Studies on the HLA-DRB1 genotypes in Japanese women with severe pre-eclampsia positive and negative for anticardiolipin antibody using a polymerase chain reaction–restriction fragment length polymorphism method

Koichi Takakuwa1,4, Keisuke Honda1, Keisuke Ishii1, Isao Hataya2, Masako Yasuda3 and Kenichi Tanaka1

1 Department of Obstetrics and Gynecology, Niigata University School of Medicine, 1-757, Asahimachi-dori, Niigata, 951-8510, 2 Department of Obstetrics and Gynecology, Nagaoka Chuo General Hospital, 2-1-5, Fukuzumi, Nagaoka City, 940-0034 and 3 Department of Obstetrics and Gynecology, Nagaoka Red Cross Hospital, 297-1, Terashima-cho, Nagaoka City, 940-2101, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The human leukocyte antigen (HLA)-DR genotype was determined in 54 Japanese women with severe pre-eclampsia in order to elucidate the relationship between HLA-DR antigen systems and pre-eclampsia. The patients were divided into two groups according to positivity for the anticardiolipin antibody (ACA), i.e. one patient group negative for ACA (n = 41) and the other patient group positive for ACA (n = 13). The frequency of each HLA-DRB1 allele in both groups was compared with that in 81 normally fertile Japanese women who had not experienced pre-eclampsia. The genotypes of HLA-DR antigens were determined using a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method. The frequency of DRB1*04 and DRB1*0403 in the patient group positive for the ACA was significantly higher compared with that in the group of normal fertile women (P< 0.05). The frequency of each HLA-DRB1 allele was not significantly different between patient group with pre-eclampsia negative for ACA and group of normal fertile women. These results suggest a difference in the immunogenetic background between the patient groups with severe pre-eclampsia positive and negative for the ACA.

Key words: anticardiolipin antibody/HLA-DR genotypes/PCR–RFLP/pre-eclampsia


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pre-eclampsia is a relatively common, yet mysterious, disease in pregnancy, of which the aetiology has not yet been fully elucidated. Considering the diversity in the immunological responses of pregnant women, aberrations of immune response have been suggested as likely causes for pathologies associated with pregnancy, such as recurrent fetal miscarriage or pre-eclampsia (Gill III, 1983Go). Genetic background has also been reported to be associated with the genesis of pre-eclampsia (Chesley and Cooper, 1986Go; Hayward et al., 1992Go). The major histocompatibility antigen complex of human beings, comprising the human leukocyte antigens (HLA), is useful in examining the immunogenetic basis of some diseases (Thomson, 1986Go, 1995Go). Thus, several studies have examined the possible association between antigens of the HLA system and the manifestation of pre-eclampsia (Redman et al., 1978Go; Simon et al., 1988Go; Kilpatrick et al., 1990Go; Omu et al., 1998Go). Although the relationship between some loci of HLA Class II antigens and pre-eclampsia has been reported (Kilpatrick et al., 1990Go; Omu et al., 1998Go), all of these reports include data using serologically defined HLA Class II antigens. Ambiguity of serologically defined HLA Class II antigens has been noted (Mytilineos et al., 1990Go; Opelz et al., 1991Go), and it is necessary to examine the genotypes of HLA Class II antigens to obtain definite conclusions. Moreover, the suggestion that autoimmune factors, such as antiphospholipid antibodies, may be involved in the genesis of pre-eclampsia has gained increasing support (Branch et al., 1985Go; El-Roeiy et al., 1991Go; Milliez et al., 1991Go; Yasuda et al., 1993Go, 1995Go). In this context, the frequency of HLA-DR alleles was evaluated by means of a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method in patients with severe pre-eclampsia, with regard to positivity for the anticardiolipin antibody (ACA), in order to elucidate the association of HLA-DR antigen systems with the development of severe pre-eclampsia.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Fifty-four patients who experienced severe pre-eclampsia were enrolled in this study for determining HLA-DRB1 genotypes. Informed consent was obtained from all patients. Pre-eclampsia was diagnosed according to the clinical criteria defined by the `International Society for the Study of Hypertension in Pregnancy' (Davey and MacGillivray, 1988Go). All of the patients had severe hypertension also diagnosed according to the same criteria. Patients who were complicated with essential hypertension or chronic renal disease were excluded from this study. As a control, 81 women who experienced at least two normal deliveries and had never experienced pre-eclampsia were examined for HLA-DR genotypes. Informed consent was obtained from all individuals. All individuals were Japanese women.

Measurement of anticardiolipin antibody
Anticardiolipin antibody was detected with an enzyme-linked immunosorbent assay (ELISA) according to a modified method (Loizou et al., 1985Go). Details of the method are described elsewhere (Yasuda et al., 1995Go). Briefly, microtitre plates were coated with cardiolipin and blocked for non-specific binding. Duplicate serum samples were added to the microtitre plates, which were then incubated for 60 min. Affinity-purified, peroxidase-conjugated goat antihuman immunoglobulin G was then added. After 60 min incubation, the plates were washed and o-phenylene diamine and hyperoxide was added as a substrate. The plates were read at 492 nm with a spectrophotometer. Control serum samples with elevated ACA (positive wells) and blank wells with ACA-negative serum (negative wells) were assayed in parallel with each plate. GPL (IgG phospholipid) units in the test serum were estimated according to the standard curve drawn from the titration of the control serum, and the threshold value was 20 GPL units of ACA.

Analyses of HLA-DRB1 Genotypes
Analysis of HLA-DRB1 genotypes was performed according to published methods (Ota et al., 1992Go). Genomic DNA was isolated by the phenol extraction of sodium dodecyl sulphate (SDS)-lysed and proteinase K-treated peripheral lymphocytes from all individuals.

Genomic DNA was amplified by polymerase chain reaction (PCR) 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, Kyoto, Japan), and 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 100 µl in a 1.5 ml Eppendorf tube, was covered with 50 µl of mineral oil to prevent evaporation and subjected to 30 cycles of 1 min for denaturing, 1 min for annealing, and 2 min for extension in an automated PCR thermal cycler (Thermal Cyclic Reactor, Toyobo Engineering Co., Tokyo, Japan). For HLA-DRB1 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 (Table IGo). The DR7, -9, -10 alleles, which have no suballeles, could be simply typed by the presence of amplified bands as DRB1*0701, DRB1*0901, DRB*1001 respectively.


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Table I. Polymerase chain reaction primers for amplification of the DRB1 genes
 
Digestion with restriction enzymes (Table IIGo)
After amplification, aliquots (6 µl) of the PCR reaction mixture were added, along with an appropriate restriction buffer and restriction enzymes, and 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, HphI and RsaI for DR3, -5, -6 and 8-DRB1.


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Table II. Restriction endonucleases for genotyping of DRB1 allele
 
Samples of the amplified DNA 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 no 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 amplified DRB1 genes previously reported (Ota et al., 1992Go).

Statistical analyses
{chi}2 analysis with Yates' correction was used to analyse any significance in the difference between the frequency of each DRB1 allele in patients with pre-eclampsia and normal fertile women. Fisher's exact probability test was used with small expected frequencies. P-values, corrected by multiplying by the number of tested alleles (Pc), were also obtained (Svejgaard et al., 1974Go). The odds ratio (OR) was calculated with a 95% confidence interval (CI).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Determination of the HLA-DRB1 alleles
The determination of the HLA-DRB1 alleles is described as an example of the PCR–RFLP method. The products of PCR amplification, using seven pairs of group-specific 5' primers and 3' primers, of a patient with pre-eclampsia are shown in Figure 1AGo. This patient was positive for HLA-DRB1*4 and DRB1*3, -5, -6, -8 alleles. The electrophoretic patterns of the polymorphic restriction fragments of DRB1*4 in this patient are shown in Figure 1BGo. One of the DRB1 alleles from this patient was determined to be DRB1*0403 by comparing the patterns of restriction fragment polymorphisms with those with the amplified DRB1*4 gene (as described by Ota et al., 1992). The electrophoretic patterns from the polymorphic restriction fragments of DRB1*3, -5, -6, -8 in this patient are shown in Figure 1CGo. The other DRB1 allele from this patient was determined to be DRB1*1302 by comparing the patterns of restriction fragment polymorphism with those of the amplified DRB1*3, -5, -6, -8 gene (as described by Ota et al., 1992).




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Figure 1. (A) The products of polymerase chain reaction amplification from one pre-eclampsia patient. Seven group-specific primers, DR2, DR4, DR9, DR1, DR7, DR10 and DRw52 associated (DR3, -5, -6 and -8) antigen-specific primers, were used to obtain only the amplified product from the DRB1 gene as shown. This patient is positive for HLA-DRB1*4 and DRB1*3, -5, -6, -8. (B) The electrophoretic patterns of the polymorphic restriction fragments of DRB1*4. One of the DRB1 alleles in this patient was determined to be DRB1*0403 by comparing the patterns of restriction fragment polymorphisms with those of the amplified DRB1*4 gene (as described by Ota et al., 1992). (C) The polymorphic restriction fragments of DRB1*3, -5, -6, -8 from the same patient. The other DRB1 allele in this patient was determined to be DRB1*1302 by comparing the patterns of restriction fragment polymorphism with those of the amplified DRB1*3, -5, -6, -8 gene (Ota et al., 1992Go).

 
Patient profiles
Out of 54 patients with severe pre-eclampsia, 41 (75.9%) were negative for ACA and 13 (24.1%) were positive for ACA. The mean age of the patient group negative for ACA was 29.1 ± 5.17 (mean ± SD) and that of the patient group positive for ACA was 31.7 ± 4.42. All of the patients had severe hypertension diagnosed according to published criteria (Davey and MacGillivray, 1988Go), and the details of the types of pre-eclampsia in the two groups are listed in Table IIIGo.


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Table III. Types of pre-eclampsia in patient groups according to positivity for anticardiolipin antibody (ACA)
 
Frequency of the HLA-DRB1 gene loci in patients with pre-eclampsia
The frequency of each HLA-DRB1 allele in patients with pre-eclampsia with negative ACA (n = 41, 82 loci), that in patients with positive ACA (n = 13, 26 loci), and that in normal fertile women (n = 81, 162 loci) are shown in Tables IV, V, VI, and VIIGoGoGoGo. No individuals in this study possessed the DRB1*03 allele (DRB1*0301 and *0302) or the the DRB1*0701 allele.


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Table IV. The frequency of the HLA-DRB1*01 and -DRB1*02 alleles among patients with pre-eclampsia, positive and negative for the anticardiolipin antibody (ACA), and the control group
 

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Table V. The frequency of the HLA-DRB1*04 allele among patients with pre-eclampsia, positive and negative for the anticardiolipin antibody (ACA), and the control group
 

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Table VI. The frequency of the HLA-DRB1*05 and -DRB1*06 alleles among patients with pre-eclampsia, positive and negative for the anticardiolipin antibody (ACA), and the control group
 

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Table VII. The frequency of the HLA-DRB1*08, -DRB1*09 and -DRB1*10 alleles among patients with pre-eclampsia, positive and negative for the anticardiolipin antibody (ACA), and normal fertile women
 
The frequency of DRB1*0403 was 15.4% (4/26 loci) in patients with positive ACA, and that in normal fertile women was 2.5 % (4/162 loci). Thus, the frequency of HLA-DRB1*0403 allele was significantly increased in patients with positive ACA compared with normal fertile women [OR 7.18, 95% CI 1.67–31.0, P < 0.05, Pc not significant (NS)]. The frequency of DRB1*04 in patients with positive ACA was 34.6% (9/26 loci), and that in normal fertile women was 15.4% (25/162 loci). Thus, the frequency of the HLA-DRB1*04 allele was significantly increased in patients with positive ACA as compared to normal fertile women (OR 2.90, 95% CI 1.16–7.23, P < 0.05, Pc NS). No significant difference was observed concerning the frequency of other DRB1 alleles between the patient group with positive ACA and normal fertile women.

There was no significant difference between the patient group with positive ACA and the patient group with negative ACA concerning the frequency of any DRB1 alleles, although that of DRB1*0403 tended to be higher in patients with positive ACA compared with that in patients with negative ACA (OR 2.80, 95% CI 0.69–11.3, P = 0.14).

No significant difference was observed between the patient group with negative ACA and normal fertile women concerning the frequency of any DRB1 alleles.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recently, it has been shown that most diseases which have an immunological or genetic background have a significant relationship with the HLA antigen system, especially HLA Class II antigens (Thomson, 1986Go, 1995Go). The reason for this is that the HLA antigen system contains a number of closely linked loci controlling a variety of immunological functions (Margulies, 1999Go).

Pre-eclampsia is a unique human disorder, although its aetiology has not yet been fully elucidated. Schuiling et al. proposed that pre-eclampsia is the consequence of an unsuccessful attack of the maternal non-specific host-defence on the implanting conceptus (Schuiling et al., 1997Go). In view of the diversity of maternal immunological response during pregnancy, aberrations in this response have been suggested as likely causes of this pathology occurring during pregnancy (Kilpatrick, 1987Go; Redman and Sargent, 1993Go; Humphrey et al., 1995Go). On the other hand, a genetic basis for pre-eclampsia has also been pointed out (Chesley and Cooper, 1986Go; Hayward et al., 1992Go).

On the basis of the above-mentioned premises, several researchers have attempted to ascertain the possible existence of an association between antigens of the HLA system and the development of pre-eclampsia using a serological method for determining HLA alleles. In an earlier study it was reported that women who were homozygous for HLA-A and -B had more severe pre-eclampsia (Redman et al., 1978Go). Simon et al. noted the association of HLA DR4 with the risk of recurrence of hypertension in pregnancy (Simon et al., 1988Go), and Kilpatrick et al. also pointed out that HLA-DR4 common to both the mother and fetus had an association with the pathogenesis of pre-eclampsia (Kilpatrick et al., 1990Go). We have also reported the possible association between HLA-DR antigens and pre-eclamptic women using a serological method (Takakuwa et al., 1996Go).

It is suggested, however, that serologically defined HLA Class II antigens may be unreliable, and that genotyping of the HLA antigen system is necessary to elucidate the relationship between the HLA Class II antigen system and various diseases (Mytilineos et al., 1990Go; Opelz et al., 1991Go). Opelz et al. reported that up to 25% of serological HLA-DR typing assignments were incorrect when compared with to a more precise DNA–RFLP method in a large, multicentre study (Opelz et al., 1991Go). They noted that the ambiguity of serological HLA-DR typing was due to the difficulty in handling B lymphocytes and the wide cross-reaction of typing reagents for HLA-DR antigens. In this context, we adopted a PCR–RFLP method, which enabled us to determine the HLA Class II genotypes very accurately, to investigate the association between pre-eclampsia and the HLA-DR antigen system.

Moreover, there is support for the suggestion that autoimmune factors such as antiphospholipid antibodies or lupus anticoagulants are generative factors in pre-eclampsia (Branch et al., 1985Go; El-Roeiy et al., 1991Go; Milliez et al., 1991Go; Yasuda et al., 1993Go, 1995Go). Gleicher and El-Roeiy have postulated a reproductive autoimmune failure syndrome in which autoimmune factors, such as lupus anticoagulants or antiphospholipid antibodies, are implicated in the genesis of unexplained recurrent fetal miscarriages as well as pre-eclampsia (Gleicher and El-Roeiy, 1988Go).

Recently, it has been noted that pre-eclampsia is associated with inherited or acquired thrombophilia (Kupferminc et al., 1999Go). Wetzka et al. pointed out that an increased expression of cyclooxygenase 1 could be involved in the pathophysiology of pre-eclamptic changes within the placental bed, which possibly caused a decreased prostacyclin:thromboxane A2 ratio followed by an increased formation of thrombi (Wetzka et al., 1997Go); antiphospholipid antibodies are considered to be one of thrombophilic factors in patients with pre-eclampsia.

Recent investigations adopting a genotyping method disclosed that a significantly high frequency of HLA Class II alleles exist in patients having recurrent fetal miscarriage positive for ACA. Christiansen et al. reported that HLA-DR3 phenotypes were significantly more frequent in ACA-positive recurrent miscarriage patients compared with healthy controls in Danish and Czech population (Christiansen et al., 1998Go). We reported that the frequency of DR*0403 was significantly higher in patients having recurrent fetal miscarriage positive for ACA, while DR*0101 was lower compared with the normal fertile women group (Hataya et al., 1998Go).

In this context, we divided the patients with severe pre-eclampsia into two groups according to positivity for ACA, and compared the frequency of each HLA-DRB1 allele in patient populations with that of normal fertile women in this study. As a result, the frequency of HLA-DRB1*0403 and -DRB1*04 was found to be significantly higher in patients positive for ACA compared with normal fertile women, although the significance was not so marked (OR 7.18, 95% CI 1.67–31.0, P < 0.05, Pc not significant: OR 2.90, 95% CI 1.16–7.23, P < 0.05, Pc not significant respectively). The patients in the current study were not included in our previous study (Hataya et al., 1998Go) and the current results suggest the same immunogenetic background between patients with recurrent fetal miscarriage positive for ACA and patients with severe pre-eclampsia positive for ACA.

On the other hand, the frequency of each HLA-DRB1 allele was not significantly different in patients with severe pre-eclampsia negative for ACA compared with normal fertile women. Wilton et al. described absence of an association between maternal HLA-DRB gene and pre-eclampsia/eclampsia (Wilton et al., 1990Go, 1991Go), and results obtained in this study are concordant with their results, as far as the patients with negative ACA are concerned.

Recently, Kilpatrick et al. reported that HLA and tumour necrosis factor genes are closely related to patient population with pre-eclampsia (Kilpatrick, 1996Go, 1999Go). Thus, further investigations of the frequency of the HLA antigen alleles in connection with immune response genes are warranted to determine new association between the antigen system and pre-eclamptic patients.


    Acknowledgments
 
This study was supported by a research grant from the Ministry of Health and Welfare of Japan, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.


    Notes
 
4 To whom correspondence should be addressed Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 3, 1999; accepted on August 26, 1999.