Study of polymorphism in methionine synthase gene by RFLP-PCR in middle Euphrates region of Iraq

Background: Autism spectrum disorders (ASDs) are a collection of neuro-developmental conditions that are usually of prenatal origin and can be diagnosed in early childhood when it is severe. Analysis of methionine synthase (rs1805087) gene polymorphism in autism patients in Northern Iran reported that methionine synthase (MTR) is responsible for the regeneration of methionine from homocysteine. Therefore, the aim of current study was to study polymorphism in methionine synthase gene by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) in middle Euphrates region of Iraq. Methods: 70 children with autism and 30 controls in middle Euphrates region of Iraq were involved in current study. Genotypes and allele frequencies were determined in patients and controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results and conclusion: Current study showed that the highest genotype in control group was AA homozygote genotype (63.33%) followed by mutant homozygote GG (26.66 %) and then heterozygote genotype AG(10%) . In autism disease, the highest genotype was GG mutant homozygote genotype (62.58%). The Rs 18 polymorphism revealed that the homozygote mutant genotype G/G had significant higher risk of autism patients (P= 0.001; OR=13.06; 95% CI= (4.27-39.95) when compared with control group. The A allele frequency was significantly associated with autism patients (P=<0.001; OR=6.72; 95% CI=(3.45-13.11). Key word: Autism, gene polymorphism, Methionine synthase, RFLP-PCR, Homozygous genotype. INTRODUCTION Autism spectrum disorders (ASDs) are a collection of neurodevelopmental conditions that are usually of prenatal origin and can be diagnosed in early childhood when it is severe [1, . Although the etiology of ASDs is unknown, many theories support an interaction of environmental and genetic factors, the genetic variants participated in ASDs, and inherited from parents to affected individuals, have been estimated to explain ~40% of ASDs risk . The Methionine synthase (MTR), methylene tetrahydrofolate reductase (MTHFR), and methionine synthase reductase (MTRR) are key enzymes participated in the folate-mediated one-carbon metabolism and involved in DNA synthesis, methylation and repair . MTR consists of five important regions including homocysteine (HCY)-binding, 5-methyltetrahydrofolate (5-methylTHF) binding, cap, cobalamin-binding and SAM-binding domains. On the other hand, MTR gene is located on chromosome 1q43. Methionine synthase, a vitamin B12-dependent enzyme is involved in the folate-mediated one-carbon metabolism. It catalyzes the methylation of homocysteine to methionine with simultaneous conversion of 5-methyl-tetrahydrofolate (5-methyl-THF) to tetrahydrofolate (THF) which is essential for nucleotide synthesis. Methionine is essential for S-adenosyl-methionine (SAM) synthesis and DNA methyltransferases (DNMTs) which transfer the methyl group from SAM to the DNA . It is reported that a polymorphism in MTR A2756G (rs1805087) leads to a change from aspartic acid to glycine at codon 919 (D919G) and it was initially thought to be associated with the lower enzyme activity followed by homocysteine elevation and DNA hypomethylation . However, some other studies revealed a modest inverse association between GG genotype (A2756G MTR) and HCY levels indicating an increased enzymatic activity of the variant genotype . Haghiri et al. [10] showed the analysis of methionine synthase (rs1805087) gene polymorphism in autism patients in Northern Iran and the results reported that methionine synthase (MTR) is responsible for the regeneration of methionine from homocysteine. The prevalence of MTR A2756G polymorphism was determined in 108 children with autism and 130 controls in northern Iran. Genotypes and allele frequencies were determined in patients and controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) . The prevalence of genotype frequencies of AA, AG and GG in autistic children were 57.41%, 22.22% and 20.37%, respectively, while in controls were 61.54%, 32.31% and 6.15%, respectively. There was significant difference between the MTR polymorphism distribution in control and patient groups. The prevalence of allele frequencies of A and G in autistic children were 0.69 and 0.31, respectively, and in controls were 0.78 and 0.22, respectively, (P=0.03). The MTR G allele conferred a 1.6-fold increased risk to autism relative to the A allele (95% CI=1.06–2.41, P=0.02). The present study was aimed to explore the association between G allele of MTR A2756G polymorphism and the increased risk of autism. MATERIAL AND METHODS 1. Patients and Control groups A case – control study that was conducted during period from May 2017to May 2018 . The study subjects comprised from 70 Autistic children who were selected from Al-Hilla, Al-Najaf, Diwaniyah and Karbala cities. In addition, blood samples of 30 individuals were collected to compare with case patients. About 2mL of venous blood sample was collected from each subject in the study into EDTA-containing tubes to be used for genetic analysis. 2. Extraction of DNA Genomic DNA of frozen blood was extracted according to DNA extraction kit protocol (Favorgen). 3. Estimation of DNA Concentration and Purity The DNA concentration of samples was estimated by using a spectrophotometer (Nanodrop). 4. PCR amplification Preparing the Primers: The Bioneer primers in table 1 were prepared depending on manufacturer instruction. To create a stock of primers, the lyophilized primer were dissolved and nucleasefree water was added to each primer to obtain master stock that will be used again to obtain a working stock. PCR mixtures: The PCR mixtures were brought together according manufacture procedure of the master mix (Promega). All the appending was done in laminar flow cabinet on the frozen cooling blocks and ice when it was necessary. The components of PCR working solutions were representated in table 1. Table 1 The components of PCR working solutions Component Amount (μl) Concentration Master Mix 2X 12.5 2X DNA Template 3 30-60 ng/μl