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Lyubchich Natalya Ivanovna, Republican specialized scientific-practice medical center of obstetrics and gynecology, Tashkent, Uzbekistan, scientific explorer E-mail: lyubchich@mail.ru
Polymorphism C677T of gene MTHFR as a factor risk for developing of preterm birth at uzbek women
Abstract: In purpose of to study of relation of MTGFR gene polymorphism (C677T) for preterm birth DNA of 235 women, which 121 of them with preterm birth in the anamnesis (main group) and 114 somatically healthy women with a physiological current of pregnancy (control group) have been investigated. Results showed that dominating risk factor of preterm birth among inherited thrombophilia is the homozygote mutation С677 Т of MTHFR gene (in 73,3% of cases), leading to reducing of enzyme activity of protein with homosystein elevation.
Keywords: preterm birth, thrombophilia, gene polimorphizm, pregnant, MTHFR
Introduction. Achievements of the XX-th century in tions, also on developing pregnancy loss and PB and there are
the field of molecular medicine, biology and medical genetics have allowed to estimate from essentially new positions патогенез such complication of pregnancy as preterm birth (PB) which remain an actual problem of modern obstetrics [2; 3; 5].
Nowadays РБ and reproductive health ofwoman are considered as a major medical and the social problem which is under closely attention of experts from leading scientific centers in the world [1, 2, 3]. On the basis of numerous researches there was established variety of the factors increasing risk of preterm birth at women. And among of them the special role is taken away to the congenital thrombophilia — he-mostasis failure, which leading toward thrombi formation in uterine-placental vessels [6; 8; 9].
The most important direction in research of thrombophilia considers studying of thrombophilic conditions at pregnant women, the analysis an influence of hemostasis system failure at risk of development on thrombosis and thromboembolia during pregnancy and labor, and also, definition of degree of participation of those or other genetic markers of thrombophilia in development on obstetrical and gynecologic complications [9; 10; 12].
Perhaps, abruptness and rapidity of development a thrombosis in placental vessels at thrombophila are the principal causes a placental insufficiency which not allowing in most cases to prevent severe consequences of thrombophilic complications at pregnant women. There have defined enough wide spectrum a genetic markers of thrombophilia, and dominating among of them in which is believed a genes of hemo-stasis system and homocystein exchange, in particularly, FV gene polymorphism (G1691 А) of blood coagulation and methylenetetrahydrofolate reductase (MTHFR) gene mutation (C677T) [1; 2; 3; 5; 7; 8].
However, MRHFR gene mutation is one of the most frequent reasons of inhereditary forms of thrombophilia at obstetrical practice. Therefore the scientist's form over the world have being deeply interested to investigate the role of this gene on the developing most obstetrical and perinatal complica-
a great number of published research works devoted to study of associations an allele variants of MTHFR gene on PB. So, according to dates published by R. L. Bick and all., (1998), there is the legible communication between heterozygous MTHFR gene mutation and PB which risk of its development increases in 2 times.
E. Brenner and all. [6, 7] have revealed this mutation at 46% of women with PB. At the same time W. H. Kutteh [3; 4; 6; 8] has not found out communication between MTHFR gene mutation (C677 T) and recurrent miscarriage. Later S. C. Guba and all. (1999), V. Kakkar and all. [11; 13] have shown obvious communication between the heterozygous mutation and recurrent miscarriage, and the risk thus rises on 2 times.
However gestation represents multistage process of interaction of mother and a fetus, hence the fruit also can influence to the stages of gestation process.
Aim of this work was estimation of association ofMTGFR gene polymorphism (C677T) for developing of preterm birth among Uzbek population.
Materials and methods. For this case-control study from February 2011 toJanuary 2014, 26 females with preterm birth were matched with 50 healthy parturients which delivered on term as controls. The study was carried out on clinical base of the Republican specialized scientific-practice medical centre of obstetrics and gynecology Ministry of public health of Republic of Uzbekistan. In this research were included women who have had preterm birth, more than 2 unexplained recurrent miscarriages, antenatal fetal death and stillbirth in anamnesis. The women described in this study were enrolled in this study between February 2011 and December 2013 and subsequently followed prospectively. DNA of 235 women, from them 121 with preterm birth in anamnesis and 114 somatically healthy women with a physiological current of pregnancy which have made control group have been investigated. Age of the observed women varied from 21 up to 35 years. To statement of the clinical diagnosis applied clinic, hemostasiologic and functional methods.
Revealing thrombophilia spent to laboratories of molecular-genetic researches of scientific research institute of hae-matology and blood transfusion under the management of PhD. K. T. Boboeva. There was determined presence of polymorphism genes of haemostasis system. DNA molecule was allocated by a standard technique [13] with some updating. Amplification of the polymorphic locus (USA) spent with use polymerase Chain reaction on programmed termocycler of firms «Applied Biosystems».
The statistical analysis of results is spent with use of a package of statistical programs OpenEpi 2009, Version 2.3.
Frequency of variants of allele and genotypes (f) calculated by the following formula:
f = n/2N and f = n/N (F1),
Where: n — occurrence of a variant (allele or a genotype), N — sample volume.
Degree of associations estimated in values of indicators of a proportion of chances odds ratio, OR, by following formula: OR = (a x d)/(b x c) (F2),
Where: a — frequency of allele (genotype) in sample of patients, b — frequency of allele (genotype) in control sample, c — the sum of frequencies of the other alleles (genotypes) in sample of patients, d — the sum of frequencies of the other alleles (genotypes) in control sample [11].
Results of research and their discussion. At the first stage our research we have carried out molecular-genetic investigations for frequency revealing of thrombophilia and its structures among of women belongs to uzbek population omen with preterm and on term birth. Thrombophilic mutations among patients of the basic group with PB are found out at 75 (62%) of them, and among women with a physiological current of pregnancy and on term birth — in 32,4% cases. Studying of dynamics of markers of thrombophilia has shown, that the most prevalence its form in general population of women with PB believed is MTHFR gene polymorphism (C677T) which is diagnosed at 63 (84,0%) patients, from them at 8 (12,7%) — homozygous, at 55 (87,3%) — the heterozygote form.
The mutation of the factor V Leiden is found out at 9 (7,4%) women with PB, and all of them have appeared carriers a heterozygote genotype. The rare mutation for Asian population G20210A of a gene prothrombin is revealed all at 3 (2,3%) women with PB, all of them also had a heterozygote genotype. Results of our work partially concurred with data of some authors [4; 6; 9; 10; 13]. The Most widespread in the European populations thrombophilic factors — polymorphism C677 T of 5,10-MTHFR gene, Leiden mutation of the factor V G1691A, a prothrombin gene mutation G20210A with the same frequency occurred among the women surveyed by us.
Thus, high frequency of inherited thrombophilia at our patients has allowed us to consider it as the major etio-patho-genetic factor of development of PB that dictates necessity of a choice of the optimal and safety preventive therapy directed on compensating of genetic infringements.
Because of the polymorphism C677T of MTHFR gene was occurred at the greatest frequency among of women with PB, they all have been undergone to the following investigation phase. We have interested on whether presence ofpolymorphism C677T of MTGFR gene for the term of occurrence of preterm birth and, ifyes, then how much it influences. With that purpose the patients of the basic group have been divided into subgroups depending on term gestation at occurrence of PB. So, 1st subgroup was made by 67 women with very early (22-27 week) and early PB (28-33 week), 57 women have included into 2nd subgroup with PB on term gestation from 34 till 37 weeks.
According to the received data, a mutation C677 T of MTHFR gene has been revealed at 63 (52,1%) women in the basic group, 55 (45,4%) from of them had heterozygous and 8 (6,6%) — a homozygous genotype of the given mutation. According to research problems we had been carried out the detailed analysis of influence of each of specified mutant allele and genotypes of the given marker on development PB in women from the main group.
Allele C and T distribution in the main group corresponded to 70,7 and 29,35% (tab. 2) accordingly. Frequency of these alleles in control group has made accordingly 81,6 and 18,4%. Thus distinctions on frequency of occurrence mutant allele in groups of patients were statistically authentic (X2=7,66; P=0,003; OR=1,9; 95% CI 1,19-2,839).
At patients from the main group there has been fixed significantly decrease in functional normal genotype C677 C on 1,3 times in comparison with the control (x2=6,87; P=0,004) (tab. 2). The reduction of frequency of occurrence of variant C677 C of gene MTGFR revealed by us in the main group allows to assume about possible «stability» of persons with this genotype to development of PB.
Significant associations were observed between functionally weakened hetero-and homozygous genotypes with occurrence of PB. Thus the most distinct association have revealed in frequency of occurrence homozygote T677T of the given polymorphism. At women of this group frequency of the given genotype was on 4 times higher, than in the control (6,6 against 1,7%, x2=3,4; P=0,03; 0R=4,0; 95% CI 0,8219,08). Rather unexpected there was an increase in a fraction heterozygous carriers of mutations C677 T among of patients of the main group. At carriers of heterozygous mutation frequency of PB was on 1,7 times more than at patients with absence of this mutation (43,6 against 33,3%). Thus, despite on rather low risk level of development of PB (0R=1,7; 95% CI 0,98-2,83), the obtained data have appeared statistically significant (x2=3,6; P=0,03).
There also have been revealed statistically significant distinctions (tab. 3, 4) at comparison of frequencies of genotypes and alleles between subgroups of women with PB and patients of control group. The frequency of677C and 677T allele at patients of 1st subgroup has made accordingly 68,7 and 31,3%, and in control group — 81,6 and 18,4%.
Prevalence carriers of allele 677T of MTHFR gene among women with very early and early PB in comparison
with women with physiologically pregnancy there has appeared statistically significant (x2=7,6; P=0,003; OR=2,3; 95% CI 1,22-3,32).
The analysis of distribution of functionally defective genotypes in these comparative samples has shown, that at individuals with presence of heterozygous genotype C/T an indicator of a parity of chances of development PB is equal to 1,5 (accordingly 43,6 and 33,3%, OR=1,5; 95% CI 0,83-2,92).
However at statistical data processing of reliability it is not received (x2=1,91; P=0,08).
Predictably, frequency of homozygous genotype T/T in a subgroup patients with PB was authentically high (9,4%), than in control group (1,7%). The calculated indicator of risk of development PB at carriers of the given genotype has appeared significantly high (x2=5,55; P=0,01; OR=5,8; 95% CI 1,134-29,61).
The similar situation has been found out also at the comparative analysis of frequency of distribution 677C and 677T allele among surveyed 2nd subgroup and control group. Frequency of mutant allele carriage in a subgroup of women with PB was statistically significantly higher, than in control group (27,2 against 18,4%, x2=3,48; P=0,03; OR=1,6; 95% CI 0,97-2,81).
At research carrying out there is received an unexpected enough result: distribution of a homozygous genotype in studied samples authentically did not differ. And there was marked some increase of frequency of the given genotype at patients of 2nd subgroup (3,5%) in comparison with control group (1,7%). Thus the risk of development of PB also was high (0R=2,0; 95% CI 0,279-14,84). However such distinction has appeared statistically insignificant (x2=0,51; P=0,2), that, probably, is connected with low frequency of the given variant of MTHFR gene mutation and with small number of sample.
At the analysis of frequency distribution of a genotype of the given gene we managed to reveal statistically significant accumulation of frequency heterozygous C677 T a genotype of MTHFR gene at the patients who have transferred PB. At carriage of this adverse genotype the risk of occurrence of thrombophilic complications at patients with PB more than on 1,8 times higher, than at women with absence of the given genotype (x2=3,2; P=0,04; OR=1,8; 95% CI 0,94-3,45).
In summary it is necessary to underline, that distinction in total frequency of adverse genotypes of mutation С677 Т of MTHFR gene in studied subgroups has appeared statistically uncertain (53,0% against 51,0%, x2=0,06; Р=0,4).
Thus, results of our researches have allowed to make following conclusions: in pathogenesis of PB at women the major place belongs to missence mutation С677 Т of MTHFR gene connected with replacement of nucleotide cytosine with the nucleotide thymine at position 677 (especially homozygous genotype Т/Т), leading to reducing of enzyme activity of protein with elevation of homosystein in plasma.
Hyperhomosysteinemia is connected with endotheli-al impairment which it is accompanied by synthesis reducing of nitric oxide, activation of markers endothelial dysfunction with increase of inflammatory factors and decrease of antiinflammatory interleukin-10 [2]. As a result of this process in the organism ofwomen there is formed latent thrombophilia, which under the influence of provoking factors (pregnancy, contraceptives, a trauma, operative interventions, smoking, etc.) leads to development various obstetrical complications including PB.
Conclusion. In the structure of the reasons of development of preterm birth at women a frequency of inherited thrombophilia makes 62%. A dominating risk factor of preterm birth among inherited thrombophilia is the homozygote mutation С677 Т of MTHFR gene (in 73,3% of cases), leading to reducing of enzyme activity of protein with homosystein elevation. And it can be considered as independent risk factor of development of obstetrical complications. Polymorphic marker С677 Т of inherited thrombophilia of MTHFR gene is significantly associated with development of PB at women. Thus especially strongly carriage of homozygous Т/Т genotype which more than on 5,5 times significantly increases occurrence of very early PB at women (x2=5,55; Р=0,01; OR=5,8; 95% CI 1,134 29,6). Timely revealing of the given mutations and carrying out of preventive standard methods of prophylaxis prior to gestation will allow to improve pregnancy outcomes and to reduce of perinatal disease and death rate, and also will be as preventive maintenance of maternal mortality and morbidity rate.
Rezume
Table 1. - The frequency of alleles and genotype distribution of C677T polymorphism of MTGFR gene among of patients with preterm birth ad control group
N The frequency of allele The frequency of genotype distribution
С Т СС СТ ТТ
абс. % абс. % абс. % абс. %
Main group: 121 70,7 29,3 58 48,0 55 45,4 8 6,6
a) Very early and early PB 64 68,7 31,3 30 47,0 28 43,6 6 9,4
b) PB 57 72,8 27,2 28 49,1 27 47,4 2 3,5
Control 114 81,6 18,4 74 65,0 38 33,3 2 1,7
Table 2. - Difference of allele and genotypes of C677T polymorph marker of MTGFR gene in main and control group
Alleles and genotypes The frequency of allele and genotypes in surveying groups Statistically distinction
Main group n=121 Control group n=114
Allele C 70,7 81,6 X2=7,66; P=0,003; OR=1,9; 95% CI 1,19-2,839
Allele T 29,3 18,4
Genotype CC 48,0 65,0 X2= 6,87; P=0,004
Genotype CT 45,4 33,3 X 2=3,6; P=0,03; OR=1,7; 95% CI 0,98-2,83
Genotype TT 6,6 1,7 X 2=3,4; P=0,03; 0R=4,0; 95% CI 0,82-19,08
Table 3. - Distinction of alleles and genotypes of polymorph marker C677T of MTGFR gene in patients from 1st subgroup and control group
Alleles and genotypes The frequency of allele and genotypes in surveyed groups Statistically distinction
Very early and early PB, n=64 Control n=114
Allele C 68,7 81,6 X2=7,6; P=0,003; 0R=2,3; 95% CI 1,22-3,32
Allele T 31,3 18,4
Genotype CC 47,0 65,0 X 2=5,49; P=0,01
Genotype CT 43,6 33,3 X 2=1,91; P=0,08; 0R=1,5; 95% CI 0,83-2,92
Genotype TT 9,4 1,7 X 2=5,55; P=0,01; 0R=5,8; 95% CI 1,134-29,61
Ttable 4. - Distinction of alleles and genotypes of polymorph marker C677T of MTGFR gene in patients from 2nd subgroup and control group
Alleles and genotypes The frequency of allele and genotypes in surveyed groups Statistically distinction
PB, n=57 Control, n=114
Allele C 72,8 81,6 X 2=3,48; P=0,03; 0R=1,6; 95% CI 0,97-2,81
Allele T 27,2 18,4
Genotype CC 49,1 65,0 X2=3,9; P=0,02
Genotype CT 47,4 33,3 X 2=3,2; p=0,04; 0R=1,8; 95% CI 0,94-3,45
Genotype TT 3,5 1,7 X 2=0,51; P=0,2; 0R=2,0; 95% CI 0,279-14,84
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