Rapid Simultaneous Genotyping Of ADH1B And ALDH2Using High-Resolution Melting Assay

Background and ObjectivesThe principal enzymes in alcohol metabolism are alcohol dehydrogenase1B (ADH1B), which metabolizes ethanol to acetaldehyde, and aldehyde dehydrogenase-2(ALDH2), which oxidizes acetaldehyde to acetate. Fast ethanol metabolism combined slow acetaldehyde metabolism leads to higher concentrations of acetaldehyde which is related to alcohol-related cancer. The polymorphism of ADH1B and ALDH2are different among ethnic groups. According to research, ADH1B and ALDH2polymorphisms are related to alcohol-related cancer in populations of Asian origin.ADH1B is situated in4q22and ALDH2is situated in12q24. The polymorphic variant143G>A in exon3of the ADH1B gene resulting from47Argâ†'His encodes a superactive subunit of ADH1B. The polymorphic variant1510G>A in exon12of the ALDH2gene results from the substitution of glutamate to lysine at residue487. The alleles that encode the active and inactive subunits are ALDH2*1and ALDH2*2, respectively. The polymorphic variants of ADH1B and ALDH2which encode enzymes with altered kinetic properties, are the most well known and are related to alcohol-related cancer. Recently, genetic variants of alcohol metabolism, including ADH1B and ALDH2polymorphisms, can partially explain susceptibility to some human disorders, such as human cancers, liver dysfunction, coronary heart disease, Alzheimer, diabetes complications and polyneuropathy. Therefore, simultaneous genotyping of these two polymorphic loci would be useful for population survey and prediction diagnosis of relevant disease in both research and clinical setting.High-resolution melting (HRM) is a new generation of mutation scanning and genotyping technology. High-resolution melting curve analysis had been proved to be high efficiency, high sensitivity, inexpensive, high-throughput, rapid and labor-saving. The melting profile of the PCR product depends on its length, GC content and GC distribution. When the fragment under certain conditions, single base changes have an impact in the melting temperature of the fragment, can be detected through the high resolution and sensitivity detection technology. A sample with mutation or SNP after denaturation, renaturation of PCR, contains four duplex species:two homologous double-stranded and two heteroduplexes. Mutations or SNP sites of heteroduplexes do not match result in melting quickly than homologous and dye is released from the product. A single base difference can be reflected in the melting temperature difference, the performance difference in the melting curve. In the process of HRM analysis, fluorescent dyes bind to double-stranded DNA with the DNA amplification. As the temperature rises and the duplex passes through its melting transition, dye is released and fluorescence intensity is reduced. All dye is released until the temperature rise to95℃. The product is heatedwhile the level of fluorescence is measured. HRM analysis is based on the basis of Tm differences and curve shapes and width of melting.Recently, a range of molecular approaches has been used for allele discrimination of polymorphisms in the ADH1B and/or ALDH2genes, such as PCR-Restriction Fragment Length Polymorphism (RFLP), Single-Strand Conformation Polymorphisms (SSCP), PCR with confronting two-pair primers (PCR-CTPP), Amplified Product Length Polymorphism(APLP) and Denaturing High Performance Liquid Chromatography(DHPLC). Each of these approaches has some limitations, for example, time-consuming, complex operation, low precision and high-cost. Comparing with the methods, HRM has some advantages as following: low-cost with saturating DNA dye without expensive probe; after PCR and then conducted a high-resolution melting analysis; high throughput; all reaction in a closed tube reducing pollution. PCR product after HRM analysis can be used for DNA sequencing as well. The advantages of HRM include the high efficiency and labor-saving benefits. This technology has been adopted in clinical chemistry, human pathology and plant sciences. In this study, we have developed a partially HRM assay capable of simultaneously detecting the two polymorphic alleles in both ADHIB and ALDH2. This method can be used in genetic counseling, epidemiology and the investigation about the association between these enzyme polymorphisms and other alcohol-related problems.Samples and Methods1. Samples:Two hundred volunteers were recruited, peripheral blood samples were obtained after informed consent to validate the accuracy of the duplex HRM assay. Genomic DNA was extracted from2ml EDTA-anticoagulated peripheral blood by the standard phenol/chloroform method.2. Molecular analysisA polymerase chain reaction (PCR) composed of two separate amplicons was designed to generate sequences containing the polymorphic site of interest in the ADHIB and ALDH2genes. Samples with different kind of genotype of ADHIB and ALDH2can be gotten from DNA sequencing.Samples with different kind of genotype of ADHIB and ALDH2were used as standards to establish and optimize HRM assay conditions.Of200unrelated samples,40of three different genotypes (*1/*1,*1/*2, and*2/*2) were randomly selected for direct DNA sequencing validation, and10samples of genotype ADH1B*1/*2and ALDH2*1/*2were randomly selected for triplicate experiments at24-hour intervals for reproducibility and stability assessment. In addition, two-fold serial dilutions of gDNA were used to assess the detection limit.3. Statistical analysisTo validate the accuracy of the method, a number of samples from each of the genotype groups detected by HRM assay were selected to perform DNA sequencing. To validate the stability of the method,10samples with ADH1B*1/*2and ALDH2*1/*2genotypes were analyzed in triplicate at24-hour intervals. The gene and genotypic frequency of Han people in Dongguan city should be analyzed and the Hardy-Weinberg equilibrium was identified by chi-square statistic. Statistical analysis was conducted with SPSS13.0software.4. Comprehensive analysis of the experimental results.ResultsAfter adjustment and optimization of the duplex PCR system, the ADH1B and ALDH2genes were efficiently amplified and easily genotyped by HRM analysis.A total of200genomic DNA samples were tested to validate this assay by blind analysis. Direct DNA sequencing was performed on samples randomly selected from each of the genotype groups detected by HRM assay. The results indicate100%concordance between the sequencing analysis and the HRM detection. The precision of HRM genotyping was assessed by comparing the retention times and calculating the coefficients of variation (CV). Statistical analysis indicated good reproducibility and stability with a CV of melting temperatures from0.021%to0.062%. To assess robustness, two ADH1B homozygous samples of G/G and A/A alleles were two-fold serially diluted to100ng,50ng,25ng,12.5ng, and6.25ng, and analyzed. All concentrations could be detected. Only6ng of genomic DNA can be detected wellThe result of the chi-square test showed that the population we selected was in Hardy-Weinberg equilibrium. The gene frequency of ADH1B and ALDH2conformed the ones reported previously, which also identified the accuracy of the method.DiscussionAlcohol consumption is recognized as a potential risk factor for alcohol-related cancer. In addition to Alcohol consumption and drinking frequency, genetic variability also makes an important contribution to variation in alcohol intake and alcohol dependency. Thus, genetic variants that influence ADH1B or ALDH2activity could be relevant for the biological actions of alcohol. Therefore, simultaneous genotyping of these two polymorphic loci would be useful for genetic counseling, epidemiology and the investigation about the association between these enzyme polymorphisms and other alcohol-related problems.High-resolution melting (HRM) analysis growing popularity is a powerful mutation-screening tool. This method is not limited by the type and the site of mutation, sequence-specific probe is unnecessary, and run directly to detect genotype of sample after PCR amplification. This method because of its simple, quick, low cost, accurate, and achieve a true operation of the closed tube was widespread concerned. HRM analysis is used both for detecting unknown mutation and known mutation only with saturating DNA dye. Because of this superiority, HRM analysis has become the hot spot of genetics and methodology.In this study, the method we present is proved to be high-throughput, high reproducibility and reliable which suitable for rapid simultaneous genotyping of all nine genotypes groups of ADH1B and ALDH2gene in a single closed tube.Of200unrelated samples, we detected eight genotype groups except the genotype group composed of ADH1B*1/*1and ALDH2*2/*2. So it is speculated that individual whose both of ADH’IB and ALDH2are inactive existed scarcely in china. Some studies suggests that ADH1B*1allele increased predisposition to suffer certain types of cancer, such asupper aerodigestive cancers and alcohol liver disease. It is well known that ALDH2-deficient in ALDH2*1/*2or ALDH2*2/*2individuals is a strong risk factor for cancer with higher concentrations of acetaldehyde. Obviously, the combination of ADH1B*2allele with ALDH2*2allele for drinkers was the most risk factor for cancer with the reason that fast ethanol metabolism combined slow acetaldehyde metabolism leads to higher concentrations of acetaldehyde in drinkers. Thus, further study is needed to study the case-control studies of both genetic polymorphisms of ADH1B Arg47His and ALDH2Glu4871ys and cancer risk.This study established a simple, low-cost, high-throughput, rapid, and highly sensitive method for genotyping ADH1B and ALDH2gene, which provides an alternative method in genetic counseling, epidemiology and the investigation about the association between these enzyme polymorphisms and other alcohol-related problems.