Human Reproduction, Vol. 18, No. 5, 985-989,
May 2003
© 2003 European Society of Human Reproduction and Embryology
Associations between patients with endometriosis and HLA class II; the analysis of HLA-DQB1 and HLA-DPB1 genotypes
Department of Obstetrics & Gynecology, Niigata University School of Medicine, 1–757, Asahimachi-dori, Niigata, 951–8510, Japan
1 To whom correspondence should be addressed. e-mail: Keisui28{at}med.niigata-u.ac.jp
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Abstract |
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BACKGROUND: Although the aetiology of endometriosis remains unclear, many immunological abnormalities involving changes in cell-mediated and humoral immunity may be associated with endometriosis. Several disorders are thought to be associated with particular HLA antigen types. This study examines the possible association between HLA-DQ and DP. METHODS: A total of 83 patients diagnosed with endometriosis following laparoscopic examination were typed for the HLA-DQB1 and DPB1 alleles using PCR–restriction fragment length polymorphism (RFLP). The HLA DQB1 and DPB1 allele frequencies in these patients and in 222 controls were compared. RESULTS: The prevalence of HLA-DQB1*0301 in the patient group was 16.3% (27/166 alleles), compared with 8.3% in the overall control group (37/444 alleles) and 7.7% in the females of the control group (18/234 alleles). Thus, the prevalence of the HLA-DQB1*0301 allele was significantly greater in patients with endometriosis, compared with the general controls [OR 2.13, 95% CI 1.25–3.64, P = 0.004 (
2 analysis), Corrected P-values; Pc = 0.049] and with the general female controls [OR 2.33, 95% CI 1.24–4.39, P = 0.008 (2 analysis), Pc; NS]. There was no significant association in the frequencies of DPB1 alleles between the patients and controls. CONCLUSIONS: The HLA systems may be involved in the aetiology of endometriosis, although further study is needed.
Key words: endometriosis/HLA-DPB1/HLA-DQB1/PCR–RFLP
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Introduction |
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Endometriosis is a gynaecological disorder of unknown aetiology and poorly understood histogenesis. Many immunological abnormalities have been associated with endometriosis; abnormalities in cell-mediated immunity, in particular, have been reported (Giudice et al., 1998
; Senturk and Arici, 1999). In rhesus monkeys with endometriosis, decreased T-cell activity was observed, and in infertile women with endometriosis, T-cell-mediated cytotoxity to autologous endometrial cells is reduced in comparison with infertile women without endometriosis (Dmowski et al., 1981). Supposing that the suppression of cellular immunity in the peritoneal space causes endometriosis, it is possible that particular types of HLA may signal these cells to decrease their cytotoxic property.
Several disorders, including Vogt–Koyanagi–Harada’s disease, insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), and pre-eclampsia, as well as recurrent miscarriages, are thought to be associated with particular HLA types (Yao et al., 1993; Shindo et al., 1994; Tisch and McDevitt, 1996; Christiansen et al., 1998; Takakuwa et al., 1999a,b), particularly HLA-DR, which is thought to be an immune response-related gene. The association between endometriosis and HLA antigens has not been proven (Moen et al., 1984; Simpson et al., 1984; Maxwell et al., 1989), and none of the previous studies using serological analysis (microcytotoxicity tests) have found a statistically significant association between endometriosis and HLA allotype frequency. In our previous study, however, we noted that the prevalence of the HLA-DRB1*1403 allele was significantly greater in patients with endometriosis than in the general controls (Ishii et al., 2002). In the present study, we applied the PCR–restriction fragment length polymorphism (RFLP) method to the genotype analysis of HLA-DQB1 alleles and DPB1 alleles in patients with endometriosis.
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Materials and methods |
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Patients and controls
A total of 83 patients diagnosed with endometriosis following laparoscopic examination were typed for the HLA-DQB1 and DPB1 antigens. All of the patients had advanced stage disease, as classified by the Revised American Society for Reproductive Medicine Classification (American Society for Reproductive Medicine, 1996
). A total of 38 patients were classified at stage IV of the disease, and 45 patients were at stage III. The patients’ mean age (±SD) was 34.4 (±3.7) years old. Patients with indicated autoimmune abnormalities (three patients positive for anti-phospholipids antibodies and four positive for anti-nuclear antibodies) were excluded from this study. A total of 44 patients suffered from infertility. The remaining patients in this cohort complained of dysmenorrhoea and/or pelvic pain. A control population of 222 healthy individuals—105 males and 117 females—from the Niigata prefecture of Japan were studied to determine the distribution of HLA-DQ and HLA-DP antigens in the general population. They were selected as controls randomly and their mean age was 33.0 (±5.0) years. All individuals in this study were Japanese, and all gave their informed consent before participating in the study.
Analysis of HLA-DQB1 and DPB1 genotypes
Analysis of HLA-DQB1 and DPB1 genotypes was performed using PCR–RFLP analysis (Nomura et al., 1991; Ota et al., 1991).
Genomic DNA isolated from peripheral lymphocytes was amplified by the PCR procedure. A 241-bp fragment from the second exon of the HLA-DQB1 gene was amplified by using DQ1-group specific primers, and a 237-bp fragment was amplified by using DQ2, DQ3 and DQ4 group-specific primers. A 299-bp fragment from the second exon of the HLA-DPB1 gene was amplified with DPB1-specific PCR primers.
Following the amplification, the PCR products were digested with appropriate restriction endonucleases (5 units) for 3 h, electrophoresed through a 12% polyacrylamide gel (Minigel apparatus AE-6450; Atto Corporation, Tokyo, Japan) and visualized by staining with ethidium bromide.
HLA-DQB1 and HLA-DPB1 genotypes were determined by comparing the restriction fragment patterns of RFLP obtained in tested individuals with those of amplified genes. The number of DQB1 alleles that could be differentiated by this method was 12, and the number of DPB1 alleles was 19. We were able to type all of the individuals who participated in this study.
Institutional review board approval was obtained for this study.
Statistical analyses
The HLA DQB1 and DPB1 allele frequencies in patients with endometriosis and in the general controls were compared using 2 analysis with Yates’ correction. Fisher’s exact probability test was used for small expected frequencies that were <5. Corrected P-values (Pc) were obtained by multiplying by the number of alleles tested for each locus (Svejgaard et al., 1974). The odds ratio (OR) was calculated with a 95% confidence interval (CI).
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Results |
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The determination of the HLA-DQB1 alleles is mentioned as an example. The products of PCR amplification, using two pairs of group-specific 5' primers and 3' primers, are shown on the left side of Figure 1. It is obvious that this person has HLA-DQB1*1 and HLA-DQB1*2,3,4. The electrophoretic patterns from the polymorphic restriction fragments of DQB1*1 and HLA-DQB1*2,3,4 are shown on the right side of Figure 1. The DQB1 alleles from this person were determined to be DQB1*0303 and HLA-DQB1*0601 by comparing the patterns of restriction fragment polymorphisms with those of the each amplified DQB1 genes.
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The frequencies of each HLA-DQB1 allele and DPB1 allele in patients with endometriosis (n = 83, 166 alleles) and in the general controls (n = 222, 444 alleles) and the female controls (n = 117, 234 alleles) are shown in Tables I and II. The values predicted by the assumption of Hardy–Weinberg equilibrium for all of the genotypes were similar to those observed in the patients as well as in the control group.
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The prevalence of HLA-DQB1*0301 in the patient group was 16.3% (27/166 alleles), compared with 8.3% in the control group (37/444 alleles) and 7.7% in the control female group (18/234 alleles). Thus, the prevalence of the HLA-DQB1*0301 allele was significantly greater in patients with endometriosis, compared with the general controls [OR 2.13, 95% CI 1.25–3.64, P = 0.004 (
2 analysis), Pc = 0.049] and with the general female controls [OR 2.33, 95% CI 1.24–4.39, P = 0.008 (2 analysis), Pc = NS]. There were no other DQB1 allele frequencies in the patient group that were significantly different from those in the control group. Also, there was no significant association in the frequencies of DPB1 alleles between the patients and controls. |
Discussion |
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One major theory regarding the hystogenesis of endometriosis holds that the implantation of endometrial fragments in conjunction with immunological and peritoneal factors stimulates cell growth (Sampson, 1927
; Olive and Schwarts, 1993). The hypothesis that an impaired immune system may result in a defect in the ability to remove menstrual debris in the peritoneum and cause endometriosis is advocated. Decreased T-cell activity was observed in rhesus monkeys with endometriosis, and infertile women with endometriosis show a reduced T-cell-mediated cytotoxity to autologous endometrium, compared with infertile women without endometriosis (Dmowski et al., 1981). The reduction in T-cell reactivities to ectopic endometrial cells could implicate the HLA antigen system in the development of endometriosis, because HLA-DR positive glandular epithelial cells and stromal cells in the ectopic endometrium were present in abundance in comparison with the eutopic endometrium (Ota and Igarashi, 1993; Chiang and Hill, 1997).
There appears to be a genetic background for the development and progression of endometriosis (Kennedy et al., 1995). The age of symptom onset was identical in non-twin sisters (Kennedy et al., 1996), and some studies have demonstrated an increased risk of endometriosis among first-degree relatives (Simpson et al., 1980; Coxhead and Thomas, 1993; Moen and Magnus, 1993). Furthermore, the Australian twin study indicated the genetic influences on endometriosis (Treloar et al., 1999). A susceptibility gene for endometriosis that is close to the HLA genes and in linkage disequilibrium with certain HLA alleles may explain the genetic basis of endometriosis.
The association between the HLA antigen system and endometriosis has yet to be fully elucidated. Previous studies typed HLA antigens using serological methods, but none of these studies showed a statistically significant association between endometriosis and HLA allotype frequency (Moen et al., 1984; Simpson et al., 1984; Maxwell et al., 1989). However, in patients with endometriosis, our previous study showed a higher frequency of HLA-DRB1*1403 allele (Ishii et al., 2002), and in the present investigation we elucidate the significantly higher prevalence of HLA-DQB1*0301. This discrepancy within our results may be due to a variety of factors. The first is that PCR–RFLP is a more useful and accurate method for HLA typing. Opelz et al. (1991) found that up to 25% of serological HLA-DR typings may be incorrect when compared with those determined by the PCR–RFLP method. The second factor accounting for the difference in results between these prior studies and our present study is that HLA allotype frequency differs between Caucasians and Japanese. The cause of this discrepancy may also be due to chance because of the small number of these samples included in this study. The frequency of DQB1*0301 is reported to be increased in cases of mycosis fungoides, mucous membrane pemphigoid and systemic sclerosis (Carrozzo et al., 2001; Hodak et al., 2001; Reveille et al., 2001; Setterfield et al., 2001), but the mechanism by which DQB1*0301 is associated with these diseases has not been fully analysed, and the peculiarity of this allele is not clear at this stage. The higher prevalence of the HLA-DQB1*0301 allele in the patient group in this study may be useful for explaining how endometriosis develops and progresses in the Japanese population. However, after correction for multiple testing, the P-value was limited to only borderline significance (0.049), although the correction of multiple testing sometimes produces results that are too conservative (i.e. type II error). Further investigations by increasing sample size (and thereby obviating the need for multiple-testing adjustment) and by replication in both Japanese and other populations, are needed to fully understand the association between HLA genes and this disease.
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Submitted on October 11, 2002; resubmitted on December 4, 2002; accepted on January 10, 2003.
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