The factor V Leiden mutation in Japanese couples with recurrent spontaneous abortion

Kazumasa Hashimoto1,2,5, Yoshie Shizusawa2, Koichiro Shimoya2,3, Kazutomo Ohashi2, Takashi Shimizu4, Chihiro Azuma2 and Yuji Murata2

1 Department of Obstetrics and Gynecology, Case Western Reserve University at Metro Health Medical Center, 2500 Metro Health Drive, Cleveland, OH 44109–1998, USA, 2 Department of Obstetrics and Gynecology, Faculty of Medicine, Osaka University 2–2, Yamadaoka, Suita, Osaka, 565-0871, 3 Department of Obstetrics and Gynecology, Osaka Police Hospital, 10–31 Kitayama-cho, Tennouji-ku, Osaka 543-8502 and 4 Shimizu Women's Clinic, 2–2–4 Minamiguchi, Takarazuka, Hyogo, 665-0011, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Thrombosis of placental vessels can be a major cause of recurrent spontaneous abortion (RSA). The factor V Leiden (FVL) mutation, a single point mutation in the factor V gene, is the most common genetic predisposition to thrombosis in European countries and the United States. However, even among Caucasian populations, the association between the FVL mutation and RSA is still controversial. The objectives of the present study were to investigate the prevalence of the FVL mutation in Japanese women who have experienced RSA and to clarify the contribution of the FVL mutation to recurrent miscarriages. A total of 52 Japanese women with a history of three or more consecutive idiopathic first trimester miscarriages and 41 of their male partners were studied. The control group consisted of 55 parous women without obstetric complications. Peripheral blood cell DNA was examined for the presence of the FVL alleles by polymerase chain reaction with Mnl I restriction fragment length polymorphisms. None of the 52 women with RSA and the 41 partners carried the mutation. We also found no subject carrying the FVL alleles in the control group. These results suggest that the FVL alleles are not concentrated in women with RSA at least to clinically significant levels and that there is no apparent association between the FVL mutation and RSA in our Japanese population.

Key words: factor V Leiden mutation/recurrent spontaneous abortion


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Venous thrombotic diathesis (such as antiphospholipid syndrome) is considered to be one of the major causes and/or risk factors of recurrent spontaneous abortion (RSA). Recent advances in molecular genetics have revealed that the factor V Leiden (FVL) mutation, a single point mutation in the factor V gene (G to A substitution at nucleotide 1691 or Arg506Gln), is the most common predisposition to thrombosis through activated protein C resistance in the Caucasian population (Bertina et al., 1994Go). The FVL mutation increases the risk for thrombosis by 5- to 10-fold in heterozygous and by 50- to 100-fold in homozygous patients. Based on the hypothesis that thrombosis is involved in RSA, the FVL mutation could be the causal factor of recurrent miscarriages in some individuals. To date, several investigations have been conducted to test this hypothesis, but their results have not established whether the FVL mutation is associated with RSA or not (Preston et al., 1996Go; Balasch et al., 1997Go; Brenner et al., 1997Go; Dizon-Townson et al., 1997Go; Grandone et al., 1997bGo; Pauer et al., 1998Go).

The FVL mutation is detectable in ~3–5% of the Caucasian population in Europe and the United States (Rees et al., 1995Go). In contrast, its prevalence is <1% in southeast Asia, the Middle East, and Africa (Rees et al., 1995Go). In accordance with the Asian statistics which include Indonesia, Taiwan, Mongolia, and Hong Kong Chinese (Rees et al., 1995Go), the prevalence is also extremely low in the Japanese (Takamiya et al., 1995Go; Ozawa et al., 1996Go; Odawara and Yamashita, 1997Go). However, the prevalence of the FVL mutation has not yet been examined in Japanese women with a history of RSA or other obstetric complications.

The aim of the present study is to clarify the association between the FVL mutation and recurrent pregnancy loss in the Japanese population, in part since the controversial results in the previous studies may be due to the racial differences in study populations. It is also possible that the frequency of the mutation may be concentrated in a population with potential thrombotic diseases, even though the basal prevalence in the general population is low. Another question is that if the FVL mutation is linked to fetal loss, which factor is responsible for the situation; maternal carriers, fetal carriers, or both? In fact, one study suggested that fetal carriers of the FVL mutation are at high risk during prenatal life (Dizon-Townson et al., 1997Go). In this study, we examined both parental peripheral blood samples instead of using fetal tissues.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The study group consisted of 52 Japanese women with a history of three or more consecutive spontaneous abortions of unexplained aetiology during the first trimester. A patient was excluded if she had a systemic disease (diabetes mellitus, thyroid disease, or systemic lupus erythematosus), antiphospholipid antibodies, luteal phase defects, uterine structural abnormalities, or she or her partner had an abnormal karyotype. No patient had a history of venous thrombosis. Peripheral blood samples were obtained from 41 of the male partners of the 52 subjects. The control group consisted of 55 women who had just experienced normal vaginal delivery or elective Caesarean section without obstetrical complications or a history of recurrent miscarriage and late fetal loss.

All of the materials were collected after obtaining informed consent. From each subject, ~1 ml of peripheral blood sample was obtained and stored at –80°C in a heparinized tube until use. Peripheral blood DNA was prepared from 75 µl with a rapid method described elsewhere (Gelhaus et al., 1995Go). A 267 base pair (bp) fragment in exon 10 of the factor V gene was amplified by the polymerase chain reaction (PCR) method with the forward primer 5'-TGCCCAGTGCTTAACAAGACCA-3' and the reverse primer 5'-TGTTATCACACTGGTGCTAA-3' (Bertina et al., 1994Go). Amplification was carried out in 10 µl of reaction mixture which contained 10 mM Tris–HCl (pH 8.3), 50 nM KCl, 1.5 mM MgCl2, 0.01% gelatin, 50 ng of template DNA, 200 µM of each dNTP, 200 nM of each primer, and 0.2 IU of Taq DNA polymerase (Boehringer Mannheim, Mannheim, Germany). The PCR was performed in a thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT, USA) under the following conditions: denaturation for 1 min at 94°C; then 35 cycles of 40 s at 94°C, 40 s at 55°C and 40 s at 72°C. A portion of the PCR products was digested with Mnl I and electrophoresed on 4% agarose gels (Nu Sieve GTG, FMC, Rockland, ME, USA) in Tris-acetate-EDTA buffer. The fragments were stained with ethidium bromide and visualized using ultraviolet light. Normal alleles with a G at nucleotide 1691 yielded 163, 67 and 37 bp fragments, whereas FVL alleles (G to A substitution) yielded 200 and 67 bp fragments.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We found no FVL mutation among 52 RSA subjects or the 41 partners, or in the 55 parous women without any obstetrical complications. In the general Japanese population, the prevalence of FVL mutation is reported to be extremely low; no such mutation was found in 304 individuals, including 13 patients with venous thrombosis (Ozawa et al., 1996Go), in 192 healthy individuals (Takamiya et al., 1995Go), in 314 patients with non-insulin-dependent diabetes mellitus and 139 non-diabetic subjects (Odawara and Yamashita, 1997Go). Our control group data are thus in accord with the previous reports. In addition, we found no clinically significant concentration of mutant alleles in the women with RSA or their partners, at least to clinically significant levels. Our results suggested that the FVL mutation is not a common cause of recurrent pregnancy loss in Japanese women. To the best of our knowledge, with regard to the incidence and frequency of the FVL mutation in the Japanese population, there is no literature which describes and demonstrates the existence of FVL mutant alleles. However, we cannot conclude that the Japanese population never carries the FVL mutations because only a limited number of patients and controls have been investigated. Thus, it would be safe to say that the frequency of the mutant allele is extremely low in Japanese people.

The possibility remains that our conclusion is not warranted, based on the relatively small sample size. However, our sample size and negative results are comparable to those of the previous studies; the prevalence of FVL mutation in women with RSA and in a control group were 0/40 and 0/25 (Dizon-Townson et al., 1997Go) and 10.7% (9/84) and 9.2% (8/87) (Pauer et al., 1998Go) respectively. To the best of our knowledge, our report is the first to describe the prevalence of the FVL mutation in women with RSA in an Asian population. Our data contribute to the clarification of the world wide distribution of the mutation in women with RSA. It is of interest that negative results with regard to the association between recurrent fetal loss and the FVL mutation have been reported in both high-risk (Dizon-Townson et al., 1997Go; Pauer et al., 1998Go) and low-risk populations, as in this study. This may indicate that there is little, if any, relationship between the FVL mutation and RSA. Only a large prospective study would enable a definite conclusion regarding this relation.

It is also a matter of controversy whether routine screening for the FVL mutation should be done to diagnose and manage patients with recurrent pregnancy losses (Balasch et al., 1997Go; Pauer et al., 1998Go). Considering the extremely low prevalence of FVL alleles in our present population in general and in patients with RSA, we feel that it is not necessarily advisable to screen for this mutation in Japanese patients with RSA at present. The screening policy in a given country should be based on that country's own population data.

In addition to early fetal miscarriage, the FVL mutation may be a factor of predisposition to other pregnancy complications (Dizon-Townson et al., 1996Go; Grandone et al., 1997aGo). The FVL mutation was more than twice as common in pregnant women with severe pre-eclampsia (8.9%) compared to those with normal blood pressure (4.2%) (Dizon-Townson et al., 1996Go). A case report suggested that the FVL mutation, together with another mutation in 5,10-methylene-tetrahydrofolate reductase, was associated with pregnancy complications such as recurrent fetal loss, intrauterine growth restriction, and coagulopathies during pregnancy (Grandone et al., 1997cGo). Further studies may resolve the question of whether the FVL mutation is a cause of such obstetrical complications in the Japanese population.


    Acknowledgments
 
This work was supported in part by Grants-in-Aid for Scientific Research (nos 20151061, 30203897, 50294062, 70283786, 80301266, and 90093478) from the Ministry of Education, Science and Culture of Japan, Tokyo, Japan.


    Notes
 
5 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Submitted on October 7, 1998; accepted on March 10, 1999.