http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–5 ! 2015 Royan institute for reproductive biomedicine research center, ACECR. DOI: 10.3109/14767058.2015.1044431
ORIGINAL ARTICLE
Thrombophilic genes alterations as risk factor for recurrent pregnancy loss
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Kamelia Farahmand1, Mehdi Totonchi2, Mehrdad Hashemi1, Fakhreddin Reyhani Sabet1, Hamid Kalantari2, Hamid Gourabi2, and Anahita Mohseni Meybodi2 1
Department of Genetics, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran and 2Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran Abstract
Keywords
Objective: The important polymorphisms leading to inherited thrombophilia are Factor V Leiden (FVL), Prothrombin G20210A and MTHFR C677T and A1298C. The frequencies also the correlation among these polymorphisms and RPL have been reported controversially in various populations. Our clinic is one of the referral centers in reproductive biomedicine in which patients in all over Iran refer to; thus the results of this study could be considered clinically beneficial. Besides, in the present study, not only the frequency of specific but also multiple thrombophilic gene alterations were compared in Iranian women with RPL and a control group. Methods: The patients group comprised 330 women with three or more consecutive RPLs. The control population included 350 women with at least one child and no history of pregnancy loss. FVL, Prothrombin G20210A and MTHFR C677T polymorphisms were analyzed by Strip assay kit. MTHFR A1298C was genotyped by PCR-RFLP. Results: The frequencies of FVL, Prothrombin G20210A, MTHFR C677T and MTHFR A1298C mutations in patients were 8.48, 4.24, 45.45 and 59.39%, and in controls were 2.86, 2.86, 34.28 and 6%, respectively. Conclusions: The present data showed that FVL, MTHFR polymorphisms also combined with thrombophilic gene mutations have a strong association with RPL.
Factor V Leiden, gene polymorphism, MTHFR, prothrombin, recurrent pregnancy loss, thrombophilia
Introduction Recurrent pregnancy loss (RPL) affects 0.4–2% of couples trying to have children and is very stressful condition for these couples. RPL is generally known as three or more consecutive fetal losses prior to 20 weeks of gestation. Several causes may leads to RPL, such as chromosomal anomalies, maternal thrombophilic defect, structural abnormalities of the uterus, endocrinological problems and autoimmune disorders [1]. However, in 50% of cases, the reason of miscarriage is unknown [2]. Thrombophilia is defined as condition in which an individual is predisposed to thromboembolism, including acquired or inherited [3]. Pregnancy is a hypercoagulable state and for the establishment of a successful pregnancy, the contact between placenta and maternal circulation is crucial. This contact is required to exchange of nutrients, gases and other metabolites. Abnormal blood clotting in the small placental blood vessels may results in RPL [2]. The most This work was performed in part of a Contributor’s duties as an employee of Royan institute for reproductive biomedicine research center, ACECR. Address for correspondence: A. Mohseni Meybodi, Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. Tel: +98-2123562711. Fax: +98-21-22306481. E-mail: [email protected]
History Received 14 March 2015 Revised 13 April 2015 Accepted 21 April 2015 Published online 2 July 2015
common mutation leads to thrombophilia is factor V Leiden (FVL). This polymorphism is the result of glutamine–arginine substitution at amino acid position 506 in the factor V molecule and may produce thrombosis by decreasing the sensitivity of factor V to inactivation by activated protein C. The role of this mutation in the etiology of RPL is not confirmed yet [4,5]. The second common polymorphism, Prothrombin G20210A, is a missense mutation which occurs in the 30 -untranslated region of Prothrombin gene and results in guanine to adenine transition at nucleotide position 20210. This mutation gives rise to increased mRNA and protein expression for Prothrombin and thus elevated levels of blood Prothrombin [6]. Methylenetetrahydrofolate reductase (MTHFR) plays a crucial role in the folate-dependent remethylation of homocysteine, by catalyzing the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. MTHFR gene polymorphisms are commonly associated with hyperhomocysteinaemia, which is a risk factor for RPL, down syndrome, venous thrombosis, placental infarction and placental abruption [7]. The most common polymorphisms of this gene, including MTHFR C677T and MTHFR A1298C, result in reduction of the activity of MTHFR enzyme, although the decrease in the enzyme activity in the case of MTHFR A1298C is less than MTHFR C677T [8]. It has been shown that women with combined thrombophilia (more than
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one inherited thrombophilic defect) have an increased risk for RPL than women with only one mutation [9]. The aim of this study is to compare the frequency of these mutations in Iranian woman with the history of RPL with fertile control group to evaluate the correlation between these polymorphisms and RPL.
J Matern Fetal Neonatal Med, Early Online: 1–5
256 bp was digested by MboII enzyme at 37 C for 18 h. Wildtype allele (1298A) had three restriction sites and after digestion produced four fragments of 176, 30, 28 and 22 bp. 1298C allele destroyed a restriction site in the amplified fragment and, therefore, was digested into three fragments of 204, 30 and 22 bp, respectively. The fragments were visualized by ethidium bromide under UV transilluminator.
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Methods Study groups
Sequencing
This case–control study was performed during 2011 and 2014. The patients group consisted of 330 women, who had three or more consecutive pregnancy losses before 20 weeks’ of gestation, with no history of full-term pregnancies. Exclusion criteria included uterine anomalies, endocrine dysfunction, chromosomal abnormalities and autoimmune disorders. The control group consisted of 350 women, with at least one live born children and no history of pregnancy loss and uncomplicated pregnancy. These women were recruited during their attendance to the Royan Institute for the family balancing sex selection. All participants gave their informed consent before inclusion to the study.
Sequencing of PCR products of MTHFR A1298C polymorphism was carried out by Fazabiotech Company (Tehran, Iran) according to Sanger method using ABI 3730XL Capillary Sequencer. Sequencing results were compared with the sequence of normal MTHFR gene obtained from the NCBI website.
FVL, Prothrombin G20210A and MTHFR C677T analysis FVL, Prothrombin G20210A and MTHFR C677T gene polymorphisms were analyzed by FV-PTH-MTHFR Strip Assay kit (Vienna lab-REF 4-260). This technology is based on polymerase chain reaction (PCR) and reverse-hybridization. This procedure included three steps, which are explained below. DNA isolation DNA was extracted from blood using a commercial kit (PAX gene blood DNA; Qiagene). PCR amplification using biotinylated primers Taq DNA polymerase diluted to 0.2 U/ul using the supplied Dilution buffer. The total PCR reaction volume was 25 ml:15 ml Amplification Mix + 5 ml (diluted Taq Pol) + 5 ml extracted DNA (less than 40 ng/ml). Mentioned reaction mix was amplified on thermal cycler using a standard three steps, 30-cycle protocol. Amplicons checked via gel electrophoresis. Hybridization Amplification products hybridized to a test strip containing allele-specific oligonucleotide probes immobilized as an array of parallel lines. Bound biotinylated sequences were detected using streptavidin-alkaline phosphatase and color substrates. MTHFR A1298C analysis MTHFR A1298C polymorphism was experimented by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The forward and reverse primers used in the PCR amplification reactions were 50 -CTTCTACCTGAA GAGCAAGTC-30 and 50 -CATGTCCACAGCATGGAG-30 , respectively. Polymerase chain reaction conditions were 95 C for 10 min, 30 cycles of 95 C for 45 s, 30 cycles of 54 C for 45 s, 30 cycles of 72 C for 45 s followed by final extension of 72 C for 5 min. The amplified fragment of
Statistical analysis Univariate odds ratio (OR) and 95% CI were estimated separately for each polymorphism. The prevalence of each polymorphism was compared between patients and controls with the use of the 2-test by SPSS software. The level of p50.05 was calculated as significance.
Results Both cases and controls were Iranian women living in all districts of Iran. The mean age of patients (30.37 years, SD 5.05) did not statistically different from that of the controls (29.88, SD 4.09) (p ¼ 0.44). The number of miscarriages among patients was ranged from 3 to 7 with average of 3.34. 270 patients had three miscarriages and 60 had four or more miscarriages, while none of the controls had abortion. Early (before 12 weeks) and late (after 12 weeks) pregnancy losses were seen in 254 and 26 patients, respectively. In addition, 50 patients showed combined early-late losses. The results are shown in Table 1. There were no homozygous cases of FVL and Prothrombin G20210A in either group. 28 patients with RPL and 10 women of the control group (8.48 versus 2.86%, S) were heterozygous for FVL. The frequency of the 1691A allele among cases and controls was 4.24 versus 1.43% (S). The heterozygous form of Prothrombin G20210A occurred in 14 patients and in 10 controls (4.24 versus 2.86%, NS). Mutant allele (A) was seen in 2.12% of patients group and 1.43% of controls (NS). Heterozygosity for the MTHFR C677T mutation was found in 114 patients and in 85 controls (34.55 versus 24.29%, S). Homozygosity for this mutation was seen in 36 RPL cases and in 35 controls (10.9 versus 10%, NS). The frequency of the 677T allele among cases and controls was 28.18 versus 22.14% (NS), respectively. The MTHFR 1298AC genotype was diagnosed in 152 patients (46.07%) and 20 controls (5.71%) (S). The homozygote genotype 1298CC was seen in 44 patients and in 1 control (13.33 versus 0.29%, S). 1298C allele occurred with a frequency of 36.36 and 3.14% among patients and controls (S), respectively. The prevalence of combined mutations (more than one mutation) in RPL women was found to be 18.79%. This frequency among control group was 2.86% (S). Table 2 shows the frequency and type of combined mutations.
Thrombophilic genes alterations and recurrent pregnancy loss
DOI: 10.3109/14767058.2015.1044431
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Table 1. Distribution of thrombophilic polymorphisms in the case and control groups. Case (n ¼ 330) Type of abortion
Normal
Heterozygote
Homozygote
Normal
Heterozygote
Homozygote
p value
Odds ratio
95% CI
FV Leiden
Early Late Early + late Total
243 10 49 302
11 16 1 28
– – – –
340
10
–
NS S NS S
1.53 54.4 0.69 3.15
0.64–3.68 19.8–149.3 0.08–5.53 1.5–6.59
Prothrombin G20210A
Early Late Early + late Total
241 25 50 316
13 1 – 14
– – – –
340
10
–
NS NS NS NS
1.83 1.36 0.32 1.50
0.79–4.25 0.16–11.05 0.01–5.56 0.65–3.44
MTHFR C677T
Early Late Early + late Total
133 21 26 180
94 3 17 114
27 2 7 36
230
85
35
S NS NS S
1.74 0.45 1.76 1.59
1.25–2.42 0.16–1.24 0.97–3.21 1.17–2.17
MTHFR A1298C
Early Late Early + late Total
104 16 14 134
109 10 33 152
41 – 3 44
329
20
1
S S S S
22.5 9.79 40.2 22.9
13.6–37.5 3.96–24.2 18.86–86.03 13.99–37.51
Type of mutations
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Control (n ¼ 350)
S: Significant, NS: Not significant.
Table 2. Distribution of combined thrombophilic mutations among groups. Combined thrombophilic mutations MTHFR C677T/A1298C MTHFR C677T/A1298C/FV G1691A MTHFR T677T/FV G1691A MTHFR C677T/FV G1691A MTHFR A1298C/FV G1691A MTHFR C1298C/FV G1691A MTHFR T677T/Prothrombin G20210A Prothrombin G20210A/FV G1691A MTHFR C677T/C1298C Total
Patients (n ¼ 330) 31 9 7 4 3 2 2 2 2 62
(9.4%) (2.73%) (2.12%) (1.21%) (0.9%) (0.6%) (0.6%) (0.6%) (0.6%) (18.79%)
Control (n ¼ 350)
p value
Odds ratio
95% CI
3 (0.86%) – – 1 (0.28%) – 2 (0.58%) 1 (0.28%) – 3 (0.86%) 10 (2.86%)
50.0001 0.03 0.05 0.19 0.18 0.95 0.53 0.28 0.7 50.0001
11.99 20.71 16.25 4.28 7.49 1.06 2.12 5.33 0.7 7.86
3.62–39.62 1.2–357.34 0.92–285.7 0.47–38.51 0.38–145.59 0.14–7.57 0.19–23.58 0.25–111.54 0.11–4.24 3.95–15.63
p50.05 was considered to be statistically significant.
Discussion Pregnancy is a hypercoagulable state due to the multifactorial setting of underlying pathophysiologic changes, because prevention or reduction of hemorrhage in this stage is necessary [3]. In some cases, the presence of thrombophilia also aggravates this problem [10]. FVL is the most common polymorphism studied for correlation with RPL. This polymorphism is very common among white population. About 3 to 7% of individuals from Northern European are heterozygous for this mutation. Heterozygosity for this polymorphism has been demonstrated to be associated with a 3- to 7-fold increase in the risk of venous thrombosis, while homozygosity confers a 50- to 100-fold increase in risk [11]. It was shown that FVL is associated with second trimester losses, because the risk of thrombosis in placental vessels increases with advanced gestational age [4]. As it has been shown in Table 1, in the present study, increased prevalence of this mutation was seen in women with only late abortions (p50.0001), but not in woman with early (p ¼ 0.33) and combined early-late (p ¼ 0.73) abortions. In addition, there was a statistical significant correlation between this mutation and women with RPL irrespective of their gestational status (p ¼ 0.002).
It has been demonstrated that Prothrombin G20210A polymorphism has high frequency among Europeans, while the low prevalence of it has been reported in Asians and other non-European populations [4]. As it has been summarized in Table 1, the difference in the prevalence of this polymorphism was not statistically significant between early (p ¼ 0.15), late (p ¼ 0.77), early-late (p ¼ 0.43) abortions, as well as all RPL cases (p ¼ 0.33) when compared to controls. Some studies have shown an association between RPL and these two polymorphisms, while others have not. Some of these results are summarized in Table 3. As it can be seen the frequencies of homozygote genotype for these polymorphisms have been reported very low. MTHFR enzyme maintains the blood homocysteine level. Elevated homocysteine concentration affects vascular endothelium and leads to placental vasculopathy, which impairs embryo development. Besides, implantation and invasion of the embryo in first trimester of pregnancy is regulating by DNA methylation. This enzyme is known to maintain the methyl pool for control of gene expression, as well [2]. In 2000, a report brought the number of polymorphisms of MTHFR gene up to 24 [12]. The first common mutation in the coding region of MTHFR gene is alanine to valine
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J Matern Fetal Neonatal Med, Early Online: 1–5
Table 3. The frequency of thrombophilic mutations and their correlation with RPL in different studies. Frequency Mutation type
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FV Leiden
Frequency
Number of Heterozygote Homozygote Number of Heterozygote Homozygote Year Country case group (%) (%) control group (%) (%) p value References 2005 2012 2013 2012 2007 2011 2014
Tunisia Turkey India Romania Greece Palestine Iran
200 543 1000 283 99 329 330
40 109 50 21 5 72 28
(20%) (20%) (5%) (7.42%) (5%) (21.9%) (8.48%)
Prothrombin G20210A 2002 2012 2005 2007 2011 2013 2014
Lebanon Romania Tunisia Greece Palestine India Iran
110 283 200 99 329 1000 330
15 8 4 5 11
MTHFR C677T
2006 2012 2013 2007 2011 2011 2014
Tunisia India India Greece Egypt Palestine Iran
200 106 200 97 70 329 330
47 26 70 61 26 151 114
(23.5%) (24.53%) (35%) (62.9%) (37.1%) (46.8%) (34.55%)
61 5 20 12 4 33 36
MTHFR A1298C
2006 2013 2011 2012 2007 2011 2014
Tunisia India Korea Turkey Greece Egypt Iran
200 200 57 543 88 70 330
65 92 21 257 37 49 152
(32.5%) (46%) (36.84%) (47.4%) (42%) (70%) (46.07%)
27 20 2 85 7 6 44
(13.64%) (2.83%) (2%) (5%) (3.3%) – 14 (4.24%)
8 (4%) 1 (0.002%) – 2 (0.71%) – 7 (2.1%) –
200 106 500 100 102 402 350
11 2 12 5 3 69 10
(5.5%) (1.9%) (2.4%) (5%) (2.9%) (17.2%) (2.86%)
– – – – – 4 (1%) –
S S S NS NS NS S
[6] [26] [4] [27] [19] [28] This study
– 2 (0.71%) – – – – –
67 100 200 101 402 500 350
2 4 9 2 17
S NS NS NS NS NS NS
[29] [27] [6] [19] [28] [4] This study
(30%) (4.72%) (10%) (12.4%) (5.7%) (9.7%) (10.9%)
200 140 300 102 136 402 350
30 21 90 57 68 177 85
(15%) (15%) (30%) (55.9%) (50%) (44%) (24.29%)
14 1 14 13 1 43 35
(7%) (0.71%) (4.6%) (12.6%) (0.7%) (10.7%) (10%)
S S S NS NS NS S
[13] [2] [14] [19] [30] [28] This study
(13.5%) (10%) (3.5%) (15.6%) (7.9%) (8.6%) (13.33%)
200 300 155 106 90 136 350
62 127 38 35 39 97 20
(31%) (42.3%) (24.5%) (33%) (43.3%) (71.3%) (5.71%)
8 (4%) 16 (5.3%) 4 (2.6%) (2.2%) 6 (6.6%) 3 1 (0.29%)
S S S S NS NS S
[13] [14] [7] [26] [19] [30] This Study
(2.99%) (4%) (4.5%) (2%) (4.2%) – 10 (2.86%)
– – – – – – –
S: Significant, NS: Not significant.
substitution at the amino acid 222 (C677T). This polymorphism is located in the catalytic domain of enzyme [13]. Heterozygosity and homozygosity for this polymorphism reduce the enzyme activity about 35 and 70%, respectively. Near 50% of the general populations are heterozygote carriers of this mutation, and the frequency of the homozygote carriers for this polymorphism has been reported between 1 and 20%, depending on the population studied [8]. For example, the high frequency for this genotype has been demonstrated among US Hispanics, Colombians and Amerindians in Brazil, while the incidences of it among Blacks have been reported less than 2%. The frequency of heterozygote genotype for this mutation varies between 8 and 20% among White populations of Australia, North America and Europe [14]. As it is demonstrated in Table 1, the correlation between this mutation and RPL in the present study was statistically significant in women with only early losses (p ¼ 0.001), while this association was not significant in late (p ¼ 0.12) and early-late (p ¼ 0.06) abortions. Moreover, the difference between the frequency of this polymorphism among all RPL cases and controls has been statistically significant (p ¼ 0.003). The second common mutation in this gene is A to C transition at nucleotide 1298 (A1298C). This mutation results in alanine to glutamate substitution in the MTHFR protein at the amino acid 429. This polymorphism found within the enzyme regulatory domain, and like the C677T mutation, leads to decreasing in
MTHFR activity, but this reduction in case of A1298C polymorphism is less than the other one. It has been shown that almost 10% of individuals are homozygous for this mutation. These individuals show 40% reduction in enzyme activity in vitro, but they do not have higher levels of plasma homocysteine than controls. Individuals with 677CT/1298AC genotype have a 40 to 50% reduction in MTHFR activity in vitro, and increased homocysteine and decreased folate levels [8,13]. The frequency of the homozygote genotype for this polymorphism has been reported between 7 and 12% among White populations of North America and Europe. This frequency is lower among Hispanics, Chinese and Asian populations [14]. In the present study, there was a statistically significant association among this polymorphism and all RPL groups including early (p50.0001), late (p50.0001), combined early-late (p50.0001) and all RPL cases (p50.0001) (Table 1). A strong association between MTHFR polymorphisms and RPL was confirmed in some studies, while others denied this association. Table 3 shows some of these results. In the present study, there was a statistically significant correlation between combined mutations (more than one thrombophilic mutation) and RPL. The same results were obtained in some studies [3,15–18], while this correlation was ignored by some others [19,20]. The frequency of MTHFR C677T/A1298C haplotype in this study was 9.4%. This frequency was reported 15.73% in the study of Poursadegh et al. [21]. Same as the findings of Friedman et al. [22],
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DOI: 10.3109/14767058.2015.1044431
Weisberg et al. [23] and Rady et al. [24], combined homozygosity for these two mutations was not observed in this study, while MTHFR C677T/C1298 C haplotype was seen in two patients (0.6%). These types of mutations were also observed by Sotiriadis et al. [19]. In our study, seven patients (2.12%) showed MTHFR C677T homozygosity associated with FVL heterozygosity, this combination was reported 1% by Androutsopoulos et al. [25]. They have also reported a frequency of 0.5% for MTHFR C677T homozygosity associated with Prothrombin G20210A heterozygosity, which was seen in two patients (0.6%) in present study and 0.8% in the study of D’Uva et al. [18]. The frequency of FVL and Prothrombin G20210A combined heterozygosity in our study was 0.6%. This frequency in the study of D’Uva et al. [18] was 0.8%. In conclusion, there was a significant higher frequency of FVL, MTHFR C677T and MTHFR A1298C polymorphisms in patients with RPL compared with healthy controls. In addition, combination of these mutations, especially combination of FVL and MTHFR mutations, significantly increases the risk of RPL. According to our data, checking all of these polymorphisms in women with the history of RPL is strongly recommended.
Acknowledgements The authors would like to express their gratitude toward Dr Zamanian, Mrs. Jalilnejad and staffs of genetic lab of Royan Institute for their technical assistance.
Declaration of interest The authors report no declarations of interest. This research was supported by reproductive biomedicine research center, Royan Institute, Tehran (project code: 629-1-91000149).
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9. Di Micco P, D’Uva M, Strina I, et al. Recurrent pregnancy loss and thrombophilia. Clin Lab 2007;53:309–14. 10. Aracic N, Roje D, Drmic Hofman I, et al. Low molecular weight heparin treatment and impact of inherited thrombophilia type in pregnancies with previous adverse outcome. J Matern Fetal Neonatal Med 2015;28:306–10. 11. Bogdanova N, Markoff A. Hereditary thrombophilic risk factors for recurrent pregnancy loss. J Community Genet 2010;1:47–53. 12. Sibani S, Christensen B, O’Ferrall E, et al. Characterization of six novel mutations in the methylenetetrahydrofolate reductase (MTHFR) gene in patients with homocystinuria. Hum Mutat 2000;15:280–7. 13. Mtiraoui N, Zammiti W, Ghazouani L, et al. Methylenetetrahydrofolate reductase C677T and A1298C polymorphism and changes in homocysteine concentrations in women with idiopathic recurrent pregnancy losses. Reproduction 2006;131: 395–401. 14. Parveen F, Tuteja M, Agrawal S. Polymorphisms in MTHFR, MTHFD, and PAI-1 and recurrent miscarriage among North Indian women. Arch Gynecol Obstet 2013;288:1171–7. 15. Brenner B, Sarig G, Weiner Z, et al. Thrombophilic polymorphisms are common in women with fetal loss without apparent cause. Thromb Haemost 1999;82:6–9. 16. Sarig G, Younis JS, Hoffman R, et al. Thrombophilia is common in women with idiopathic pregnancy loss and is associated with late pregnancy wastage. Fertil Steril 2002;77:342–7. 17. Preston FE, Rosendaal FR, Walker ID, et al. Increased fetal loss in women with heritable thrombophilia. Lancet 1996;348:913–16. 18. D’Uva M, Di Micco P, Strina I, et al. Etiology of hypercoagulable state in women with recurrent fetal loss without other causes of miscarriage from Southern Italy: new clinical target for antithrombotic therapy. Biologics 2008;2:897–902. 19. Sotiriadis A, Vartholomatos G, Pavlou M, et al. Combined thrombophilic mutations in women with unexplained recurrent miscarriage. Am J Reprod Immunol 2007;57:133–41. 20. Jivraj S, Rai R, Underwood J, Regan L. Genetic thrombophilic mutations among couples with recurrent miscarriage. Hum Reprod 2006;21:1161–5. 21. Poursadegh Zonouzi A, Chaparzadeh N, Asghari Estiar M, et al. Methylenetetrahydrofolate reductase C677T and A1298C mutations in women with recurrent spontaneous abortions in the northwest of Iran. ISRN Obstet Gynecol 2012;2012:945486. 22. Friedman G, Goldschmidt N, Friedlander Y, et al. A common mutation A1298C in human methylenetetrahydrofolate reductase gene: association with plasma total homocysteine and folate concentrations. J Nutr 1999;129:1656–61. 23. Weisberg I, Tran P, Christensen B, et al. A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab 1998; 64:169–72. 24. Rady PL, Tyring SK, Hudnall SD, et al. Methylenetetrahydrofolate reductase (MTHFR): the incidence of mutations C677T and A1298C in the Ashkenazi Jewish population. Am J Med Genet 1999;86:380–4. 25. Androutsopoulos G, Mougiou A, Karakantza M, et al. Combined inherited thrombophilia and adverse pregnancy outcome. Clin Exp Obstet Gynecol 2007;34:236–8. 26. Ozdemir O, Yenicesu GI, Silan F, et al. Recurrent pregnancy loss and its relation to combined parental thrombophilic gene mutations. Genet Test Mol Biomarkers 2012;16:279–86. 27. Mierla D, Szmal C, Neagos D, et al. Association of prothrombin (A20210G) and factor V Leiden (A506G) with recurrent pregnancy loss. Maedica (Buchar) 2012;7:222–6. 28. Abu-Asab NS, Ayesh SK, Ateeq RO, et al. Association of inherited thrombophilia with recurrent pregnancy loss in palestinian women. Obstet Gynecol Int 2011;2011:689684. 29. Finan RR, Tamim H, Ameen G, et al. Prevalence of factor V G1691A (factor V-Leiden) and prothrombin G20210A gene mutations in a recurrent miscarriage population. Am J Hematol 2002;71:300–5. 30. Settin A, Elshazli R, Salama A, ElBaz R. Methylenetetrahydrofolate reductase gene polymorphisms in Egyptian women with unexplained recurrent pregnancy loss. Genet Test Mol Biomarkers 2011;15:887–92.
ORIGINAL ARTICLE
Thrombophilic genes alterations as risk factor for recurrent pregnancy loss
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Nyu Medical Center on 07/13/15 For personal use only.
Kamelia Farahmand1, Mehdi Totonchi2, Mehrdad Hashemi1, Fakhreddin Reyhani Sabet1, Hamid Kalantari2, Hamid Gourabi2, and Anahita Mohseni Meybodi2 1
Department of Genetics, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran and 2Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran Abstract
Keywords
Objective: The important polymorphisms leading to inherited thrombophilia are Factor V Leiden (FVL), Prothrombin G20210A and MTHFR C677T and A1298C. The frequencies also the correlation among these polymorphisms and RPL have been reported controversially in various populations. Our clinic is one of the referral centers in reproductive biomedicine in which patients in all over Iran refer to; thus the results of this study could be considered clinically beneficial. Besides, in the present study, not only the frequency of specific but also multiple thrombophilic gene alterations were compared in Iranian women with RPL and a control group. Methods: The patients group comprised 330 women with three or more consecutive RPLs. The control population included 350 women with at least one child and no history of pregnancy loss. FVL, Prothrombin G20210A and MTHFR C677T polymorphisms were analyzed by Strip assay kit. MTHFR A1298C was genotyped by PCR-RFLP. Results: The frequencies of FVL, Prothrombin G20210A, MTHFR C677T and MTHFR A1298C mutations in patients were 8.48, 4.24, 45.45 and 59.39%, and in controls were 2.86, 2.86, 34.28 and 6%, respectively. Conclusions: The present data showed that FVL, MTHFR polymorphisms also combined with thrombophilic gene mutations have a strong association with RPL.
Factor V Leiden, gene polymorphism, MTHFR, prothrombin, recurrent pregnancy loss, thrombophilia
Introduction Recurrent pregnancy loss (RPL) affects 0.4–2% of couples trying to have children and is very stressful condition for these couples. RPL is generally known as three or more consecutive fetal losses prior to 20 weeks of gestation. Several causes may leads to RPL, such as chromosomal anomalies, maternal thrombophilic defect, structural abnormalities of the uterus, endocrinological problems and autoimmune disorders [1]. However, in 50% of cases, the reason of miscarriage is unknown [2]. Thrombophilia is defined as condition in which an individual is predisposed to thromboembolism, including acquired or inherited [3]. Pregnancy is a hypercoagulable state and for the establishment of a successful pregnancy, the contact between placenta and maternal circulation is crucial. This contact is required to exchange of nutrients, gases and other metabolites. Abnormal blood clotting in the small placental blood vessels may results in RPL [2]. The most This work was performed in part of a Contributor’s duties as an employee of Royan institute for reproductive biomedicine research center, ACECR. Address for correspondence: A. Mohseni Meybodi, Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. Tel: +98-2123562711. Fax: +98-21-22306481. E-mail: [email protected]
History Received 14 March 2015 Revised 13 April 2015 Accepted 21 April 2015 Published online 2 July 2015
common mutation leads to thrombophilia is factor V Leiden (FVL). This polymorphism is the result of glutamine–arginine substitution at amino acid position 506 in the factor V molecule and may produce thrombosis by decreasing the sensitivity of factor V to inactivation by activated protein C. The role of this mutation in the etiology of RPL is not confirmed yet [4,5]. The second common polymorphism, Prothrombin G20210A, is a missense mutation which occurs in the 30 -untranslated region of Prothrombin gene and results in guanine to adenine transition at nucleotide position 20210. This mutation gives rise to increased mRNA and protein expression for Prothrombin and thus elevated levels of blood Prothrombin [6]. Methylenetetrahydrofolate reductase (MTHFR) plays a crucial role in the folate-dependent remethylation of homocysteine, by catalyzing the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. MTHFR gene polymorphisms are commonly associated with hyperhomocysteinaemia, which is a risk factor for RPL, down syndrome, venous thrombosis, placental infarction and placental abruption [7]. The most common polymorphisms of this gene, including MTHFR C677T and MTHFR A1298C, result in reduction of the activity of MTHFR enzyme, although the decrease in the enzyme activity in the case of MTHFR A1298C is less than MTHFR C677T [8]. It has been shown that women with combined thrombophilia (more than
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one inherited thrombophilic defect) have an increased risk for RPL than women with only one mutation [9]. The aim of this study is to compare the frequency of these mutations in Iranian woman with the history of RPL with fertile control group to evaluate the correlation between these polymorphisms and RPL.
J Matern Fetal Neonatal Med, Early Online: 1–5
256 bp was digested by MboII enzyme at 37 C for 18 h. Wildtype allele (1298A) had three restriction sites and after digestion produced four fragments of 176, 30, 28 and 22 bp. 1298C allele destroyed a restriction site in the amplified fragment and, therefore, was digested into three fragments of 204, 30 and 22 bp, respectively. The fragments were visualized by ethidium bromide under UV transilluminator.
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Methods Study groups
Sequencing
This case–control study was performed during 2011 and 2014. The patients group consisted of 330 women, who had three or more consecutive pregnancy losses before 20 weeks’ of gestation, with no history of full-term pregnancies. Exclusion criteria included uterine anomalies, endocrine dysfunction, chromosomal abnormalities and autoimmune disorders. The control group consisted of 350 women, with at least one live born children and no history of pregnancy loss and uncomplicated pregnancy. These women were recruited during their attendance to the Royan Institute for the family balancing sex selection. All participants gave their informed consent before inclusion to the study.
Sequencing of PCR products of MTHFR A1298C polymorphism was carried out by Fazabiotech Company (Tehran, Iran) according to Sanger method using ABI 3730XL Capillary Sequencer. Sequencing results were compared with the sequence of normal MTHFR gene obtained from the NCBI website.
FVL, Prothrombin G20210A and MTHFR C677T analysis FVL, Prothrombin G20210A and MTHFR C677T gene polymorphisms were analyzed by FV-PTH-MTHFR Strip Assay kit (Vienna lab-REF 4-260). This technology is based on polymerase chain reaction (PCR) and reverse-hybridization. This procedure included three steps, which are explained below. DNA isolation DNA was extracted from blood using a commercial kit (PAX gene blood DNA; Qiagene). PCR amplification using biotinylated primers Taq DNA polymerase diluted to 0.2 U/ul using the supplied Dilution buffer. The total PCR reaction volume was 25 ml:15 ml Amplification Mix + 5 ml (diluted Taq Pol) + 5 ml extracted DNA (less than 40 ng/ml). Mentioned reaction mix was amplified on thermal cycler using a standard three steps, 30-cycle protocol. Amplicons checked via gel electrophoresis. Hybridization Amplification products hybridized to a test strip containing allele-specific oligonucleotide probes immobilized as an array of parallel lines. Bound biotinylated sequences were detected using streptavidin-alkaline phosphatase and color substrates. MTHFR A1298C analysis MTHFR A1298C polymorphism was experimented by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The forward and reverse primers used in the PCR amplification reactions were 50 -CTTCTACCTGAA GAGCAAGTC-30 and 50 -CATGTCCACAGCATGGAG-30 , respectively. Polymerase chain reaction conditions were 95 C for 10 min, 30 cycles of 95 C for 45 s, 30 cycles of 54 C for 45 s, 30 cycles of 72 C for 45 s followed by final extension of 72 C for 5 min. The amplified fragment of
Statistical analysis Univariate odds ratio (OR) and 95% CI were estimated separately for each polymorphism. The prevalence of each polymorphism was compared between patients and controls with the use of the 2-test by SPSS software. The level of p50.05 was calculated as significance.
Results Both cases and controls were Iranian women living in all districts of Iran. The mean age of patients (30.37 years, SD 5.05) did not statistically different from that of the controls (29.88, SD 4.09) (p ¼ 0.44). The number of miscarriages among patients was ranged from 3 to 7 with average of 3.34. 270 patients had three miscarriages and 60 had four or more miscarriages, while none of the controls had abortion. Early (before 12 weeks) and late (after 12 weeks) pregnancy losses were seen in 254 and 26 patients, respectively. In addition, 50 patients showed combined early-late losses. The results are shown in Table 1. There were no homozygous cases of FVL and Prothrombin G20210A in either group. 28 patients with RPL and 10 women of the control group (8.48 versus 2.86%, S) were heterozygous for FVL. The frequency of the 1691A allele among cases and controls was 4.24 versus 1.43% (S). The heterozygous form of Prothrombin G20210A occurred in 14 patients and in 10 controls (4.24 versus 2.86%, NS). Mutant allele (A) was seen in 2.12% of patients group and 1.43% of controls (NS). Heterozygosity for the MTHFR C677T mutation was found in 114 patients and in 85 controls (34.55 versus 24.29%, S). Homozygosity for this mutation was seen in 36 RPL cases and in 35 controls (10.9 versus 10%, NS). The frequency of the 677T allele among cases and controls was 28.18 versus 22.14% (NS), respectively. The MTHFR 1298AC genotype was diagnosed in 152 patients (46.07%) and 20 controls (5.71%) (S). The homozygote genotype 1298CC was seen in 44 patients and in 1 control (13.33 versus 0.29%, S). 1298C allele occurred with a frequency of 36.36 and 3.14% among patients and controls (S), respectively. The prevalence of combined mutations (more than one mutation) in RPL women was found to be 18.79%. This frequency among control group was 2.86% (S). Table 2 shows the frequency and type of combined mutations.
Thrombophilic genes alterations and recurrent pregnancy loss
DOI: 10.3109/14767058.2015.1044431
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Table 1. Distribution of thrombophilic polymorphisms in the case and control groups. Case (n ¼ 330) Type of abortion
Normal
Heterozygote
Homozygote
Normal
Heterozygote
Homozygote
p value
Odds ratio
95% CI
FV Leiden
Early Late Early + late Total
243 10 49 302
11 16 1 28
– – – –
340
10
–
NS S NS S
1.53 54.4 0.69 3.15
0.64–3.68 19.8–149.3 0.08–5.53 1.5–6.59
Prothrombin G20210A
Early Late Early + late Total
241 25 50 316
13 1 – 14
– – – –
340
10
–
NS NS NS NS
1.83 1.36 0.32 1.50
0.79–4.25 0.16–11.05 0.01–5.56 0.65–3.44
MTHFR C677T
Early Late Early + late Total
133 21 26 180
94 3 17 114
27 2 7 36
230
85
35
S NS NS S
1.74 0.45 1.76 1.59
1.25–2.42 0.16–1.24 0.97–3.21 1.17–2.17
MTHFR A1298C
Early Late Early + late Total
104 16 14 134
109 10 33 152
41 – 3 44
329
20
1
S S S S
22.5 9.79 40.2 22.9
13.6–37.5 3.96–24.2 18.86–86.03 13.99–37.51
Type of mutations
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Control (n ¼ 350)
S: Significant, NS: Not significant.
Table 2. Distribution of combined thrombophilic mutations among groups. Combined thrombophilic mutations MTHFR C677T/A1298C MTHFR C677T/A1298C/FV G1691A MTHFR T677T/FV G1691A MTHFR C677T/FV G1691A MTHFR A1298C/FV G1691A MTHFR C1298C/FV G1691A MTHFR T677T/Prothrombin G20210A Prothrombin G20210A/FV G1691A MTHFR C677T/C1298C Total
Patients (n ¼ 330) 31 9 7 4 3 2 2 2 2 62
(9.4%) (2.73%) (2.12%) (1.21%) (0.9%) (0.6%) (0.6%) (0.6%) (0.6%) (18.79%)
Control (n ¼ 350)
p value
Odds ratio
95% CI
3 (0.86%) – – 1 (0.28%) – 2 (0.58%) 1 (0.28%) – 3 (0.86%) 10 (2.86%)
50.0001 0.03 0.05 0.19 0.18 0.95 0.53 0.28 0.7 50.0001
11.99 20.71 16.25 4.28 7.49 1.06 2.12 5.33 0.7 7.86
3.62–39.62 1.2–357.34 0.92–285.7 0.47–38.51 0.38–145.59 0.14–7.57 0.19–23.58 0.25–111.54 0.11–4.24 3.95–15.63
p50.05 was considered to be statistically significant.
Discussion Pregnancy is a hypercoagulable state due to the multifactorial setting of underlying pathophysiologic changes, because prevention or reduction of hemorrhage in this stage is necessary [3]. In some cases, the presence of thrombophilia also aggravates this problem [10]. FVL is the most common polymorphism studied for correlation with RPL. This polymorphism is very common among white population. About 3 to 7% of individuals from Northern European are heterozygous for this mutation. Heterozygosity for this polymorphism has been demonstrated to be associated with a 3- to 7-fold increase in the risk of venous thrombosis, while homozygosity confers a 50- to 100-fold increase in risk [11]. It was shown that FVL is associated with second trimester losses, because the risk of thrombosis in placental vessels increases with advanced gestational age [4]. As it has been shown in Table 1, in the present study, increased prevalence of this mutation was seen in women with only late abortions (p50.0001), but not in woman with early (p ¼ 0.33) and combined early-late (p ¼ 0.73) abortions. In addition, there was a statistical significant correlation between this mutation and women with RPL irrespective of their gestational status (p ¼ 0.002).
It has been demonstrated that Prothrombin G20210A polymorphism has high frequency among Europeans, while the low prevalence of it has been reported in Asians and other non-European populations [4]. As it has been summarized in Table 1, the difference in the prevalence of this polymorphism was not statistically significant between early (p ¼ 0.15), late (p ¼ 0.77), early-late (p ¼ 0.43) abortions, as well as all RPL cases (p ¼ 0.33) when compared to controls. Some studies have shown an association between RPL and these two polymorphisms, while others have not. Some of these results are summarized in Table 3. As it can be seen the frequencies of homozygote genotype for these polymorphisms have been reported very low. MTHFR enzyme maintains the blood homocysteine level. Elevated homocysteine concentration affects vascular endothelium and leads to placental vasculopathy, which impairs embryo development. Besides, implantation and invasion of the embryo in first trimester of pregnancy is regulating by DNA methylation. This enzyme is known to maintain the methyl pool for control of gene expression, as well [2]. In 2000, a report brought the number of polymorphisms of MTHFR gene up to 24 [12]. The first common mutation in the coding region of MTHFR gene is alanine to valine
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J Matern Fetal Neonatal Med, Early Online: 1–5
Table 3. The frequency of thrombophilic mutations and their correlation with RPL in different studies. Frequency Mutation type
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FV Leiden
Frequency
Number of Heterozygote Homozygote Number of Heterozygote Homozygote Year Country case group (%) (%) control group (%) (%) p value References 2005 2012 2013 2012 2007 2011 2014
Tunisia Turkey India Romania Greece Palestine Iran
200 543 1000 283 99 329 330
40 109 50 21 5 72 28
(20%) (20%) (5%) (7.42%) (5%) (21.9%) (8.48%)
Prothrombin G20210A 2002 2012 2005 2007 2011 2013 2014
Lebanon Romania Tunisia Greece Palestine India Iran
110 283 200 99 329 1000 330
15 8 4 5 11
MTHFR C677T
2006 2012 2013 2007 2011 2011 2014
Tunisia India India Greece Egypt Palestine Iran
200 106 200 97 70 329 330
47 26 70 61 26 151 114
(23.5%) (24.53%) (35%) (62.9%) (37.1%) (46.8%) (34.55%)
61 5 20 12 4 33 36
MTHFR A1298C
2006 2013 2011 2012 2007 2011 2014
Tunisia India Korea Turkey Greece Egypt Iran
200 200 57 543 88 70 330
65 92 21 257 37 49 152
(32.5%) (46%) (36.84%) (47.4%) (42%) (70%) (46.07%)
27 20 2 85 7 6 44
(13.64%) (2.83%) (2%) (5%) (3.3%) – 14 (4.24%)
8 (4%) 1 (0.002%) – 2 (0.71%) – 7 (2.1%) –
200 106 500 100 102 402 350
11 2 12 5 3 69 10
(5.5%) (1.9%) (2.4%) (5%) (2.9%) (17.2%) (2.86%)
– – – – – 4 (1%) –
S S S NS NS NS S
[6] [26] [4] [27] [19] [28] This study
– 2 (0.71%) – – – – –
67 100 200 101 402 500 350
2 4 9 2 17
S NS NS NS NS NS NS
[29] [27] [6] [19] [28] [4] This study
(30%) (4.72%) (10%) (12.4%) (5.7%) (9.7%) (10.9%)
200 140 300 102 136 402 350
30 21 90 57 68 177 85
(15%) (15%) (30%) (55.9%) (50%) (44%) (24.29%)
14 1 14 13 1 43 35
(7%) (0.71%) (4.6%) (12.6%) (0.7%) (10.7%) (10%)
S S S NS NS NS S
[13] [2] [14] [19] [30] [28] This study
(13.5%) (10%) (3.5%) (15.6%) (7.9%) (8.6%) (13.33%)
200 300 155 106 90 136 350
62 127 38 35 39 97 20
(31%) (42.3%) (24.5%) (33%) (43.3%) (71.3%) (5.71%)
8 (4%) 16 (5.3%) 4 (2.6%) (2.2%) 6 (6.6%) 3 1 (0.29%)
S S S S NS NS S
[13] [14] [7] [26] [19] [30] This Study
(2.99%) (4%) (4.5%) (2%) (4.2%) – 10 (2.86%)
– – – – – – –
S: Significant, NS: Not significant.
substitution at the amino acid 222 (C677T). This polymorphism is located in the catalytic domain of enzyme [13]. Heterozygosity and homozygosity for this polymorphism reduce the enzyme activity about 35 and 70%, respectively. Near 50% of the general populations are heterozygote carriers of this mutation, and the frequency of the homozygote carriers for this polymorphism has been reported between 1 and 20%, depending on the population studied [8]. For example, the high frequency for this genotype has been demonstrated among US Hispanics, Colombians and Amerindians in Brazil, while the incidences of it among Blacks have been reported less than 2%. The frequency of heterozygote genotype for this mutation varies between 8 and 20% among White populations of Australia, North America and Europe [14]. As it is demonstrated in Table 1, the correlation between this mutation and RPL in the present study was statistically significant in women with only early losses (p ¼ 0.001), while this association was not significant in late (p ¼ 0.12) and early-late (p ¼ 0.06) abortions. Moreover, the difference between the frequency of this polymorphism among all RPL cases and controls has been statistically significant (p ¼ 0.003). The second common mutation in this gene is A to C transition at nucleotide 1298 (A1298C). This mutation results in alanine to glutamate substitution in the MTHFR protein at the amino acid 429. This polymorphism found within the enzyme regulatory domain, and like the C677T mutation, leads to decreasing in
MTHFR activity, but this reduction in case of A1298C polymorphism is less than the other one. It has been shown that almost 10% of individuals are homozygous for this mutation. These individuals show 40% reduction in enzyme activity in vitro, but they do not have higher levels of plasma homocysteine than controls. Individuals with 677CT/1298AC genotype have a 40 to 50% reduction in MTHFR activity in vitro, and increased homocysteine and decreased folate levels [8,13]. The frequency of the homozygote genotype for this polymorphism has been reported between 7 and 12% among White populations of North America and Europe. This frequency is lower among Hispanics, Chinese and Asian populations [14]. In the present study, there was a statistically significant association among this polymorphism and all RPL groups including early (p50.0001), late (p50.0001), combined early-late (p50.0001) and all RPL cases (p50.0001) (Table 1). A strong association between MTHFR polymorphisms and RPL was confirmed in some studies, while others denied this association. Table 3 shows some of these results. In the present study, there was a statistically significant correlation between combined mutations (more than one thrombophilic mutation) and RPL. The same results were obtained in some studies [3,15–18], while this correlation was ignored by some others [19,20]. The frequency of MTHFR C677T/A1298C haplotype in this study was 9.4%. This frequency was reported 15.73% in the study of Poursadegh et al. [21]. Same as the findings of Friedman et al. [22],
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DOI: 10.3109/14767058.2015.1044431
Weisberg et al. [23] and Rady et al. [24], combined homozygosity for these two mutations was not observed in this study, while MTHFR C677T/C1298 C haplotype was seen in two patients (0.6%). These types of mutations were also observed by Sotiriadis et al. [19]. In our study, seven patients (2.12%) showed MTHFR C677T homozygosity associated with FVL heterozygosity, this combination was reported 1% by Androutsopoulos et al. [25]. They have also reported a frequency of 0.5% for MTHFR C677T homozygosity associated with Prothrombin G20210A heterozygosity, which was seen in two patients (0.6%) in present study and 0.8% in the study of D’Uva et al. [18]. The frequency of FVL and Prothrombin G20210A combined heterozygosity in our study was 0.6%. This frequency in the study of D’Uva et al. [18] was 0.8%. In conclusion, there was a significant higher frequency of FVL, MTHFR C677T and MTHFR A1298C polymorphisms in patients with RPL compared with healthy controls. In addition, combination of these mutations, especially combination of FVL and MTHFR mutations, significantly increases the risk of RPL. According to our data, checking all of these polymorphisms in women with the history of RPL is strongly recommended.
Acknowledgements The authors would like to express their gratitude toward Dr Zamanian, Mrs. Jalilnejad and staffs of genetic lab of Royan Institute for their technical assistance.
Declaration of interest The authors report no declarations of interest. This research was supported by reproductive biomedicine research center, Royan Institute, Tehran (project code: 629-1-91000149).
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