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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Key words: endometriosis/HLA-DRB1 genotypes/PCR/RFLP
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Introduction |
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Several diseases, for example VogtKoyanagiHarada'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 PCRrestriction fragment length polymorphism (PCRRFLP) method to the genotype analysis of HLA-DRB1 alleles in patients with endometriosis.
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Materials and methods |
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Analysis of HLA-DRB1 genotypes
Analysis of HLA-DRB1 alleles was performed using PCRRFLP 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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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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Discussion |
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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, PCRRFLP 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 PCRRFLP 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 PCRRFLP 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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Notes |
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Submitted on January 29, 2001; resubmitted on July 13, 2001
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References |
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accepted on November 2, 2001.