Development Of Genotyping Methods For Major Alcohol-metabolizing Enzyme Genes

Objective:The principal enzymes involed in alcohol metabolism are alcohol dehydrogenase‐2(ADH2), which metabolizes ethanol to acetaldehyde, and aldehyde dehydrogenase‐2(ALDH2), which oxidizes toxic acetaldehyde to non‐toxic acetate. As ADH2and ALDH2have gene polymorphisms, G143A locus (rs1229984)of ADH2gene and G1510A locus (rs671) of ALDH2gene, which results in a different activity for alcohol‐related metabolic enzymes. These locus are associated with some alcohol‐related diseases.We have established a simple, economical, fast and high‐accuracy genotyping medthod for rs1229984locus and rs671locus, and it is conducive to promoting alcohol‐related metabolic enzymes genotyping carried out in the health sector. It will help people to know their own alcohol metabolic enzymes ability, drinking healthy to reduce the incidence of alcohol‐related diseases.Method:1. For rs671locus: the same100cases were tested in the following five methods. They are all based on polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP), while there are some differences in template preparation, PCR primer design‐ation and modification, restriction endonuclease selection, and electrophoresis methods, etc.(1) Method1: Amplifing the PCR products spanning1510G>A locus by a pair of primers F1, R1,they were completely matched with the template sequences which is extracted and purified from buccal swabs. The length of PCR products is535bp. The wild‐type ALDH2*1allele (G1510) PCR products contain the sequence recognized by restriction endonuclease TspRⅠ(NNCASTGNN↓) near polymorphic site, while the mutant allele ALDH2*2(A1510) PCR products don’t. Furthermore, both of them contain another recognize site within their downstream sequence. Then PCR products was digested by TspRⅠfor5min at65℃before electrophoresis on4%(w/v) agarose gel and the genotypes separated. The535bp mutant fragment ALDH2*2/ALDH2*2was cleaved into two fragments of405.5and129.5bp. The wild‐type fragment ALDH2*1/ALDH2*1was cleaved into three fragments of255,150.5and129.5bp. So the heterozygous ALDH2*1/ALDH2*2was cleaved into four fragments of405.5,255,150.5,129.5bp.(2) Method2: Same with the method1in addition to the PCR templates. The templates were taken from buccal swabs sample processing solution.(3) Method3: Amplifing PCR products spanning1510G>A locus by the semi‐nested PCR. The first round of PCR used primers F1, R1as were used in method1. The second round of PCR used F2which were completely matched with the template sequences while R1was the other primer. The template taken from buccal swabs sample processing solution. The length of PCR products is467bp. The wild‐type ALDH2*1allele (G1510) PCR products contain the polymorphic site with the sequence(NNCASTGNN↓) can recognize by drestriction endonuclease TspRⅠ, while the mutant allele ALDH2*2(A1510) PCR products don’t. Furthermore, both of them contain another recognize site within their downstream sequence. Then PCR products was digested by TspR Ⅰ for5min at65℃before electrophoresis on4%(w/v) agarose gel and the genotypes separated. The467bp mutant fragment ALDH2*2/ALDH2*2was cleaved into two fragments of337.5and129.5bp. The wild‐type fragment ALDH2*1/ALDH2*1was cleaved into three fragments of255,129.5and82.5bp. So the heterozygous ALDH2*1/ALDH2*2was cleaved into four fragments of337.5,255,129.5and82.5bp.(4) Method4: Amplifing PCR products spanning1510G>A locus by the nested PCR, using the template taken from buccal swabs sample processing solution. The first round of PCR used primers F2, R1which were completely matched with the template sequences. The second round of PCR used F3, R3. Primer F3was matched with the template while R3was not. Primer R3is at length of56nt with two mutations near the3’end of the primer. It is in order to introducing HinfⅠrecognition site（GA↓NTC）in the vicinity of the polymorphic site of the wild‐type allele. Furthermore, we added a33nt joints at the5’end to increase the resolution.The length of PCR products is372bp. The wild‐type ALDH2*1allele (G1510) PCR products contain the polymorphic locus with the sequence that can recognize by drestriction endonuclease HinfⅠ, while the mutant allele ALDH2*2(A1510) PCR products don’t. In addition, both of them contain another recognize site within their upstream sequence. Then PCR products was digested by Hinf Ⅰ for5min at37℃before electrophoresis on4%(w/v) agarose gel and the genotypes separated. The372bp mutant fragment ALDH2*2/ALDH2*2was cleaved into two fragments of290.5and81.5bp. The wild‐type fragment ALDH2*1/ALDH2*1was cleaved into three fragments of236,81.5and54.5bp. So the heterozygous ALDH2*1/ALDH2*2was cleaved into four fragments of290.5,236,81.5and54.5bp.(5)Methods5: Amplifing the PCR products spanning1510G>A locus by a pair of primers F3, R3which were used in method4. The template was extracted and purified from buccal swabs. The length of PCR products is372bp, and was digested by HinfⅠfor5min at37℃before electrophoresis on10%polyacrylamide gel and the genotypes separated. The genotyping results were the same as method4.2.Using samples as ALDH2*1/*1、ALDH2*2/*2genetype to construct recombinant plasmids which contained ALDH2*1/*1or ALDH2*2/*2allele by T‐A clone and verified by sequencing.3. For rs1229984locus: the same100cases were test in the method. PCR products spanning143G>A locus were amplified by the semi‐nested PCR, using the template taken from buccal swabs sample processing solution. The first round of PCR used primers F5, R6which were completely matched with the template sequences. The second round of PCR used F6, R6. Primer R6was matched with the template while F6was not. Primer F6is at length of59nt with one mutation near the3’end of the primer. It is in order to introducing HhaⅠrecognition site（GC↓GC）in the vicinity of the polymorphic site of the mutant allele. Furthermore, we added a39nt joints at the5’end to increase the resolution. The length of PCR products is393bp. The wild‐type ALDH2*2(G143)PCR products contain the polymorphic loci with the sequence that can recognize by drestriction endonuclease HhaⅠ, while the mutant allele ALDH2*1allele (A143) PCR products haven’t. In addition, both of them contain another recognize site within their downstream sequences. Then PCR products was digested by HhaⅠfor5min at37℃before electrophoresis on4%(w/v) agarose gel and the genotypes separated. The393bp mutant fragment ALDH2*2/ALDH2*2was cleaved into two fragments of286and107bp. The wild‐type fragment ALDH2*1/ALDH2*1was cleaved into three fragments of277,107and59bp. So the heterozygous ALDH2*1/ALDH2*2was cleaved into four fragments of286,277,107and59bp.4. ALDH2gene (rs671) and ADH2gene (rs1229984) of Guangdong population have been genetypied by method4and ADH2semi‐nested PCR–RFLP, then analysing the frequencies for Hardy‐Weinberg equilibrium by SPSS v19.0.Results:1. We have established five modified gene typing methods based on the RFLP‐PCR for ALDH2gene (G1510A). Each method can obtain the expected target fragment, PCR products were digested within10minutes completely, and the results were very clear and consistent with theoretical expectations. The results of the five methods of these100samples were exactly the same. And esch method has selected three samples of three genotypes randomly to sequence to verify the result.2. Successfully cloned ALDH2*1(wild type gene), ALDH2*2(mutant gene), and obtained recombinant plasmids pMD18‐T‐ALDH2*1, pMD18‐T‐ALDH2*2.3. We have established a rapid gene typing method for ADH2gene (G143A).100samples were tested by the new method. The PCR products were amplified well and the gene typing results were consistent with theoretical expectations. And three samples of three genotypes o were selected randomly to sequence to verify the result.4. There are346samples for genotyping ALDH2,181men,165women. The results showed that, ALDH2*1/*1, ALDH2*1/*2, ALDH2*2/*2genotype frequencies were0.51,0.44,0.05respectively. G allele had frequency of0.727, and0.273for the A allele. And the Hardy‐Weinberg equilibrium test results for ALDH2is2=3.40, P=0.183which satisfied the condition P>0.05.For the same346samples, ADH2genotyping results show thay ADH2*1/*1, ADH2*1/*2, ADH2*2/*2genotype frequencies corresponding respectively0.10,0.47,0.43. A allele was0.66, and the frequency of allele G was0.34. And the Hardy‐Weinberg equilibrium test results for ADH2gene frequencies is2=0.88, P=0.643833which satisfied the condition P>0.05.5. The rs1229984, rs671loci genotype combinations in Guangdong population are ADH2(GG)/ALDH2(GG)、 ADH2(GG)/ALDH2(GA)、 ADH2(GG)/ALDH2(AA)、ADH2(GA)/ALDH2(GG)、 ADH2(GA)/ALDH2(GA)、 ADH2(GA)/ALDH2(AA)、ADH2(AA)/ALDH2(GG)、ADH2(AA)/ALDH2(GA)、ADH2(AA)/ALDH2(AA) and the frequencies were0.052、0.043、0.006、0.272、0.176、0.02、0.185、0.217、0.029。.6. For rs671locus, the ALDH2genotypes frequency chi‐square test results between Guangdong and Yunnan populations,2values was8.893, P values were0.012, P>0.005, which means there had no significant difference between these two populations; For Guangzhou compared with Luoyang，Hunan，Sichuan populations, the ALDH2genotypes frequency chi‐square test results were2=15.043,61.618,16.619, P <0.005；the ALDH2allele genotypes frequency chi‐square test results were2=13.760,25.531,15.293, P <0.005；which means there are significant differences between these populations.For rs1229984locus, the ALDH2genotypes frequencies chi‐square test results of Guangdong and Taixing populations,2values was0.891, P values was0.604, P>0.005, which means there had no significant difference between these two populations; For Guangzhou compared with Yunnan, Luoyang, Elunchun populations the2values were13.174,11.503,54.444, P <0.005, which means there are significant differences between these populations；the ADH2allele genotypes frequencies chi‐square test results of Guangdong compared with Luoyang and Taixing populations,2values were6.111,0.697, P values were0.013,0.404, P>0.005, which means there had no significant difference between these two populations; For Guangzhou compared with Yunnan and Elunchun populations the2values were12.147,52.501, P <0.005, which means there are significant differences between these populations.Conclusion: 1. We have established five modified gene typing method based on the RFLP‐PCR for ALDH2gene (G1510A). The classical PCR‐RFLP method is easy to operate. Each method contains a control polymorphic site beside the polymorphic site to avoid misjudgment. The digestion reaction can be done within5‐10minutes, thereby reducing the time of gene typing greatly. The modified methods are fast and economical with operating time from3h~5h30min, cost2.5~15.0yuan.2. Successfully obtained the recombinant plasmids pMD18‐T‐ALDH2*1, pMD18‐T‐ALDH2*2, as a standard type for gene typing kit.3. We have established nest‐PCR‐RFLP for ADH2gene (G143A), which contains a control polymorphic site beside the polymorphic site to avoid misjudgment and with the operating time of3h, cost3yuan.4. The rs671locus gene frequency between male and female in Guangdong population has no significant difference.(2=2.700, P=0.259). The1229984loci gene frequency between male and female in Guangdong population has no significant difference.(2=1.814, P=0.404).5. People who carry a high risk of alcohol‐related diseases ALDH2*2allele, like (ALDH2*1/*2, ALDH2*2/*2) up to the ratio of49.0%in Guangdong population.6. For rs671genotype and allele frequency distribution analysis, there has no significant difference between Guangdong and Yunnan populations; and there has a significant difference between Guangdong, Luoyang, Hunan and Sichuan populations. For rs1229984genotype frequencies distribution analysis, there has no significant difference between Guangdong and Taixing populations; and there has a significant difference between Guangdong, Yunnan, Luoyang and Elunchun populations. And for allele genotypes frequencies distribution analysis, there has no significant difference between Guangdong, Luoyang and Taixing populations; and there has a significant difference between Guangdong, Yunnan and Elunchun populations.