactions.

Most studies have defined individuals at high risk through the presence of at least one alcoholic parent. This was usually the father because of concern over the environmental consequences of excessive alcohol consumption by a pregnant mother, i.e., the fetal alcohol effect. Whenever possible, subjects with multiple alcoholic relatives (high-density families) are selected (Yuan et al., 1996). It is also important to control for the effects of comorbidity among alcoholic probands and family members (Hill and Neiswanger, 1997) and to examine multiple phenotypes simultaneously, e.g., level of response to alcohol and P300 amplitude.

BIOLOGICAL PHENOTYPES ASSOCIATED WITH INDIVIDUALS AT HIGH RISK FOR DEVELOPING ALCOHOL-RELATED DISORDERS

State of Knowledge (Shirley Y. Hill, Ph.D. and Marc A. Schuckit, M.D.)

A number of biological characteristics have been associated with a family history of alcoholism. These are briefly outlined below. Space constraints do not allow for a detailed review of the specific studies, and the interested reader should carefully review the sample size and characteristics and appropriateness of the research methods for each study before drawing conclusions.

Neurotransmitter-Related Systems

Opioids - A possible relationship has been suggested between endogenous opioids and alcohol consumption. Activation or blockade of the opioid system has been shown to alter alcohol consumption in animals (Reid and Hubbell, 1990). Lower beta-endorphin-like immunoreactivity has been reported in family history positive (FHP) individuals (Gianoulakis et al., 1996), and there is evidence of a greater increase in beta endorphins following alcohol consumption in high-risk subjects (Peterson, et al., 1996). Proposed mechanisms for this relationship are complex and include differences in concentrations of opiate peptides, possible actions of opiate-producing neurons inhibiting CRH production, interactions between inherent levels of opiates and reactivity of the hypothalamic pituitary adrenal system, and innate activity levels (Gianoulakis et al., 1996; George et al., 1991).

Serotonin (5-HT) - In general, increased serotonergic functioning decreases alcohol consumption, and decreased serotonergic functioning increases consumption in animals and humans. There are at least 14 mammalian 5-HT receptors, and the relationships between them and alcohol consumption are complex. Although several of the receptor subtypes have been reported to be altered in alcohol dependence, including 5HT_1B (Crabbe et al., 1996), 5-HT_2C (Pandey et al., 1996; George et al., 1997), and 5-HT₃ (Grant, 1995; Sellers et al., 1994), it is unclear whether they are altered in individuals at risk.

GABA - GABA is an inhibitory neurotransmitter that has been demonstrated to mediate some of the effects of alcohol (Volicer and Biagioni, 1982). The GABA_A receptor system is responsive to the effects of alcohol at clinically relevant doses, and some studies have reported that alcohol-dependent individuals have lower levels of activity of this receptor (Parsian et al., 1997). Alcoholics might have an increased prevalence of at least one GABA_A receptor gene (a₃ ) (Parsian et al., 1997), and additional evidence indicates a decrease in the a₁ subunit (Hoffman and Tabakoff, 1996). Limited data are available on FHPs and FHNs, and what is available is contradictory (Moss et al., 1990; Cowley et al., 1996).

Dopamine (DA) - Some aspects of the reinforcement of behavior involves the brain=s DA pathways (Hoffman and Tabakoff, 1996), with evidence from animals of the importance of DA activity in the rewarding effects of acute alcohol administration (Hodge et al., 1994; Pfeffer and Sampson, 1988). Although the initial report of a population-based association between alcoholism and the dopamine D2 receptor locus (DRD2; Blum et al., 1990) generated considerable interest, evidence for the presence of either association and/or linkage of the alcoholic phenotype to allelic variation in DRD2 remains controversial (Gelernter et al., 1993; Turner et al., 1997). It has recently been suggested that the nature of the control group may determine whether significant population-based associations are found (Neiswanger et al., 1995).

Only limited data regarding DA are available on FHPs and FHNs. One study noted an increase in the DA metabolite homovanillic acid in the younger sons of substance-abusing fathers (Gabel et al., 1995), while another (Schuckit et al., 1981) noted no differences between FHPs and FHNs in dopamine beta hydroxylase, an enzyme that metabolizes DA.

Adenylyl Cyclase (AC) - AC is part of a complex biological system composed of at least three membrane-bound proteins, including cell membrane receptors, G (guanine nucleotide binding) proteins, and the AC enzyme. The G proteins operate by coupling with cell membrane receptors, resulting in changes in c-AMP. There are two forms of G proteins, one that stimulates (G_s) and one than inhibits (G_i). The c-AMP is a Asecond messenger @ that has numerous effects within the cell, including gene expression. The activity of AC can be measured by the production of c-AMP under baseline conditions or following chemical stimulation.

Alcoholics have reduced c-AMP in platelets and lymphocytes after stimulation (Parsian et al., 1996), with subsequent recovery after multiple generations in cell culture (Nagy et al., 1988). It has been proposed that these findings may result from a G_sa protein (Wand et al., 1994). Greater activity levels of G_sa have been observed in red blood cells and lymphocytes of FHP subjects with no group differences on various measures of G_i (Wand et al., 1994).

Monoamine Oxidase (MAO) Activity - MAO is a mitochondrial enzyme responsible for the degradation of biological monoamines. There are multiple forms of MAO (A and B), with the levels of each under genetic control (Hsu et al., 1996). In some studies, alcoholics were reported to demonstrate lower MAO-B activity, especially if the antisocial personality is present (Anthenelli et al., 1995), while other investigators reported no major differences between alcoholics and controls. Lower (Schuckit et al., 1982) and higher (Lex et al., 1993) activity of MAO-B have been reported in the relatives of alcoholics. Unfortunately, many studies did not appropriately control for the effects of smoking on MAO activity.

Alcohol is primarily metabolized by the enzyme alcohol dehydrogenase (ADH), with smaller amounts broken down by cytochrome P450 as part of the microsomal ethanol oxidizing system (MEOS), especially at higher blood alcohol concentrations. The remainder of this drug is metabolized by catalase and additional enzymatic and nonenzymatic pathways (Lieber, in press). Each of these mechanisms produces acetaldehyde, which is rapidly metabolized by the enzyme aldehyde dehydrogenase (ALDH). Both ADH and ALDH are under genetic control.

Alcohol Dehydrogenase (ADH) - There are at least seven different ADH-related genes producing at least eight forms of this enzyme, with ADH2 (for which there are at least three different alleles) and ADH3 (for which there are at least two different alleles) having greatest clinical relevance (Kitson and Weiner, 1996). Some forms of ADH2 (ADH2-2) and ADH3 (ADH3-1) are associated with a faster rate of metabolism of alcohol (Higuchi et al., 1996). A negative relationship between ADH2-2 and alcohol consumption or the risk of alcoholism has been reported in Asians and Israelis (Higuchi et al., 1996; Neumark et al., in press). The presence of ADH3-1 has been associated with a lower risk for alcoholism in the Chinese, along with an increased risk for liver or pancreatic disease among drinkers from England (Thomasson et al., 1991; Day et al., 1991).

Additional Alcohol-Metabolizing Systems - At least one gene affecting the cytochrome P450 system has been reported to interact with the ALDH-2 allele in affecting the risk for liver disease (Tanaka et al., 1997). It has been proposed that variations in catalase might be related to alcohol consumption (Koechling et al., 1995).

Aldehyde Dehydrogenase (ALDH) - There is some indication that low levels of acetaldehyde may be stimulating and reinforcing (Amit et al., 1980). High levels are known to produce skin flushing, rapidly changing blood pressure, nausea and vomiting, and a rapid pulse. There are at least two forms or isoenzymes of ALDH, the major mechanism of metabolism of the acetaldehyde produced from ADH action on alcohol (Wall and Ehlers, 1995). The mitochondrial ALDH2 is biologically active at low acetaldehyde levels, and has at least two variants controlled by genes on chromosome 12 (Higuchi et al., 1996). ALDH2-1 codes for a functional form of this enzyme, while ALDH2-2 is biologically inactive. Individuals who are homozygotes for ALDH2-2 experience an intense flushing after consuming alcohol, whereas heterozygotes flush, but not with great intensity. ALDH2-2 homozygotes represent 5-10% of the Asian population and have close to 0% risk for alcoholism (Higuchi et al., 1996). Heterozygotes represent 30-40% of Asians and show significantly lower risk for alcoholism than individuals homozygote for ALDH2-1. The impact of the ALDH2-2 heterozygote state is affected by environmental factors, including living in heavier drinking environments or demonstrating the antisocial personality disorder.

Hormones 

No large-scale comparisons of people at high and low risk for alcoholism have demonstrated baseline differences in cortisol (Schuckit, 1984), prolactin (Schuckit et al., 1983), or ACTH (Schuckit et al., 1988). Although there is one report of higher levels of thyroid-stimulating hormone (TSH) in FHPs versus controls (Moss et al., 1986), there is no reported increase in TSH following stimulation with thyrotropin-releasing hormone (Monteiro et al., 1990).

In some studies, FHPs are more likely to demonstrate problems with attention, organizational skills, abstracting, planning, reflectivity, and impulsivity, while also showing increased evidence of motor activity or diminished standing steadiness (Gillen and Hesselbrock, 1992; Peterson et al., 1992). There are, however, a relatively large number of studies reporting a general absence of group differences in cognitive functioning (Workman-Daniels and Hesselbrock, 1997). Similarly, there are no clear findings on FHP/FHN differences in IQ (Hesselbrock et al., 1991). The differences in the findings across studies might be related to characteristics of the specific populations evaluated. The most fruitful area for future research appears to be in measures of planning, impulsivity, and abstracting abilities.

It has been hypothesized that FHPs might have an increased cardiovascular response to stress (Newlin and Thomson, 1990) along with an ability of alcohol to improve this condition (Finn et al., 1990). This idea has been corroborated in a limited number of studies, although there are a number of inconsistent results.

It has been demonstrated that detoxified alcoholics and FHP children have a reduced amplitude of the scalp-positive wave that peaks approximately 300 msec after a rare but anticipated event (P300) (Begleiter et al., 1984). The relationship of a family history of alcoholism to a reduced P300 amplitude was demonstrated in two small follow-up studies wherein increased rates of substance-related problems were observed in those FHP children with reduced P300 amplitudes (Berman et al., 1993; Hill et al., 1995). The heritability of P300 amplitude (Aston and Hill, 1990; O= Connor et al, 1994) suggests that reduced-amplitude P300 could be transmitted from alcoholic parent to offspring.

In contrast to the P300 studies, the results of EEG studies have been more inconsistent. Most studies specifically addressing possible differences between FHP and FHN groups have been negative. The three exceptions have noted that FHP was associated with higher amounts of fast activity (Gabrielli et al., 1982) or more fast alpha (Bauer and Hesselbrock, 1993; Ehlers and Schuckit, 1991).

Alcohol challenge studies compare FHPs and FHNs on responses to alcohol as measured by subjective feelings, motor performance, and/or physiologic measures (Schuckit, 1994). Genetic factors appear to be involved in the level of response to alcohol, because identical twins are more similar than fraternal twins on this characteristic (Heath and Martin, 1992; Rose et al., 1994), and because animals can be bred to show high or low alcohol reactions. The ability of a low level of response to alcohol to predict alcoholism is supported by the finding that a low level of response to alcohol at age 20 significantly predicted an increased likelihood for a subsequent diagnosis of alcohol abuse or dependence in 453 men. (Schuckit and Smith, 1996) and by a 10-year follow-up of about 100 men in Denmark (Volavka et al., 1996).

There are many more cross-sectional studies of the level of response to alcohol. A meta-analysis of 10 studies revealed a lower subjective response to alcohol in FHPs (Pollock, 1992). This study also determined that the differences occurred only after active alcohol challenge, not placebo, and was seen at both rising and falling blood alcohol concentrations. Although there are some studies that did not observe this phenomenon (e.g., de Wit and McCracken, 1990), they tended to use small samples, relatively low blood alcohol concentrations, or a prolonged multistep procedure for the administering alcohol.

Most studies have reported a lower level of alcohol-induced changes in body sway in FHPs (Schuckit et al., 1996). Compared to the differences in subjective level of intoxication, the data on standing steadiness after consuming alcohol are less plentiful and consistent.

Less persistent alcohol-related changes have been reported for P300 latency of FHPs (Schuckit et al., 1988), along with less of a decrease in the fast alpha component of the cortical EEG (Ehlers and Schuckit, 1991). In contrast, other investigators have shown greater EEG changes for FHPs, especially during increasing blood alcohol concentrations (Bauer and Hesselbrock, 1993; Cohen et al., 1993). Differences among studies include analysis of different electrode positions, frequency bands, and levels of alcohol consumption. Some investigators have observed that the higher the density of alcoholism in families, the greater is the decrease in alpha following consumption of alcohol (Volavka et al., 1996). However, a less intense decrease in alpha with alcohol consumption at 19 years of age predicted alcohol dependence 10 years later; this relationship was also observed for beta activity (Volavka et al., 1996).

There are a number of other biological measures that have been shown to differ after alcohol consumption as a function of family history. A decreased endocrine response to alcohol in FHPs has been reported for cortisol, ACTH, and prolactin (Schuckit et al., 1996), and FHPs appear to be resistant to the ability of alcohol to blunt the ACTH response to CRF (Waltman et al., 1994). Although some studies have reported alcohol-associated increases in heart rate in FHPs males (Conrod et al., 1997), this finding has not been observed in females or in other studies (Bauer and Hesselbrock, 1993). FHPs, especially those with multigenerational family histories of alcoholism, are more likely to show a greater stress-dampening effect after consuming alcohol (Finn and Pihl, 1987; Levenson et al., 1987). FHPs demonstrate a greater increase in beta-endorphin-like material following alcohol consumption (Peterson et al., 1996).

The specificity of response to an alcohol challenge has been examined by comparisons with those induced by IV diazepam. In contrast to alcohol consumption, FHPs demonstrated no diminished response to diazepam on subjective high, body sway, or several hormones (Schuckit et al., 1991). However, in another study, FHPs administered logarithmically increasing doses of IV diazepam showed significantly higher levels of high and euphoric feelings, and decreased changes in memory, self-rating of sedation, and several biological measures (Cowley et al., 1992, 1994).

Accurate family history needs to be determined, especially quantification along the dimensions of number of relatives with alcohol-related disorders, degree of relatedness to those individuals, severity of alcoholism, and classification of families as unilineal or bilineal.

Comorbidity of major psychiatric disorders and the antisocial personality disorder (ASPD) is an important consideration in these studies. One strategy to deal with this problem is to investigate individuals with only a diagnosis of alcohol abuse or dependence in order to define genes specific to alcohol-related disorders. Another strategy is to include comorbidity as a specific variable in order to increase the likelihood of generalization to the general population.

Studies which are limited to investigations of single domains, though informative, may have less potential to explain major portions of the variance in outcome among high-risk individuals. Combining information concerning such diverse areas of inquiry as temperament and information processing characteristics will have greater likelihood of explaining more of the variance in outcome among high-risk populations than either alone.

Gene expression has been shown to vary with development. Behavioral markers relevant in childhood (e.g., Attention Deficit Hyperactivity Disorder, P300 amplitude) may not be relevant in adulthood and vice versa. Hence, alcohol-related, age-specific genes are likely, necessitating the study of genes and neurobehavioral markers with developmental trajectories.

Longitudinal studies are required to determine the relationships between possible phenotypes of the risk for alcoholism and the "endpoint " phenotype of alcohol dependence/abuse.

Alcohol challenge studies can provide potentially unique and informative biologic phenotypes that are defined by specific changes accompanying the consumption of alcoholic beverages. Placebo and active drug controls are necessary to insure specificity.

There is a need for much more research in defining biological phenotypes in high-risk samples.

PSYCHOSOCIAL PHENOTYPES ASSOCIATED WITH INDIVIDUALS AT HIGH RISK
FOR DEVELOPING ALCOHOL-RELATED DISORDERS

State of Knowledge (Kenneth J. Sher, Ph.D.)

The classification of alcohol-related disorders has challenged researchers and clinicians for many years. There appears to be a general consensus in the research community that alcohol-related consequences (negative life events consistent with alcohol abuse) and alcohol dependence are the central phenomena in the pathological involvement with alcohol. The formal diagnosis of alcohol abuse or dependence has been characterized as little more than acknowledging relatively severe symptoms related to drinking, and the general issue of whether alcohol-related disorders are best conceptualized as categories or continua is unresolved.

Attempts to explain the heterogeneity associated with alcohol-related disorders have characterized various subtypes based on drinking patterns and symptoms (Jellinek, 1960), personality (Knight, 1937), family history (Penick et al., 1978), age of onset (Buydens-Branchey et al., 1989), course over the lifespan (Zucker, 1987), and coexisting psychopathology (Winokur et al., 1971). Although there appears to be consistent evidence for at least two clusters, broadly termed a personality disorder cluster and a neurotic cluster (Morey and Blashfield, 1981), it is unclear if these sources of variation are best conceptualized as clusters of discrete subtypes or as multiple dimensions.

The development of pathological alcohol consumption involves pharmacological vulnerability (sensitivity, development of tolerance), alcohol-related expectancies, affect regulation, and socialization processes (and associated impulsivity and antisocial behavior). Moreover, the trait of pathological alcohol consumption involves both severity and chronicity, and expressions of this phenotype can be influenced by various broad dispositional variables, such as temperament and general motives for alcohol use, as well as shorter-term situational variables (state characteristics) that tend to promote or inhibit consumption.

Alcohol-related disorders appear to be primarily a disorder of late adolescence and young adulthood, with the highest prevalence occurring in the 18-29 age group (Grant et al., 1994). This finding occurs for both abuse and dependence diagnoses and for gender and race. These findings suggest that alcohol-related disorders may be developmentally limited (Zucker, 1987) and that chronicity may be of importance. Concurrent with the normative decrease in alcohol-related disorders in early adulthood are major changes in social roles, e.g., decreases in the prevalence of heavy drinking as a function of marriage, pregnancy, and full-time employment and homemaker status (Bachman et al., 1997). It is clear that alcohol-related disorders are embedded within a social-developmental context, including psychological comorbidity.

There are a number of ways of representing or modeling the complexity of the course of pathological alcohol involvement over time. Autoregressive models assume that behavior assessed at one time is a function of behavior at a preceding time; advantages and disadvantages of this approach for alcohol involvement have been discussed (Sher and Wood, 1997; Windle, 1997). A relatively new class of latent-variable models which decomposes each measurement into a state and trait component (Steyer et al., 1992) might be particularly useful for quantifying the persistence of pathological alcohol involvement and for distinguishing between variables that have transient and enduring effects.

There is a great deal of arbitrariness concerning the conceptual and operational definition of alcohol use disorders. A new conceptual framework for the diagnosis of pathological alcohol involvement is required, one that is more dimensional, more developmental, and that incorporates chronicity and a range of contextual, personality, and psychopathological variables.

Further development of statistical modeling approaches that emphasize change over time should be encouraged. These include various forms of growth curve, latent variable, and state-trait techniques. Thorough specification of the functional (process) relationships between various components of the models are necessary to further understanding.

DESCRIPTION OF PORTFOLIO FOR HIGH RISK FOR DEVELOPING ALCOHOL- RELATED DISORDERS (Ellen Witt, Ph.D. and Kendall J. Bryant, Ph.D.)

The Division of Basic Research supports thirteen grants that are directly related to the identification of neurobiological and behavioral markers of alcoholism risk. Historically, the approach to investigate vulnerability to alcoholism has been family studies. A variety of physiological and biological measures have been used to distinguish individuals at high risk, e.g., those with a positive family history for alcoholism from individuals at low risk, e.g., those with a negative family history for alcoholism. The most widely studied neurobiological marker is brain electrical activity, including electroencephalographic (EEG) and event-related potential (ERP) measures. Currently, five grants are studying EEG/ERP measures in conjunction with personality, neuropsychological, and other cognitive measures. One study is also examining biobehavioral mechanisms underlying the relationship between disinhibited/antisocial personality and alcohol abuse using a combination of self-report questionnaires and behavioral-psychophysiological measures in standard conditioning paradigms.

Other biological markers, such as alcohol sensitivity/tolerance, body sway, cortisol and ACTH levels, and G proteins, are also being examined in high- and low-risk individuals. These grants are focussed on young (3-11 years) and/or adolescent (12-18 years) children of alcoholics and have incorporated studies targeted at female children of alcoholics. Two grants are longitudinal in nature in that they have been following cohorts of young and adult children of alcoholics, one of which will extend to a 20-year follow-up at the end of the current grant period. Longitudinal prospective studies are particularly useful because only these types of designs can provide information about the predictive ability of potential markers.

Four grants, including two Career Development Awards, are focussed on risk markers in populations of Asians and Native Americans. A unique feature of these studies is that genetic markers are used, specifically alleles of the alcohol dehydrogenase and aldehyde dehydrogenase enzymes, in addition to electrophysiological, biological, and behavioral measures, to identify individuals at high risk for developing alcoholism. One study is measuring candidate genes from the dopaminergic system (DRD2 and DRD4) as potential markers. Once genes for alcoholism are identified, the integration of molecular genetic, environmental, biological, and behavioral measures in a longitudinal prospective design will allow us to selectively predict susceptibility to alcoholism.

Three additional grants are located within the Treatment Research Branch of the Division of Clinical and Prevention Research and involve comparisons of high-risk and low-risk individuals; total amount of FY97 funds for these three grants is $678,100.

The following table provides a summary of these grants by mechanism, number of grants, and expenditures for FY97.

Genetic Basis for Alcoholism in Humans

TWIN AND ADOPTION STUDIES OF ALCOHOLISM

 State of Knowledge (Matt McGue, Ph.D.)

Twin and adoption studies have been used to estimate the separate contribution of shared environmental and genetic factors in the familial transmission of alcoholism. Adoption studies provide the most direct method of assessing the separate contribution of genetic and shared environmental factors to alcoholism risk, i.e., an individual removed from her or his biological parents in infancy and placed with nonbiologically-related adoptive parents will share only genetic factors with her or his biological relatives and shared environmental factors with her or his adoptive relatives. In twin studies, the similarity of reared-together, genetically identical monozygotic (MZ) twins is compared with the similarity of reared-together, genetically nonidentical dizygotic (DZ) twins, who share an average of 50% of their segregating genes.

Genetic Factors Influence Risk of Alcoholism in Men 

Almost every published adoption study has reported a significantly higher rate of alcoholism among the reared-away sons of alcoholics than among the reared-away sons of nonalcoholics (e.g., Cadoret et al., 1985). These findings are consistent with those from twin studies wherein the MZ concordance is consistently higher than the DZ concordance (e.g., Kendler et al., 1997).

Adoption and twin studies have been less consistent in women, with only two of five adoption studies demonstrating a significant biological parent effect, and significantly greater MZ than DZ concordance for alcoholism in women was observed in only three of six studies. However, significant effects have been observed in the best designed studies (Bohman et al., 1981; Kendler et al., 1994).

Large-scale twin surveys of community samples have documented genetic influences on adult alcohol consumption patterns in Australia (Heath and Martin, 1994), Finland (Kaprio et al., 1992), Sweden (Medlund et al., 1977), United Kingdom (Clifford et al., 1984), and United States (Carmelli et al., 1993). Genetic influences appear to be present in women and men. An alcohol challenge resulted in heritable ratings of intoxication (Neale and Martin, 1989) and body sway (Martin, 1990). Inconsistent findings have been reported for adolescent twin studies of alcohol use (Loehlin, 1972; Koopmans and Boomsma, 1996).

In studies of male twins, heritability estimates are remarkably consistent and range from 50-60% of the variance in alcoholism liability (Kendler et al., 1997). The precise magnitude of genetic influence in women is indeterminate. Cloninger and colleagues (1981) have reported heritability estimates of 90% for the liability of Type II alcoholism and less than 40% for the liability of Type I alcoholism. Many of the key findings of Cloninger and colleagues (1981) have been independently replicated (Sigvardsson et al., 1996). Few twin studies have investigated subtype heritability, and the results have been inconsistent.

Personality (Sher and Trull, 1994), psychopathology (Helzer and Pryzbeck, 1988), psychophysiological markers (Begleiter et al., 1984), and alcohol sensitivity (Schuckit, 1994) are each heritable and each have been associated with alcoholism risk. Although there have been few adoption and twin studies designed to characterize such mediators, Heath and colleagues (1997) demonstrated heritable variation in twin personality and psychopathology that did not explain all of the genetic influences on alcoholism risk.

Another complexity is the direction of causal influence. For example, in a large cross-section study of female twins, genetic factors were shown to contribute substantially to the comorbidity of major depression and alcoholism (Kendler et al., 1993), but without longitudinal observations, the authors were unable to determine whether the common genetic influence reflected an effect of alcoholism on depression risk, or conversely, an effect of depression on alcoholism risk.

Twin and adoption studies of alcoholism support the existence of environmental as well as genetic contributions to alcoholism risk.

It is possible that environmental liabilities associated with being reared by an alcoholic parent are contributory to the development of alcoholism. The results appear to be inconsistent, with U.S. adoption studies demonstrating an association between adoptee alcoholism and history of alcoholism in the adoptive family (Cadoret et al., 1987), while Danish (Goodwin et al., 1973) and Swedish (Cloninger et al., 1981) studies failed to demonstrate increased risk. Alternative designs (e.g., offspring of twins, twin-family, full and half-sibling) can be used to investigate environmental contributions not confounded by genetic effects.

One way that genetic and environmental factors might combine to influence alcoholism risk is synergistically. Genotype-environment interaction refers to a differential sensitivity of genotypes to environmental influences, i.e., an individual is more likely to develop alcoholism when a vulnerability is inherited and rearing takes place in a provocative environment. For example, in male adoptees with relatively low genetic risk, rearing status was unrelated to rate of severe Type I alcoholism, but among individuals with relatively high genetic risk, being reared in a provocative environment significantly increased the likelihood of developing severe Type I alcoholism (Sigvardsson et al., 1996). Genotype-environment correlation may provide another model for the joint influence of genetic and environmental factors on alcoholism.

Inheritance of alcoholism in non-Caucasians is understudied.

Similarities and differences should be determined among alcoholics ascertained with different strategies, e.g., alcoholics ascertained from community surveys may be different than those ascertained from alcoholism treatment programs.

It is uncertain whether alcoholism liability is less heritable in women than men, and it is important to determine whether the genetic factors underlying risk of alcoholism in women are the same as those in men.

It would be useful to determine if there are etiologically distinct forms of alcoholism, distinguishable in part by their pattern of heritability and in part by their pattern of concordance among genetically identical individuals.

It is important to characterize phenotypes and processes that mediate relationships among gene products and overt behavior.

Emphasis should be placed on determining critical environmental factors and how they combine with genetic factors to influence alcoholism risk.

DESCRIPTION OF PORTFOLIO FOR TWIN STUDIES

Division of Clinical and Prevention Research (Kendall J. Bryant, Ph.D.)

There are seven active grants and two with no-cost extensions for a total of $3.4M dollars administered within the Division of Clinical and Prevention Research in FY97. Of these grants, seven are investigator-initiated awards (R01) and two are K awards. Eight of the grants are managed within the Prevention Research Branch and one within the Health Services Branch. Individual grants costs range from $200 to 900K.

Virginia Registry, cohorts of adolescent female, twin pairs and their parents (N=1,785 pairs, N=2,163)

Finnish Adolescent (3,000 twin pairs)

Twin Family Assessments (675 families)

Australian Adolescent Twin Pairs (N=4,500 twin pairs)

Vietnam Twin Era Adult Twins (n=2,400, N=400 Harvard sample)

Current research portfolio includes research on the following topics.

A generation of behavioral genetic research has established the importance of a genetic influence for alcoholism in males. Less is known about the extent to which genetic factors influence women's drinking behaviors and the existence of differential mediators and moderators of genetic risk. Studies are being carried out to replicate and extend past research findings.

New methodology is being developed and tested to understand models for the natural history of alcohol abuse. These methodologies focus on developmental longitudinal models, identification of periods of differential risk for genetic and environmental influences, and the understanding and modeling of trajectories to alcohol abuse and dependence from childhood to adulthood. New methodologies employ simulation studies using high-risk samples, causal modeling using a variety of improved estimation procedures, and understanding of reciprocal causation (gene-environment correlation and interaction).

Of central importance to understanding the development of alcohol abuse and dependence and related behavioral problems is the interaction of early precursors of the predisposition toward alcohol problems and the environment.

Researchers are articulating conceptual models that identify broad personality components as precursors to alcohol problems which involve genotype-environment interactions. These interactions are hypothesized to be the mechanisms underlying transition from adolescent personality to adult psychopathology. Dimensions that have been studied include emotional reactivity, socialization, self-control, and other co-occurring disorders such as ADD, hyperactivity, etc.

Future Issues Include:

(1) Improving measurement of mediators and moderators of gene x environment interactions; (2) improving longitudinal statistical models - for example, the integration of latent-growth models with latent class and related genetic models; and (3) understanding the role of the environment - for example, better measurement of sibling and peer influence on alcohol availability.

Implications for Prevention 

Identification environmental influences and their interaction with genetic susceptibility is critical to knowing how to develop and implement preventive interventions. For example, while onset of alcohol drinking is largely under environmental control, latency to problem drinking behavior, once drinking behavior has begun, is primarily genetic. Clearly, greater prevention efforts should be directed at delaying initiation of drinking in adolescents. Other issues include the appropriateness of genetic counseling for individuals with family histories of alcoholism and the delivery of appropriate advice based on developing scientific evidence.

How can behavioral genetic information inform treatment: (1) studies of twin populations may identify the relative environmental and genetic components of the treatment process including treatment-seeking behavior and comorbid substance use (e.g., tobacco), (2) targeted pharmacotherapy can be developed with identification of genetically controlled biological mechanisms underlying alcoholism, and (3) articulating the phenotype for alcoholism (typologies) may improve treatment matching outcomes.

Division of Biometry and Epidemiology (Page Chiapella, Ph.D.)

The Division of Biometry and Epidemiology portfolio consists of two grants at a current direct cost of $601,592 (one grant currently has a no-cost extension). The portfolio uses population-based twins to evaluate genetics and social factors and their interactions in the development and temporal consistency (maintenance) of drinking and alcohol-related problems. Females are a special focus and are substantially over-represented in the study populations. Male/female comparisons are also emphasized. Health and social consequences of genetically-related alcohol dependence are additional areas covered by the grants. Extensive information has been collected on subjects, including data on early lifetime experiences; illicit substance use; and detailed history of psychopathology, assessed using an adaptation of the SSAGA interview (designed by COGA) which includes the core DSM-based diagnosis components and a comprehensive array of measures of alcohol-related behaviors. Long-term longitudinal follow-up is an important tool used in evaluating development and comorbidites of alcohol-related problems. Many study subjects have been followed for over 15 years. DNA has been obtained from over 3,000 subjects for future genotyping to identify genetic risk and protective factors.

HETEROGENEITY OF HUMAN PHENOTYPES ASSOCIATED
WITH ALCOHOL-RELATED DISORDERS

State of Knowledge (C. Robert Cloninger, M.D.)

In the general population, most of the variation related to alcohol use and associated problems is explained by frequency of drinking and only a small amount of variance is attributable to differences in alcohol dependence. Factor analytic studies (Muthen et al., 1993;1995) of current drinkers (N=22,102) in the National Health Interview Survey revealed two factors, approximating those of alcohol abuse and dependence as described in DSM-IV and ICD-10. Factor 1 corresponded to alcohol abuse and explained most of the variability among current drinkers; it was distinguished by consuming larger amounts than intended and hazardous drinking, such as driving a car after drinking or creating a hazard for others by drinking. Factor 2 corresponded to alcohol dependence; it was distinguished by wanting to cut down, giving up important activities to drink, continuing despite knowledge of problems from drinking, drinking to relieve or avoid withdrawal symptoms, and recurrent arrests or police trouble from drinking. Individual symptoms of abuse without dependence were more common (10-25%) than symptoms of dependence (< 3%). These general population results are similar to the results of Partanen and associates (1966) for a group of twins unselected for alcoholism in Finland.

In families of alcoholics, drinking is sufficiently frequent that most individual differences are attributable to differences in the severity of dependence. Bucholz and colleagues (1996) used a latent-class analysis to obtain a four-class solution for both men and women: (1) non-problem drinkers; (2) mild alcoholics with persistent desire to stop, tolerance, and blackouts; (3) moderate alcoholics with social, health, and emotional problems; and (4) severe alcoholics with withdrawal, inability to stop, craving, plus health and emotional problems. Individuals in classes 3 and 4 usually met DSM-IIIR criteria for alcohol dependence, as did the men in class 2. Bucholz and colleagues (1996) concluded that these classes represent gradients in severity, not distinct subtypes. Even though most of the variability in the symptoms of alcoholics is explained by a single severity dimension related to dependence, about 20% can be usefully explained by subtyping alcoholics into two subtypes.

The first multivariate classification of alcoholic subtypes related to genetic and neurobiological differences was the distinction between Type 1 and Type 2 alcoholism (Cloninger et al., 1981). Type 1 alcoholism was characterized by onset of alcohol-related problems after 25 years of age, infrequent fighting and arrests when drinking, infrequent spontaneous alcohol seeking, frequent loss of control, and frequent guilt and fear about alcohol dependence, whereas Type 2 alcoholism was characterized by onset of alcohol-related problems before 25 years of age, frequent fighting and arrests when drinking, frequent spontaneous alcohol seeking, infrequent loss of control, and infrequent guilt and fear about alcohol dependence (Cloninger, 1987). There were also differences in personality traits: Type 2 is high in novelty seeking and low in harm avoidance and reward dependence; Type 1 is low in novelty seeking and high in harm avoidance and reward dependence. High novelty seeking and low harm avoidance have been shown to predict early-onset alcohol abuse by Cloninger and colleagues (1988), resulting in the formulation that early onset alcoholism is frequent in boys with antisocial or explosive (borderline) temperaments. A recent review by Howard and colleagues (1997) concluded that novelty seeking predicts early-onset alcoholism, criminality, and other substance abuse.

Another approach to the derivation and validation of a multivariate subtyping of alcoholism has been carried out by Babor and colleagues (1992). Type A is characterized by later onset, fewer childhood risk factors such as hyperactivity or conduct disorder, less severe dependence, fewer alcohol-related problems, less treatment for alcoholism, and less psychopathology. Type B is characterized by early onset, childhood conduct disorder, more severe dependence, more treatment, and more psychopathology. The Type A versus B distinction has been replicated (Schuckit et al., 1995). There are similarities between Type 1 and Type A and between Type 2 and Type B.

Several features emerge repeatedly as robust indicators of phenotypic heterogeneity, including age of onset, gender, childhood conduct disorder, antisocial personality disorder, severity of dependence, and polysubstance abuse. Each of these variables has been advocated as the preferred basis for subgrouping alcoholics. However, the overlap among such univariate subdivisions is imperfect, and there is no consensus on the optimal criterion for calibrating a multivariate classification.

Longitudinal developmental studies are needed with personality and psychopathology measured along with alcohol consumption. Developmental studies in families (especially those of twins) and birth cohorts are particularly important.

It would be informative to study personality, alcohol and other drug use, and other kinds of lifestyle choices, e.g., exercise and diet. Such comparisons would help to distinguish what is specific for alcohol and particular drugs and what is nonspecific for various forms of dependence or maladaptive behavior.

Phenotypic specification should include quantitative measures of personality, dependence, quantity and frequency of alcohol consumption, and measures of other associated psychopathology.

ALCOHOLISM COMORBIDITY WITH PSYCHIATRIC DISORDERS

 State of Knowledge (Kathleen R. Merikangas, Ph.D.)

Epidemiologic surveys using cross-sectional and retrospective assessments indicate that substantial cormorbidity exists between alcoholism and the most common psychiatric disorders in the general adult and adolescent populations (Kessler et al., 1997; Regier et al., 1990), suggesting that comorbidity is a major source of heterogeneity in alcohol-related disorders. Moreover, heritability of alcoholism (at least in males) is greater when an individual also has a cormorbid psychiatric diagnosis (Pickens et al., 1995). Alcoholism is most strongly associated with antisocial personality disorder and drug abuse, but its relationships to other forms of psychopathology has become increasingly evident.

If an association exists between alcoholism and any other psychiatric disorder, there are two possible mechanisms by which the association may occur: (1) one disorder causes or predisposes the individual to the other disorder, or (2) both conditions share underlying etiologic factors (Merikangas and Gelernter, 1990). In the causal explanation, comorbid relationships are frequently examined by assessing the temporal order of onset for each condition. In the shared etiology explanation, a common genetic predisposition underlying alcoholism and a given comorbid disorder is often proposed. In that there are numerous pathways of association, it is not surprising that there has been little scientific consensus.

Although the majority of previous research on this topic has been based on clinically derived samples, other paradigms have been increasingly applied to understand the mechanisms of comorbidity. There are data available from large epidemiological investigations, family studies, and twin and adoption studies.

Although clinical investigators have reported significant amounts (33-67%) of major depression in treated alcoholics (Roy et al., 1991; Davidson, 1995), it has also been reported that the depression can remit after 2-4 weeks of abstinence (Davidson, 1995). Moreover, others have shown remission of depressive symptoms during a 4-week period of abstinence among pure alcoholics and alcoholics with secondary depression, whereas depressive symptoms persisted in both pure depression and depression with secondary alcoholism (Brown et al., 1995).

The majority of clinical studies report a positive association between bipolar disorder and excessive alcohol consumption (Schuckit, 1979), especially during manic or mixed phases. Bipolar disorder is commonly antecedent to alcoholism (Merikangas and Gelernter, 1990).

The association between alcoholism and mood disorders has also been observed in large-scale epidemiologic surveys (Kessler et al., 1997). Consistent with the findings in clinical samples, alcoholism is more strongly associated with bipolar disorder than with major depression.

Some investigators have concluded that alcoholism and mood disorders are transmitted independently (Maier et al, 1994), while others are not certain (Grant and Pickering, 1997). Considering the lack of strong and consistent evidence for the cross-transmission of pure forms of mood disorders and alcoholism in families, shared etiology seems to be an unlikely explanation for the majority of cases of comorbid alcoholism and depression (Merikangas and Gelernter, 1990). Although major depression usually appears to be a consequence of alcoholism (Merikangas and Gelernter, 1990), the onset of depression has been shown to precede the onset of alcoholism in 78% of female twins with comorbid alcohol problems or dependence (Prescott et al., 1997).

Anxiety disorders have been diagnosed in 44% of alcoholics, and 25% of patients with anxiety disorders have met criteria for alcoholism (Kushner et al., 1990). Lewinsohn and colleagues (1997) reported a significant association between anxiety disorders and alcohol abuse/ dependence as early as mid-teens, confirming the association reported among adults in epidemiologic surveys (Kessler et al., 1997). Knop et al., (1993) reported that anxiety symptoms in childhood were a moderate predictor of alcoholism in adulthood. Social phobias and agor-aphobia are most strongly associated with alcoholism in treated samples (Kushner et al., 1990). Although a self-medication model appears to be the most widely accepted explanation for the association between alcoholism and anxiety, consistent empirical support is lacking (Wesner, 1990).

Family studies of probands with anxiety disorders show a significant increase in the risk of alcoholism among first-degree relatives (Leckman et al., 1983). Likewise, family studies of alcoholic probands have also reported increased rates of anxiety among relatives (Merikangas et al., 1985). Maier and colleagues (1994) have observed patterns of cosegregation for alcoholism and panic disorder, suggesting shared susceptibility factors. In contrast, Schuckit and colleagues (1995) failed to demonstrate an association between alcohol disorders and anxiety.

High-risk studies have noted that anxiety symptoms or disorders were more common among offspring of alcoholics than normal or psychiatric controls (Bidaut-Russell, 1994). An increased risk of alcoholism among adult relatives of children with anxiety disorder has been reported (Last et al., 1991), and the presence of an anxiety disorder has been shown to discriminate between offspring of alcoholic and nonalcoholic fathers (Knop et al., 1993).

There are some data to suggest that alcoholism varies in its association with specific subtypes of anxiety, with familial patterns suggesting that social phobia and alcoholism are transmitted independently, whereas panic disorder and alcoholism comorbidity may be partially attributable to shared factors (Maier and Merikangas, 1996).

Results of the Epidemiological Catchment Area study revealed that the prevalence rate of ASPD among people with comorbid alcohol abuse is about 14% (Robins et al., 1988). While the sex distribution of antisocial behaviors favors males overall, when a female is an alcoholic, she is much more likely to have comorbid antisocial behaviors (23-fold risk in females compared to 11-fold risk in males; Robins et al., 1988). The odds ratio for alcoholism and ASPD is 8.3 for men and 17.0 for women in the National Comorbidity Survey (Kessler et al., 1997).

There have only been a few studies of the familial aggregation of alcoholism and antisocial behavior. Cloninger and Reich (1983) found only a slight increase of alcoholism among the relatives of probands with ASPD and no elevation in risk for ASPD in the relatives of alcoholics. Merikangas and colleagues (1985) have suggested that alcoholism and ASPD or conduct disorder may have shared vulnerability, or that nongenetic environmental factors (e.g., family disruption) contribute to these associations.

Comorbidity of Alcoholism with Drug Abuse 

Substantial comorbidity has also been observed between alcoholism and drug abuse/dependence in clinical and epidemiologic samples (Robins et al., 1988), and findings of the National Comorbidity Survey indicate that alcoholism is positively associated with drug dependence, with odds ratios ranging from 9.8 for men to 15.8 for women (Kessler et al., 1997).

The familial aggregation of alcoholism and drug abuse has been well established (McGue, 1994; Gordon, 1994). Controlled family studies of alcoholic probands reveal a two-fold increased risk of drug abuse among relatives compared to those of controls. Examination of the familial aggregation of alcoholism and drug abuse suggests independence of these disorders (Mirin et al., 1991). Moreover, findings from twin studies also suggest independence of genetic factors predisposing to alcohol and drug disorders (Jang et al., 1995).

The major implication of comorbidity for genetic studies is its role in phenotypic characterization. Accurate classification of comorbidity as a source of heterogeneity with alcoholism requires understanding the nature of the association between particular psychiatric disorders (or symptoms or traits). Twin and family studies are useful methods to distinguish between alternative models of association between two or more disorders.

Prospective longitudinal studies, particularly those at high risk, provide a necessary source of information on the evolution of psychopathology and alcoholism. Such designs enable discrimination between alcoholism resulting from primary psychopathology, psychopathology induced by prolonged alcohol consumption, or both.

COLLABORATIVE STUDY ON THE GENETICS OF ALCOHOLISM (COGA)

State of Knowledge (Theodore Reich, M.D.)

The Collaborative Study on the Genetics of Alcoholism (COGA) began eight years ago and is a six-center program to detect and map susceptibility genes for alcohol dependence and related phenotypes.

Probands for COGA were ascertained from inpatient and outpatient treatment units if their families included a sibship of at least three individuals and if parents were available for study. Probands were excluded if they were intravenous drug users, had AIDS, or had a terminal nonalcohol-related illness. The phenotypes of families that included three or more affected first-degree relatives with alcohol dependence were studied more intensely and were used for genetic linkage and association studies. Control families were randomly ascertained without respect to diagnosis and included two available parents and three offspring over the age of 14 years.

Adult lifetime psychiatric status was assessed by direct interview with the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA). It was used to make diagnoses of alcohol dependence, alcoholism, alcohol abuse or harmful use by Feighner, DSM III, and DSM III-R criteria. A close approximation to a diagnosis of alcohol dependence by DSM IV and ICD 10 criteria was also possible. Semi-structured interviews for age-appropriate, lifetime psychiatric diagnosis of children and adolescents (C-SSAGA, A-SSAGA) was also accomplished and accompanied by an interview for parents (P-SSAGA) in order for symptoms of psychopathology in juveniles to be assessed. A semi-structured interview, Family History Assessment, was also develop to assess the presence of psychiatric disorders in relatives.

Phenotypic assessment included measures that have been correlated with susceptibility to alcohol dependence. Personality traits were assessed using the Tridimensional Personality Questionnaire (TPQ) and the Zuckerman Sensation Seeking Scale.

Cooperative families that included three or more members with alcohol dependence were selected for more intensive phenotypic and genotypic study if they were not bilineal. Blood was drawn from adult and juvenile members for the transformation of lymphocytes into lymphoblastoid cell lines that were cryopreserved (adults) and for the direct extraction of DNA (juveniles). Biochemical assays were conducted for adenylate cyclase and monoamine oxidase activity. Probands and relatives, including juveniles, completed neuropsychological and neurophysiological studies. Since these measures are heritable they were studied as correlated quantitative phenotypic markers of susceptibility to alcohol dependence. An alcohol challenge protocol was implemented at the San Diego COGA site to study the effects of controlled exposure to alcohol in 18-30 year-old offspring of alcohol-dependent probands.

Twelve hundred and seventeen families of alcohol-dependent probands, including 7,847 adults (18+) and 1,047 juveniles (7+) have been personally interviewed with the age-appropriate SSAGA. A random sample of 234 control families made up of two parents and at least three children (14+) have also been ascertained and administered the SSAGA. Everyone was also administered selected personality trait-assessment instruments (TPQ and Zuckerman).

Cooperative families with three or more interviewed alcohol-dependent, first-degree members were accepted for more intensive assessment and inclusion into the molecular genetics database (N= 346 families, 4,164 individuals). Members of these families were recruited for a battery of neuropsychological tests, assessment of EEGs and ERPs, and blood samples for the production of cell lines (adults), DNA samples (adults and juveniles), and biochemistry tests. Members of control families participated in the neurophysiology protocol and had blood drawn for the preparation of DNA.

The assessment of 263 informative families is complete. An informative sample for genetic linkage and association studies of alcohol dependence, ERP phenotype, and personality traits was selected to conduct an initial genome-wide linkage analysis (N=105 families). In order to replicate initial findings of genetic linkage and association, a secondary "replication" sample (N=157 families) was also selected for study.

Two hundred and ninety-one markers with an average heterozygosity of 0.72 and an average intermarker distance of 13.8 cM were genotyped. There were seven intermarker regions greater than 30 cM and 28 greater than 20 cM. Most markers were tri- or tetranucleotide repeat polymorphisms.

Alcohol Dependence (COGA Criteria) - The COGA criteria requires a diagnosis of definite alcoholism by Feighner criteria and alcohol dependence by the DSM III-R criteria. The two-point linkage analyses revealed that 24 loci provided evidence for linkage at the p<0.01 level. Multipoint analysis identified chromosomal regions suggestive of linkage with the alcohol-dependent phenotype on chromosomes 1, 2, and 7. Using the SIBPHASE program, the maximum lod score for a region on chromosome 1 was 2.93, on chromosome 7 was 3.49, and on chromosome 2 was 1.81. There was suggestive evidence for a protective locus on chromosome 4 near the alcohol dehydrogenase genes, for which protective effects have been reported in Asian populations.

Alcohol Dependence (ICD 10 Criteria), Withdrawal from Alcohol, and Severe Dependence - Analysis with two-point methods provided evidence for linkage at the p < 0.01 level on chromosomes 1, 7, 11, and 16 for regions that showed linkage to more than one phenotype. Regions on chromosome 1 and 16 showed evidence for linkage with alcohol dependence defined by the COGA and ICD 10 criteria. The levels of significance, however, for linkage with the withdrawal and latent class phenotypes was greater than for the diagnostic criteria (COGA or ICD 10) over all of the chromosomal regions identified. Multipoint lod scores greater than 2 were observed for the alcohol dependence defined by COGA criteria on chromosomes 1 and 7 and with alcohol dependence defined by ICD 10 criteria on chromosomes 8 and 16. Evidence of linkage with the withdrawal phenotype was more widely distributed (chromosomes 7, 10, 16, and 18). The highest lod score was 4.6 on chromosome 16 for severe alcohol dependence defined by latent-class analysis.

Event-Related Brain Potentials - Multipoint quantitative linkage analyses revealed lod scores greater than 2 on chromosomes 2, 5, 6, and 11. The strongest signals were found on chromosomes 2 (lod score = 3.28) and 6 (lod score = 3.41).

The genome survey included markers in or near several candidate genes implicated in the etiology of alcohol dependence. No evidence for linkage with the dopamine D2 receptor gene (DRD2) was found. Detailed analyses including family-based association tests were also negative. GABA receptor genes on chromosome 4 are near the region for which protective factors have been reported. No evidence of linkage was found for GABA receptor genes on chromosome 5, and there was only weak evidence for linkage with two-point methods on chromosome 15 near the region that includes GABA receptor genes. No evidence for linkage was found on the X chromosome in the region of the monoamine loci (MAO B).

A five-year follow-up study has begun this year in COGA. Probands, control families, and families that were included in the genetic linkage studies are being followed-up to detect incident cases, reduce diagnostic error by repeated measurement, and determine the predictive validation of putative causal factors.

Gaps in Knowledge and Research Opportunities 

An additional sample of 250-350 families will greatly increase the power to study a narrowly defined severe form of alcohol dependence. It will probably be necessary to continue to use treatment populations as a source of probands for the study of severe dependence.

The number of African American (17%) and Hispanic (5%) families is too small for detection of genetic heterogeneity when compared with the Caucasian population.

The study of affected individuals and their families in population isolates increases the power to detect and map genes for common complex disorders.

It is likely that several strategies, each implemented by separate research groups, will be necessary to completely characterize genes that determine alcohol dependence and other alcohol-related phenotypes. It is strongly recommended that a consensus to produce standardization of assessment be forged among investigators active in genetic studies to permit phenotypic comparability.

It is important to compare genetic results in families ascertained from community samples with those families ascertained from treatment centers to determine whether findings can be generalized.

Multidomain assessment takes advantage of heritable variables that are correlated with susceptibility to develop alcohol dependence. These variables, such as personality dimensions or ERPs, may reflect underlying causal "endophenotypes" and hence be suitable candidates for study.

Much more development in genetic analytic techniques is required to accept multiple correlated qualitative, quantitative, and compound phenotypic measures that are produced in the multidomain studies of alcohol-related phenotypes.

Phenotypic data and DNA from the COGA samples will become publicly available in September 1999. If other large genetic studies also contributed their data to a national archival database, increasingly large sample would accrue, providing material for many experiments and offering powerful tests of hypotheses in a timely manner.

It is probable that there will be a continuing need for large samples of affected sib and nonsib relative pairs to identify genes of moderate effect, such as in alcohol-related disorders. Multicenter collaborations provide a cost-effective and useful means of achieving these data.

LINKAGE AND ASSOCIATION PORTFOLIO

(Robert W., Karp, Ph.D.)

Linkage and association studies assess the influence of individual genes on phenotypes of interest. Identification of specific genes influencing predisposition to alcoholism, by virtue of identifying the corresponding gene products, will contribute greatly to elucidation of the physiological mechanisms predisposing to alcoholism. By permitting control for variation at known relevant genes, it will also aid in the design of studies of environmental factors. Over the past ten years, the enormous development of the human genome map, as well as new advances in statistical analysis of genetic data, have stimulated efforts to map genes underlying complex, genetically heterogeneous disorders, such as alcoholism. NIAAA's principal linkage study of alcoholism is the multicenter Collaborative Study on the Genetics of Alcoholism (COGA). This study is mapping genes influencing several categorical definitions of alcoholism, as well as a more quantitative model based on latent-class analysis of the phenotypic measures. COGA is also mapping genes influencing electrophysiological traits correlated with alcoholism. Several other smaller scale studies on independent populations, employing differing ascertainment schemes, have recently been funded.

While linkage studies have the advantage of presupposing no hypotheses of specific genes influencing a trait, the power of such studies to detect genes of only moderate effect can be limited for polygenic, heterogeneous disorders. When well-characterized polymorphisms exist in known genes, association studies have the power to detect genes of even modest effect. The COGA investigators are studying associations of a number of candidate loci with the various phenotypes they are studying. A number of additional smaller studies are investigating associations of genes, whose products are involved in various neurotransmitter pathways or alcohol metabolism, with alcohol dependence. As the number of mapped human genes and well-characterized polymorphisms undergoes a dramatic increase during the new few years, NIAAA's investment in association studies seems likely to undergo a similar increase.

There are 10 grants, totaling $7,481,000 in FY97.

  ALCOHOL-RELATED BEHAVIORAL GENETICS IN ANIMALS

A view of alcoholism as a developmental process with multiple biological origins, multiple precipitating environmental events, and complex processes maintaining the disorder predicts that it will be difficult to identify an individual phenotype for study in animals that captures all relevant dimensions.

SELECTIVE BREEDING, SPECIALIZED STRAINS, AND NEOCLASSICAL APPROACHES TO THE ANALYSIS OF ALCOHOL-RELATED PHENOTYPES

State of Knowledge (Bruce C. Dudek, Ph.D.)

Selective breeding was used initially to demonstrate that alcohol-related behavioral phenotypes were heritable. It was also proposed as a means for uncovering neurobiological or neuro-endocrine mechanisms for individual differences in behavioral phenotypes (McClearn, 1979). Major selective breeding programs have established the following alcohol-related phenotypes: acute alcohol responses, alcohol tolerance/dependence withdrawal, and alcohol preference.

There are some advantages to using selected lines that suggest they will have continued utility. For example, selected breeding provides a wide divergence of phenotypes beyond the extremes found in the base population. The use of such extreme phenotypes has resulted in improved pharmacotherapies for excessive alcohol consumption (Froelich and Li, 1993). Other advantages include: selected lines capture most of the relevant genetic variation; genetic variation is usually derived from a wide array of progenitor strains; heterogeneous background; availability of relatively large population sizes permitting examination of multivariate correlation structure while controlling for the selected phenotype; production of extreme populations can produce unexpected, but informative outcomes (Phillips and Dudek, 1983; Dudek and Phillips, 1989); enables simpler comparisons than characterizing batteries of inbred strains or large arrays of QTL-based, marker-differentiated stocks; and provides an ideal model system for tests of behavioral theories (Stewart et al., 1993). Selective breeding is also an ideal strategy for gene mapping, with lower power required (Haley et al., 1994), considerable generalizability, and can be viewed as a control condition against which genotypic-based, selected QTL lines are compared.

Difficulties with selected lines can result form using nonreplicated lines, relatively low population sizes, and common breeding bottlenecks. The replicated within-family, bidirectional design seems to mitigate these difficulties (Crabbe, 1989). Henderson (1997) has concluded that the presence of a genetic correlation is questionable unless selected lines differ by 2-3 phenotypic SDs on the correlated character.

Quantitative genetic approaches have been used successfully, i.e., full Mendelian (Markel et al., 1995) and diallel cross (Dudek et al., 1991). These genetic procedures have demonstrated simple polygenic (< 10 genes) control for determining acute alcohol-response phenotypes.

These strains have proven to be good tools for the evaluation of genetic correlations and as foundation populations for QTL studies (Crabbe et al., 1994; Buck et al., 1997).

Traditional congenic methodology involves repeated backcrossing wherein a monogenically controlled phenotypic variant is "moved" onto a background of another strain (typically inbred). Phenotype-based congenic approach has been used by Dudek and Tritto (1995) to move alleles for the stimulant response to alcohol onto a nonresponsive background. Similarly, Erwin and colleagues (1997) transferred alleles for low alcohol consumption onto a non-drinking background.

Marker-based breeding schemes involve a mapped QTL and can be used in the genetic analysis of alcohol-related phenotypes (Dudek and Tritto, 1995). Using polymorphic flanking markers, spaced closely enough to make recombination unlikely, it is possible to move a QTL onto another background (Markel et al., 1997). Marker-assisted selection uses pairs of flanking polymorphic PCR-identified markers to fix to homozygosity (Bennett et al., 1997).

This methodology has been proposed for alcohol-related phenotypes (Belknap et al., 1997). Mass selection is utilized, beginning typically with a F2 population, where QTL mapping has already been accomplished. Markers linked to the putative QTL are tracked through a few generations of selection, and this process can provide a strong verification strategy for QTL regions.

Relatively little is know of the genetic architecture of alcohol-related phenotypes, especially related to epistasis.

Neurobiological characterization of selected lines is limited and should be expanded to include anatomy and development.

It would be a useful strategy to select lines divergent in specific neurobiological characteristics and determine their relationships to alcohol-related behavior.

Selection for more than one alcohol-related phenotype (tandem selection) in all possible high/low combinations could be a useful and informative strategy.

It is important to study complex phenotypes, even though large sample sizes may be required. Utilization of Recombinant Inbred Strains may enable between-group comparisons, thereby reducing the sample size required.

It is important to begin to evaluate alcohol-related neurobiological phenomena identified as relevant for genetic influences within a developmental framework.

QUANTITATIVE TRAIT LOCI FOR ALCOHOL-RELATED BEHAVIORS

State of Knowledge (Thomas E. Johnson, Ph.D.)

 A quantitative trait is one whose phenotypic variation is continuous (rather than discrete) and is determined by the segregation of many genes. Most of the genetic variation in alcohol action is specified by several genes that are typically defined as a QTL (quantitative trait locus).

The genetic map of the mouse is densely covered with highly polymorphic loci, facilitating the mapping of QTLs. The ability to perform controlled matings and to collect large numbers of offspring of known genotype make the mouse and rat favorable organisms for performing initial studies to localize QTLs.

QTL mapping for alcohol-related phenotypes has most frequently used BXD (C57BL/6J by DBA/2J) RI series because of its large size, relatively dense molecular map, and the finding that virtually all alcohol-related traits differ in the two parental strains. Many provisional QTLs have been identified, but few are statistically significant after appropriate correction for multiple comparisons (Belknap, 1996; Lander and Kruglyak, 1995).

Provisional QTLs were first reported for alcohol-induced ambulatory ataxia, 1-bottle alcohol preference, and withdrawal severity (Gora-Maslak et al., 1991). Subsequent provisional QTLs have been reported for alcohol preference (Rodriguez et al., 1995; Phillips et al., 1994), behavioral activation (Phillips et al., 1996), conditioned place preference (Cunningham, 1995), and alcohol sensitivity (Rodriguez et al., 1995).

Only two studies have reported QTLs that satisfy rigorous criteria for statistical significance. These studies involved whole-genome scans, mapping preference for alcohol (Melo et al., 1995) and initial sensitivity to alcohol (Markel et al., 1997).

Although many rodent lines have been selected for differential responses to alcohol, relatively few studies have examined QTLs underlying their alcohol actions, e.g., Markel et al. (1996) and Erwin et al. (1997).

Problems associated with phenotype assessment include lack of normality, truncation, low replicability, and measuring one trait may preclude measuring another.

Follow-up studies in F2 or backcross populations have been suggested (Johnson et al., 1992), but only infrequently have provisional QTL results been confirmed by additional studies (Buck et al., 1997; Markel et al., 1997).

The degree of detectable association between a marker and a phenotype is based on the size of the effect of the QTL on the phenotype and on the genetic distance between the QTL and the marker. If the QTL is close to the marker and has a large effect, then detection and mapping of the QTL can be performed easily and accurately using methods such as regression (McClearn et al., 1992). If the QTL is not close to the marker gene, then recombination between the marker and the QTL will lead to a lower level of association which will result in a lower "effect size" being attributed to the QTL. More complex and statistically optimal interval mapping methodologies are then required (Markel et al., 1996). A major concern in whole-genome scans for identifying QTLs is that this type of mapping requires many independent statistical tests, requiring appropriate corrections for multiple comparisons (Lander and Kruglyak, 1995).

The small number of RIs typically used in mapping studies significantly limits the ability to detect QTLs and the certainty with which QTLs can be localized. Additional resources should be devoted to validation and resolution of position of these provisional QTLs.

The mapping of genes in crosses between outbred selected lines should be encouraged. The many selected lines of mice and rats should be used for identifying the genetic differences leading to the observed phenotypic differences. Since these lines were derived from heterogeneous stocks (in most cases), they are a richer source of allelic variation than are crosses between any two inbred strains.

It is important to identify genes underlying the QTLs for alcohol actions. So far, few genes underlying QTLs have been identified in any system and certainly none have been confirmed in the area of alcohol actions. Although the approach of choice is candidate gene assessment, the ultimate proof probably involves knockin type gene replacement.

KNOCKOUTS AND ANTISENSE TECHNOLOGY IN THE STUDY OF ALCOHOL-
RELATED BEHAVIORAL PHENOTYPES

 State of Knowledge (Tamara J. Phillips, Ph.D.)

A transgenic animal possesses a gene that has been specifically altered to over- or underexpress a particular gene product. The term knockout generally applies to a gene that has been altered in such a way that its function is entirely deleted (Hiller-Sturmhofel et al., 1995). There has been only limited use of transgenic and knockout mice in alcohol research.

Male transgenic mice overexpressing human transforming growth factor a (TGFa) maintained elevated aggressive behavior in the presence of ethanol, except at the highest dose administered (Hilakivi-Clarke et al., 1993). Greater sensitivity to the sedative effects of ethanol suggest that failure to respond to ethanol in an aggression test could not be explained by reduced ethanol sensitivity. In a follow-up investigation, castration markedly attenuated the difference between control and transgenic mice in intermale aggressive behavior, and ethanol had no effects on aggression (Hilakivi-Clarke and Goldberg, 1995). Overexpression transgenics for insulin-like growth factor-I (IGF-I) were less sensitive to the sedative effects of ethanol, while overexpressed IGF-binding protein 1 mice were more sensitive than controls (Pucilowski et al., 1996). Non-transgenic controls developed substantial tolerance to ethanol, while transgenic mice developed very little, and the overexpression transgenic for IGF-binding protein 1 developed significantly more ethanol tolerance.

A null mutant strain lacking the gamma isoform of protein kinase C has been shown to display reduced sensitivity to both ethanol sedation and hypothermia, but not to flunitrazepam or pentobarbital (Harris et al., 1995). These behavioral results are consistent with an alteration in GABA_A receptor function found in brain tissue from the mutant mice in response to ethanol, but not to flunitrazepam or pentobarbital. Homanics and colleagues (1997) reported that a knockout mouse for the a6 subunit of the GABA_A did not differ in ethanol, pentobarbital or general anesthetic sensitivity relative to wildtype controls.

Crabbe and colleagues (1996) found that mice lacking a functional copy of the gene coding for serotonin_1B receptors (5-HT_1B) drank twice as much ethanol in a free-choice drinking condition relative to wildtype controls. The two genotypes consumed equivalent amounts of saccharin, sucrose, and quinine and showed equivalent preferences in choice experiments for these substances suggesting that taste and caloric factors could not account for the differential ethanol consumption. Furthermore, the magnitude of ethanol conditioned taste aversion to a preferred sodium chloride solution was equivalent in wildtype and knockout mice (Risinger et al., 1996), suggesting equivalent sensitivity to ethanol's aversive effects. Ethanol-associated conditioned place preference was less well developed in 5-HT_1B knockout mice, but these mice demonstrated a larger stimulant response to ethanol (Risinger et al., 1996). Knockout mice were less sensitive to the ataxic effects of ethanol, developed less ethanol ataxia tolerance, and were not differentially sensitive to ethanol withdrawal compared to the wildtype (Crabbe et al., 1996).

Dopamine D₄ receptor gene knockout mice are supersensitive to the locomotor stimulant effects of ethanol, methamphetamine, and cocaine (Rubinstein et al., 1997).

One advantage of these strategies is to study the effects of genes which are thought to be candidates for modulating sensitivity to a variety of ethanol's effects. Moreover, the combined effects of > 2 altered genes can be examined. Another advantage is that there may be the opportunity to study compensatory processes in mice with gene alterations present during virtually all of development. With inducible knockouts, mice in which the gene is knocked out later in development or in adulthood enables examination of the effects of a developmentally lethal knockout as well as studying the effects of the knockout without possible developmental compensatory changes. By introducing a mutation into a particular cellular compartment (Gu et al., 1994), a tissue-specific knockout allows determination of the influence of alteration of gene expression in a specific tissue or brain area.

Concern has been expressed that genetic background effects may alter the phenotypic expression of the knockout (Schauwecker and Steward, 1997). Another concern is compensation by other genes for the missing, under-, or overexpressed gene (Crawley, 1996). In that alcohol-related characteristics are polygenically determined, a given gene in a set of genes influencing a trait may not have a large effect on its own. It has been known for some time that foreign genes in transgenic mice may be integrated at abnormal chromosomal positions and expressed in inappropriate tissues (Lacy et al., 1983).

A complementary approach to transgenic methods for examining the effects of a specific gene product on behavior is antisense RNA technology (Hiller-Sturmhofel et al., 1995). This technology can temporarily reduce or prevent the expression of a gene by inhibiting the translation of mRNA into its respective protein, using an "antisense" molecule. Although this technology has not reached the stage of being routine, an antisense oligonucleotide to c-fos, administered intracerebroventricularly in mice, blocked ethanol tolerance that was maintained by arginine vasopressin (Szabo et al., 1996).

Advantages of this procedure include the impermanence of the translational disruption so that animals can be examined in the normal and abnormal expression state, ability to perform the translational disruption in fully developed animals, savings in cost and time relative to knockout production, and the possibility of therapeutic potential (Hiller-Sturmhofel et al., 1995). Additional advantages are that the procedure can be applied to different species, anatomic specificity is attainted more easily, and specific antisense oligonucleotides are available commercially. Difficulties include delivery of sufficient quantities of the antisense oligonucleotide, particularly to the brain; short lifespan of antisense oligonucleotides; toxicity; and antisense has been found to trigger extreme immune responses.

Knockouts already available at the Jackson Laboratory (Induced Mutant Resource) could be used to study candidate genes in the regions of ethanol-related QTLs.

Increased development of double, triple, etc. knockout mice and determination of resulting effects on ethanol-related phenotypes.

Inducible knockout and tissue-specific knockout strategies should be investigated in ethanol-related research. 

Create more knockouts on isogenic backgrounds that would be informative for ethanol-related phenotypes.

Identification of genes using unselected forward mutagenesis screens should be encouraged.

Increase support for a rescue or gene knockin strategy (Gerlai, 1996) that expresses the protein of the target gene, thereby replacing the function of the mutant gene and restoring the wildtype phenotype.

Antisense oligonucleotide technology could be extremely informative for ethanol-related research.

It may be useful to explore the utility of viral-mediated gene transfer for ethanol-related research.

INVERTEBRATE MODELS OF ETHANOL-RELATED PHENOTYPES
AND ASSOCIATED GENOTYPES

State of Knowledge (Ulrike Heberlein, Ph.D.)

Although the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans have been extremely useful in deciphering the molecular mechanisms underlying development and behavior, these model systems have not yet been used extensively in the context of ethanol. The primary goals of genetic analysis in Drosophila and C. elegans are to identify mutations that disrupt particular ethanol-related phenotypes, to identify the disrupted gene, and to dissect the reward mechanisms by which the gene functions in mediating ethanol-related behavior.

Research has clearly demonstrated that the genes and biochemical pathways have been conserved during evolution, and many genes first identified in Drosophila have provided major insights into vertebrate and human development and disease. Flies have a relatively sophisticated nervous system and are capable of many complex behaviors. More than 80 years of intense genetic analysis has laid the foundation for classical genetic analysis by the generation of multiple tools.

Alcohol Metabolism and Toxicity - The natural environment of Drosophila melanogaster includes fermenting plant materials, which often contain high levels of ethanol and other alcohols (> 3%; McKechnie and Morgan, 1982). Drosophila is quite resistant to the toxic effects of ethanol and can degrade ethanol efficiently for use as an energy source or substrate for lipid biosynthesis (Geer et al., 1993). The enzyme alcohol dehydrogenase (ADH) plays a key role in ensuring both metabolic use and detoxification from ethanol; it is encoded by a single structural gene. Several heritable characteristics have been associated with increased ethano l resistance: levels of ADH (Geer et al., 1989) and glycerol-3-phosphate oxidase (McKechnie and Geer, 1986) and lipid constitution of membranes (Geer et al., 1993). However, a clear causal relationship has not been established, and a direct genetic approach to dissect the underlying mechanisms has not yet been undertaken.

Ethanol Preference - Drosophila larvae with high ADH activity prefer ethanol-supplemented agar, whereas larvae lacking ADH avoid agar containing 8% or more ethanol (Depiereux et al., 1985). Acetaldehyde is toxic to Drosophila larvae, and it has been suggested that ethanol avoidance correlates with both the relative rate of acetaldehyde production and relative aldehyde sensitivity. Thus, as in humans, genetic changes that alter ethanol metabolism affect ethanol preference in Drosophila.

Ethanol Intoxication - Assays to measure intoxication have been developed (Weber and Diggins, 1990). Degree of loss of postural control has been shown to be dependent upon the concentration of ethanol. Reasonable dose-response curves have been obtained, with severe intoxication associated with ethanol concentrations of 35 mM or 160 mg/dl.

Ethanol Tolerance - The term "tolerance" has been used for many decades to describe the increased resistance of fly strains that exist naturally or that have been bred for many generations for increased ability to tolerate food containing toxic levels of ethanol (3-9%; Geer et al, 1993). It would be more appropriate, in the context of alcohol research, to use the term "resistance" and reserve the term "tolerance" for increase in resistance caused by multiple exposures to ethanol in the absence of genetic change. Studies of ethanol tolerance have been conducted but not yet published.

Genetic Screens for Mutations that Alter Ethanol-Induced Behaviors - Genetic screens for mutations that alter the sensitivity to ethanol intoxication and the ability of flies to develop tolerance have been carried out. Mutants carrying a disruption of a single locus (or in very few loci) have been generated by chemical mutagenesis or transposon insertion. Among many thousands of such mutant flies, those mutations that alter ethanol-induced behaviors have been isolated by selection. This genetic approach can result in the isolation of any and all genes that, when mutated, will lead to altered ethanol-related behaviors.

The nematode C. elegans is an attractive organism in which to address questions related to nervous system function and behavior. It has a simple nervous system containing exactly 302 neurons, each of them can be identified in the live, semi-transparent animal, and the complete circuitry of the nervous system is known (White et al., 1986). Despite its simple anatomy, C. elegans has an array of classical neurotransmitters similar to that found in vertebrate nervous systems. C. elegans also provides a system with great potential for genetic analysis. Its generation time is only three days, and the small size of the adult (1.5 mm) allows easy cultivation in the laboratory. In addition, its genome is the smallest of any eukaryote (10⁸ bp), and its complete sequence is targeted for completion in 1998.

Several mutations have been isolated that result in altered sensitivity to volatile anesthetics (Morgan et al., 1996). When tested, a subset of these mutants also displayed altered sensitivity (increased in some and reduced in others) to the anesthetic effects of ethanol (Morgan and Sedensky, 1995). These studies suggest that the effects of ethanol and volatile anesthetics on C. elegans may be mediated by an overlapping set of gene products. Although a molecular characterization of these genes has been initiated, a systematic genetic screen for mutations that specifically alter ethanol-induced behaviors has not been carried out with C. elegans.

It is critical to increase the number of investigators in the invertebrate field that study ethanol-related phenotypes and to increase collaborations between investigators using different experimental systems.

It is important to increase the number of ethanol-related phenotypes that are studied.

Success in identifying genes in flies and nematodes that disrupt ethanol-related phenotypes provides at least two potential benefits. First, vertebrate and human studies could be facilitated through the generation of potential candidate genes. Second, tools could emerge to study the mechanisms of ethanol-induced phenotypes in invertebrates with the potential for extrapolation to higher systems.

It is important to establish the effects of ethanol on neurotransmission in invertebrates and to identify the specific receptor systems affected by ethanol. In addition, the effects of various mutants on neurotransmission in the absence and presence of ethanol warrant more study.

ALCOHOL-RELATED BEHAVIORAL PHENOTYPES
AND LINKAGE IN ANIMALS
(Robert W. Karp, Ph.D.)

NIAAA has a history of more than 20 years of support of selective breeding studies of alcohol-related behavior in rats and mice. These studies have provided critical evidence for heritability of a wide array of traits such as consumption preference, sedation, locomotor activation, withdrawal, and hypothermia. New selections are currently in progress for high and low extremes of consumption preference (mice), withdrawal intensity (mice), and acute functional tolerance (mice). The major use of selectively bred lines has been for studies of behavior and neuropharmacological traits correlated with the trait on which a given line was selected. Such correlations can suggest behavioral and physiological mechanisms mediating the selected traits. They encompass a very broad range of sophisticated behavioral, neurochemical, and neurophysiological measures. More recently, selectively bred lines have come into increasing use for QTL (Quantitative Trait Locus) mapping (see below).

In similar fashion to the burgeoning knowledge of the human genome, the ongoing explosion in knowledge of the mouse (and more recently, the rat) genome has permitted the mapping of Quantitative Trait Loci, the multiple genes influencing traits which vary in a continuous, quantitative (as opposed to dichotomous) fashion, and which are not transmitted according to Mendelian models. QTL mapping, carried to its logical conclusion of gene cloning and identification, provides an obvious prospect for elucidation of physiological pathways mediating the traits under study. In addition, it affords a rigorous examination of the biological significance of correlations among multiple traits, by allowing a precise determination of whether such traits are influenced by overlapping sets of genes. Most importantly, QTL mapping permits a direct transfer of genetic knowledge gained from more easily manipulated animal systems to humans, by means of isolation of human homologues of animal genes.

NIAAA's investment in QTL mapping has expanded rapidly in recent years, so that it now accounts for more than 40% of the investment in animal genetics. While the earlier QTL mapping studies concentrated on traits most easily measured (e.g., consumption preference, sensitivity to sedation, withdrawal intensity), more recent studies are looking at more behaviorally complex measures (e.g., conditioned place preference, operant responding, taste reactivity), as well as neurochemical measures. The most advanced QTL mapping studies (consumption preference, sedation sensitivity, and withdrawal intensity) have each mapped several QTLs with a resolution of a few cM, and are now engaged in increasing their mapping resolution, in preparation for testing candidate genes residing within the QTL map intervals.

In addition to selective breeding and QTL mapping, which rely on naturally occurring genetic variation, animal genetic studies afford the use of artificially induced mutations. Site-directed mutagenesis in the mouse, colloquially known as gene knockouts, provides a powerful method for testing the influence of specific genes of interest on a wide variety of physiological and behavioral traits. The first extensive use of this technique to analyze behavior was on learning and memory. Early knockout studies were technically limited to the relatively crude device of germline null mutations. When such mutations cause alterations in behavioral phenotypes, it is difficult to distinguish whether these alterations are due to an acute role for the missing gene product in the behavioral process itself, or to a role in the previous development of some part of the body required for the behavior. Interpretation of such studies has also been confounded by modifying effects of inhomogeneous genetic backgrounds. More sophisticated versions of this technique (e.g., tissue-specific knockouts, inducible knockouts) have recently been developed to avoid these shortcomings. Gene knockouts have only recently been introduced into the alcohol field, and NIAAA's investment in this methodology is still relatively modest. NIAAA-supported investigators are currently studying the effects of knockouts of a protein kinase C isoform, an adenylate cyclase isoform, the 5HT1b receptor, B-endorphin, and subunits of the GABA-A receptor on various aspects of sensitivity to, consumption of, and motivation by alcohol.

In the absence of hypotheses about specific genes influencing traits of interest, mutagenesis screens can allow the identification of such genes and their corresponding products. This method has long been a mainstay of genetic research on viruses, bacteria, fungi, fruit fly Drosophila melanogaster, and soil nematode Caenorhabditis elegans. Recent research has demonstrated an astonishing level of evolutionary conservation of neurodevelopmental and neurophysiological mechanisms among Drosophila, Caenorhabditis, and mammals. There is accordingly reason to suspect that physiological mechanisms of certain responses to alcohol may display a similar level of conservation. Given assays for, for example, sensitivity and tolerance to alcohol in these invertebrate species, it is a relatively straightforward (if labor intensive) matter to isolate mutations influencing these traits and identify the corresponding genes. These genes may possibly implicate known physiological pathways. Even if they do not, powerful genetic techniques are available in these organisms to identify genes mediating other steps in the pathways leading to the phenotypes under study. Mammalian homologues of these genes can be readily isolated, and tested for a role in analogous alcohol-related behavior in mammals.

NIAAA has a very small investment (one grant and one fellowship) in screens for Drosophila mutations affecting alcohol-induced ataxia and tolerance to this effect. NIAAA has previously funded a similar nematode project, but this project was not funded during 1997. The investigators have isolated mutations, cloned several genes, and identified a mammalian homologue of at least one of them. A critical test of the relevance of mammalian homologues of these genes to mammalian alcohol-related behavior remains to be performed. Pending the outcome of such tests, it may prove desirable to increase NIAAA's investment in invertebrate mutagenesis screens in the future.

It is only comparatively recent that mutagenesis screens have been applied to the analysis of behavior in mice. The notable success of a screen for mutants affecting circadian rhythms has stimulated widespread interest in screens for a wide variety of developmental and behavioral mutants. NIAAA supports no mouse mutagenesis screens as yet (having received no applications for such a project). An investment in such projects merits serious consideration.

There are 41 awards totaling $10.2M in the animal genetics portfolio, subdivided into the following categories.

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