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Am. J. Hum. Genet.

43:744-748, 1988

Genotypes of Alcohol-Metabolizing Enzymes in Japanese with


Alcohol Liver Diseases: A Strong Association of the Usual
Caucasian-Type Aldehyde Dehydrogenase Gene (ALDH21)
with the Disease
Akitaka Shibuya and Akira Yoshida
Department of Biochemical Genetics, Beckman Research Institute of the City of Hope, Duarte, CA

Summary
Genetic polymorphisms of two major alcohol-metabolizing enzymes-i.e., one of the class I alcohol de-
hydrogenase isozymes (ADH2) and the mitochondrial aldehyde dehydrogenase (ALDH2)-exist in Japanese
and other Orientals but not in Caucasians. Liver ADH activity of about 90% of Orientals is much higher
than that of most Caucasians, while approximately 50% of Orientals lack the ALDH2 activity. The
genetic differences have been implicated in the high incidence of alcohol sensitivity observed in Orientals.
We determined, by means of hybridization of genomic DNA samples with allele-specific synthetic oligonu-
cleotide probes, genotypes of the ADH2 and the ALDH2 loci of Japanese with alcoholic liver diseases and
of control subjects. No significant difference between the patient and control groups was found in the
ADH2 genotypes. A remarkable genetic difference between the two groups was found in the ALDH2 locus.
The frequency of the typical (Caucasian-type) ALDH2 gene was found to be .65 and that of the atypical
(Oriental type) ALDHI gene was .35 in the controls, while these were .93 and .07, respectively, in the pa-
tients. Thus, most (20 of 23) of the Japanese patients were homozygous Caucasian type ALDH1/
ALDH2, only three were heterozygous ALDH2/ALDH2, and none of the patients were homozygous Orien-
tal type ALDH2/ALDHI. The results indicate that Japanese with the atypical ALDH2 allele are at a much
lower risk in developing the alcoholic liver diseases than are those with homozygous, usual (Caucasian-
type) ALDH21ALDH2, presumably owing to their sensitivity to alcohol intoxication.

Introduction
isozymes, which are homo- and heterodimers consist-
Individual and racial differences in alcoholic intoxica- ing of three types of subunits, a, I, and y (Smith et
tion are thought to be related to genetic differences in al. 1973). The three subunits are controlled by three
the enzymes involved in alcohol metabolism. The ox- nonallelic genes, i.e., ADH1 for a, ADH2 for 13, and
idative pathway of ethanol in the human liver is cata- ADH3 for y (Smith et al. 1973). Nearly 90% of Orien-
lyzed primarily by the class I alcohol dehydrogenase tals are "atypical," and their livers exhibit much higher
(alcohol: NAD + oxidoreductase; E.C.1.1.1.1; abbrevi- ADH activity than do those of typical Caucasians
ation ADH) and aldehyde dehydrogenase (aldehyde: (Stamatoyannopoulos et al. 1975). It has been suggested
NAD + oxidoreductase; E.C.1.2.1.3; abbreviation that a higher incidence of alcohol sensitivity in Orien-
ALDH). The human liver contains several class I ADH tals (50%-90%) than in Caucasians (about 10%) could
be due to the rapid acetaldehyde formation by the more
active, atypical ADH2 isozymes (Stamatoyannopoulos
Received June 1, 1988; revision received June 9, 1988. et al. 1975).
Address for correspondence and reprints: A. Yoshida, Department Human livers contain two major ALDH isozymes,
of Biochemical Genetics, Beckman Research Institute of the City of
Hope, Duarte, CA 91010. i.e., cytosolic ALDH1 and mitochondrial ALDH2
i 1988 by The American Society of Human Genetics. All rights reserved. (Greenfield and Pietruszko 1977; Ikawa et al. 1983).
0002-9297/88/4305-0022$02.00 Approximately 50% of Orientals lack the activity of
744
Alcohol-metabolizing Enzymes in Japanese 745

ALDH2 (Goedde et al. 1979; Teng 1981), which has pital in Japan, as follows: alcoholic fatty liver (two pa-
a low Km for acetaldehyde, and hence is considered to tients), alcoholic fibrosis (five patients), alcoholic liver
play a major role in acetaldehyde oxidation in the liver. cirrhosis (15 patients) and alcoholic liver cirrhosis ac-
The alcohol sensitivity observed in the majority of companied by hepatocellular carcinoma (one patient).
Orientals may be due to the absence of ALDH2 activ-
ity and to consequent accumulation of acetaldehyde in Genotype Determination
the body (Goedde et al. 1979; Teng 1981; Kogame and DNA samples were prepared from fresh whole blood
Mizoi 1985). by the method of Blin and Stafford (1976). The DNA
The alcohol sensitivity might discourage individuals samples ("'10 gg each) were either doubly digested by
from drinking, thus decreasing the risk of alcohol- EcoRI and PvuII for genotyping the ADH2, locus or
related problems. It has been reported that most of the singly digested by PstI for genotyping the ALDH2 lo-
Japanese with alcoholic liver disease had the usual Cau- cus. The restriction fragments were separated by aga-
casian-type ALDH2 isozyme (Kanayama et al. 1983; rose-gel electrophoresis. The first in-gel hybridization
Yoshihara et al. 1983). The ADH2 types were not de- was carried out using the 32P-labeled oligonucleotide
termined in these studies. To elucidate the genetic back- probe specific to the usual allele (i.e., ADH1 or
ground of alcohol sensitivity, alcoholic liver diseases, ALDH'). The hybridized gel was treated with alkaline
and other alcohol-related problems, it would be im- solution to remove the first probe, and the gel was rehy-
portant to determine the genotypes of the ADH and bridized with the P32-labeled oligonucleotide probe
ALDH loci of patients and controls. Owing to both specific to the atypical allele (i.e., ADH2 or ALDH2).
the difficulty in obtaining liver samples and the techni- The details of the procedures, including the conditions
cal problems in genotyping, the studies have been seri- of agarose-gel electrophoresis, hybridization, washing,
ously hindered. and rehybridization, have been described elsewhere (Hsu
We previously determined the molecular differences et al. 1987; Ikuta et al., in press). The sequences of
between the usual ADH' and the atypical ADH2 and synthetic oligonucleotide are given in the figure legends.
the differences between the usual ALDH' and the atyp- The oligonucleotides were labeled at the 5' end with
ical ALDH2. The atypical ADH2 gene has a nucleo- [y32p] ATP and T4 polynucleotide kinase by the stan-
tide base transition G/C A/T, and the gene product, dard method (Maniatis et al. 1982).
i.e., the atypical 2 subunit, contains an Arg--His sub-
stitution at the 47th position from the NH2-terminal Results and Discussion
(Ikuta et al. 1986). The atypical ALDH2 gene has a
nucleotide base transition G/C- A/T, and the gene The genotypes of ADH2 and ALDH2 loci were de-
product, i.e., the atypical ALDH2 protein, contains a termined by the hybridization of genomic DNA sam-
Glu-*Lys substitution at the 14th position from the ples by using the allele-specific synthetic oligonucleo-
COOH-terminal (Yoshida et al. 1984; Hsu et al. 1985). tide probes. Examples of hybridization profiles are
Utilizing a pair of allele-specific synthetic oligonucleo- shown in figures 1 and 2. On the basis of positive or
tides, one complementary to the usual gene and the negative hybridization of the targeted restriction frag-
other complementary to the atypical gene, we devel- ments to the probes, the genotypes of ADH2 and
oped the method for determining the genotypes of the ALDH2 loci can be unequivocally determined. The
ADH2 locus and the ALDH2 locus (Hsu et al. 1987; results shown in tables 1 and 2 are compatible with
Ikuta et al., in press). The present paper reports the the Hardy-Weinberg expectation, confirming the valid-
genotypes of the two loci in Japanese patients with al- ity of the genotyping method.
coholic liver diseases and in control subjects. In the 49 control subjects (98 chromosomes) exam-
ined, the frequency of the usual ADH1 gene is .29 and
Material and Methods that of the atypical ADH2 gene is .71; in the 23 pa-
tients examined, the respective allele frequencies are .26
Sample Sources for the ADH1 and .74. The difference in the gene fre-
Forty-nine healthy unrelated Japanese and 23 un- quency between the two groups is statistically not
related patients with alcoholic liver diseases were the significant (P > .40). The deviation from Hardy-
subjects ofthe study. On the basis of histological criteria Weinberg expectation observed in the patients is also
(Takeuchi et al. 1987), the patients were diagnosed at statistically not significant. On the basis of their elec-
the Department of Pathology, Kitasato University Hos- trophoretic or isoelectric-focusing examinations of liver
746 Shibuya and Yoshida

A 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 1011
2.0Kb-
1.8Kb-

B
Kb ::~

2.0Kb-

Figure I Hybridization of human genomic DNAs with the


oligonucleotide probes for the ADH2 locus. DNA samples were
digested with EcoRI and PVUII, electrophoresed in 0.9% agarose Figure 2 Hybridization of human genomic DNAs with the
gel, and hybridized with oligonucleotide probe-i, which is complemen- oligonucleotide probes for the ALDH2 locus. DNA samples were
tary to the usual ALDH2 allele (A) and were washed out and rehy- digested with PstI, electrophoresed in 0.9% agarose gel, and hybrid-
bridized with oligonucleotide probe-2, which is complementary to ized with oligonucleotide probe-i, which is complementary to the
the atypical ALDH2 allele (B). Probe-1: 3' C-CGT-ATG-TGA- usual ALDH' allele (A) and were washed out and rehybridized with

CTT*CAC-TTT-TG 5'. Probe-2: 5'G*GCA*TAC*ACT*AAA*GTG- oligonucleotide probe-2, which is complementary to the atypical
AAA-AC 3' (Position of base substitution is underlined.) Sample 11 ALDH 2allele (B). Probe-i: 3' CCT-TAG-ACA-GCG-TGT-CTA-CTG
is identified to be homozygous usual 1-1; samples 2 and 10 are 5' Probe-2: 5' GGA-ATC-TGT-CAC-ACT-GAT-GAC 3'. (Position
identified to be heterozygous atypical 1-2; and samples 1 and 3-9 of the base substitution is underlined.) Samples 1, 4, and 8 are identi-

are identified to be homozygous atypical 2-2. fied to be homozygous usual 1-i; samples 2, 3, 5-7, and 9 are

identified to be heterozygous atypical 1-2; and samples 10 and 11

are identified to be homozygous atypical 2-2.

samples, previous investigators roughly estimated the


ADH2 gene frequency as .61-.74 in Japanese (Stama- subjects are at a low risk of alcoholism and other
toyannopoulos et al. 1975; Harada et al. 1980; Yin et alcohol-related problems.
al. 1984; Kogame and Mizoi 1985). There is a paradoxical relationship between alcohol
A remarkable difference between the controls and sensitivity and alcohol-related problems in American
the patients is observed in the ALDH2 genotypes. In Indians and Eskimos. These populations and Orien-
the control group, 21 of 49 are homozygous usual 1-1
(i.e., Caucasian type), 22 are heterozygous atypical 1-2,
and six are homozygous atypical 2-2. In the patient Table I
group, 20 of 23 are homozygous usual 1-1, three are
Genotypes of the ALDH2 Locus in Patients With Alcoholic
heterozygous atypical 1-2, and none are homozygous Liver Disease and in Controls
atypical 2-2. In the control group the frequency of the
usual ALDH1 gene is calculated to be .65 and that of GENOTYPES GENE FREQUENCY
the atypical ALDH2 gene is .35, while these are .93 1-1 1-2 2-2 ADH' ADH'
and .07, respectively, in the patients. The difference in Control (n = 49):
gene frequency between the two groups is statistically Observed ........ 4 20 25 .29 .71
significant (P < .005). The frequency of the atypical Calculated aa .... 4.1 20.2 24.7 ... ...
ALDH2 was null in Caucasian DNA samples exam- Patient (n = 23):
ined (unpublished observation). Observed ........ 3 6 14 .26 .74
The results clearly indicate that both the homozy- Calculated aa ... . 1.9 9.5 11.6
Calculated bb .... 1.6 8.9 12.6
gous atypical ALDH 2/ALDH2 subjects, who lack ac-
tive ALDH2 isozyme, and the heterozygous atypical NOTE. -The difference in the gene frequency between the con-
ALDH2/ALDH2 subjects, who have diminished ALDH2 trols and patients is statistically not significant (P > .4).
a
Based on Hardy-Weinberg equilibrium, using the gene frequen-
activity, are far less predisposed to alcoholic liver dis- cies of the control group.
eases, presumably owing to their alcohol sensitivity. On b Based on Hardy-Weinberg equilibrium, using the gene frequen-
the same reasoning, it is expected that these atypical cies of the patients' group.
Alcohol-metabolizing Enzymes in Japanese 747

Table 2 lation, Kyushu University, for providing us with the control


DNA samples. This work was supported by National Insti-
Genotypes of the ALDH2 Locus in Patients with tutes of Health grant AA05763.
Alcoholic Liver Diseases and in Controls
GENOTYPES GENE FREQUENCY
References
1-1 1-2 2-2 ADH2 ADH2
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