![aa2 hf result: -1 aa2 hf result: -1](https://media1.popsugar-assets.com/files/thumbor/15hf0Iz3jtIYuPxEkWc6REU7wMs/fit-in/1200x630/filters:format_auto-!!-:strip_icc-!!-:fill-!white!-/2019/12/05/902/n/1922153/a5b44c385de96aeae55a41.56507222_.jpg)
The LCLs (established by Epstein–Barr virus transformation of peripheral blood mononuclear cells) were acquired from Coriell biorepositories and cultured in RPMI-1640 (Merck Millipore, cat. Our results in this study serve as a proof of principle for considering inter-individual differences as an essential parameter before using the LCLs for studies like SNP-mediated gene expression analysis or epigenetic modifications. We incubated the cells with water or various physiological and nonphysiological concentrations of ethanol and characterized the cell lines for ALDH2 promoter methylation in the negative regulatory region, the positive regulatory region and the dense CpG island spanning through the core promoter along with ALDH2 protein expression. We acquired six LCLs, two each consisting of AA, GA or GG at SNP rs886205 position. The NHGRI repository at coriell institute is a collection of lymphoblastoid cell lines (LCLs) from multiple ethnicities used for HapMap Project, HapMap3 Project and 1000 Genomes Project and serves as an important source to study genetic variations in a range of human populations. Therefore our aim was to establish an in vitro system to study distinct rs886205 mediated recruitment of methylation machinery under basal conditions as well as in the course of alcohol intoxication. Luciferase reporter assays using ALDH2 promoter construct showed higher transcriptional activity in the insert with AA genotype as compared to GG genotype. The kinetics of mean methylation also differed in the patients with A and G genotype. In the positive regulatory fragment AA genotype patients showed lower mean methylation than AG/GG patients on day one of alcohol withdrawal. Patients with AA genotype show higher ALDH2 protein expression in comparison to GG/AG patients. The patients with AA genotype and not with AG/GG showed a decrease in mean methylation of negative regulatory fragment from day 1 to day 7 of alcohol withdrawal. Our Previous findings from the methylation analysis in alcohol-dependent patients revealed an interaction between methylation levels and SNP rs886205. Two important fragments on the ALDH2 promoter: a negative regulatory element and a positive regulatory element harboring a nuclear receptor response element (NRRE) have been reported. Among these previously reported SNPs, we focused on SNP rs886205 because of prevalence in the German population and its association with esophageal carcinoma in moderate to heavy alcohol consumers. In the past, numerous studies were performed in various populations and ethnicities leading to the discovery of the multiple SNPs in alcohol metabolizing enzymes. These observations formed the basis for studies to understand genetic mechanisms of addiction biology. Although all these substances induce the reward circuitry in most individuals, only a few get addicted. The limbic system plays a central role in mechanisms of addiction towards alcohol, nicotine and psychoactive drugs like heroin and cocaine. Investigating genetic variations is important to understand the contribution of inter-individual differences in the manifestation of physiological and psychological disorders. Our findings show the limitations of the usage of lymphoblastoid cell lines due to the unavoidable background genetic differences that may influence the effect of SNP. Although ALDH2 protein expression seemed to be driven by the rs886205 genotype, results were not in consensus with data from the patient cohorts. ResultsĭNA methylation showed significant differences not only based on genotype but also due to the different background of the cells owing to their origin from different individuals. We measured the promoter methylation of ALDH2 by using bisulfite sequencing and quantified protein expression of ALDH2 by western blot to compare the cell lines with the previous findings in patients. To study the DNA–protein interactions involved in rs886205 mediated regulation of ALDH2, we chose lymphoblastoid cell lines harboring AA/GA/GG genotype and acquired two for each genotype from National Human Genome Research Institute repository. Previously, we found an interaction of the SNP with the methylation of promoter regions as well as the protein levels of ALDH2 in alcohol-dependent patients. SNP rs886205 (A/G) located in the aldehyde dehydrogenase 2 (ALDH2) promoter is associated with esophageal carcinoma in alcohol-dependent patients.
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Single nucleotide polymorphisms (SNPs) are widely linked to the susceptibility and penetrance of diseases.