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Association of MTHFR and MTRR genes with the development of antiphospholipid syndrome in pregnant women...
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Kamalov Zaynitdin Sayfutdinovich, Head of the Laboratory of Immunoregulation of the Institute of Immunology of the Academy of Sciences of the Republic of Uzbekistan, MD, Professor E-mail: zay_kamal@ramdler.ru Akramhodzhaeva Dilfuza Shakarimovna, Junior Scientific Researcher of the Institute of Immunology of the Academy of Sciences of the Republic of Uzbekistan.
Khegai Tatiana Rudolfovna, Head of the Laboratory of Human Genomics of the Institute of Immunology of the Academy of Sciences of the Republic of Uzbekistan, MD Zakhidova Nadira Erkinovna, Junior Scientific Researcher of the Laboratory of Human Genomics of the Institute of Immunology of the Academy of Sciences of the Republic of Uzbekistan
Association of MTHFR and MTRR genes with the development of antiphospholipid syndrome in pregnant women of Uzbek population
Abstract: By comparing the frequencies of genotypes studied polymorphic markers were installed by us the genetic association of folate cycle genes to the development of antiphospholipid syndrome. The results of this study demonstrate the relationship risk of antiphospholipid syndrome in the Uzbek population with carriage of the G allele of rs180139 polymorphism, and AG genotype of rs180139 polymorphism of MTRR gene. According to the allelic variants of MTHFR gene of rs1801133 polymorphism was significantly significant differences in the distribution of genotype frequencies were found.
Keywords: Pregnancy complications, antiphospholipid syndrome (APS), folate cycle genes, allele frequency, polymorphism of genes, genetic association.
The pregnancy complications, according to the report of the WHO Study Group, are one of the most important health problems and often the direct cause of maternal and perinatal morbidity and mortality. Received to date data on the molecular mechanisms of this disease development allow us to consider it as a multifactorial condition, the development of which is determined by the interaction of certain hereditary and environmental factors. Numerous studies on this issue have shown that the basis of many types of obstetric pathology is a generalized microangiopathy and thrombophilia associated with autoimmune disorders, defects of angio-genesis and invasion of trophoblast, hyperhomocysteinemia and hereditary disorders of hemostasis [1-7].
In the last decade attention of scientists and clinicians turned to the problem of development of antiphospholipid syndrome — an autoimmune disease characterized by recurrent thrombosis, initial miscarriage, thrombocytopenia and persistent antiphospholipid antibodies [10].
Among the many risk factors for APS, open today, an important role in the pathogenesis of this disease takes a genetic component, and in particular antigen HLA system (Human Leucocites Antigen), a gene factor 5 (FV, 1691G> A, rs6025) and prothrombin (FII,
20210G> A, rs1799963), and the C677T polymorphism (rs1801133) of methylenetetrahydrofolate reductase gene (MTHFR) [11].
The MTHFR gene encodes N5, N10-methylenetetrahydrofo-late reductase — a key enzyme in folate cycle, catalyzing the recovery of N5, N10-methylenetetrahydrofolate to N5-methyltetrahydro-folate, which is a donor of the methyl group in the reverse conversion reaction (remethylation) ofhomocysteine to methionine.
The polymorphism of C677T (rs1801133) methylenetetrahydrofolate reductase (MTHFR), located in the coding region of the gene MTHFR corresponding amino acid substitution of alanine residue for a valine residue at amino acid position 222 of the amino acid sequence of the protein. The peculiarity of this amino acid substitution is to reduce of termostability MTHFR, whereupon its enzymatic activity decreases. Deficiency of MTHFR enzyme activity leads to a deficiency N5-methyltetrahydrofolate, resulting in decreased speed remethylation homocysteine, and as a result, developing hyperhomocysteinemia. Accumulating in the circulating blood, homocysteine due to its cytotoxicity strikes the inner wall of the blood vessel that ultimately leads to the activation of the coagulation cascade reactions and hemostasis system to increase the risk of atherosclerosis and thrombosis. Along with this, the
Section 7. Medical science
MTHFR deficiency contributes to teratogenic (damaging the fetus) and mutagenic (DNA damaging) action due to violations of the processes of methylation [11-14].
The MTRR gene encodes a cytoplasmic enzyme methionine synthase reductase takes part in the biochemical reactions involving the transfer of a methyl group, and including, in the inverse transformation (remethylation) homocysteine to methionine. The function of the MTRR reductive methylation-dependent methionine synthase cobalamin (MTR), which catalyzes the methyl group transfer reaction with N5-methyltetrahydrofolate to homocysteine.
The Polymorphism of A66G (rs180139) metioninsintazare-ductse (MTRR) in the coding region of the gene corresponds to the replacement of MTRR residue isoleucine (Ile) at methionine residue (Met) at position 22 the amino acid sequence of the protein.
As a result of such changes in the primary structure of protein conformational rearrangement occurs which results in a decrease in enzymatic activity. As a result, the efficiency falls reductive methylation methionine synthase, which for its part, leads to a decrease in the efficiency remethylation homocysteine. Thus, MTRR deficiency contributes to teratogenic (damaging the fruit) and mutagenic (DNA damaging) action due to violations of methylation processes [12-14].
However, it should be noted that the results of studies on this issue, often contradictory, that may be due to ethnic heterogeneity and/or clinical heterogeneity of the patient population, the small number of samples, incorrect selection of the control group, as well as the ethnic specificity of hereditary predisposition to the disease. The frequency of polymorphisms related to the metabolism of folate and homocysteine levels, varies considerably among different ethnic groups, which can currently be validated population screening using genotyping. So far, I have not been analyzed, which would estimate the frequency of polymorphisms of genes involved in the metabolism of folate and homocysteine in the Uzbek population. The present study is an attempt to estimate the frequency of polymorphic gene MTHFR and MTRR in the Uzbek population to find out the existence of a legitimate connection between the development of antiphospholipid syndrome and impaired DNA methylation due to deficiency of folate cycle enzymes.
Material and methods
The study included 62 subjects of the Uzbek population of both sexes aged 20 to 65 years. The test persons conditionally divided into
2 groups: a group with APS (28 people) and a group of healthy subjects (34 pers.), Matched by age and sex.
Isolation of DNA from peripheral blood leukocytes was performed by the standard method. Genotyping was performed by amplifying the relevant regions of the genome methods qPCR (RG-6000, Australia) and pyrosequencing PyroMark Q24 (Qia-gen, Germany).
Statistical results of the study treatment was carried out with the help of software packages «SPSS 13», «PLINK» and «Haploview 4.2», forming haplotypes and evaluate their association with APS syndrome was carried out using «THESIAS» program (version 2.0)
Results and discussion
The distribution of the genotypes studied polymorphisms were tested for compliance with the expected Hardy-Weinberg equilibrium using Fisher's exact tests (Weir, 1995). For comparison, the allele and genotype frequencies between the groups analyzed using Pearson criterion x2 adjusted Ieytsa or Fisher's exact test. To assess the association of polymorphisms of genes with the pathological phenotype calculates «odds ratio» — OR. To determine the nature of the data distribution using the Shapiro-Wilk test statistics. For the analysis of quantitative traits when comparing two independent samples with normal distribution using analysis of variance, with deviation from the normal distribution — Mann-Whitney (Glanz, 1999). For each polymorphism and haplotypes were calculated OR, the magnitude P, and the 95% confidence interval. Differences were considered statistically significant at P <0.05.
Among 2 studied polymorphisms the deviation from Hardy-Weinberg equilibrium among both cases and was not found among controls (Table 1).
The analysis of the frequency distribution of alleles for polymorphisms rs1801133 and rs180139 gene folate cycle in a group with APS and in the control sample revealed a statistically significant difference (table 2) between them. The frequency of the G allele polymorphism rs180139 MTRR gene was significantly significantly higher in patients with APS compared to the control group (x2 = 19.51; OR = 2.41; P = 0.02), which may be considered in the development of its risk APS syndrome (table 2). The analysis of allelic frequencies of polymorphisms of MTHFR gene rs1801133 was significantly significant differences were not found.
Table 1. - Hardy-Weinberg equilibrium test for cases and controls in the APS Group "+" (28 people) and APS "-" (34 people)
CHROM SNP GROUP A1 A2 x2 P
1 rs1801133 Case T C 0.04 0.85
1 rs1801133 Control T C 0.21 0.64
17 rs180139 Case G A 0.2 0.66
17 rs180139 Control G A 2.94 0.09
Table 2. — Distribution of allele frequencies of rs1801133 and rs180139 polymorphisms Group APS "+" (28 people) and APS "-" (34 people)
Alleles Cases Controls x2 P OR 95% CI
n = 28 n = 34
rs1801133 Allele C 0.661 0.794 2.80 0.09 0.50 0.23-1.13
Allele T 0.339 0.206 1.98 0.88-4.44
rs180139 Allele A 0.554 0.750 19.51 0.02 0.41 0.19-0.88
Allele G 0.446 0.250 2.41 1.13-5.18
When comparing the frequencies of the genotypes studied drome. Analysis of genotypic associations showed that the greatest polymorphic markers were installed by us the genetic association risk of antiphospholipid syndrome is caused by heterozygous AG of folate cycle genes to the development of antiphospholipid syn- genotype of rs180139 polymorphism MTRR gene (x2 = 6.92; OR
Association of MTHFR and MTRR genes with the development of antiphospholipid syndrome in pregnant women...
= 3.21; P = 0.03). According to the allelic variants of MTHFR ferences in the distribution of genotype frequencies were not degene polymorphism rs1801133 was significantly significant dif- tected (Table 3).
Table 3. — distribution of genotypes frequency of rs1801133 and rs180139 polymorphisms Group APS "+" (28 people) and APS "-" (34 people).
SNP Genotypes Cases Controls x2 P OR 95% CI
n = 28 n = 34
rs1801133 Genotype C/C 0.429 0.618 2.94 0.23 0.46 0.17-1.29
Genotype C/T 0.464 0.353 1.59 0.57-4.42
Genotype T/T 0.107 0.029 3.96 0.39-40.38
rs180139 Genotype A/A 0.286 0.618 6.92 0.03 0.25 0.08-0.72
Genotype A/G 0.536 0.265 3.21 1.11-9.29
Genotype G/G 0.179 0.118 1.63 0.39-6.76
The Haplotypic analysis of the studied loci showed fairly significant association with the development of the APS.
Thus, the results of this study demonstrate the relationship risk of antiphospholipid syndrome in the Uzbek population with carriage of the G allele of rs180139 polymorphism (x2 = 5.29; OR = 2.42; P = 0.02), and AG genotype of rs180139 polymorphism of MTRR gene (x2 = 6.92; OR = 3.21; P = 0.03). Statistically significant associations rs1801133 polymorphism of the gene has been identified MTHFR c development of antiphospholipid syndrome.
Conclusions:
1. The greatest risk of antiphospholipid syndrome in the Uzbek population is due to carriage of the G allele polymorphism rs180139 (x2 = 5.29; OR = 2.42; P = 0.02) and genotype AG rs180139 polymorphism of MTRR gene (x2 = 6.92; OR = 3.21; P = 0.03).
2. Statistically significant associations of rs1801133 polymorphism of MTHFR gene development of antiphospholipid syndrome have been identified.
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