Materials and Methods
Forty-seven women who had experienced severe preeclampsia were enrolled between July 1994 and June 1999. Informed consent was obtained from each participant. Preeclampsia was diagnosed according to clinical criteria defined by the International Society for the Study of Hypertension in Pregnancy.16 All women had severe hypertension, diagnosed according to the same criteria. Women with essential hypertension, chronic renal disease, and autoimmune diseases, such as systemic lupus erythematosus, or who were positive for antiphospholipid antibodies, were excluded.
The HLA-DQB1 and HLA-DPB1 genotypes of the women were determined by PCR-RFLP. As controls, 85 women who had at least two normal deliveries and had never had preeclampsia were examined for HLA-DQB1 and HLA-DPB1 genotypes after informed consent was obtained. The control population was selected randomly during the same period. All subjects were Japanese women. Blood samples were collected from them within 2 weeks of delivery.
Analyses of HLA-DQB1 genotypes were done according to methods described by Nomura et al,17 and analyses of HLA-DPB1 genotypes were done according to the methods described by Ota et al.18 The PCR primers used in this study are listed in Table 1.
Genomic DNA was isolated from peripheral lymphocytes. A 241-bp fragment from the second exon of the HLA-DQB1 gene was amplified using DQ1 group-specific primers, and a 237-bp fragment was amplified using DQ2, 3, and 4 group-specific primers. A 299-bp fragment at the second exon of the HLA-DPB1 gene was amplified using DPB1-specific primers (Table 1). After amplification, aliquots of the reaction mixture were digested with the restriction endonucleases listed in Table 2. HLA-DQB1 genotypes were determined by comparing the restriction fragment patterns with those of amplified DQB1 genes, as reported by Nomura et al.17 HLA-DPB1 genotypes were determined by comparing the restriction fragment patterns to those of amplified DPB1 genes, as reported by Ota et al.18
χ2 analysis with Yates correction was used to analyze significant differences between frequency of each HLA-DQB1 allele or HLA-DPB1 allele in women with preeclampsia and controls. Fisher exact probability test was used with small expected frequencies. Odds ratios (OR) and 95% confidence intervals (CI) were calculated. χ2 analysis with Yates correction was also used to analyze any significant differences in the incidence of homozygosity of the HLA-DQB1 or HLA-DPB1 alleles between patient and control groups. A nonpaired t test was used to establish whether there was significant difference in the mean age between the preeclamptic and control groups.
Demographic data of preeclamptic women and controls are shown in Table 3. The mean age of preeclamptic women was significantly lower than that of normal fertile women (28.7 ± 4.82 versus 30.8 ± 3.37, P < .005 by nonpaired t test). Of 47 preeclamptic women, 35 (74.5%) were nulliparas (the index deliveries were their first), and the remaining 12 women had one delivery each. Five of those 12 had preeclampsia and seven had normal pregnancies. Controls had two or more deliveries.
Frequencies of each HLA-DQB1 allele in women with severe preeclampsia (n = 47, 94 loci) and in normal fertile women (n = 85, 170 loci) are shown in Table 4. The frequency of the HLA-DQB1*04 allele in preeclamptic women was 21.3% (20 of 94 loci) and in normal fertile women was 11.2% (19 of 170 loci). Thus, the HLA-DQB1*04 allele frequency was statistically significantly higher in preeclamptic women compared with normal fertile women (P < .05 by χ2 test, OR 2.15, 95% CI 1.08, 4.27). The frequency of other HLA-DQB1 alleles was not statistically significantly different between groups.
The frequencies of each HLA-DPB1 allele in women with severe preeclampsia and normal fertile women are shown in Table 5. The frequency of each HLA-DPB1 genotype in the subject group was not significantly different than in controls. The incidence of individuals homozygous for the HLA-DQB1 or HLA-DPB1 alleles was not statistically significantly different between groups (Table 6).
It is clear that the HLA antigen system more than any other genetic marker is associated with disease susceptibility.5 That system contains many closely linked loci that control several immunologic functions, in which determination of cell-surface molecules, immune response differences, and possibly other functions of cell-cell regulation are included.19 The abundant polymorphism of HLA antigen systems, which is genetically inherited by children from their parents, also led us to seek association between preeclampsia and HLA antigen systems because a genetic background in preeclampsia has been reported.20,21
Earlier studies that used serologic methods found significant differences in the distribution of HLA-A, -B, -C, and -DR antigens between preeclamptic women and control subjects.6,7
A molecular biologic procedure was developed recently for determining types of HLA antigens more precisely, especially HLA class II antigens. The association between antigen systems and preeclamptic patients also was investigated. Wilton et al8,9 reported an absence of association between the maternal HLA-DRB genes and preeclampsia or eclampsia. We recently reported the association between HLA-DRB1 genotype and preeclampsia, using PCR-RFLP.10
Autoimmune factors such as antiphospholipid antibodies or lupus anticoagulants are believed to be generative factors in recurrent miscarriages22,23 and preeclampsia.11–13 Investigations using genotyping methods found that women who had recurrent miscarriages and were positive for antiphospholipid antibodies had significantly more frequent HLA class II alleles.14,15
We found that patients with severe preeclampsia, who were positive for antiphospholipid antibodies had an HLA-DRB1 genotype association, whereas patients who were negative for antiphospholipid antibodies had no association.10 Thus, it is possible that women with preeclampsia who are positive for antiphospholipid antibodies have different immunogenetic backgrounds than women who are negative for antiphospholipid antibodies. Consequently, we excluded women who were positive for antiphospholipid antibodies from the current study.
The frequency of DQB1*04 in our subjects was significantly higher compared with that of the control group, indicating that the HLA-DQB1*04 allele might be involved in preeclampsia. A recent investigation showed the critical influence of specific HLA-DQ polymorphisms in establishing the nature of bound antigens, thereby influencing the potential immune repertoire.24
Various authors have reported a possible effect of predominance of Th1 over Th2 in the T cell repertoire, in the genesis of diversity in reproductive failures.25,26 Tumor necrosis factor-α, one of the Th1-type cytokines, is now believed to influence the pathogenesis of preeclampsia.4,27 Kilpatrick3 reported that HLA and tumor necrosis factor genes are closely related in women with it.
Aberrations in immune mechanisms, such as predominance of Th1 against Th2 repertoire, followed by increased production of tumor necrosis factor α, could be important in the genesis of preeclampsia in women with HLA-DQB1*04. Examination of amino acid residues from the DQB1 alleles has shown that glutamic acid at position 70 and aspartic acid at position 71 are specific to the DQB1*04 allele,28 the frequency of which was higher in our preeclamptic subjects. Those amino acid residues are on an alpha helix sheet at the bottom of the antigen binding groove, and a specially processed epitope on a foreign or self-triggered antigen might have specific, unusual affinity for the DQB1 molecule with DQB1*04 specificities, leading to generation of aberrant immune reactions in preeclamptic women.
The number of subjects analyzed in this study was insufficient, and it is necessary to increase the sample size in further investigations. Details of the association of the HLA-DQB1*04 allele and preeclampsia remain unknown, and further investigations into frequency of HLA antigen alleles in connection with immune response genes are warranted to associate antigen systems and preeclamptic women.
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