Comparison of SNP Polymorphism of Alcohol Metabolizing Related Enzyme Genes in Intoxicated Individuals and Nonalcoholic Population

Research Article

Austin J Forensic Sci Criminol. 2015; 2(3): 1031.

Comparison of SNP Polymorphism of Alcohol Metabolizing Related Enzyme Genes in Intoxicated Individuals and Nonalcoholic Population

Li Li*, Ping Xiang, Yan Liu, Yan Shi, Yuan Lin and Zhenmin Zhao

Shanghai Key Laboratory of Forensic Medicine, Institute of Forensic Science, China

*Corresponding author: Li Li, Shanghai Key Laboratory of Forensic Medicine, Institute of Forensic Science, Ministry of Justice, 200063, China

Received: July 16, 2015; Accepted: August 08, 2015; Published: August 11, 2015

Abstract

Objective: To compare the distributions of SNP allele and genotype about alcohol dehydrogenase 2 (ADH2), aldehyde dehydrogenase 2 (ALDH2) and CYP2E1 in intoxicated individuals and nonalcoholic population.

Methods: 100 individuals who were penalized duo to intoxicated driving were genotyped for 40 SNPs of ADH2,ALDH2 and CYP2E1 using multiplex PCR and Iplex chemistry on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer and compared with a reference group of 99 blood donors. Allele frequencies and genotype frequencies of 40 SNP loci were calculated and compared in the two groups.

Results: Among the 40 SNP loci, seven SNPs (e.g. rs698, rs2241894, rs1789915, rs13306164, rs671, rs28371746 and rs2515641) were polymorphic in the intoxicated population, but only six SNPs (e.g. rs698, rs2241894, rs13306164, rs671, rs28371746 and rs2515641) were polymorphic in the control individuals. Two SNP loci (rs671 in and rs2515641) were found to have a significant difference in frequency distribution between the two populations (p<0.01).

Conclusion: Among the above 40 SNP loci of ADH2,ALDH2 and CYP2E1 genes, rs671 and rs2515641 in the intoxicated population were found to differ in allele and genotype frequency distribution from the nonalcoholic population and may be related to alcohol intoxication.

Keywords: ADH; ALDH; CYP2E1; Single nucleotide polymorphism; Genetic polymorphism

Introduction

The metabolism of drug shows inter-individual variation and inter-ethnic variation. Because of the difference of Drug-Metabolizing Enzyme (DMEs) activity, the individuals can be divided into Poor Metabolizers (PMs), Extensive Metabolizers (EMs) and Ultra-Rapid Metabolizers (UMs) [1,2]. Therefore, it is usually a hard work to interpret drug related poisoning or death in forensic science [3]. Now punitive measures against people who drink and drive have been strictly enforced. In china, current standard for judging drunk drinking and drunken drinking is mainly based on the concentration of ethanol in blood. The driver whose alcohol concentration above 0.2mg/mL in blood is convicted of drunk drinking and the driver whose alcohol concentration above 0.8mg/mL in blood is convicted of drunken drinking, but, except for alcohol dehydrogenase (ADH), aldehyde dehydrogenase 2 (ALDH2) and cytochrome P450 2E1 enzyme (CYP2E1) has also been found to be involved in alcohol metabolism [4-7], which means that ALDH2 and CYP2E1 with gene polymorphism may also play dominant role in alcohol metabolism. Individuals with distinct genotypes are different in alcohol tolerance and show unequal behavioral responses after drinking [8]. Because of high concentration of acetaldehyde in blood transformed from ethanol, poor metabolism of acetaldehyde may lead to traffic accident even if the individual’s alcohol concentration below 0.2mg/mL in blood. The individual differences are mainly caused by the gene polymorphisms of ADH, ALDH and CYP2E1. It was confirmed that the presence of the less-active form of alcohol dehydrogenase- 1B encoded by ADH1B*1/*1 and active form of ALDH2 encoded by ALDH2*1/*1 increases the risk of alcoholism in East Asians [9]. SNPs were found to be related to the polymorphism of enzyme activity [8], base deletions, insertions, substitutions or transversions in genes of ADH, ALDH2 and CYP2E1 may lead to changes of amino acid sequences and result in enzyme activities lost or decreased or increased. In order to investigate SNP polymorphism of alcohol metabolizing- related enzyme genes in Chinese Han population, we genetyped 40 SNPs of ADH2, ALDH2 and CYP2E1 based on multiplex amplification and matrix-assisted laser desorption/ionization timeof- flight mass spectrometry (MALDI-TOF MS) [10,11] and compared the SNP polymorphism in intoxicated population and nonalcoholic population.

Materials and Methods

Selection of SNP loci and design of primers

Corresponding to sequences of genes coding for ADH2, ALDH2 and CYP2E1, 40 SNP loci (Table 1) were selected via NCBI website: https://www.ncbi.nlm.nih.gov/. Forty groups of primes were designed via Mass ARRAY Assay Design software (Sequenom, Inc.). Each group has three primes, including a pair of PCR primes and a single base extension prime (Table 2). Primers were synthesized by Shanghai Biological Engineering Technology Corporation.

Table 1: 40 SNPs in Alcohol Metabolizing Related Enzyme Genes.

Table 2: 40 groups of primes used for SNP genotyping.

DNA extraction

Blood samples were collected from 100 unrelated Han individuals who were penalized duo to intoxicated driving. Another 99 blood samples for control were collected from nonalcoholic males. DNA was extracted according to the instructions of the blood genomic DNA Mini Kit (Sangon Biotech, Shanghai, China).

Multiplex PCR amplification

Aliquots of 1 μL DNA were amplified in a total volume of 5μL. Each reaction contained 0.625μL PCR buffer (10×) (Qiagen GmbH), 0.325μL 25 mmol/L MgCl2, 1μL dNTP (2.5mmol/L) (Tatara Inc.), 0.1μL of HotStarTaq polymerase (5U/μL), 0.95μL H2O, and 1μL the designed primers at their optimized concentrations. Using a Gene Amp PCR System 9700 (Applied Biosystems, Norwalk, CT), the reaction mixtures were incubated at 94°C for 15 min and then cycled 45 times through desaturation at 94deg;C for 20 s, annealing at 56deg;C for 30 s and extension at 72deg;C for 60 s and finally incubated at 72deg;C for 3 min. No-template controls were carried along in every plate to exclude contaminations.

SAP process

After PCR, the products were treated with hrimp alkaline phosphatase (SAP) to remove excess dNTPs. This dephosphorylation reaction contained 0.3μL SAP (1U/μL), 0.17μL SAP buffer (10×), 1.53μL H2O (Sequenom, Inc.) was carried out at 37deg;C for 40 min, and 85deg;C for 15 min.

Primer extension reactions

The PCR products were used as templates for the primer extension reactions. Extension reactions (final volume, 9μL) contained 0.2μL iPlex buffer (10×), 0.1μL iPlex termination mix, 0.0205μL iPlex enzyme, 0.7395μL H2O (all from Sequenom, Inc.), and 0.94μL extension primers at optimized concentrations. On a Gene Amp PCR System 9700 (Applied Biosystems, Norwalk, CT), extension reactions were performed at 94deg;C for 30 s followed by 40 cycles (94deg;C for 5 s, followed by 5 cycles of 52deg;C for 5 s, 80deg;C for 5 s); and finally 72deg;C for 3 min. The final nucleotide extension products were treated with a cationic exchange resin (AG® 50W-X8 Resin; Bio-Rad Inc.) for 30 min to remove salts. All reactions, including PCR amplification, shrimp alkaline phosphatase treatment, and base extension, were performed in 384 microtiter plates (Sequenom Inc.).

MALDI-TOF-MS

The reaction products were spotted onto the Mass ARRAY SpectroCHIP with an auto-spot arm (Sequenom, Inc.). The target plate was then inserted into the MALDI-TOF mass spectrometer of Mass ARRAY compact System (Sequenom, Inc.), and analysis was performed with 180 nitrogen laser shots for each sample. The mass range of the MS instrument was set at 3920–12023 Da. SNP loci was genotyped by Mass ARRAY Type Analyzer software version 4.0 (Sequenom, Inc.).

Statistical analysis

The data were analyzed with SPSS 13.0. The statistical information included genotype frequency, allele frequency and p values.

Results and Discussion

Evaluation of the MALDI-TOF MS genotyping assay

To evaluate the established SNP genotyping assay, we analyzed 100 genomic DNA samples from individuals of Chinese origin who had previously been genotyped for some of the SNPs by TaqMan assay and got consistent results.

Polymorphisms of SNPs

Among the 40 SNP loci in intoxicated population, three SNPs (rs698, rs2241894, rs1789915) in ADH3 gene, two SNPs (rs13306164, rs671) in ALDH2 gene, two SNPs (rs28371746, rs2515641) in CYP2E1 gene were found to be polymorphic, i.e. the minor allele frequency(MAF) of each SNP locus was above 1%, while others were not ( the MAF of them was below 1%). Among the 40 SNP loci in control population, the seven SNPs except rs1789915 were found to be polymorphic. The observed genotype and allele frequencies in intoxicated population and control population were shown in table 3 and 4 respectively.

Table 3: Genotype frequencies of 40 SNPs in intoxicated population (n=100) and control population (n=99).

Table 4: Allele frequencies of seven polymorphic SNPs in intoxicated population (n=100) and control population (n=99).

Population comparison

The allele frequencies of seven polymorphic SNPs (rs698, rs2241894, rs1789915, rs13306164, rs671, rs28371746, rs2515641) in intoxicated population were compared to the data about the control population. It’s found that two SNPs (rs671 in and rs2515641) with a significant difference in frequency distribution between intoxicated population and control population (p<0.01, (Table 5) might be related to alcohol intoxication.

Table 5: Comparison of allele frequency of seven polymorphic SNPs in intoxicated population (n=100) and control population (n=99).

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Citation: Li L, Xiang P, Liu Y, Shi Y, Lin Y and Zhao Z. Comparison of SNP Polymorphism of Alcohol Metabolizing Related Enzyme Genes in Intoxicated Individuals and Nonalcoholic Population. Austin J Forensic Sci Criminol. 2015; 2(3): 1031. ISSN : 2380-0801