MTHFR 677 C< T and 1298 A>C polymorphisms increases the risk of recurrent abortion in the Iraqi woman

Background: The C677T and A1298C polymorphism mutations in the methylenetetrahydrofolate reductase (MTHFR) gene will be investigated in a multi-abortion study. Aim: To determine mutation (SNP) in the methyl tetrahydrofolate reductase (MTHFR) Gene with multiple abortion. Methods: “We nominated two hundred patients for this study in three groups: the study group, The rst group included 50 women with a history of 1 or 2 missed rst trimester Abortions and fty to control group the last group which consisted of one hundred Patients with a history more than two missed abortion. Anticoagulants human blood tests such as (protein C, protein S, and lupus) as well as general serum tests IgG and IgM for (Cytomegalovirus, Toxoplasma gondii, Rubella virus, Anti-cardiolipin antibodies and anti-phospholipid antibody) were performed. In addition, screening of the maternal MTHFR C667T and MTHFR a 1298C mutation was determined by PCR. Result: all common serum test for study population (CMV, Toxo, Rubella, ACA and APL) IgG and IgM, also anticoagulants human blood test (protein C, protein S and Lupus) is negative. The frequency of heterozygous (genotype) A1298C and C677T was similar. The distribution of MTHFR, C677T and A1298C genotypes show signicantly differences P ≤ 0.05; OR= (95%CI) between patients with multiple abortions and control subjects. Conclusions: the result suggestion MTHFR A>C 1298 and C< T 677 polymorphisms might be associated with multiple abortion in the examined population.


Introduction
The World Health Organization (WHO) de nes miscarriage as the loss of embryo or fetus weighing less than 500 grams, which match up to approximately (20)(21)(22) weeks of pregnancy [1]. The classi cation of spontaneous abortion as early and late (<12 weeks) and (≥ 12 weeks) respectively [2]. In recent years, several studies have assessed the correlation of the methylenetetrahydrofolate reductase (MTHFR) and the hygienic risk of multiple abortions between C677T and A1298C polymorphisms [3,4]. Methylenetetrahydrofolate is a key enzyme of folate/homocysteine pathway [5,6] MTHFR which performs a vital role in folate-dependent homocysteine metabolic rate. Mutations in MTHFR have been reported as the major causes of hyperhomocysteinemia [7]. Hyperhomocysteinaemia alone may pose a mild risk, but in conjunction with other factors causing vascular lesions it will increase the risk of disease. Encoding gene variants of the homocysteine (hcy) metabolism enzyme or, using up important substrates or cofactors for those enzymes, which include vitamin B12, vitamin B6and folic acid\folate (FA), may result in the rise plasma homocysteine levels [8]. 'The active form of FA in an organism is derived from a reduced reaction of FA into tethrahydrofolate, tethrahydrofolic acid, (THF), with the latter being the true coenzyme of MTHFR. Metabolism of folate is essential for proper cellular function. Within the folate track, methylenetetrahydrofolate reductase (MTHFR) reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate: a methyl donor for re-methylation of homocysteine to methionine" [9] as shown in gure1 [10].
MTHFR stimulates irreversible transformation of 5,10-methyentethrahydrofolate CH3-THF to 5--CH3-THF. The normal activity of MTHFR assistances to maintain methionine and folate in the bloodstream at steady levels, preventing Hcy aggregation [11]. The A1298C and C677T SNP of the MTHFR gene, in convinced conditions, may lead to an increase in homocysteineemia and plasma homocysteine, which can lead endothelial grievance in blood vessel. This may lead to increase thromboembolic possibility, which lead to stimulate an impediment of the placental vessels in pregnant women, which are the procreating outcomes in repeated abortions. Thus, Recurring PRLs are regarded as risk factors for hyperhomocysteinemia. Hyperhomocysteinemia may be demonstrated in women with RPLs. [12].
Enzymatic activity of MTHFR may be minimize due to the Polymorphisms in the gene encoding it. The variation C677T leads to a replacement of a cytosine C into a thymine T at situation 677 in exon 4 of the MTHFR gene. In position 222, at protein level (p.Ala>Val), this genetic variant provides a path to amino acid substitution in place [13]. A further MTHFR polymorphism linked to a reduction in enzyme activity is an adenine replacement with cytosine (A1298C) in position 1,298 [14,15]. in position 429 at the protein level (p.429Glu>Ala) variant ignites do to the substitution of a glutamate G with an alanine [16]. It has been established that the A1298C and C677T polymorphisms in heterozygous genotype, laterally with a folate de ciency, initiate increased level of Hcy plasmatic [17]. However, many studies have endeavored to nding the relationship between the genetic variations of the thromboembolic risk and MTHFR gene, which in turn brings about risk of RPL in pregnant women. The aim is to investigate the correlation between the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphism in multiple-abortion.

Materials And Methods
Two hundred women (suffer from recurrent miscarriages were recruited as the subject of this study and all of them visited the Alburaq medical laboratory from January 2018 to August 2020. They were ask about their medical history and undertook the physical examination of genital system. Group of study are divided in two groups: The rst group involved 50 women with a history of one or two missed trimester abortions, wherever the reason was unidenti ed. The second study group involved 150 cases with a history of more than two missed abortions. The age of all the women in our study ranging between 18-40 years. All causes of abortions in the rst trimester were made after 5, 10 me-THF excluding other possible causes of multiple abortion, such as a common serum tests IgG and IgM for (Cytomegalovirus, Toxoplasma gondii, Rubella virus, Anti-cardiolipin antibodies and anti-phospholipid antibody) besides an Anticoagulants human blood test such as (protein C, protein S, lupus) was carried out. After separating the blood serum from the patient study, PCR was used to determine the presence of the mutations of MTHFR C667T and A1298C. Different groups compared the results.

Genotyping
Qiagen DNA extraction kit (Qiagen, USA) was applied to extract genomic DNA from 2 ml peripheral venous anticoagulant blood, and then stored at -80 °C. MTHFR genotyping was determined after PCR ampli cation. The procedure includes three steps: (1) DNA isolation, (2) PCR ampli cation using biotinylated primers, (3) hybridization of ampli cation products to a test strip containing allele-speci c oligonucleotide probes immobilization as an array of parallel lines, bound biotinylated sequences are detected using streptavidin-alkaline phosphatase and color substrate. The probes and primers were  Analysis of statistics SPSS Version 20 was used to carry out statistical data analysis (SPSS Inc., Chicago, IL, USA). In the Pearson chi-square test and at a 95% con dence interval the mutation frequencies in MTHFR mutation between the case study and the control group have been analyzed (95 % CIs). P values < 0.05 considered signi cant.

Results
Among 200 (150 miscarriage women, 50 control ) women with a mean age of 29 years (range, 18-40 years), all of them had both IgM and IgG test results as common serum tests IgG and IgM for (Cytomegalovirus, Toxoplasma gondii, Rubella virus, Anticardiolipin antibodies, and anti-phospholipid antibody) also an Anticoagulants human blood test such as (protein C, protein S, lupus) are negative, The differences between Toxoplasma, Rubella, ACA, APL, and CMV IgM IgG rates were found to be statistically no signi cant". Similarly, the differences between protein C, protein S, and lupus differences rates were determined to be statistically no signi cant (p<0.01). in addition, from the table (2) explain There are no signi cant differences between the studied groups) patient and control). This supports the fact that the studied groups are homogeneous .where when (sig) p = 0,000 this experiment is repeated 100 times, and every time the researcher rejects the null theory (the arithmetic mean is equal) there is no single decision of his decisions is wrong. This means that the data study is real  Whereas, statistically signi cant difference (P ≤ 0.05) was detected in the frequency of MTHFR SNPs C677T and A1289C in women as show in table (4). Combined heterozygosity and homozygosity of MTHFR polymorphisms for two SNP was a common phenomenon in the women suffer abortion more than two times was (29 %) of women .also combined heterozygosity for both SNPs in all studied groups was observed. Combined 677TT/1298AC ,677CT/1298CC or 1298CC/ 677TTgenotypes, which contain three or four mutant alleles, were also identi ed in our study groups, 15% from the cases study show combined heterozygosity in both SNP This refer to the heterozygosis is more risk.
However, there was a substantial difference between the multiple abortion and control groups when the detected frequencies of the 677CT/1298AC and 677TT/1298AA genotypes were combined; this indicates that these genotypes are involved in multiple abortion pathogenesis.

Discussion
It remains unclear how genetically modi ed substances contribute to the onset of multiple abortions. One of the key enzymes in folate metabolism, the MTHFR gene is found on chromosome 1 (1p36.3), which contains 12 exons [18]. MTHFR can in uence gene expression via DNA methylation by participating in the methionine cycle. Dozen MTHFR gene mutations have been identi ed so far, there are still more, and more diseases linked to new mutations [19]. has been reported that C677T polymorphism of MTHFR gene is strictly related to birth defects hypertension, Alzheimer's, atherosclerosis, heart disease and hormone metabolism, miscarriage [20]. That attracted a lot of attention. The risk of these diseases can be reduced by exogenous folic acid supplementation.
"The MTHFR C677T allele frequencies and genotype distribution found in our patient group agree well with those reported in a previous investigation of many populations". But there is no previous research on the MTHFR C677T and A1298C polymorphism in Iraqi patient.
We observed a very high frequency of MTHFR 1298C and 677T alleles in the multiple abortion group this agreement with (Henrik, 2002) [21]. Frosst et al. found of CT heterozygote exhibited ∼30% enzyme activity, which was signi cantly higher than TT homozygote that (almost 65% of normal enzyme activity) and CC homozygote (normal enzyme activity), MTHFR c.677C>T can have an effect on enzyme activity, according to this study. In addition, the c.677C>T allele of the MTHFR gene has been linked to mild hyperhomocysteinemia and RPL in some studies [22,23].
The frequency of the c.1298 C allele in multiple abortion women was signi cantly higher than control group, and the risk factor for sensitivity was presented by the C allele.
Based on the current ndings, it is evident in the rst or second trimester that the MTHFR versions in C677T and A1298C in uence miscarriage predisposition. Analyzing them for diagnostic purposes may be advantageous. The daily consumption of Folic acid however remains an important treatment for pregnant women to reduce, among other complications, the risk of increasing plasma homocysteine [17][18].
Our ndings have a signi cant clinical signi cance for him: properly targeted folic acid supplements (active folate form) can help to prevent abortion. By catalyzing HCY re-methylation into methionine and thus lowering plasma HCY levels, targeted folate supplements could minimize thrombosis-related pregnancy loss.  Figure 1 methyltetrahydrofolate (mTHF) by methylenetetrahydrofolate reductase (MTHFR). 5-mTHF is demethylated to complete the folate cycle by donating a carbon into the methionine cycle through the methylation of homocysteine (hCYS) by methionine synthase and its cofactor vitamin B 12".

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