Volume 14, Issue 5 (5-2016)                   IJRM 2016, 14(5): 329-334 | Back to browse issues page

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Arabkhazaeli N, Ghanaat K, Hashemi-Soteh M B. H1299R in coagulation Factor V and Glu429Ala in MTHFR genes in recurrent pregnancy loss in Sari, Mazandaran. IJRM. 2016; 14 (5) :329-334
URL: http://ijrm.ssu.ac.ir/article-1-748-en.html
1- Department of Genetic, Faculty of Science, Damghan Branch, Islamic Azad University, Damghan, Iran
2- Department of Clinical Biochemistry and Genetics, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
3- Department of Clinical Biochemistry and Genetics, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran , Hashemisoteh@gmail.com
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Recurrent pregnancy loss (RPL) appears a significant clinical problem affecting approximately 2% of women (1). RPL pathophysiology is poorly understood. Different factors, including genetic, immune defect, infection, anatomical as well as thrombophilia have been presumed as RPL causes (2, 3). Many recent studies have examined the mutations incidence in specific thrombophilia factor genes in women with unexplained pregnancy loss (4, 5).
Mutations in coagulation factor V gene are among the most common causes for venous thrombosis and also for pregnancy complications, such as RPLs. One of the well-known mutation in factor V gene is (A1691G; R506Q). FV is inactivated by protein C (APC), so the amount and activity of FV is regulated by APC protein (6). Factor V Leiden mutation causes APC resistance which is associated with increased risk of venous thromboembolism (VTE) and RPL (7). In 1996, another nucleotide change in exon 13 of FV gene (A4070G; His1299Arg), known as R2, has identified and linked to hereditary thrombophilia (8). Previous studies have shown that His1299Arg change will increase 2-3 folds the venous thromboembolism (VTE) risk (8-10).
The MTHFR enzyme plays important roles in folates metabolism (11). In the MTHFR enzyme, C677T (rs1801133) and A1298C (rs1801131) single-nucleotide polymorphisms are two most important polymorphisms that affect folate and total homocysteine status. The MTHFRC677T, a cytosine (C) to a thymine (T) substitution at position 677 (Ala222Val), causes impaired folate binding and reduced activity of the MTHFR enzyme (12, 13). The activity of MTHFR enzyme is reduced by 35% in people who are 677CT carriers and by 70% among 677TT carriers, while the effect of A1298C polymorphism has not been demonstrated consistently (14). Two MTHFR polymorphic variants, C677T and A1298C (Glu429A) were analyzed in association with different disorders such as cancer, vascular disease, neural tube defects, hyperhomocysteinemia, as well as RPL (3, 12, 15-18).
The prevalence of these mutations varies among different populations and ethnic groups. To date, no comprehensive study has established on the relationship between H1299R and A1298C and RPL in Iranian populations. Present study tried to evaluate the association between H1299R and A1298C polymorphisms in Mazandarani ethnic's women with RPL from northern Iran.
Materials and methods
This case-control study was performed in Sari, capital city of Mazandaran province during the January 2013 to December 2013. Prior to enrollment, all patients were given an explanation of study nature, and written informed consent was obtained from all individuals. The study was approved by the Research Ethics Committee at the Islamic Azad University, Damghan Branch.
RPL was defined as two or more spontaneous consecutive abortions at 5-20 weeks of gestation. The miscarriage history of women with RPL was examined and cases with anatomic, chromosomal, hormonal, autoimmune or infectious causes were excluded from this study. The study comprised of 100 women with RPL aged 20-45 years and 100 healthy fertile controls aged 27-44 years.
Genotyping of the Factor V A4070G variant
The primers for the PCR reaction to analyze the A4070G (H1299R) polymorphism were: forward (5-GCA GAC AGT CAT CTC TCC AGA CCT-3) and reverse (5-CTC TGG AGG AGT TGA TGT TTG TCC-3) as previously described (19). PCR conditions included one step initial denaturation (94oC for 3 min), 35 cycles (94oC for 45 sec, 63.6oC for 40 sec and 72oC for 40 sec) and a final extension at 72oC for 5 min. PCR products were then electrophoresed in a 1.5% agarose gel (Fermentas, Germany). Amplified PCR products were subjected to enzymatic digestion with RsaI restriction enzyme (Fermentas, Germany) for 16 hr at 37oC and visualized after separation by 3% agarose gel electrophoresis followed by staining with ethidium bromide (Figure 1).
Genotyping of the MTHFR A1298C variant
Venous blood collected from all participants was used to isolate genomic DNA restriction fragment length polymorphism analysis of polymerase chain reaction amplified fragments (PCR-RFLP) as previously described (20). Briefly, for the A1298C polymorphism, the primers were: forward (5-CTTCTACCTGAAGAGCAAGTC-3) and reverse (5-CATGTCCACAGCATGGAG-3), amplifying a 256 bp fragment as previously described (21). PCR conditions were included one step initial denaturation at 93oC for 3 min, followed by 35 cycles (94oC for 50 sec, 61oC for 40 sec and 72oC for 40 sec) and a final extension at 72oC for 5 min. Amplified PCR products were subjected to enzymatic digestion with Sat1 (Fnu4h1) for 16 hr at 37oC and visualized after separation by 3% agarose gel electrophoresis followed by staining with ethidium bromide (Figure 2).
Statistical analysis
Statistical analysis was performed using SPSS software version 16 and was analyzed using descriptive statistics and c2 test. Statistical significance was set at p<0.05.
Frequencies of Factor V A4070G polymorphism
The VA4070G polymorphism was determined based on product size-band. The PCR amplification of FV gene including A4070G produced a 1142 bp fragment followed by RsaI restriction digestion. Enzyme digestion creates two fragments, 1012 bp and 130 bp in normal alleles (A), and three fragments, 130bp, 436bp and 576bp in mutant alleles (G) respectively (Figure 1). The genotypic and allelic frequencies of factor V A4070G polymorphisms in women with RPL and control group are demonstrated in table I. According to table I, the genotypic frequencies of factor V A4070Gin cases were 95% for AA, 5% for AG, without any GG genotype, and 91% for AA, 5% for AG and none for GG in control group respectively. These results showed that 5 women from cases and 9 women from control group were heterozygous for A4070G of factor V. The frequencies of A allele between RPL and control groups were 97.5% (0.975) and 95.5% (0.955) respectively. Also the G allele frequencies in these groups were observed 2.5% (0.025) and 4.5% (0.045) respectively. Statistical analysis showed no significant difference between two groups (p=0.4) (Table I).

Frequencies A1298C polymorphism
The PCR product for MTHFR gene A1298C polymorphism was 256 bp. Four pieces, including 22 bp, 28 bp, 30 bp and 176 bp were created after digestion with SatI (Fnu4h1) restriction enzyme in normal alleles (A), but three pieces, including 22 bp, 30 bp and 204 bp were expected in mutant alleles (C) respectively (Figure 2). The genotypic frequency of MTHFR gene A1298C was 100% for AA and no (0%) frequency was achieved for AC or CC genotypes in cases. Also, 100% frequency was seen for AA genotype and none (0%) for AC or CC for control group respectively. The frequency of A allele in A1298C polymorphism among the women with RPL and controls was1.0 and 0.0 for C respectively (Table I).

Table I. Genotype and allele frequency of factor V A4070G and MTHFR A1298C polymorphisms in patients and controls women from Mazandaran province northern Iran (n=100 in each group).

For A4070G, the frequency of A allele between RPL and control groups were 97.5% (0.975) and 95.5% (0.955) respectively. Also the frequencies of the G allele in these two groups were observed 2.5% (0.025) and 4.5% (0.045) respectively. Statistical analysis showed no significant difference (p≤0.05) between two groups (p=0.4)

Figure 1.PCR-RFLP for factor V A4070G Polymorphism using RsaI restriction enzyme digestion. A3% agarose gel electrophoresis showed, while lane 1 represents AA genotype, lane 2 and 3 represent heterozygous (A/G) samples and lane 4 is an undigested sample with 1142 bp PCR product. Lane 5 shows a 100 bp plus DNA marker.

Figure 2. PCR-RFLP for MTHFR A1298C Polymorphism using SatI restriction enzyme digestion. Lane 1 to 5 represent AA genotypes that a 256 bp fragment digest to 176 and 30,28 and 22 bp fragments, respectively, while lane 7 shows an undigested PCR product. Lane 6 shows a 100 bp DNA marker on a 3% agarose gel electrophoresis.

RPL is a multifactorial process and thrombophilic defect (22). This study was designed to determine the association between two polymorphisms in F V and MTHFR genes and RPL for the first time in Mazandaran province, northern Iran. Several investigations have reported an association between the MTHFR A1298C and Factor V H1299R (A4070G) polymorphisms with recurrent spontaneous abortion, whereas some studies have rejected this correlation (23-26).
Factor V has three polymorphisms, G1691A, A4070G (H1299R) and A5279G. Beside G1691A, factor V Leiden, several studies have shown that A4070G polymorphism also could cause thrombophilia and play a role in coagulation factor V deficiency, but factor V Leiden (G1691A) is well known as a risk factor. Although Zammiti et al have rejected the association between Factor V H1299R (A4070G) polymorphisms with recurrent spontaneous abortion, but they verified the correlation between homozygousity of G/G in A4070G with increased risk of recurrent abortion after 8 wks of pregnancy (p>0.0002) (25, 26).
In current study, the frequency of heterozygous AG genotype for factor V A4070G was observed 5% in patients and 9% in control groups, that have not shown significantly association with recurrent abortion (p=0.267). Also low level frequency of G allele in patients (2.5%) and controls (4.5%) (Table I) showed no statistically difference between patients and controls in women from northern Iran. These results are consistent with the results achieved by Coulam et al that rejected the association between A4070G with recurrent miscarriage (27).
A1298C polymorphism in the MTHFR gene is believed that changes the MTHFR enzyme activity (28). The Effect of C677T polymorphism in MTHFR enzyme activity was already studied and reported (27, 29, 30), however, a few studies have been done on A1298C polymorphism in our population to date. It already has shown that hyperhomocysteinemia and homozygous for MTHFR gene polymorphism, are risk factors for spontaneous abortion, whereas some researchers have rejected the relation between MTHFR polymorphisms and recurrent miscarriage (31-33).
Wang et al indicated that A1298C polymorphism is not significantly different between RPL and control group, but AA genotype frequencies among women with RPL is significantly lower than control group (34). In one study, Mtiraoui et al reported that the prevalence of AAin A1298C in RPL group was significantly higher than the control, but in three separate studies, Hohlagschwandtner et al, Khaleghparast et al, and Poursadegh et al showed no relationship between C677T or A1298C polymorphisms in MTHFR with recurrent spontaneous abortion (35-38). Khaleghparast et al in a similar study in Iran reported that frequencies of AA genotype in A1298C MTHFR were 100% and the frequencies of AC or CC genotype were achieved zero (37).
Our results indicate that the MTHFR A1298C and Factor V A4070G polymorphisms are not significantly correlated with RPL in our population, women from Mazandaran province, northern Iran.
This study was supported by Mazandaran University of Medical Sciences, Iran and Islamic Azad University, Damghan Branch. We would like to thank the people from Medical Genetic laboratory in Medical faculty of Mazandaran University of Medical Sciences.
Conflict of interest
There is no conflicts of interest in this study.
Type of Study: Original Article |

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