Human Reproduction, Vol. 17, No. 3, 560-563,
March 2002
© 2002 European Society of Human Reproduction and Embryology
Studies on the human leukocyte antigen-DR in patients with endometriosis: genotyping of HLA-DRB1 alleles
Department of Obstetrics and Gynecology, Niigata University School of Medicine, 1-757, Asahimachi-dori, Niigata, 951-8510, Japan
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Abstract |
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BACKGROUND: An increasing number of reports suggest that endometriosis is associated with abnormal immune function involving changes in both cell-mediated and humoral immunity, although the aetiology of the disease remains undefined. The human leukocyte antigen system (HLA) is known to play a role in the aetiology of a number of diseases. This study examines the possible association between the HLA-DR and endometriosis. METHODS: Eighty-three patients diagnosed with endometriosis by laparoscopic examination were typed for HLA-DR antigens using a PCR–restriction fragment length polymorphism analysis. The frequency of HLA-DR genotypes in this patient population was compared with that in a population of general controls. RESULTS: The incidence of HLA-DRB1*1403 in the patient group was 6.0% (10/166 alleles) compared with 1.4% in the control group (6/444 alleles), and the incidence of the HLA-DRB1*1403 allele was significantly greater in patients with endometriosis compared with the control population [odds ratio 4.68, 95% confidence interval 1.67–13.09, P = 0.0013 (
2 analysis), (corrected P values: Pc = 0.0494)]. No significant difference was found between the two groups with regards to the frequency of the other HLA-DR alleles. CONCLUSIONS: The results from this study may be helpful to implicate a role of HLA-DR antigens in the development of endometriosis, although further investigation is required.
Key words: endometriosis/HLA-DRB1 genotypes/PCR/RFLP
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Endometriosis is a gynaecological disorder of unknown aetiology and poorly understood histogenesis. In particular, abnormalities in cell-mediated immunity and humoral immunity including autoimmunity have been reported (Giudice et al., 1998
; Senturk and Arici, 1999).
Several diseases, for example Vogt–Koyanagi–Harada's disease, insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus and pre-eclampsia, as well as recurrent miscarriages, are thought to be associated with particular human leukocyte antigens (HLA) (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. An association between endometriosis and HLA-DR has not yet 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-DR allotypes frequency. However, in this study, we applied the PCR–restriction fragment length polymorphism (PCR–RFLP) method to the genotype analysis of HLA-DRB1 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 upon laparoscopic examination were typed for the HLA-DRB1 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, 1997
). Thirty-eight patients were classified as stage IV of the disease and 45 patients were stage III. Patients with indicated autoimmune abnormalities (three patients with anti-phospholipid antibodies-positive and four with anti-nuclear antibodies-positive) were excluded from this study. Forty-four patients suffered from infertility. The remaining patients in this cohort complained of dysmenorrhoea and/or pelvic pain. A control population of 222 healthy individuals from the Niigata region of Japan, 105 males and 117 females, were studied to determine the distribution of HLA-DR antigens in the general population. All individuals in this study were Japanese and all gave their informed consent before participating in the study.
Analysis of HLA-DRB1 genotypes
Analysis of HLA-DRB1 alleles was performed using PCR–RFLP analysis (Ota et al., 1992). Genomic DNA was isolated following phenol extraction of sodium dodecyl sulphate (SDS)-lysed and proteinase K-treated peripheral lymphocytes. Genomic DNA (1 µg) was amplified in a 100 µl reaction volume, which included 2.5 units of Taq DNA polymerase (TaKaRa Taq; Takara Shuzo Co. Ltd, Kyoto, Japan), 250 nmol/l of each primer, 25 nmol/l of each deooxynucleotide triphosphate and 10 µl of PCR buffer. The cycling conditions consisted of 30 cycles at 94°C for 1 min, 62°C for 1 min, and 72°C for 2 min (Thermal Cyclic Reacter; Toyobo Engineering Co., Tokyo, Japan). Seven specific primers, DR1, DR2, DR4, DR7, DR9, DR10 and DRw52-associated (DR3, –5, –6 and –8) antigen-specific primers were used to amplify the DRB1 gene alleles (Table I). The DR7, –9 and –10 alleles, which have no suballeles, were typed by the presence of the amplified bands DRB1*0701, DRB1*0901 and DRB1*1001 respectively.
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Following 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) and visualized by staining with ethidium bromide. Ava II and Pst I were used to digest amplified DR1-DRB1; Fok I, Cfr13 I and Hph I for DR2-DRB1; Sac II, Ava II, Hinf I, Hae II, Hph I and Mnl I for DR4-DRB1; and Ava II, Fok I, Kpn I, Hae II, Cfr13 I, SfaN I, Sac II, BsaJ I, Apa I, Hph I and Rsa I for DR3, 5, 6 and 8-DRB1.
HLA-DRB1 genotypes were determined by comparing the restriction fragment patterns with those of amplified DRB1 genes, as previously described (Ota et al., 1992). The number of alleles that could be differentiated by this method was 38, and we were able to type all of the individuals who participated in this study.
Statistical analysis
HLA DRB1 allele frequencies in patients with endometriosis and in healthy controls were compared using 2 analysis with Yate's correction. Fisher's exact probability test was used for small expected frequencies of <5. Corrected P-values were obtained by multiplying by the number of alleles tested for each locus (Pc) (Svejgaard et al., 1974), because the number of alleles typed in this study was as many as 38. The odds ratio (OR) was calculated to a 95% confidence interval (CI).
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Results |
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The frequencies of each HLA-DRB1 allele in patients with endometriosis (n = 83, 166 alleles), in the general population (n = 222, 444 alleles) and the general female population (n = 117, 234 alleles) are shown in Table II
. The values predicted by assumption of Hardy–Weinberg equilibrium for all the genotypes were similar to those observed in the patients group as well as in the control group.
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No individuals in this study possessed the DRB1*03 allele (DRB1*0301 and *0302) or the DRB1*0701 allele. The incidence of HLA-DRB1*1403 in the patient group was 6.0% (10/166 alleles), compared with 1.4% in the control group (6/444 alleles) and 1.7% in the control female group (4/234 alleles). Thus, the incidence of the HLA-DRB1*1403 allele was significantly greater in patients with endometriosis compared with the general population [OR 4.68, 95% CI 1.67–13.09, P = 0.0013 (
2 analysis), Pc = 0.0494] and with the general female population [OR 3.69, 95% CI 1.14–11.96, P = 0.0207 (2 analysis), Pc not significant]. There were no other DRB1 allele frequencies in the patient group that were significantly different from those in the control group. |
Discussion |
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Endometriosis is a common disease of the reproductive system, but does not have a defined pathogenesis. The aetiology of endometriosis has recently been the subject of intense research, and several immunological factors have been identified as important to the disease process. The use of endometrial material together with components of the immune system in implantation experiments (Sampson, 1927
; Olive and Schwarts, 1993) was the basis for one of the key theories to explain the histogenesis of endometriosis. This theory proposes that an impaired immune system may adversely affect the body's ability to remove menstrual debris from the peritoneum, thereby resulting in endometriosis. In addition, cellular immunity to autologous endometrium in patients with endometriosis appears to be reduced. Decreased T cell activity was observed in rhesus monkeys with endometriosis, and infertile women with endometriosis show a reduced T cell-mediated cytotoxicity to autologous endometrial cells compared with infertile women without endometriosis (Dmowski et al., 1981). The ratio of helper T lymphocytes to suppresser T lymphocytes is increased in the peripheral blood (Badawy et al., 1987) and in the peritoneal fluid (Dmowski et al., 1994) of women with endometriosis. Conversely, some studies have reported that cellular immune activity is unaffected in the peripheral blood and peritoneal lesions of patients with endometriosis (Gleicher et al., 1984; Hill et al., 1988; Mettler et al., 1996). These abnormalities in T cell reactivities to ectopic endometrium 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 compared with those in the eutopic endometrium (Ota and Igarashi, 1993; Chiang and Hill, 1997).
There appears to be a genetic basis for the development and progression of endometriosis (Kennedy et al., 1995). In one study, the age of symptom onset was identical in non-twin sisters (Kennedy et al., 1996). Other studies have demonstrated an increased risk of endometriosis amongst first degree relatives (Simpson et al., 1980; Coxhead and Thomas, 1993; Moen and Magnus, 1993). A susceptibility gene for endometriosis which is close to HLA-DR and in linkage disequilibrium with certain HLA-DR alleles could explain the genetic basis.
The association between the HLA antigen system and endometriosis has yet to be fully elucidated. Previous studies typed HLA-DR antigens using serological methods, but none of these studies showed a statistically significant association between endometriosis and HLA-DR allotypes frequency (Moen et al. 1984; Simpson et al. 1984; Maxwell et al. 1989). This discrepancy with our results may be due to several factors. Firstly, PCR–RFLP is a more useful and accurate method for HLA typing than serological methods, particularly with respect to HLA-DR antigens. Opelz described that how up to 25% of serological HLA-DR typings may be incorrect when compared with the more accurate PCR–RFLP method (Opelz et al. 1991). The second factor accounting for the difference in results between this and prior studies, is that the frequency of HLA-DR allotypes differs in Caucasian and Japanese populations. This study used PCR–RFLP to show the association of a certain HLA-DRB1 genotype, HLA-DRB1*1403, in the endometriosis patient group and in the general population. There is no report of HLA-DRB1*1403 being associated with any immunological conditions, and the peculiarity of this allele is not clear at this stage. The higher incidence of the HLA-DRB1*1403 allele in the patient group in this study may be helpful to explain how endometriosis develops and progresses in the Japanese population. Further investigation is required to fully understand the association between HLA genes and this disease, for the incidence of DRB1*1403 accounts for only 6% of the patient population.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We are grateful to Katsunori Kashima, Hiroshi Adachi, Masahiko Higashino, Takumi Kurabayashi (Niigata University School of Medicine), Isao Hataya (Nagaoka General Hospital) and Masaki Tamura (Takeyama Hospital).
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Notes |
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1 To whom correspondence should be addressed. E-mail: keisui28{at}med.niigata-u.ac.jp
Submitted on January 29, 2001; resubmitted on July 13, 2001
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accepted on November 2, 2001.
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