Israel, Yedy

Tu, Guang-chou

Clinical Chemistry and Laboratory Medicine (CCLM)

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A New Approach for the Rapid Detection of Common and Atypical Aldehyde Dehydrogenase Alleles

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Tu and Israel: AJdehyde dehydrogenase genotyping 591Eur. J. Clin. Chem. Clin. Bioehem.Vol. 31, 1993, pp. 591-594© 1993 Walter de Gruyter & Co.Berlin · New YorkA New Approach for the Rapid Detection of Commonand Atypical Aldehyde Dehydrogenase AllelesBy Guang-chou Tu1 and Yedy Israel1'21 Addiction Research Foundation of Ontario, Toronto, Ontario, Canada2 Departments of Pharmacology and Medicine, University of Toronto, Toronto, Ontario, Canada(Received February 3/May 27, 1993)Summary: A strong protection against the development of alcoholism is exerted by a point mutation in thegene coding for low Km aldehyde dehydrogenase (ALDH), i. e. ALDH2. We report a non-radioactive methodfor determining the common and atypical human mitochondrial aldehyde dehydrogenase (ALDH2) genotypes.This method is based on the fact that the base change (G —> A) in Exon 12 of the ALDH2 gene abolishes anEcoSl I restriction site (CTGAAG —> CTAAAG). A GC-clamp attached oligonucleotide was designed toyield a 176 base pair product by the polymerase chain reaction. After amplification, the resulting fragmentcontaining the normal nucleotide sequence is cut by EcoSl I into two segments (131 base pairs + 45 basepairs) while the fragment containing the mutated sequence remains intact (176 base pairs). These are visualizedby staining with ethidium bromide on agarose gels without blotting, hybridization or autoradiography.IntroductionA single base substitution (G/A) in Exon 12 of mi-tochondrial aldehyde dehydrogenase (ALDHk)1) genecauses the loss of the activity of the enzyme coded(1). Individuals carrying this gene mutation are unableto rapidly detoxify acetaldehyde produced in the me-tabolism of ethanol. When alcohol is consumed bythese individuals, blood acetaldehyde concentrationsare markedly increased, reaching values which leadto the development of facial flushing, hypotension,tachycardia, nausea and in some cases vomiting (2, 3).Racial differences in ALDH2 alleles between Cauca-sians and Orientals have been found. Approximately30 — 50% of Orientals have the mutant gene, whilevirtually all Caucasians examined thus far have theusual alleles (4, 5).The existence of the ALDH2 gene mutation has beenshown to drastically lower the prevalence of alcohol') Enzymes: Aldehyde dehydrogenase (aldehyde : NAD* oxi-doreductase, EC 1.2.1.3)Eco 57 I (restriction endonuclease, EC 00.00.00.0)dependence. In Japan, only 5% of alcohol dependentpatients present the mutation compared with about40% for the normal population (6). Thus, the muta-tion exerts an 80 — 90% reduction in the risk of be-coming an alcoholic. In Taiwan, the protection is ofthe order of 75% (7).In order to elucidate the possible association betweenALDH2 genotypes, alcohol sensitivity, alcoholism,and other alcohol-related morbidities, it is importantto develop a simple and reliable genotyping method.In 1987, Hsu et al. (8) first developed a method fordetermining human ALDH2 genotypes. These inves-tigators used two 21-base synthetic oligonucleotides,one complementary to the usual ALDH2 gene andthe other complementary to the atypical ALDH2 gene,as .specific probes for in-gel hybridization analysis ofhuman genomic DNA. Under appropriate hybridi-zation conditions, these two probes hybridize to theirspecific complementary alleles and thus allow thegenotyping of the ALDH2 locus.In 1989, Crabb et al. (9) developed a method forALDH2 genotyping based on the amplification ofEur. J. Clin. Chem. Clin. Biochem. / Vol. 31,1993 / No. 9592 Tu and Israel: Aldehyde dehydrogenase genotypinggenomic DNA by the polymerase chain reaction(PCR). This method is faster and requires smalleramounts of DNA (2 μ§ or less) than methods thatuse probing of Southern blots of genomic DNA di-gested with restriction endonuclease. Singh et al. (5)and Enomoto et al. (10) improved the method byemploying the dot-blot and slot-blot hybridization.The above methods are time-consuming and requirethe use of radioactive phosphorous (32P). We havedeveloped a simple non-radioactive method in whichthe gene fragment containing either the mutated orthe normal sequence is amplified by the PCR reactionand is then cut by a new restriction enzyme (EcoSl I)1)that recognizes only the normal (CT(/AAG), but notthe mutated (CTAAAG) nucleotide sequence. Follow-ing enzymatic digestion, separation by electrophoresisand staining by ethidium bromide, two distinct bandsfrom the fragment containing the normal sequence,can be seen4 on agarose gels. The mutated sequence,not recognized by Eco511, yields only one band.Materials and MethodsWhole blood and hair root samplesSamples of whole blood and hair roots were collected from 12individuals working at the University of Toronto. The volunteernumber is indicated by the number or the subscript in figures2-4. [Chinese (#1-5, #8-9), Caucasians (#6, #12), Chi-leans (#7, #11), Indian (#10)].Genomic DNAGenomic DNAs were prepared from either 50 μΐ of wholeblood or 10 hair roots as described in a previous paper (11).The samples were dissolved in 20 μΐ of buffer (10 mmol/1 Tris-HC1, pH 8.0 and 1 mmol/1 EDTA).OligonucleotidesTwo polymerase chain reaction (PCR) primers and two ALDH2allele-specific probes were synthesized on an Applied Biosys-tems 380B DNA synthesizer. The base sequences of two ALDH2allele-specific 32P-probes are the same as these used by Hsu etal. (8), Crabb et al. (9) and O 'Dowdei al. (11). The base sequenceof PCR primer #1 (23 mer) and primer #2 (60 mer) are: 5'-CAAATTACAGGGTCAACTGCTAT-3' and 5'-CCGCGCCCGCCGCCCCGCGCCCCCCCGCCCGCCCCGCGCTCCACACTCACAGTTTTCAC-3', respectively.Polymerase chain reactionA 10 μΐ aliquot of the genomic DNA, extracted from eitherwhole blood or hair roots as indicated above, was mixed with100 pmol of each primer and submitted to the polymerase chainreaction using GeneAmp PCR Reagent kit with Ampli TaqDNA Polymerase (Perkin-Elmer Cetus). The amplification tem-perature conditions were as follows: 94 °C for 5 min followedby 30 cycles consisting of l min at 94 °C, l min at 55 °C, andl min at 72 °C; the final extension was 7 min at 72 °C. Afteramplification, 10 μΐ of each reaction mixture were submitted toagarose gel electrophoresis analysis or enzymatic digestion.EcoSl I digestionA 10 μΐ aliquot of the PCR reaction mixture was mixed with2 μΐ of buffer (100 mmol/1 Tris-HCl, pH 7.5,100 mmol/1 MgCI2),1 μΐ of 0.2 mmol/1 S-adenosyl-Z,-methonine, 1 μΐ of 2 g/l bovineserum albumin, 5.5 μΐ of distilled w^ter and 0.5 μΐ of EcoSl I(4 units/μΐ, Fermentas, Lithuania; distributed by New EnglandBiolabs). The digestion was carried out at 37 °C for l h andstopped by adding 1 μΐ of 0.5 mol/1 EDTA. After digestion thefragments were separated on a 1.5% or 2% agarose gel andstained with ethidium bromide.Slot-blot-hybridizati nA 10 μΐ aliquot of the PCR reaction mixture was submitted tothe slot-blot hybridization described by Enomoto et al. (10) ona Hybri-slot Manifold (BRL Life Technologies Inc., MD 20877,U. S. A.). After blotting, the duplicate nitrocellulose filters weretreated in a Stratalinker UV Crosslinker (Stratagene, CA 92037,U.S.A.), then probed respectively with two allele-specific 32Poligonucleotides, for ALDH2 and ALDH2, as described by Hsuet al. (8), using the prehybridization and hybridization condi-tions outlined by Crabb et al. (9). Each filter was exposed toKodak XAR-5 film for several hours, with one intensifyingscreen.Results and DiscussionAlthough the recognition sequence for EcoSl I is inExon 12 where the Oriental point mutation occurs,the cutting point of the restriction enzyme is located6 to 8 nucleotides downstream from the exon (fig. 1).In order to distinguish a digested PCR amplifiedfragment from an undigested one, it was necessary toamplify a PCR fragment at least 50 base pairs longerthan Exon 12. Unfortunately, except for the first 10bases, the sequence of intron 12 of the ALDH2 geneis not available. Thus it is not possible to design aprimer that is fully complementary to the sequenceof intron 12.To solve this problem we designed a primer by ex-tending a 41 base pair of GC-clamp at the 5'-end ofa 19 mer oligonucleotide which is complementary tothe exon 12-intron 12 known sequence of the ALDH2gene (fig. 1). Using this primer (designated as Primer#2) and the other primer (designated as Primer #1,see fig. 1) a 176 base pair fragment was amplifiedfrom genomic DNA by the PCR and visualized withethidium bromide on the agarose gel (fig. 2).After digestion with EcoSl I, the 176 base pair frag-ment from 10 subjects (including 5 Orientals) werefound to be cut into two segments (131 base pairsand 45 base pairs), while the fragment from subject4 remained intact as one fragment and the fragmentfrom subject 5 was partially (40%) cut as shown infigure 3 (the 45 base pair bands overlap with the 60pair primer #2 bands).These results show that sample 4 was from an indi-vidual who carries a homozygous atypical ALDH2Eur. J. Clin. Chem. Clin. Biochem. / Vol. 31,1993 / No. 9Tu and Israel: Aldehyde dehydrogenase genotyping 593Intron 11 Exon 12PrimerC A A A T T A C A G G G T C A A C T G C T A TG T T T A A T G T C C C A G T T G A C G A T AG A T G T G T T T G G A G C C C A G T C A C C C T T T G G T G G C T A C A A G A T G T CCT AC AC A A ACCT C G G G T C A G T G G G A A A C C A C C G A T G T TC T AC AG•Exon 12·Allele-specif ic probes Cutting point4G G G G A G T G G C C G G G A G T T G G G C G A G T A C G G G C T G C A G G C A T A C A C T Q A A G T G A A A A C T G T G A G T G T G GC C C C T C A C C G G C C C T G A A C C C G C T C A T G C C C G A C G T C C G T A T G T G A C TT C A C T T T T G A C A C T C A C A C CtCutting pointPrimerGC clampAldehyde dehydrogenase adele specific probesALDH2 1 : 5' GT T T T C ACT TC AGT GT AT GCCALDH22: 5' G G C A T AC A C T A A A G T G A A A AC5'...C T G A A G (N)16Y... 3'3'...G A C T T C (N)14... 5'Eco57l recognition sequenceFig. 1. Structure of the common ALDH2 gene in the region amplified by the polymerase chain reaction. Two oligonucleotides,primer #1 and primer #2, serve to initiate DNA synthesis of the 176 base pair fragment. The nucleotide bases in boldtype indicate the position of the mutation in the atypical allele. The two allele-specific probes are those described by Hsuet al. (8) and by Crabb et al. (9). The site of the single base change in the oligonucleotide ALDH| is indicated by anarrow. The oligonucleotides anneal to complementary DNA strands.200 Base pairs -100 Base pairs-N1 N2 N3 N4 N5 N6 N7 Νβ L N9N1 0N1 1N1 2Fig. 2. The genomic DNA from whole blood or hair rootsamples was amplified, using the primers described inMaterials and Methods, and subjected to electropho-resis in 2% agarose gel, followed by ethidium bromidestaining. Laue L is 100 base pair ladder. Lanes Nj-^are samples from subjects 1 — 12 (not digested withrestriction enzyme). The major band generated corre-sponds to the 176 base pair product.gene (ASDHi/ALDHi), sample 5 from the individualwho carries the heterozygous ALDH2 gene (ALDHi/ALDHi) and samples 1—3 and 6 — 12 were takenfrom the individuals who carry the common homo-zygous ALDH2 gene (ALDH|/ALDHi).In order to confirm the new method, the PCR prod-ucts were transferred to nitrocellulose by the slot-blottechnique and the filters were hybridized with the twospecific ALDH2 allele 32P-labelled probes. After wash-ing, the filters were exposed to X-ray film. As shownin figure 4 the DNA amplified from sample 4 wasonly recognized by probe ALDHi, the DNA amplifiedfrom sample 1—3 and 6 — 12 were only recognizedwith probe ALDHi, and the DNA amplified fromsample 5 was recognized with both the probe forALDHi and the probe for ALDHi. The new methodgives the same results as the Southern blot radioactivemethod (5, 8-11).According to the EcoSl I manufacturer's information,the cleavage of DNA by this restriction enzyme isnever quantitative. In addition to the 131 base pairband, all subjects (except 4 and 5) show a very weakundigested band at the location of the 176 base pairband on the agarose gel (fig. 3). This may be thereason for the fact that for sample 5 (heterozygote)about 40% of the 176 base pair fragment was foundto be cut instead of 50%. However, this characteristicof the enzyme does not affect the genotype of thesamples.Eur. J. Clin. Chem. din. Biochem. / Vol. 31,1993 / No. 9594 Tu and Israel: Aldehyde dehydrogenase genotyping200 Base pairs100 Base pairsm.L 0, D2 N3 D3 D4 D5 L 06 D7 N7-*— 200 Base pairs«—100 Base pairsFig. 3. The polymerase chain reaction (PCR) products were digested with restriction enzyme Eco511 as described in Materialsand Methods, subjected to electrophoresis in 2% agarose gel (or 1.5% on the extreme right), followed by ethidiumbromide staining. Lane L is a 100 base pair ladder. N = non-digested PCR product, D = Eco571 digested PCR product.ALDHjprobeALDH* probe1 2 3 4 5 6 7 8 9 10 11 12| t | 0 0 0 0 3 (I 0 0Fig. 4. The polymerase chain reaction (PCR) products wereslot blotted on nitrocellulose filters as described byEnomoto et al. (10), hybridized with two 32P^labelledallele-specific probes, ALDHi and ALDH?, describedin figure 1, followed by autoradiography.In conclusion, we have developed a new approach forgenotyping the common and the atypical aldehydedehydrogenase (ALDH2) alleles, which(i) is considerably faster and simpler than presentlyavailable methods,(ii) does not use a radioactive isotope and(iii) requires very minute amounts of DNA, whichcan be obtained from a drop of whole blood orfrom hair roots.This new approach should be of value in the geno-typing of populations for epidemiological and geneticstudies.References1. Hsu, L. C, Tani, K., Fujiyoshi, T, Kurachi, K. & Yoshida,A. (1985) Cloning of cDNA's for human aldehyde dehy-drogenase 1 and 2. Proc. Natl. Acad. Sei. USA 82, 3771 -3775.2. Mizoi, Y., Ijiri, L, Tatsuno, Y, Kijima, T, Fujiwara, S. &Adachi, J. (1979) Relationship between facial flushing andblood acetaldehyde levels after alcohol intake. Pharmacol.Biochem. Behav. 10, 303-311.3. Harada, S., Agarwal, D. P. & Goedde, H. W. (1981) Al-dehyde dehydrogenase deficiency as cause of facial flushingreaction to alcohol in the Japanese. Lancet //, 982.4. Shibuya, A. & Yoshida, A. (1988) Frequency of the atypicalaldehyde dehydrogenase-2 gene (ALDH?) in Japanese andCaucasians. Am. J. Hum. Genet. 43, 741-743.5. Singh, S., Fritze, G., Fang, B., Harada, S., Paik, Y. K.,Eckey, R., Agarwal, D. P. & Goedde, H. W. (1989) Inher-itance of mitochondrial aldehyde dehydrogenase: Genotyp-ing in Chinese, Japanese and South Korean families revealsdominance of the mutant allele. Hum. Genet. 83, 119-6. Harada, S., Agarwal, D. P. & Goedde, H. W. (1985) Al-dehyde dehydrogenase polymorphism and alcohol metab-olism in alcoholics. Alcohol 2, 391-392.7. Thomasson, H. R., Edenberg, H. J., Crabb, D. W., Mai,X-L., Jerome, R. E., Li, T-K., Wang, S-P., Lin, Y-T, Lu,R-B. & Yin, S-J. (1991) Alcohol and aldehyde dehydrogen-ase genotypes and alcoholism in Chinese men. Am. J. Hum.Genet. 48, 667-681.8. Hsu, L. C, Bendel, R. E. & Yoshida, A. (1987) Directdetection of usual and atypical alleles on the human alde-hyde dehydrogenase-2 (ALDH2) locus. Am. J. Hum. Genet.47,996-1001.9. Crabb, D. W., Edenberg, H. J., Bosron, W. F. & Li, T-K.(1989) Genotypes for aldehyde dehydrogenase deficiencyand alcohol sensitivity. The inactive ALDHi allele is dom-inant. J. Clin. Invest. 83, 314-316.10. Enomoto, N., Takase, S., Takada, N. & Takada, A. (1990)Alcoholic liver disease in heterozygotes of mutant andnormal aldehyde dehydrogenase-2 genes. Hepatol. 73,1071-1075.O'Dpwd, B. P., Rothhammer, F. & Israel, Y. (1990) Gen-otyping of mitochondrial aldehyde dehydrogenase locus ofnative American Indians. Alcoholism: Clin. Exp. Res 14531-533.Dr. Yedy IsraelPrimary Mechanisms DepartmentAddiction Research Foundation33 Russell StreetToronto, Ontario M5S 2S1Canada11Eur. J. Clin. Chem. Clin. Biochem. / Vol. 31,1993 / No. 9

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A New Approach for the Rapid Detection of Common and Atypical Aldehyde Dehydrogenase Alleles

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