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Akramhodzhaeva Dilfuza Shakarimovna, Junior Scientific Researcher of the Institute of Immunology of the Academy Kamalov Zaynitdin Sayfutdinovich, Head of the Laboratory of Immunoregulation of the Institute of Immunology of the Academy MD, Professor
E-mail: [email protected] Khegai Tatiana Rudolfovna, Head of the Laboratory of Human Genomics of the Institute of Immunology of the Academy MD Zakhidova Nadira Erkinovna, Junior Research Assistant, of the Laboratory of Human Genomics of the Institute of Immunology of the Academy MD of Sciences of the Republic of Uzbekistan
The role of genes of the folate cycle in the development of antiphospholipid syndrome in the Uzbek population
Abstract: When comparing of frequencies of genotypes of the studied polymorphic markers we have been established the genetic associations of the genes of folate cycle 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 A alleles of rs1805087 polymorphism, and rs1805087 polymorphism of AA genotypes and AA genotype of the rs1801131 polymorphic marker.
Keywords: Antiphospholipid syndrome (APS), the genes of folate cycle, allele frequency, polymorphism of genes, genetic association.
Antiphospholipid syndrome (APS) — clinical and laboratory syndrome characterized by venous and arterial thrombosis, pathology of pregnancy and some other less common clinical manifestations and laboratory disorders, pathogenesis associated with the synthesis of antiphospholipid antibodies (aPL) [1; 2].
APS — a complex and insufficiently developed problem. This is explained by the heterogeneity of pathogenetic mechanisms that underlie the APS, the lack of reliable clinical and laboratorial indicators to predict the risk of recurrence of thrombosis. Currently, there are no generally accepted international standards of the treatment tactics of patients with various forms of APS and the proposed recommendations are based primarily on the results of the "open" tests or retrospective analysis of outcomes of the disease [3-11]. The approaches to prevention and treatment of atherosclerotic vascular lesions, often develops in patients with APS are not enough studied [12; 3].
In the population, according to the American authors, APS occurs in 5% of cases [15]. In our country, such studies have not been undertaken. Antiphospholipid syndrome is observed in women 2-5 times more often than in men, and, if the initial ratio of the number of patients with APS women and men is 4: 1, then the sec-
ondary form of the disease, this figure rises to 7: 1, which is probably due to greater susceptibility ofwomen to systemic connective tissue diseases [14]. The investigations of HLA antigens (human leucoc-ites antigen) showed that in patients with APS often than in the population found HLA: DR4, DR7, DRw53, suggesting a possible genetic predisposition to the disease [13]. The literature describes familial cases of APS, constituting, according to some authors, up to 2% [14]. It is possible that there are two forms of the disease: sporadic and familial.
The genes of the folate cycle involved in processes of remeth-ylation may also be considered as potential candidates genes in the development of the APS syndrome.
The Violation of the processes ofremethylation (formation ofme-thionine from homocysteine), which occurs because of the MTHFR enzyme MTRR deficiency and leads to the development of a number of pathological conditions, such as atherosclerosis; atherothrombosis; cleft neural tube defect; heart attacks and disruption of chromosome segregation in oogenesis. Methylation of DNA is a methyl group to join in the composition cytosine CpG-dinucleotide in C5 position of the cytosine ring.
Section 7. Medical science
The MTHFR gene encodes N5, N10-methylenetetrahydrofo-late reductase — a key enzyme in the folate cycle, catalyzing the recovery of N5, N10-methylenetetrahydrofolate to N5-methyltetra-hydrofolate, which is a donor of the methyl group in the reverse conversion reaction (remethylation) of homocysteine to methionine.
The polymorphism ofA1298C (rs1801131) methylenetetrahy-drofolate reductase (MTHFR), located in the MTHFR gene coding region corresponds to the substitution of glutamic acid residue (Glu) to residue of alanine (Ala) at position 429 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 lowering its enzymatic activity by 65%. Consequently, the carrier genotypes 1298AS and 1298SS carry a high risk of hyperhomocysteinemia and as a consequence, to an increased risk of thrombus formation. Along with this, the MTHFR deficiency contributes to teratogenic (damaging the fruit) and mutagenic (DNA damaging) action due to violations of methylation processes. The frequency of polymorphisms in European populations is 25-30%.
The MTR gene encodes a cytoplasmic cobalamin (vitamin B12) - dependent enzyme methionine synthase, catalyzes the reaction remethylation homocysteine. In this reaction, the methyl group donor advocates N5-methyltetrahydrofolate. The transfer of the methyl group to homocysteine is carried out in two stages. First, the methyl group is accepted cobalamin (methyl group substituted by a cyano group) to give methyl cobalamin-methionine synthase and, only then, a methyl group is transferred to homocysteine. This enzymatic reaction is one of the most important parts of the folate metabolism. On the one hand it provides the methionine concentration required to implement the many vital reactions of methylation of nucleic acids, proteins, lipids and others. On the other hand, due to this reaction, the concentration of homocysteine in plasma is maintained in a physiologically acceptable range (up to 15ng/ml).
The polymorphism ofA2756G (rs1805087) Methionine synthase (MTR) in the coding region of the gene corresponds MTR replacing an aspartic acid residue (Asp) residue for glycine (Gly) at position 919 the amino acid sequence of the enzyme. This substitution leads to changes in the structure of the enzyme, entailing a reduction of its catalytic activity. Thus, the carrier of the mutant genotype GG and AG associated with deficiency of enzymatic activity of methionine synthase, leading to hypercysteinemia, and eventually to an increased tendency to thrombosis. Carriage of the G
allele, due to methionine deficiency also contributes to teratogenic (damaging the fruit) and mutagenic (DNA damaging) action. The frequency of polymorphisms in European populations: 20-25%.
The frequency of polymorphisms related to the metabolism of the folate and homocysteine levels, varies considerably among different ethnic groups, which can currently be validated population screening using genotyping. So far, it has 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 genes MTHFR and MTR in the Uzbek population and to find out the existence of a legitimate relation between the development of antiphospholipid syndrome and impaired DNA methylation due to deficiency of the enzymes of folate cycle.
Material and methods. The study included 62 subjects of the Uzbek population both genders aged 20 to 65 years. The test persons conditionally divided into 2 groups: a group with APS (28 people) and a group ofhealthy subjects (34 people), matched by age and gender.
The DNA extraction from peripheral blood leukocytes was performed by the standard method. Genotyping was performed by amplifying the relevant regions ofthe genome methods qPCR (RG-6000, Australia) and pyrosequencing PyroMark Q24 (Qiagen, Germany).
The statistical results of the study were carried out with the help ofa package program «SPSS 13», «PLINK» and «Haploview 4.2».
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 wit Ieyts adjusting 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 we used the Shapiro-Wilk test statistics. For the analysis of quantitative traits when comparing two independent samples with normal distribution we used the analysis of variance, with deviation from the normal distribution — Mann-Whitney (Glanz, 1999). For each polymorphism and haplotypes were calculated OR, the P magnitude, 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 controls was not found (Table 1).
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 rs1801131 case C A 0.27 0.6
1 rs1801131 control C A 2.86 0.09
1 rs1805087 case G A 0.01 0.92
1 rs1805087 control G A 1.87 0.17
The analysis of the frequency distribution of alleles at rs1801131 and rs1805087 polymorphisms of genes of the folate cycle in a group with APS and in the control sample revealed between statistically significant differences (table 2) Since the value of the relative risk of the A allele of rs1805087 polymorphism was 3.86 (P = 0.0008), in while the G allele of this polymorphism can be seen as a protective in the development of this disease (x2 = =11.19; OR = 0.26; P = 0.0008). The Analysis of allelic frequencies rs1801131 polymorphism of MTHFR gene, reliably significant differences were not found.
When comparing the frequencies of the genotypes studied polymorphic markers there has been found the genetic association
of folate cycle genes to the development of antiphospholipid syndrome. Analysis of genotypic associations showed that the greatest risk of antiphospholipid syndrome is caused by homozygous AA genotype of rs1805087 polymorphism (x2 = 9.29; OR = 3.76; P = 0.01). Other relevant genotypes in the development of this pathology were: AA genotype of the rs1801131 polymorphic marker (x2 = 6.29; OR = 3.71; P = 0.04). Heterozygous combination of alleles AC (OR = 0.29), as well as the genotype CC (OR = 0.79), AG (OR = 0.77) and GG (OR = 0.09) of these polymorphisms showed no significant association with the development of antiphospholipid syndrome (P <0.05) (Table 3).
Table 2. - Distribution of allele frequencies of rs1801131 and rs1805087 polymorphisms in APS Group "+" (28 people) and APS "-" (34 people)
Alleles Cases Controls x2 P OR 95% CI
n = 28 n = 34
rs1801131 AlleleA 0.768 0.603 3.82 0.05 2.18 0.99-4.79
Allele C 0.232 0.397 0.46 0.21-1.01
rs1805087 Allele A 0.804 0.515 11.19 0.0008 3.86 1.71-8.70
Allele G 0.196 0.485 0.26 0.12-0.58
Table 3. - Distribution of genotypes frequencies of rs1801131 and rs1805087 polymorphisms in APS Group "+" (28 people) and APS "-" (34 people)
SNP Genotypes cases controls x2 P OR 95% CI
n = 28 n = 34
rs1801131 Genotype A/A 0.607 0.294 6.29 0.04 3.71 1.29-10.68
GenotypeA/C 0.321 0.618 0.29 0.10-0.84
Genotype C/C 0.071 0.088 0.79 0.12-5.12
rs1805087 Genotype A/A 0.643 0.324 9.29 0.01 3.76 1.31-10.81
Genotype A/G 0.321 0.382 0.77 0.27-2.19
Genotype G/G 0.036 0.294 0.09 0.01-0.75
Thus, the results of this study demonstrate the relationship risk of antiphospholipid syndrome in the Uzbek population with carriage of A alleles of rs1805087 polymorphism (x2 = 11.19; OR = 3.86; P = 0.0008), as well as the AA genotypes of rs 1805087 polymorphism (x2 = 9.29; OR = 3.76; P = 0.01) and the AA genotype of the rs1801131 polymorphic marker (x2 = 6.29; OR = 3.71; P = 0.04).
Conclusions:
1. The greatest risk of antiphospholipid syndrome in the Uzbek population is due to homozygous AA genotype of
rs1805087 polymorphism (X2 = 9.29; OR = 3.76; P = 0.01). Other relevant genotypes in the development of this pathology were: AA genotype of the rs1801131 polymorphic marker (x2 = 6.29; OR = 3.71; P = 0.04)
2. The combination of heterozygous alleles AC (OR = 0.29), as well as the CC genotypes (OR = 0.79), AG (OR = 0.77) and GG (OR = 0.09) of these polymorphisms showed no significant polymorphisms association with the development of antiphospholipid syndrome (P <0.05).
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