Molecular genetic studies of HLA-DRB1 alleles in patients with unexplained recurrent abortion in the Japanese population

Koichi Takakuwa1,3, Hiroshi Adachi1, Isao Hataya2, Keisuke Ishii1, Masaki Tamura1 and Kenichi Tanaka1

1 Department of Obstetrics and Gynecology, Niigata University School of Medicine, Niigata and 2 Department of Obstetrics and Gynecology, Nagaoka Chuo General Hospital, Nagaoka City, Japan

3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, 1-757, Asahimachi-dori, Niigata, 951-8510, Japan. e-mail: obgy{at}med.niigata-u.ac.jp


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUD: Recently, evidence that HLA antigens are markers for recurrent spontaneous abortion has gained increased attention. Although the association between HLA class II antigens and patients with unexplained recurrent abortion was elucidated by a large population study in a Caucasian population, such analyses have been conducted in only a small Japanese population. The aim of the present study was to determine whether HLA-DR antigens are associated with patient populations with unexplained recurrent abortion in the Japanese population. METHODS: HLA-DRB1 genotypes were determined using a PCR-restriction fragment length polymorphism (PCR-RFLP) method in 93 patients with unexplained recurrent abortion (79 primary recurrent aborters and 14 secondary recurrent aborters) and in 115 normal fertile women. The rate of possession of each HLA-DRB1 genotype was compared among the three populations. RESULTS: The rate of possession of the HLA-DRB1*1502 in patients with secondary recurrent abortion was significantly higher (P < 0.01 after correction for multiple comparisons) compared with the control, fertile women. The rate of possession of HLA-DRB1*1502 was also higher in patients with primary recurrent abortions than in controls, but the difference was not statistically significant after correction. CONCLUSIONS: These findings suggest that HLA-DRB1*1502 might be a risk allele for unexplained recurrent abortion in the Japanese population.

Key words: genotype/HLA-DR/PCR-RFLP/unexplained recurrent abortion


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As antigens expressed on the surface of fetal or placental tissues possibly induce the allo-immune response of the mother, recurrent spontaneous abortion—especially that of unknown aetiology—has been assumed to be caused by an immunological defect that elicits maternal allogeneic reactions against the fetus (Gill, 1983Go).

One group (Wegmann, 1987Go; Wegmann et al., 1993Go) focused on the production of a diversity of cytokines by maternal immune-competent cells in decidual tissues, and proposed an immunotrophic theory, which was followed by the ‘Th1/Th2 paradigms’ theory (Guilbert, 1996Go; Raghupathy, 1997Go; Raghupathy et al., 1999Go; Chaouat et al., 2002Go). Such immunological mechanisms for the maintenance of successful pregnancy suggest the implication of HLA antigen systems in the genesis of human abortions.

DNA analyses showed a lack of significant compatibility between patient couples compared with normal fertile couples (Christiansen et al., 1989Go; Takakuwa et al., 1992Go; Ober et al., 1993Go; Wagenknecht et al., 1997Go), although one of these groups (Ober et al., 1993Go) pointed out the possibility of significant compatibility of HLA-DQA1 and DQB1 alleles between patients and aborted fetuses using a PCR-sequence-specific oligonucleotides (SSO) method.

Recently, the association between HLA class II antigens and patients with unexplained recurrent abortion was elucidated by a large population study in a Caucasian population (Christiansen et al., 1994Go, 1996, 1999). The analyses, however, have been conducted in only a small population in Japan (Sasaki et al., 1997Go; Takakuwa et al., 1999Go). In this context, the frequency of HLA-DRB1 alleles were examined in a significant number of patients with unexplained recurrent abortion, using a PCR-restriction fragment length polymorphism (PCR-RFLP) method, in order to elucidate the association of HLA-DR antigens with unexplained recurrent abortion patients in the Japanese population.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients and controls
The HLA-DR antigen genotypes were analysed in 93 patients with unexplained recurrent abortion. All patients underwent three or more consecutive first-trimester spontaneous abortions with the same partner. Among the 93 patients, 79 had no other pregnancy history (primary recurrent aborters), and the remaining 14 had experienced one delivery prior to consecutive abortion (secondary aborters). None of the patients indicated abnormalities in these systemic analyses, such as chromosomal abnormalities, Müllerian anomalies, hormonal deficiencies, infectious diseases, metabolic disorders or autoimmune abnormalities. A total of 115 normal fertile women who had experienced at least two full-term deliveries, and had never had abortions, were studied as a control population. All individuals in this study were Japanese, and all provided their informed consent.

Analyses of HLA-DRB1 genotypes
Analyses of HLA-DRB1 genotypes were performed using the PCR-RFLP method (Ota et al., 1992Go). The use of the method was validated by these authors.

Genomic DNAs, extracted by phenol extraction of sodium dodecyl sulphate (SDS)-lysed and proteinase K-treated peripheral lymphocytes from each individual, were amplified using the PCR procedure with 2.5 units of Taq DNA polymerase (Takara Co. Ltd, Kyoto, Japan). The reaction mixture, which contained 1 µmol/l each of the PCR 3' and 5' primers, 1 µg of genomic DNA, 10 µl of dNTP mixture (Takara Co. Ltd), a PCR reaction buffer (10 mmol/l Tris–HCl, 50 mmol/l KCl, 1.5 mmol/l MgCl2), and distilled water, to make a total volume of 100 µl in a 500 µl Eppendorf tube, was covered with 50 µl of mineral oil to prevent evaporation and subjected to 30 cycles of 1 min for denaturing (94°C), 1 min for annealing (60°C), and 2 min for extension (72°C) in an automated PCR thermal cycler (Thermal Cyclic Reactor, Toyobo Engineering Co., Tokyo, Japan).

For typing, seven group-specific primers, DR1, DR2, DR4, DR7, DR9, DR10 and DRw52-associated (DR3, -5, -6 and -8) antigen-specific primers, were used to obtain only the amplified product from the DRB1 gene. The DR7, -9, -10 alleles, which have no suballeles (DRB1*0701 and 0702 have the same nucleotide sequences in their {beta}1 domain exons), were simply typed by the presence of amplified bands as DRB1*0701 or 0702, DRB1*0901 and DRB*1001 respectively.

After amplification, aliquots (6 µl) of the reaction mixture, together with an appropriate restriction buffer and restriction enzymes, were incubated for 1–3 h.

AvaII and PstI were used for digestion of the amplified DR1-DRB1, FokI, Cfr13I and HphI for DR2-DRB1, SacII, AvaII, HinfI, HaeII, HphI and MnlI for DR4-DRB1, AvaII, FokI, KpnI, HaeII, Cfr13I, SfaNI, SacII, BsaJI, ApaI and HphI for DR3, 5, 6 and 8-DRB1.

Samples of the amplified DNAs cleaved by restriction enzymes were subjected to electrophoresis using a 12% polyacrylamide horizontal gel in a minigel apparatus (AE-6450; Atto Corporation, Tokyo, Japan). Cleavage or non-cleavage of amplified fragments was detected by staining with ethidium bromide.

HLA-DRB1 genotypes were determined by comparing the restriction fragment patterns to those of the amplified DRB1 genes as reported previously (Ota et al., 1992Go).

Statistical analyses
A {chi}2 analysis with Yates’ correction was used to analyse any significance in the difference between the rates of possession of each HLA-DRB1 allele in patients with recurrent abortion and those in the control population. A two-tailed Fisher’s exact probability test was used with small expected frequencies. P-values, corrected by multiplying by the number of tested alleles (n = 27) (Pc), were obtained using a previously published method (Svejgaard et al., 1974Go). Odds ratios (OR) were calculated with a 95% confidence interval (CI), using the method of Woolf (Woolf et al., 1955Go).


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 Results
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Determination of the HLA-DRB1 alleles
The determination of the HLA-DRB1 alleles is mentioned as an example. The products of PCR amplification, using seven pairs of group-specific 5' primers and 3' primers, of a patient with recurrent abortion are shown in Figure 1. It is clear that this patient has HLA-DRB1*2 and DRB1*3,5,6,8 (left part of Figure 1). The electrophoretic patterns of the polymorphic restriction fragments of DRB1*2 in this patient are shown in the upper right part of Figure 1. One of the DRB1 alleles from this patient was determined to be DRB1*1502 by comparing the patterns of restriction fragment polymorphisms with those with the amplified DRB1*2 gene, as described previously (Ota et al., 1992Go). The electrophoretic patterns from the polymorphic restriction fragments of DRB1*3,5,6,8 in this patient are shown in the lower right part of Figure 1. The other DRB1 allele from this patient was determined to be DRB1*1403 by comparing the patterns of restriction fragment polymorphism to those of the amplified DRB1*3,5,6,8 gene (Ota et al., 1992Go).



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Figure 1. Left panel: Products of PCR amplification using seven pairs of group-specific 5' primers and 3' primers from one patient. Seven group-specific primers, DR1, DR2, DR4, DR7, DR9, DR10, and DRw52-associated (DR3, -5, -6 and -8) antigen-specific primers, were used to obtain only the amplified product from the DRB1 gene. The figure over each lane indicates each group-specific primer. It is clear that this patient has HLA-DRB1*2 and DRB1*3,5,6,8. Upper right panel: Electrophoretic patterns of the polymorphic restriction fragments of DRB1*2. Restricted endonucleases for digestion are described over each lane. One of the DRB1 alleles in this patient was determined to be DRB1*1502 by comparing the patterns of restriction fragment polymorphisms to those with the amplified DRB1*2 gene, as described previously (Ota et al., 1992). Lower right panel: Electrophoretic patterns of polymorphic restriction fragments of DRB1*3,5,6,8. Restricted endonucleases used for digestion are described over each lane. The other DRB1 allele in this patient was determined to be DRB1*1403 by comparing the patterns of restriction fragment polymorphism to those with the amplified DRB1*3,5,6,8 gene (Ota et al., 1992).

 
Rate of possession of each HLA-DRB1 allele
The rates of possession of each HLA-DRB1 allele in patients with unexplained primary recurrent abortion (n = 79), patients with unexplained secondary recurrent abortion (n = 14) and normal fertile women (n = 115) are shown in Tables I, II and III. No individuals in the present study had the DRB1*03 allele (DRB1*0301 and *0302) or the DRB1*07 allele (DRB1*0701 and *0702), which is due to the rarity of these alleles in Japanese population (Hashimoto et al., 1994Go).


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Table I. The number and rate of individuals possessing the respective HLA-DRB1 genotypes (HLA-DRB1*01 and *04) in primary recurrent aborters, secondary recurrent aborters and control individuals
 

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Table II. The number and rate of individuals possessing the respective HLA-DRB1 genotypes (HLA-DRB1*08, *09, *10, *11 and *12) in primary recurrent aborters, secondary recurrent aborters and control individuals
 

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Table III. The number and rate of individuals possessing the respective HLA-DRB1 genotypes (HLA-DRB1*13, and *14, *15 and *16) in primary recurrent aborters, secondary recurrent aborters and control individuals
 
No significant differences were observed concerning the rate of each genotype of DRB1*01, *04, *08, *09, *10, *11, *12, *13, *14 and *16 among the patients with primary recurrent abortion, those with secondary recurrent abortion, and the control population (Tables I, II and III).

The rate of possession of the HLA-DRB1*1502 allele in the patients with secondary recurrent abortion was 57.1% (8/14 patients), while that in the normal fertile women group was 15.7% (18/115 patients). Thus, the rate of possession of the HLA-DRB1*1502 allele was significantly higher in the secondary recurrent abortion group compared with that in the normal fertile women group (OR, 7.19; 95% CI, 2.23–23.2; P < 0.0001; Pc < 0.01) (Table III). The rate of possession of the HLA-DRB1*1502 allele in the patients with primary recurrent abortion was 27.8% (22/79 patients), which was higher compared with that in the normal fertile women group (OR, 2.08; 95% CI, 1.03–4.20; P < 0.05). However, after adjustment for the number of tested alleles (Pc), no significant difference was observed (Table III). In general, the rate of possession of the HLA-DRB1*1502 allele in patients with primary and secondary recurrent abortion was higher compared with that in normal fertile women (32.3 versus 15.7%, OR, 2.57; 95% CI, 1.32–4.99; P < 0.005; Pc = 0.081, not significant) (Table III).


    Discussion
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 Abstract
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 Materials and methods
 Results
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 References
 
In the present study, the PCR-RFLP method was used to examine the rate of possession of HLA-DRB1 alleles in patients with unexplained recurrent abortion. The PCR-RFLP method was adopted for this study because there are many serologically undetermined or ambiguous class II antigens, evidenced by the fact that upwards of 25% of serological HLA-DR typing assignments were incorrect when compared with a more precise DNA-RFLP method reported in a multicentre study (Opelz et al., 1991Go). The method enables the simple and accurate determination of HLA class II genotypes, as well as class II serotypes, which have crucial biological functions.

It has been recognized for many years that the HLA system plays an important role in the aetiology of a number of diseases (Thomson, 1986Go, 1995; Nepom and Erlich, 1991Go), and it is also possible that HLA systems are implicated in the genesis of human abortions. In particular, evidence that HLA antigens are markers for recurrent spontaneous abortion has gained increased attention. It has been reported (Christiansen et al., 1994Go, 1996, 1999) that maternal HLA DR1, DR3 and DR10 were genetic markers for pregnancy loss in a large population study of Danish recurrent spontaneous abortion patients. With regard to the Japanese population, some investigators (including the present authors) reported a relationship between certain HLA class II genotypes and patient populations with unexplained recurrent abortion (Sasaki et al., 1997Go; Takakuwa et al., 1999Go). However, the size of the populations in these studies was very small compared with the present series. The increase in the rate of HLA-DRB1*1502 possession in the patient population in the present study was considered to indicate the possibility that HLA-DRB1*1502 is a risk allele for recurrent spontaneous abortion.

The appropriate selection of a control population in such a case–control study is crucial. In the present study, 115 normal fertile women were used as a control population. The frequency of each HLA-DRB1 genotype in this control group (data not shown) was not significantly different compared with that in 916 Japanese unrelated individuals reported previously (Hashimoto et al., 1994Go). Thus, the frequency of the HLA-DRB1 allele in the control group did not deviate from that of the total population.

In the present series, the population of unexplained recurrent aborters consisted of primary recurrent aborters and secondary recurrent aborters, and an increase in the rate of possession of HLA-DRB1*1502 was observed in both populations, though the immunological mechanisms responsible for the recurrent abortion in both populations may differ. The reason for such findings remains unknown.

The precise mechanisms underlying the association of most diseases with particular MHC haplotypes are not yet fully understood, though several possibilities exist. One of the first such reports was of the so-called ‘immunotrophic theory’, whereby some cytokines produced by maternal cells that recognize fetal antigens promote the proliferation of trophoblastic cells and sustain pregnancy continuation (Wegmann, 1987Go; Wegmann et al., 1993Go). Moreover, some investigators demonstrated the importance of a T helper 2 (Th2) bias for normal pregnancy, indicating the crucial role of the activation of maternal humoral immunity following recognition of fetal antigens during pregnancy (Guilbert et al., 1996Go; Raghupathy, 1997Go; Raghupathy et al., 1999Go; Chaouat et al., 2002Go). Fetal tissue expresses HLA-DR antigens as early as the ninth week of gestation, and these may elicit maternal immune responses (Trebichavsky and Nyklicek, 1992Go). MHC class II molecules, especially HLA-DR molecules, bind peptides derived from the degradation of proteins ingested by MHC class II-expressing antigen-presenting cells (APC), and display them at the cell surface for recognition by CD4-positive T lymphocytes (Margulies, 1999Go). In the decidua, CD4-positive T lymphocytes expressing HLA-DR antigens are reported to be activated during early pregnancy (Chao et al., 1999Go).

Thus, it is possible that the increased rate of possession of the HLA-DRB1*1502 allele in patients with unexplained recurrent spontaneous abortion may have implications for the lack of recognition of fetal antigens by the maternal immune system.

It is also possible that some genes, which have linkage disequilibrium with certain HLA class II haplotypes, are responsible for the genesis of recurrent spontaneous abortion. Although placental cells, which are in contact with maternal blood or tissue, are devoid of HLA class II antigens, HLA class I antigens, such as HLA-C or HLA-G antigens are expressed on the cells (King et al., 1996Go; Hammer et al., 1997Go). It was reported that the expression of HLA class I antigens is controlled by the transporter associated with antigen processing (TAP) genes and proteosome genes in the human placenta, and these genes have strong linkage disequilibrium with HLA-DR regions (van Endert et al., 1992Go; Roby et al., 1994Go). Thus, HLA-DR antigens may be implicated in the genesis of recurrent spontaneous abortions. All of these hypotheses, however, require further investigation.

The association between HLA-DRB1*1502 and unexplained recurrent spontaneous abortions in the present study was not very strong, and a new independent study must be conducted in order to provide conclusive proof of any such association.


    Acknowledgements
 
These studies were supported by a research grant from the Ministry of Health, Welfare and Labor of Japan, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.


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Submitted on July 8, 2002; resubmitted on November 4, 2002; accepted on January 9, 2003.