Department of Obstetrics & Gynecology, Niigata University School of Medicine, 1757, Asahimachi-dori, Niigata, 9518510, 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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Key words: endometriosis/HLA-DPB1/HLA-DQB1/PCRRFLP
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Introduction |
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Several disorders, including VogtKoyanagiHaradas 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 PCRrestriction 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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Analysis of HLA-DQB1 and DPB1 genotypes
Analysis of HLA-DQB1 and DPB1 genotypes was performed using PCRRFLP 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. Fishers 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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Discussion |
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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 PCRRFLP 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 PCRRFLP 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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References |
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Carrozzo, M., Fasano, M.E., Broccoletti, R., Carbone, M., Cozzani, E., Rendine, S., Roggero, S., Parodi, A. and Gandolfo, S. (2001) HLA-DQB1 alleles in Italian patients with mucous membrane pemphigoid predominantly affecting the oral cavity. Br. J. Dermatol., 145, 805808.[CrossRef][ISI][Medline]
Chiang, C.M. and Hill, J.A. (1997) Localization of T cells, Interferon-Gamma and HLA-DR in eutopic and ectopic human endometrium. Gynecol. Obstet. Invest., 43, 245250.[ISI][Medline]
Christiansen, O.B., Ulcova-Gaiiova, Z., Mohapeloa, H. and Krauz, V. (1998) Studies on associations between human leukocyte antigen (HLA) class II alleles and antiphospholipid antibodies in Danish and Czech women with recurrent miscarriages. Hum. Reprod., 13, 33263331[Abstract]
Coxhead, D. and Thomas, E.J. (1993) Familial inheritance of endometriosis in a British population: A case control study. J. Obstet. Gynecol., 13, 4244.
Dmowski, W.P., Steele, R.W. and Baker, G.F. (1981) Deficient cellular immunity in endometriosis. Am. J. Obstet. Gynecol., 141, 377383.[ISI][Medline]
Giudice, L.C., Tazuke, S.I. and Swiersz, l. (1998) Status of current research on endometriosis. J. Reprod. Med., 43, 252262.[ISI][Medline]
Hodak, E., Lapidoth, M., Kohn, K., David, D., Brautbar, B., Kfir, K., Narinski, N., Safirman, S., Maron, M. and Klein, K. (2001) Mycosis fungoides: HLA class II associations among Ashkenazi and non-Ashkenazi Jewish patients. Br. J. Dermatol., 145, 974980[CrossRef][ISI][Medline]
Ishii, K., Takakuwa, K., Mitsui, T. and Kenichi, T. (2002) Studies on the human leukocyte antigen-DR in patients with endometriosis: genotyping of HLA-DRB1 alleles. Hum. Reprod., 17, 560563.
Kennedy, S., Mardon, H. and Barlow, D. (1995) Familial endometriosis. J. Assist. Reprod. Genet., 12, 3234.[ISI][Medline]
Kennedy, S.H., Hadfield, R., Mardon, H.J. and Barlow, D. (1996) Age of onset of pain symptoms in non-twin sisters concordant for endometriosis. Hum. Reprod., 11, 101103.
Maxwell, C., Kilpatrick, D.C., Haining, R. and Smith, S.K. (1989) No HLA-DR specificity is associated with endometriosis. Tissue Antigens, 34, 145147.[ISI][Medline]
Moen, M., Bratlie, A. and Moen, T. (1984) Distribution of HLA-antigens among patients with endometriosis. Acta. Obstet. Gynecol. Scand. Suppl., 123, 2527.[Medline]
Moen, M.H. and Magnus, P. (1993) The familial risk of endometriosis. Acta. Obstet. Gynecol. Scand., 72, 560564.
Nomura, N., Ota, M., Tsuji, K. and Inoko, H. (1991) HLA-DQB1 genotyping by a modified PCR-RFLP method combined with group-specific primers. Tissue Antigens, 38, 5359.[ISI][Medline]
Olive, D.L. and Schwarts, L.B. (1993) Endometriosis. N. Engl. J. Med., 328, 17591769.
Opelz, G., Mytilineos, J., Scherer, S., Dunckley, H., Trejaut, J., Chapman, J., Middleton, D., Savage, D., Fischer, O., Bignon, J.D. et al. (1991) Survival of DNA HLA-DR typed and matched cadaver kidney transplants. Lancet, 338, 461463[CrossRef][ISI][Medline]
Ota, M., Seki, T., Nomura, N., Sugimura, K., Mizuki, N., Fukushima, H., Tsuji, K. and Inoko, H. (1991) Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping. Tissue Antigens, 38, 6071[ISI][Medline]
Ota, H. and Igarashi, S. (1993) Expression of major histocompatibility complex class II antigen in endometriotic tissue in patients with endometriosis and adenomyosis. Fertil. Steril., 60, 834838.[ISI][Medline]
Reveille, J.D., Fischbach, M., Mcnearney, T., Friedman, A.W., Aguilar, M.B., Lisse J., Fritzler, M.J., Ahn, C. and Arnett, F.C. (2001) Systemic sclerosis in 3 US ethnic groups: a comparison of clinical, sociodemographic, serologic, and immunogenetic determinants. Semin. Arthritis Rheum., 30, 332346.[CrossRef][ISI][Medline]
Sampson, J.A. (1927) Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am. J. Obstet. Gynecol., 14, 422469.
Senturk, L.M. and Arici, A.(1999) Immunology of endometriosis. J. Reprod. Immunol., 43, 6783.[CrossRef][ISI][Medline]
Setterfield, J., Theron, J., Vaughan, R.W., Welsh, K.I., Mallon, E., Wojnarowska, F., Challacombe, S.J. and Black, M.M. (2001) Mucous membrane pemphigoid: HLA-DQB1*0301 is associated with all clinical sites of involvement and may be linked to antibasement membrane IgG production. Br. J. Dermatol., 145, 406414.[CrossRef][ISI][Medline]
Shindo, Y., Ohno, S., Yamamoto, T,. Nakamura, S. and Inoko, H. (1994) Complete Association of the HLA-DRB1*04 and DQB1*04 Alleles with Vogt-Koyanagi-Haradas Disease. Hum. Immnol., 39, 169176.[CrossRef][ISI][Medline]
Simpson, J.L., Elias, S., Malinak, L.R. and Buttram, V.C. (1980) Heritable aspects of endometriosis. I. Genetic studies. Am. J. Obstet. Gynecol., 137, 327331.[ISI][Medline]
Simpson, J.L., Malinak, L.R., Elias, S., Carson, S.A. and Radvany, R.A. (1984) HLA associations in endometriosis. Am. J. Obstet. Gynecol., 148, 395397.[ISI][Medline]
Svejgaard, A., Jersild, C., Nielsen, S. and Bodmer, W.F. (1974) HLA antigens and disease: statistical and genetical consideration. Tissue Antigens, 4, 9598.[ISI][Medline]
Takakuwa, K., Hataya, I., Arakawa, M., Kikuchi, A., Higashino, M., Yasuda, M., Kurabayashi, T. and Tanaka, K. (1999a) Possible Susceptibility of the HLA-DPB1*04 Alleles to Unexplained Recurrent Abortion: Analysis by Means of Polymerase Chain ReactionRestricted Fragment Length Polymorphism Method. Am. J. Reprod. Immunol., 42, 233239.[ISI][Medline]
Takakuwa, K., Honda, K., Ishii, K., Hataya, I., Yasuda, M. and Tanaka, K. (1999b) Studies on the HLA-DRB1 genotypes in Japanese women with severe pre-eclampsia positive and negative for anticardiolipin antibody using a polymerase chain reactionrestricted fragment length polymorphism method. Hum. Reprod., 14, 29802986.
Tisch, R. and McDevitt, H. (1996) Insulin-Dependent Diabetes Mellitus. Cell, 85, 291297.[ISI][Medline]
Treloar, S.A., OConnor, D.T., OConnor, V.M. and Martin, N.G. (1999) Genetic influences on endometriosis in an Australian twin sample. Fertil. Steril., 71, 701710.[CrossRef][ISI][Medline]
Yao, Z., Kimura, A., Hartung, K., Haas, P.J., Volgger, A., Brunnler, G., Bonisch, J. and Albert, E.D. (1993) Polymorphism of the DQA1 promoter region (QAP) and DRB1, QAP, DQA1, DQB1 haplotypes in systemic lupus erythematosus. Immunogenetics, 38, 421429.[ISI][Medline]
Submitted on October 11, 2002; resubmitted on December 4, 2002; accepted on January 10, 2003.