Proposal for Thesis Research in Partial Fulfillment of the Requirements for the Degree of Master of Science in Biomedical Sciences

Faculty of Science Rangsit University

Title: ROLE OF ALDH GENOTYPES AND SUSCEPTIBILITY TO RISKS OF ALCOHOLRELATED DISEASE IN THAI POPULATION

Submitted by: Date of Submission: Expected Date of Completion:

Miss Sirikorn Phokham ID 506290 December 1, 2011 May 31, 2012 Major advisor Member

Thesis Advisory Committee: Wantica Kruanamkam, Ph.D.

Asst.Prof Jintana Siriwarasai, Ph.D. Member

CHAPTER I

INTRODUCTION

Alcohol dependence (AD) is a complex trait that has significant genetic influences but exhibits great clinical and etiological heterogeneity. Identification of specific genes that contribute to susceptibility to develop AD has been a focus of intense research but progress has been slow. Many candidate genes have been examined in association. The genes coding for alcohol metabolizing enzymes are clearly relevant. Two sets of genes code for enzymes in the oxidative pathway of ethanol metabolism: the alcohol dehydrogenases (ADH) convert ethanol into acetaldehyde and the aldehyde dehydrogenases (ALDH) transform acetaldehyde into acetic acid, which can then be easily excreted (1-3). Several significant associations with AD have been reported for subsets of alcohol metabolism genes such as ADH1B, ADH1C and ALDH2 which primarily found in Asian populations. Many groups of investigators reported significantly higher risk of alcoholism were associated with subjects having ADH1B*1, ADH1C*2, and ALDH2*1 alleles. Genes that code for ALDH enzymes are located on several different chromosomes. Nineteen putatively functional genes and 3 pseudogenes in the ALDH gene superfamily have been identified to be encoding ALDH isozymes, but only two of them, ALDH1 (ALDH1A1, 9q21.13, cytosolic isozyme) and ALDH2 (12q24.2, mitochondrial isozyme) are thought to be significantly involved in acetaldehyde oxidation (4-5). The ALDH2 gene is highly expressed in liver and stomach with a very high affinity for acetaldehyde and plays a central role in human acetaldehyde metabolism. The ALDH1 gene also has high affinity for acetaldehyde. A functional polymorphism of the ALDH2 gene, ALDH2*2 has lower enzyme activity than the wild-type allele. This mutant allele is mainly present in Asians populations (approximately 30%). Although homozygous individuals have no ALDH2 activity; heterozygous individuals maintain 30 to 50% activity. The deficient

ALDH2*2 allele is associated with decreasing risk of AD or alcoholism in Asian populations (67). Two genetic variants are known in ALDH1A1, ALDH1A1*2 (a 17 bp deletion in the promoter region present in many populations) and ALDH1A1*3 (a 3 bp insertion in the promoter region present only in populations of African descent). Both have protective effects against AD in African-Americans, and ALDH1A1*2 were further reported to be protective in a Native American sample. Overall, ALDH enzymes exhibit genetic polymorphism and ethnic variation. The functional polymorphisms of ALDH with protective effects against AD are mainly reported in Asian and Native American populations, or in African-Americans. In Thailand, there is very limited data related these issues (8, 45). Therefore, this research will study the association between genetic polymorphisms of ALDH and alcohol consumption influencing on risks of alcohol-induced liver and metabolic diseases.

Objectives
1. The primary objective is to test for association of polymorphism of ALDH gene and

clinical outcome of liver diseases in a sample of Thai population. 2. The secondary objective is to clarify the prevalence of polymorphism of ALDH gene in a

sample of Thai population.

CHAPTER II LITERATURE REVIEW

Ethanol is a 2-carbonchain alcohol; the chemical formula is CH3CH2OH (Fig. 1). It is ubiquitous throughout the world and is a leading cause of morbidity across cultures. Ethanol is the most common psychoactive drug used by children and adolescents in many countries and is one of the most commonly abused drugs in the world. Its pleasures are very widely acknowledged and form a bond of community for the majority of adults. References to those pleasures form a kind of physical and mental disorientation are viewed with bemused affection. Alcoholic beverages are a standard anxiety-reliever at social gatherings, and those who refuse to consume ethanol run the risk of being social outcasts. Yet some people feel concern about consuming a substance which causes physical and mental disorientation. Although the effects appear to be temporary, one could easily wonder about the physiological changes being wrought on the brain. And one could wonder whether the temporary effects are really desirable and whether the brain really escapes permanent harm. Because ethanol enters tissues in proportion to water content it is far more prevalent in the brain than in muscle or fat.

Figure 1 Three dimensional model (A) and chemical formula of ethanol (B). Health benefits are frequently claimed for alcohol when consumed in moderation. Most of the claimed benefits are associated with reducing cardiovascular disease. People often abstain from alcohol due to interaction with prescription drugs or other health-related reasons. The poor health of many abstainers should not be taken as proof of the beneficial effects of ethanol. Alcohol consumption is also the leading risk factor for disease burden in both developing and developed countries, including Thailand. The global burden related to alcohol consumption, both

in terms of morbidity and mortality, is considerable. During the past decade, reporting from WHO showed that Thailand ranked top ten in the world for overall alcohol consumption in concurrent with increased adverse effects.

Alcoholic beverages Ethanol is the principal psychoactive constituent in alcoholic beverages, with depressant effects on the central nervous system. It has a complex mode of action and affects multiple systems in the brain; most notably ethanol acts as an agonist to the gamma-aminobutyric acid (GABA) receptors. Similar psychoactive include those which also interact with GABA receptors, such as gamma-hydroxybutyric acid (GHB). Ethanol is metabolized by the body as an energyproviding carbohydrate nutrient, as it metabolizes into acetyl CoA, an intermediate common with glucose metabolism that can be used for energy in the citric acid cycle or for biosynthesis (9-10). Alcoholic beverages vary considerably in their ethanol content and in the foodstuffs from which they are produced. Most alcoholic beverages can be broadly classified as fermented beverages, beverages made by the action of yeast on sugary foodstuffs, or as distilled beverages, beverages whose preparation involves concentrating the ethanol in fermented beverages by distillation. The ethanol content of a beverage is usually measured in terms of the volume fraction of ethanol in the beverage, expressed either as a percentage or in alcoholic proof units. Fermented beverages can be broadly classified by the foodstuff from which they are fermented. Beers are made from cereal grains or other starchy materials, wines and ciders from fruit juices, and meads from honey. Cultures around the world have made fermented beverages from numerous other foodstuffs, and local and national names for various fermented beverages abound. Distilled beverages are made by distilling fermented beverages. Broad categories of distilled beverages include whiskeys, distilled from fermented cereal grains; brandies, distilled from fermented fruit juices, and rum, distilled from fermented molasses or sugarcane juice.

Vodka and similar neutral grain spirits can be distilled from any fermented material (grain or potatoes are most common); these spirits are so thoroughly distilled that no tastes from the particular starting material remain. Numerous other spirits and liqueurs are prepared by infusing flavors from fruits, herbs, and spices into distilled spirits. A traditional example is gin, which is created by infusing juniper berries into a neutral grain alcohol. In a few beverages, ethanol is concentrated by means other than distillation. Applejack is traditionally made by freeze distillation, by which water is frozen out of fermented apple cider, leaving a more ethanol-rich liquid behind. Ice beer is also freeze-distilled, with beer as the base beverage. Fortified wines are prepared by adding brandy or some other distilled spirit to partially-fermented wine. This kills the yeast and conserves some of the sugar in grape juice; such beverages are not only more ethanol-rich, but are often sweeter than other wines. Alcoholic beverages are sometimes used in cooking, not only for their inherent flavors, but also because the alcohol dissolves hydrophobic flavor compounds which water cannot.

Ethanol metabolism Ethanol has a volume of distribution (0.6 L/kg) and is readily distributed throughout the body. The primary route of absorption is oral, although it can be absorbed by inhalation and even percutaneously. It is well recognized that ethanol can be absorbed unchanged along the whole length of the digestive tract, that absorption takes place rapidly from the stomach (about 2o %), and most rapidly from the small gut (about 80 %). The peak plasma concentrations typically occur 30-60 minutes after ingestion. The rate of absorption after drinking is affected by several factors, for example the volume, concentration (10-20 % solutions are most rapidly absorbed) and nature of the alcoholic drink, the presence or absence of food in the stomach, rate of gastric emptying, permeability of the gastric mucosa, intestinal tissues and individual variations. After absorption into the blood-stream, alcohol is distributed quickly throughout the total body water

and is subsequently metabolized at a steady rate. A method which makes use of this property of ethanol has been successfully employed for measuring the total body water volume in human subjects. After equilibration, normally 1-1.5 h after drinking, the alcohol concentration in any body fluid depends on the water content of that fluid (11). Over 90% of the absorbed alcohol is metabolized in the body, yielding some 7 kcal/g on complete oxidation to carbon dioxide and water, with a concomitant fall in the respiratory quotient; the remainder is excreted unchanged in the urine, expired air and sweat. The main site of metabolism of ethanol is the liver, although some other tissues, for example kidney, muscle, lung, intestine and possibly even the brain, may metabolize smaller quantities. Figure 2 shows the main pathways of ethanol metabolism. It is generally believed that the rate-limiting step in the metabolism of alcohol is its conversion to acetaldehyde, a reaction catalyzed by the zinc-containing enzyme, alcohol dehydrogenase (ADH). This process occurs chiefly in the soluble cytoplasm of liver cells, with nicotinamide adenine dinucleotide (NAD) acting as the hydrogen acceptor. However, particularly in alcoholics, some ethanol may be oxidized by the peroxidase-xanthine oxidase-catalase system, and possibly other oxidases both in liver and plasma (12). Small amounts of alcohol may also be converted to ethyl glucuronide, ethyl sulphate and other esters, and excreted in the urine. The acetaldehyde formed in the first oxidative step in the metabolism of ethanol is converted to acetate, a reaction catalyzed by aldehyde dehydrogenase (ALDH), utilizing NAD as cofactor, and eventually to acetyl-CoA, the Krebs cycle, and other reactions. Acetaldehyde can also be converted to other substances, such as acetoin and hydroxyketohexanoic acid. The availability of unreduced NAD in the cell sap seems to be important in alcohol metabolism, since NADH competes with NAD for binding sites on ADH and, in sufficient concentration, may inhibit the rate of ethanol dehydration. The NADH formed in the oxidative steps must be continually reoxidized to NAD for alcohol oxidation to proceed. In the cytoplasm of liver cells

the reoxidation of NADH may be coupled with the reduction of pyruvate to lactate, elongation and saturation of fatty acids; but perhaps the most important route for reoxidation of the NADH involves the mitochondrial flavoprotein-cytochrome electron transfer system coupled with oxidative phosphorylation. The carrier of the hydrogen equivalents from NADH across the relatively impermeable mitochondrial membranes for oxidation in the intra-mitochondria1 compartment may be substances like malate, glutamate, -glycerophosphate and hydroxybutyrate (13).

Figure 2 Pathways of alcohol (ethanol) metabolism in man. ADH, alcohol dehydrogenase; CYP2E1, microsomal P4502E1; ALDH, aldehyde dehydrogenase.

Factors influencing rate of metabolism In theory, the B vitamins, nicotinamide and riboflavin, participate in the metabolism of alcohol, for they are implicated in the formation of NAD and flavinadenine dinucleotide (FAD)

respectively. It is also possible that ascorbic acid (vitamin C) and the tocopherols (vitamin E), which may be involved in oxidationreduction reactions, affect alcohol oxidation, and it has also been reported that pyridoxine increases the rate of alcohol metabolism in man. Sugars, which yield pyruvate and participate in fatty acid synthesis, may also facilitate ethanol oxidation by means of NADH reoxidation pathways. There have been many claims that administration of vitamins and sugars can increase the rate of sobering up in man and laboratory animals, but the published results are equivocal. Caffeine and strong black coffee, dietary factors, physical exercise, environmental temperature changes, thyroid hormones, oxygen therapy and various drugs - for example oral antidiabetic agents - have all been suggested from time to time as being capable of affecting the rate of alcohol metabolism in man (11).

Long term effects Ethanol within the human body is converted into acetaldehyde by ADH and then into acetic acid by ALDH. The product of the first step of this breakdown, acetaldehyde, is more toxic than ethanol. Acetaldehyde is linked to most of the clinical effects of alcohol. It has been shown to increase the risk of developing cirrhosis of the liver, multiple forms of cancer, and alcoholism. Some individuals have less-effective forms of one or both of the metabolizing enzymes and can experience more-severe symptoms from ethanol consumption than others. Conversely, those who have acquired alcohol tolerance have a greater quantity of these enzymes, and metabolize ethanol more rapidly. The adverse effects of long-term excessive use of alcohol are similar to those seen with other sedative-hypnotics (apart from organ toxicity which is much more problematic with alcohol). Withdrawal effects and dependence are also almost identical. Alcohol at moderate levels has some positive and negative effects on health. The negative effects include increased risk of liver diseases, oropharyngeal cancer, esophageal cancer and pancreatitis (14-16). Conversely moderate intake of alcohol may have some beneficial effects on gastritis and

cholelithiasis. Chronic alcohol misuse and abuse has serious effects on physical and mental health. Chronic excess alcohol intake, or alcohol dependence, can lead to a wide range of neuropsychiatric or neurological impairment, cardiovascular disease, liver disease, and malignant neoplasms. The psychiatric disorders which are associated with alcoholism include major depression, dysthymia, mania, hypomania, panic disorder, phobias, generalized anxiety disorder, personality disorders, schizophrenia, suicide, neurologic deficits and brain damage. Alcohol dependence is associated with hypertension, coronary heart disease, and ischemic stroke, cancer of the respiratory system, but also cancers of the digestive system, liver, breast and ovaries. Heavy drinking is associated with liver disease, such as cirrhosis (Fig. 3).

Figure 3 Long-term effect of ethanol

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Genes and alcohol dependence Alcohol dependence (AD) is a complex trait that has significant genetic influences but exhibits great clinical and etiological heterogeneity. Identification of specific genes that contribute to susceptibility to develop AD has been a focus of intense research but progress has been slow. Many candidate genes have been examined in association. The genes coding for alcohol metabolizing enzymes are clearly relevant and the most studied. Two sets of genes code for enzymes in the oxidative pathway of ethanol metabolism: the alcohol dehydrogenases (ADH) convert ethanol into acetaldehyde and the aldehyde dehydrogenases (ALDH) transform acetaldehyde into acetic acid which can then be easily excreted. Several significant associations with AD have been reported for subsets of alcohol metabolism genes (e.g. ADH1B, ADH1C, ALDH2), primarily in East Asian populations. A meta-analysis including different ethnic groups reported a significantly higher risk of alcoholism was associated with subjects having ADH1B*1 (odds ratio, OR = 2.23), ADH1C*2 (OR = 1.91), and ALDH2*1 (OR = 4.35) alleles, but only in East Asian populations (17-20). There are 7 human ADH genes cluster in a small region (approximately 365 kb) on chromosome 4 q21q24. It has been suggested that 3 genes are class I (ADH1A,1B, 1C) and 1 genes is class II (ADH4), which code for primary enzymes for conversion of ethanol to acetaldehyde are involved in ethanol oxidation in vivo. The class I enzymes are mainly expressed in liver and contribute about 70% of the total ethanol oxidizing capacity. Most prior studies have focused on class I ADH genes, especially ADH1B and ADH1C, which have known functional polymorphisms in their coding regions. The functional variants of allele ADH1B*2 (His47) and ADH1B*3 (Cys369) have high enzyme activity and unusually rapid conversion of ethanol to acetaldehyde. This causes facial flushing and aversive effects after consuming alcohol and is protective against AD. These variants have markedly different frequencies in different ethnic groups. ADH1B*2 allele is common in Asian populations (approximately 90%) but is much less common in Caucasians.

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ADH1B*3 has been found in African and Native American populations. Results of meta-analysis for ADH1B*2 comparing homozygous versus heterozygous genotypes of ADH1B*2 with AD exhibited an OR of 5 in Han-Chinese and Japanese, but an OR of 2 in Europeans. Positive association of ADH1B*3 with AD was also found in African and Native American populations. Three functional polymorphisms have been identified for ADH1C. The wild-type ADH1C*1 has about twice the enzyme activity of ADH1C*2 and is very common in Asian and African populations (approximately 90%), whereas the allele frequency is about 50% in Caucasians. Some studies report protective effects of ADH1C*1 against AD in Asian samples and a Native American sample. Other studies report no association of ADH1C*1 with AD in European samples. Strong linkage disequilibrium (LD, i.e., the nonrandom association of alleles at two or more loci) has been observed between ADH1B*2 and ADH1C*1, so whether there is an independent effect of ADH1C on AD is unclear. Recently, functional polymorphisms were identified in the class II ADH4 gene; one variant in the promoter region of ADH4 (C-75A) alters expression level. Some studies reported association of ADH4 with AD in European-Americans and another sample of European- and African-Americans (21-24). The class IV ADH7 gene, which is mainly expressed in upper digestive tract, codes for the enzyme with the highest activity for oxidizing retinol. Previous studies suggest that ADH7 has an epistatic effect with ADH1B for protection against AD in Taiwanese Han and European populations (25-26). Genes that code for ALDH enzymes are located on several different chromosomes. Nineteen putatively functional genes and 3 pseudogenes in the ALDH gene superfamily have been identified to be encoding ALDH isozymes, but only two of them, ALDH1 (ALDH1A1, 9q21.13, cytosolic isozyme) and ALDH2 (12q24.2, mitochondrial isozyme) are thought to be significantly involved in acetaldehyde oxidation. The ALDH2 gene is highly expressed in liver and stomach with a very high affinity for acetaldehyde and plays a central role in human

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acetaldehyde metabolism. The ALDH1 gene also has high affinity for acetaldehyde. A functional polymorphism of the ALDH2 gene, ALDH2*2 (Lys487RAA) has lower enzyme activity than the wild-type allele. This mutant allele is mainly present in East Asians (approximately 30%). Although homozygous individuals have no ALDH2 activity; heterozygous individuals maintain 30 to 50% activity. The deficient ALDH2*2 allele is associated with decreasing risk of AD or alcoholism in Asian populations. In addition, individuals with the combination of a fast form of ADH1B and slow form of ALDH2 had particularly reduced risk for AD in Han-Chinese samples. Two genetic variants are known in ALDH1A1, ALDH1A1*2 (a 17 bp deletion in the promoter region present in many populations) and ALDH1A1*3 (a 3 bp insertion in the promoter region present only in populations of African descent). Both have protective effects against AD in African-Americans, and ALDH1A1*2 were further reported to be protective in a Native American sample (27-28).

Aldehyde dehydrogenase Human ALDH superfamily comprises 10 families which have been mapped to 11 chromosomes (Table 1). Amino acid sequence identities are 40% between families and 60% or higher between the subfamilies. The superfamily contains divergently related enzymes that metabolize a wide spectrum of endogenous and exogenous aldehydes. Cytosolic ALDH1A1 and mitochondrial ALDH2 primarily contribute to oxidation of acetaldehyde in vivo, an immediate metabolite of ethanol. The genes of ALDH1A1 and ALDH2 have been mapped to chromosomes 9q21 and 12q24, respectively. The allelic variant, ALDH2*2, caused by a G/A transition resulting in substitution of glutamic acid by lysine at position 487, occurs 1624% in the Han Chinese, Japanese and Koreans but very rare in the Caucasians, black populations and American Indians (29-36).

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Table 1 Properties of human aldehyde dehydrogenases (ALDH).

Human ALDH1A1 and ALDH2E exhibit KM for acetaldehyde in the micromolar to submicromolar range at a near physiological pH and a cytoplasmic NAD+ concentration. The catalytic efficiencies at low substrate concentrations for both ALDH isozymes are, particularly the mitochondrial ALDH2E, much greater than those of ADH isozymes, indicating that acetaldehyde produced from ethanol by the ADH can be efficiently eliminated by the ALDH in cells. X-ray crystallographic studies show that human ADH and ALDH have a similar hydrophobic substrate-binding pocket 510 wide and 1220 long. The much lower KM for ALDH may be in part due to the formation of a covalent thiohemiacetal intermediate in catalysis. The mutant ALDH2K appears inactive in vivo due to its extremely high KM for NAD+ and a much lower Vmax. Recently, a molecular model proposed by Weiner and coworkers predicts that ALDH2 activity in ALDH2*1/*2 heterozygotes would account for 25% that of normal ALDH2*1/*1 homozygotes, which is based on a dimer-of-the-dimers structure and the half of-the-site reactivity of the genetically engineered recombinant enzymes (37). It requires further studies to compare 14

the activities and protein contents of ALDH2 in human livers with 3 different ALDH2 genotypes to corroborate the proposed molecular mechanism for partial negative dominance of ALDH2*2. Higuchi et al. surveyed 1300 Japanese alcohol-dependent patients and found no single subject identified with ALDH2*2/*2, suggesting that homozygosity of variant ALDH2*2 may fully protect against developing alcoholism (38). In contrast, the frequencies of heterozygous ALDH*1/*2 genotype varied from 2.5% to 13% for the Japanese alcoholics and from 10% to 18% for the Han Chinese alcoholics surveyed from different registry periods with a trend of being higher frequencies in more recent years. These findings suggest that the heterozygosity can only afford partial protection against alcoholism, permitting other biological and socio-cultural factors to have an influence in development of the disease (39-43). Unexpectedly, a case of one homozygous ALDH2*2/*2 alcohol dependent was discovered in a survey study with 420 patients in Taiwan. This patient displayed a unique drinking pattern to accommodate his inborn error of acetaldehyde metabolism.

Aldehyde dehydrogenase 2 (ALDH2) deficiencies Japanese studies demonstrated that ALDH2 deficiency reduced the quantity and frequency of alcohol consumption by men and the risk of alcoholism. This effect was confirmed in other Asian populations by the observation that individuals who were alcoholic or who had alcoholic liver disease rarely had ALDH2 deficiency or an ALDH2*2 allele. In Japan about 41% of controls were ALDH2 deficient, while only 25% of alcoholics were ALDH2 deficient. In Taiwanese males the frequency of the ALDH2*2 allele was 30% in a non-alcoholic control group and 6% in alcoholics. Similar results were reported by other research groups. The protective effect of being heterozygous for ALDH2*2 appears to be decreasing over time in Japan, i.e. the frequency of ALDH2*2 heterozygotes among alcoholics is increasing, presumably because of environmental and cultural changes (44). However, ALDH2*2 homozygotes are nearly

15

absolutely protected against alcoholism, presumably because of the severity of their flushing. This phenomenon has been observed in other countries (China and Pacific islands; with a somewhat different prevalence of the ALDH2*2 allele, as well as in Asians living in Canada, suggesting that the flush reaction is protective against alcoholism for biochemical rather than cultural reasons(47-48). ALDH2 deficiency may be a two-edged sword for the reasons mentioned earlier for ADH2*2, since individuals with mild flushing who can tolerate heavy drinking may suffer from the hepatic effects of elevated acetaldehyde concentrations. There has been one small study suggesting that ALDH2*2 heterozygotes who drink heavily develop alcoholic hepatitis at a lower cumulative alcohol consumption than those with active ALDH2. When the results of several studies were combined the prevalence of ALDH2*2 was substantially higher in the alcoholic patients with cirrhosis than in those without cirrhosis (49-50). With the apparent increase in the number of alcoholics heterozygous for ALDH2*2 in Japan, ALDH2 deficiency may become an important risk factor in that population.

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CHAPTER III

MATERIALS AND METHODS

Sample size:

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Base on the study of Boonyaphiphat and colleague in 2002, the allele frequencies for ALDH2*1 = 0.9. The power of these study was 80% with a significance level () of 0.05 (twotailed). From the formula: N = Z2pq/d2 N = (1.96 )2 (0.90)(0.10)/(0.05)2 N = 138.29 N = 140 cases

N = number of subjects Z = the coefficient from the table of standard normal distribution p q d Subjects 1. One hundred and forty cases of patients with no drinking alcohol within 3 years will be enrolled as control group. 1.1 Inclusion criteria: Male 30 - 60 years old No liver diseases, no hypertension, no hyperlipidemia, and no = the value of the proportion as a decimal percent = 1-p = acceptable range of error

diabetes mellitus No medical treatments related with liver function

1.2 Exclusion criteria:

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Active liver diseases, hypertension, hyperlipidemia, and

diabetes mellitus Treatment with medicines related liver function 2. One hundred and forty cases of patients with alcoholic drinking within 3 years will be enrolled as study group. 2.1 Inclusion criteria: Male 30 - 60 years old 2.2 Exclusion criteria: Were diagnoses as liver diseases, hypertension,

hyperlipidemia, or diabetes mellitus Treatment with medicines related liver function Study procedures Baseline studies and clinical management On presentation, a venipuncture will be performed with the collection of 5 ml of blood into vacutainer containing EDTA, 5 ml of blood into a vacutainer with no anticoagulant. The blood collected in the EDTA-containing tube will be used to perform a genotyping. The blood collected in the vacutainer that does not contain anticoagulant will be used for liver function, lipid profiles and fasting blood sugar.

Laboratory works ALDH genotyping: A DNA will be extracted from white blood cells in EDTA blood and will be assay for ALDH genotypes by Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR RFLP).

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Blood chemistries: Serum concentrations of total bilirubin, aspartate amino-transferase, alanine aminotransferase, albumin, total protein, glucose, total cholesterol, triglyceride, HDLcholesterol, LDL-cholesterol will be measured with an automated chemistry analyzer.

CHAPTER IV

RESULTS

DATA ANALYSIS

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For continuous variables with symmetric distribution, the results will be presented as mean + standard error (SE), as well as medians and ranges (minimum, maximum). For continuous variables with skewed distribution, the results will be expressed as the median and range from the twenty-fifth to the seventy-fifth percentile. For dichotomous variables, the results will be expressed as proportions. The distributions of continuous variables will be assessed for normality and where distributions are skewed, power, log, or other appropriate transformations will be applied. Univariate analysis of baseline variables between groups will be performed by using the unpaired Students t test or the Kruskal-Wallis test. Multivariate analysis will be conducted by using analysis of variance for continuous dependent variables and logistic regression for binary dependent variables. All tests will be two-tailed; a level of 0.05 will be used to indicate statistical significance. The SPSS for Windows will be used for statistical computations.

SIGNIFICANT OF THE RESEARCH This study will provide new information about the prevalence of ALDH genes in Thai population. In addition, the result will establish the relationship between ALDH genes and clinical finding in control group compare with study group.

CHAPTER V

DISCUSSION TIME SCHEDULE (1) Preparing the subjects for control group and study group JanFeb

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2010 (2) Preparing the instruments and reagent kits (3) Study 140 cases of control group and 140 cases of study group (4) Data analysis (5) Thesis writing Mar 2010 AprAug 2010 Sep 2010 OctNov 2010

ADVISORS COMMENTS ____________________________________________________ ____________________________________________________ __________________________________ ______________________________________________ ______________________________________________ ______________________________________________ ______________________________________________ ______________________________________________ 22

 (Signature of advisor)

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APPENDICES

30

Appendix A Faculty of Science, Rungsit University Consent to Participate in a Research Study

The purpose of this consent form is to provide you with the information you need to decide whether you will participate in this research study. Title: ROLE OF ALDH GENOTYPES AND SUSCEPTIBILITY TO RISKS OF ALCOHOL-RELATED DISEASE IN THAI POPULATION Study purpose: You are invited to participate in a research study to understand how ALDH gene related with alcoholism and risks of alcohol-related diseases.

o You are participating in this study because you are study group o You are participating in this study because you are control group
Study procedures: On visiting days, beside of the blood samples needed for routine medical care, an additional 2 teaspoon (10 mL) of peripheral blood will be drawn by venipuncture for research purposes. Study risks: Your participation in this study involves the risks that when blood is drawn from a vein in the arm, there may be some temporary discomfort and the minimal risk of local bruising, infection or blockage of the vein. Suitable precautions will be taken to avoid these risks. The amount of blood to be drawn is small, and will not have any effect on health. Study benefits: You may not derive any benefit personally from this study. Benefits to society or to others may include the development of a better understanding to the association between ALDH gene and alcoholism in Thai population. Costs: There will be no costs to you for participation in this study, including the costs of laboratory tests. Compensation: For participation in this study, you will receive 150 Baht for transportation. Confidentiality: All samples and data are coded and are handled anonymously. If the results of the study are published or presented at a medical or scientific meeting, you will not be identified. Research

31

findings will not be part of your medical record. Every effort will be made to protect the confidentiality of your records, but absolute confidentiality cannot be guaranteed. By signing this document you grant permission for information about you obtained during the study to be made available to: Participation is Voluntary: The participation of you or your family member in this study is completely voluntary. You can refuse participation for yourself or your family member, or withdraw him, her or yourself from the study at any time, and such decision will not affect the medical care that either you or your family member receives at the Rungsit University, now or in the future. Signing this form does not waive any of the legal rights of your family member. Any new findings which might affect your willingness to continue in the study will be communicated to you. Questions: If you have any questions, please ask, and we will do our best to answer them. If you have additional questions or problems in the future, you can reach the Principal Investigator, Miss Sirikorn Pokham, Faculty of Science, Rungsit University, Pathumtani.

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Appendix B

Statement of Consent
I have discussed this study with _Miss Sirikorn Pokham_ to my satisfaction. I understand that the participation of me with is voluntary and that I can withdraw myself from the study at any time without prejudice. I have read the above and agree for myself to enter this research study. Signing this form does not waive any of the legal rights of me. I have been informed that if I believe that I have sustained injury as a result of participating in this research study, I may contact the Principal Investigator, Miss Sirikorn Pokham so that I can review the matter and identify the medical resources which may be available to me. I understand that: a) The Rungsit University will furnish that emergency medical care determined to be necessary by the medical staff of this hospital for my family member; b) I or my family member will be responsible for the cost of such care, either personally or through medical insurance or other form of medical coverage; c) No monetary compensation for wages lost as a result of injury will be paid to my family member by Rungsit University, and; d) I will receive a copy of this consent form. Signatures: _________________________________________________________________ Participant Date _________________________________________________________________ Investigator Eliciting Consent Date _________________________________________________________________ Witness Date The solicitation of subjects into this study has been approved by the Ethical Committee, Faculty of Science, Rungsit University, Pathumtani.

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Appendix C

ALDH STUDY

Control Group

ROLE OF ALDH GENOTYPES AND SUSCEPTIBILITY TO RISKS OF ALCOHOLRELATED DISEASE IN THAI POPULATION

CONFIDENTIAL CASE RECORD FORM

FACULTY OF SCIENCE, RUNGSIT UNIVERSITY, PATHUMTANI

Investigator: . ALDH Study Number: Hospital Number: Admission Number: __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __

34

DEMOGRAPHIC DATA Name________________________________________ Age __________ yrs Address _______________________________________________________ Sex M F Weight __ __ . __ kg Height __ . __ __ cm

Date visited to hospital: __ __ / __ __ / __ __ dd mm yy INCLUSION CRITERIA 1) Male, age 30 to 60 years 2) No liver diseases, no hypertension, no hyperlipidemia, and no diabetes mellitus 3) No medical treatments related with liver function 4) Signed informed consent EXCLUSION CRITERIA 1) Absence of signed informed consent 2) Active liver diseases, hypertension, hyperlipidemia, and diabetes mellitus 3) Treatment with medicines related liver function [ ] YES [ ] NO [ ] YES [ ] NO [ ] YES [ ] NO [ ] YES [ ] NO [ ] YES [ ] NO [ ] YES [ ] NO [ ] YES [ ] NO

_________________________ Study group member signature

Date: __ __ / __ __ / __ __

VITAL SIGNS ON ENROLLMENT Temperature __ __ . __ oC B.P. __ __ __ / __ __ __ mm Hg 35 Pulse __ __ __ bpm Respiratory rate __ __ / min

PHYSICAL EXAMINATION Normal 1) Dermatologic 2) HEENT 3) Respiratory 4) Cardiovascular 5) Digestive 6) Endocrine/Metabolic 7) Hematological 8) Urological/Reproductive 9) Neurological/Psychological 10) Musculoskeletal 11) Other [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] Abnormal If abnormal, describe:

[ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________

_________________________ Study group member signature

Date: __ __ / __ __ / __ __

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LABORATORY DATA (I) Schedule Date (dd/mm/yy) RBC Hb HCT MCV MCH MCHC RDW WBC Neutrophils Lymphocytes Monocytes Basophils Eosinophils Platelet count __/__/__

_________________________ Study group member signature

Date: __ __ / __ __ / __ __

LABORATORY DATA (II) Schedule Date (dd/mm/yy) Glucose BUN Creatinine __/__/__

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Direct Bili. Total Bili. Albumin Globulin AST ALT ALP LDH GGT

LABORATORY DATA (III) PCR-RFLP ALDH Genotype

_________________________ Study group member signature

Date: __ __ / __ __ / __ __

Appendix D

ALDH STUDY

Study Group

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ROLE OF ALDH GENOTYPES AND SUSCEPTIBILITY TO RISKS OF ALCOHOLRELATED DISEASE IN THAI POPULATION

CONFIDENTIAL CASE RECORD FORM

FACULTY OF SCIENCE, RUNGSIT UNIVERSITY, PATHUMTANI

Investigator: . ALDH Study Number: Hospital Number: Admission Number: __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __ __

DEMOGRAPHIC DATA Name________________________________________ Age __________ yrs Address _______________________________________________________ Sex M F Weight __ __ . __ kg Height __ . __ __ cm

Date visited to hospital: __ __ / __ __ / __ __ dd mm yy INCLUSION CRITERIA

39

1) Male, age 30 to 60 years 2) No liver diseases, no hypertension, no hyperlipidemia, and no diabetes mellitus 3) No medical treatments related with liver function 4) Signed informed consent EXCLUSION CRITERIA 1) Absence of signed informed consent 2) Active liver diseases, hypertension, hyperlipidemia, and diabetes mellitus 3) Treatment with medicines related liver function

[ ] YES [ ] NO [ ] YES [ ] NO [ ] YES [ ] NO [ ] YES [ ] NO

[ ] YES [ ] NO [ ] YES [ ] NO [ ] YES [ ] NO

_________________________ Study group member signature

Date: __ __ / __ __ / __ __

VITAL SIGNS ON ENROLLMENT Temperature __ __ . __ oC B.P. __ __ __ / __ __ __ mm Hg Pulse __ __ __ bpm Respiratory rate __ __ / min

PHYSICAL EXAMINATION Normal 1) Dermatologic 2) HEENT 3) Respiratory 4) Cardiovascular 5) Digestive [ ] [ ] [ ] [ ] [ ] Abnormal If abnormal, describe:

[ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________

40

6) Endocrine/Metabolic 7) Hematological 8) Urological/Reproductive 9) Neurological/Psychological 10) Musculoskeletal 11) Other

[ ] [ ] [ ] [ ] [ ] [ ]

[ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________ [ ] _________________________

_________________________ Study group member signature

Date: __ __ / __ __ / __ __

LABORATORY DATA (I) Schedule Date (dd/mm/yy) RBC Hb HCT MCV MCH MCHC RDW WBC Neutrophils Lymphocytes 41 __/__/__

Monocytes Basophils Eosinophils Platelet count

_________________________ Study group member signature

Date: __ __ / __ __ / __ __

LABORATORY DATA (II) Schedule Date (dd/mm/yy) Glucose BUN Creatinine Direct Bili. Total Bili. Albumin Globulin AST ALT ALP LDH GGT __/__/__

LABORATORY DATA (III)

42

PCR-RFLP ALDH Genotype

_________________________ Study group member signature

Date: __ __ / __ __ / __ __

43