International Scholarly Research Network
ISRN Obstetrics and Gynecology
Volume 2012, Article ID 945486, 6 pages
MethylenetetrahydrofolateReductase C677T and A1298C
Mutations inWomenwithRecurrentSpontaneous Abortionsin
Ahmad PoursadeghZonouzi,1Nader Chaparzadeh,1Mehrdad Asghari Estiar,2
Mahzad Mehrzad Sadaghiani,3LayaFarzadi,3AliehGhasemzadeh,3
1Department of Cellular and Molecular Biology, Faculty of Sciences, Azarbaijan University of Tarbiat Moallem, Tabriz, Iran
2Students’ Scientific Research Center & Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences,
3Department of Obstetrics and Gynecology, Women’s Reproductive Health Research Center, Tabriz University of Medical Sciences,
4School of Medicine, University of Liverpool, Merseyside, Liverpool L69 3GE, UK
5Tuberculosis and Lung Disease Research Center and Department of Medical Genetics, Faculty of Medicine,
Tabriz University of Medical Sciences, Tabriz 51656 38464, Iran
6Tabriz Genetic Analysis Center (TGAC), Tabriz University of Medical Sciences, Tabriz 51656 38464, Iran
Correspondence should be addressed to Ebrahim Sakhinia, email@example.com
Received 26 August 2012; Accepted 8 October 2012
Academic Editors: C. Escudero and M. T. Sanseverino
Copyright © 2012 Ahmad Poursadegh Zonouzi et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
Introduction. Recurrent spontaneous abortion (RSA) is a significant obstetrical complication that may occur during pregnancy.
Various studies in recent years have indicated that two common mutations (C677T and A1298C) of the methylenetetrahydrofolate
reductase (MTHFR) gene are risk factor for RSA. This study was carried out to determine the influence of (C677T and A1298C)
of the methylenetetrahydrofolate reductase (MTHFR) gene mutations with RSA. Materials and Methods. A total of 139 women
were included in this study: 89 women with two or more consecutive miscarriages and 50 healthy controls. Total genomic
DNA was isolated from blood leukocytes. To determine the frequency of the two common C677T and A1298C MTHFR gene
mutations in the patients and controls, we used two methods, amplification refractory mutation system-PCR and PCR-restriction
fragment length polymorphism. Results. There is no significant difference in the prevalence of 677T/T genotype among women
with RSA and healthy controls (P = 0.285). Also no statistically significant difference in the frequency of A1298C MTHFR gene
mutation was detected between the two groups (P = 0.175 ). Conclusion. In conclusion, the results indicate that the Amplification
Refractory Mutation System-PCR method was in complete concordance with the results obtained by standard PCR-restriction
fragment length polymorphism method. The results also show no significant difference in MTHFR C677T/A1298C genotype
distribution among the two groups; therefore, further studies on larger population and other genetic variants to better understand
the pathobiology of RSA are needed.
Recurrent spontaneous abortion (RSA) represents a signif-
icant clinical problem, which an estimated 1% to 5% of
all women of reproductive age experience [1, 2]. However,
the pathogenesis of RSA is complicated, and the cause in
40%–50% of the cases is not well understood . Genetic,
anatomic, endocrine, immunologic, infectious, and environ-
mental factors have been proposed as causes of RSA [3, 4].
Various reports have postulated thrombophilia as a risk for
way resulting in an elevation of homocyteine level in plasma
2 ISRN Obstetrics and Gynecology
(hyperhomocysteinemia) has been regarded as a cause
of Thrombophilia . Methylenetetrahydrofolate reductase
(MTHFR) is one of the main regulatory enzymes in the
metabolism of homocysteine that catalyses the reduction of
5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate
. Mutations in MTHFR gene lead to decreased activity of
enzyme and hyperhomocysteinemia, which induces platelet
aggregation through promotion of endothelial oxidative
damage . Although several mutations within the MTHFR
gene were described, C677T and A1298C mutations are
the two most common mutations . C677T transition
is a missense mutation in the exon 4 of this gene, which
converts an alanine to a valine codon (at codon 222) in
the N-terminal catalytic domain of the protein leading to
a thermolabile protein, with decreased enzymatic activity
. The second mutation is MTHFR A1298C, that is,
associated with decreased activity of enzyme, but not with
thermolability. A1298C transversion is a point mutation in
exon 7, characterized by a glutamate to alanine substitution
(at codon 429) within the C-terminal regulatory domain
of the protein . Numerous investigations have been
performed on the incidence of MTHFR C677T and A1298C
mutations in women with RSA [9, 12–17]. Some of these
studies have demonstrated a relationship between these
mutations and RSA [9, 10, 12, 13, 18], whereas others have
been unable to confirm these results [14, 19]. Thus, the role
of MTHFR C677T and A1298C mutations in RSA is still
Here, we evaluated the prevalence of MTHFR C677T
and A1298C mutations in RSA patients in the northwest of
Iran and compared it with healthy controls using two meth-
ods, PCR-restriction fragment length polymorphism (PCR-
RFLP) and Amplification Refractory Mutation System-PCR
2.1. Patients. We enrolled 89 patients with a history of first
trimester RSA from many different regions of Northwest
of Iran that were all referred by obstetricians. The patients
with chromosomal abnormality, uterine anomalies, genital
infections, and endocrinological disorder were excluded
from the study. Fifty healthy women with at least two live
births from the same region were selected as a control group.
Informed consent was obtained from all participants before
30.17 and 31.54 years, respectively. The average abortion in
the cases was 2.94 (ranged from 2 to 7), and the average
successful pregnancy in the controls was 2.2 (ranged from
2 to 4).
2.2. Extraction of Genomic DNA. Peripheral blood samples
(5µL) were obtained from all cases and controls in tubes
containing EDTA. Total genomic DNA was extracted from
leukocytes by a standard salting-out protocol as previously
by spectrophotometer and adjusted to 100ng/µL by adding
double distillation H2O and then stored in −20◦C.
2.3. Genotype Screening. We applied two methods, PCR-
RFLP and ARMS-PCR, for identification of MTHFR C677T
and A1298C gene mutations.
2.3.1. PCR-RFLP-Based Screening. To perform PCR-RFLP,
each DNA sample was amplified by polymerase chain
reaction using primers that are shown in Table 1 . PCR
was carried out on 25µL volume, in an eppendorf thermal
cycler (eppendorf, Germany). Initial denaturation at 94◦C
for 5min was followed by 35 cycles of denaturation at
95◦C for 40s; annealing at 58◦C (MTHFR C677T) and
62◦C (MTHFR A1298C) and extension at 72◦C for 40s
and final extension at 72◦C for 7min were performed. PCR
amplifications were then confirmed by electrophoresis on
2% agarose gel, and after staining with ethidium bromide,
viewed with ultraviolet light. Finally, the amplified products
were digested by restriction enzymes that are shown in
Table 1  and the digested fragments were separated by
electrophoresis on 8% polyacrylamide gels that stained with
2.3.2. ARMS-PCR-Based Screening. ARMS-PCR was per-
formed using three primers for each mutation, one forward
primer and two reverse primers specific for the wild type
and mutant alleles that are shown in Table 2 [12, 22]. PCR
was carried out on 20µL volume, in an eppendorf thermal
cycler (eppendorf, Germany). First denaturation step (96◦C,
2min) was followed by 10 cycles of denaturation (96◦C,
15s) and annealing/extension (65◦C, 60s), and a final 20
cycles of denaturation (96◦C, 10s), annealing (61◦C, 50s),
and extension (72◦C, 30s) . The PCR products were
2.4. Statistical Analysis. Statistical analysis was performed
using SPSS version 16. The genotype distributions of each
mutation, the frequency of heterozygous and homozygous
were compared between patients and controls with Pearson’s
Chi-square test. A P value of <0.05 was regarded as
significant. The homozygote and heterozygote genotypes of
each group were unified as a new group and then odds ratios
and 95% confidence intervals were calculated.
3.1. Comparison of PCR-RFLP and ARMS-PCR Methods.
Analysing MTHFR gene mutations among the RSA and con-
trol samples by using PCR-RFLP and ARMS-PCR methods
clarified that there was no difference between these two
3.2. Genotype Distribution of C677T and A1298C MTHFR
Gene Mutations in Case and Control Groups. The genotype
distribution of each MTHFR mutations in patients and
controls are shown in (Figure 1).
The frequency of 677C/T genotype MTHFR gene was
33.70% in patients and 44% in controls while the frequency
ISRN Obstetrics and Gynecology3
Mutation Sequence of primers
Wild type HeterozygoteMutant
198 Hinf1198 198/175/23175/23
163 MboII 56/31/30/28/18 84/56/31/30/28/1884/31/30/18
Table 2: Primer pairs used for screening of MTHFR mutations by ARMS-PCR.
MutationForward primer Reverse primer
5-TGC TGT TGG AAG GTG CAA GAT-3
RW 5-GCG TGA TGA TGA AAT CGG-3
RM 5-GCG TGA TGA TGA AAT CGA-3
Table 3: MTHFR C677T/A1298C genotype combinations.
Cases (n = 89) Controls (n = 50)
(Figure 2). The frequency of 677T allele was 23.59% in
patients and 24% in controls (Figure 3).
There is no significant difference in the prevalence of
677T/T genotype among women with RSA and healthy
controls (P = 0.285). The frequency of 1298A/C genotype
MTHFR gene was 51.68% in patients and 68% in controls.
The frequency of homozygote genotype was 8.98% in
patients and 6% in controls. In contrast with the heterozy-
gote genotype, the frequency of homozygote genotype was
higher in RSA patients compared with the control group.
The frequencies of 1298C allele were 34.83% in patients
and 40% in controls. No statistically significant difference
in the frequency of A1298C MTHFR gene mutation was
detectedbetweenthetwogroups(P = 0.17).Thefrequencies
of MTHFR 677T and MTHFR 1298C alleles were (23.4%,
34.8%) in patients and (24%, 40%) in controls, respectively
(Figure 4). The total mutant allele frequencies were 29.21%
in women experiencing RSA and 32% in fertile controls.
were represented in both groups (Table 3).
The frequencies of 677CT/1298AC combined heterozy-
gosity in patients were 15.73% and 32% in the control
group. Our findings indicated that combined MTHFR
C677T/A1298C genotype distribution has no statistically
significant differences. The odds ratios (ORs) of the MTHFR
677C/T (OR = 0.69; 95% confidence interval (CI) = 0.33–
1.42) and the MTHFR 1298A/C (OR = 0.50; 95% CI = 0.23–
Although most researchers generally use the PCR-RFLP
method for detection of gene mutations, here we have
and ARMS-PCR methods. We applied the two methods of
PCR-RFLP and ARMS-PCR for detection of genotypes. The
results from PCR-RFLP method were the same as ARMS-
PCR method. Therefore, due to the fact that it is relatively
easy to perform low costs, and the possibility of detecting
a larger number of samples, we proposed ARMS-PCR as
a useful method that could be used as an alternative for
identification of these mutations.
Many pieces of literature have discussed the matter
that MTHFR gene mutations might be a risk factor for
recurrent spontaneous abortions [9, 10, 12, 13, 18]; hence,
we investigated the prevalence of C677T and A1298C, two
common MTHFR gene mutations in Northwestern Iran, to
determine whether these mutations related with RSA. The
compared in the two studied groups. It is clear from Table 3
and Figure 2 that the heterozygosity in nucleotide 677th of
the MTHFR gene has no statistically significant difference
among the two groups. However, homozygosity for 677T
allele of the MTHFR gene in women with RSA was higher
than healthy controls, which were concordant with previous
reports [9, 10, 18]. The total frequency of 677T alleles for
MTHFR gene 677T was also compared between women
experiencing RSA with fertile control women (23.59%,
24%), respectively. On the whole, our data has indicated no
statistically significant difference in the prevalence of C677T
4 ISRN Obstetrics and Gynecology
Figure 1: Distribution of the MTHFR gene mutations genotypes
(a) MTHFR C677T and (b) MTHFR A1298C. In each graph,
columns show wild type, heterozygote, homozygote, and total
mutant allele frequencies, respectively.
mutation between the two groups. These observations are
in contrast with a previous report on the literature [14,
19]. This difference may be explained by differences in
the populations or by using low numbers of samples. On
the other hand, our findings suggest that C677T mutation
probably has no significant role in the etiology of first-
trimester RSA in patients in the Northwest of Iran or that
other hyper coagulant gene mutations may have a role in
RSA. Furthermore, the frequency of A1298C MTHFR gene
mutation was also compared in patients and healthy women.
As shown in Table 3 and Figure 2, the frequency of 1298A/C
genotype MTHFR gene in patients was less than the control
group. The frequency of 1298C/C genotype was higher in
patients with RSA compared with the control group, in
contrast with the heterozygote genotype. The total frequency
Figure 2: Frequency of heterozygous genotypes for MTHFR C677T
and MTHFR A1298C among women experiencing RSA compared
with fertile control women.
TT genotype CC genotype
Figure 3: Frequency of homozygous genotypes for MTHFR C677T
and MTHFR A1298C among women experiencing RSA compared
with fertile control women.
of MTHFR 677T
of mutant alleles
Figure 4: Frequency of mutant alleles for MTHFR, (677T and
1298C) alleles, and total mutant allele among women experiencing
RSA compared with fertile control women.
ISRN Obstetrics and Gynecology5
of 1298T alleles for MTHFR gene 677T were also compared
between women experiencing RSA compared with the fertile
women in the control group (34.83%, 40%), respectively
(Figure 2). This was in accordance with earlier investigations
[13, 15], which reported no association between MTHFR
1298A/C and RSA. Findings of previous studies have shown
that the presence of combined C677T/A1298C genotypes
highly increased the risk of RSA . Therefore, prevalence
comparison of 677CT/1298AC compound heterozygosity
in patients and healthy controls showed no significant
In conclusion, our results showed no significant variations in
MTHFR C677T and A1298C genotype distribution among
patients who suffered from RSA and controls. Further
studies on larger population may be needed. To better
understand the pathobiology of RSA disease, we need to
identify novel genetic variants and the interactive effects of
these variants with each other and the environment. Due
to the fact that, it is possibile to detect a large number
of samples and low costs, we proposed ARMS-PCR as a
useful method that could be used for identification of these
to Dr. Siavash Dastmalchi, Dr. Morteza Ghojazadeh, and
Meysam Dolati for their great helps in this paper.
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