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Frequencies of HLA-DRB1 and HLA-DQB1 alleles in patients suffering from vitiligo
23 June 1997 - Poster presentations
P.5.04.03
promoter analysis of the matrix metalloprotelnase RASI-1, a novel autoantlgen In rheumatold arthritis
M. Mueller. S. Mauch, R. Sedlacek, U. Krawinkel. Departmentof fmmunofog~! University of Konstanz, Konstanz, Germany introduction: The matrix metalloproteinase (MMP) RASI-1 was detected as a novel autoantigen in Rheumatoid Arthritis (RA) (1). Many MMPs seem to play a critical role in the pathology of systemic autoimmune diseases such as FiA (2, 3). In order to study the transcriptional regulation of RASI-1 we have isolated the complete rasi-7 gene and analyzed its 5’8anking sequence. This study allows first insights into possible regulatory mechanisms controlling the expression of RASI-1. Materials and Methods: We have isolated the complete rasi-I gene from a human genomic DNA library. The promoter/enhancer region has been sequenced and the transcription start site was identified by primer extension analysis. Potential DNA-binding sites for transcription factors were detected by database analysis. Fteaulta: We have found many potential DNA-binding sites for transcription factors, several of which are typical for matrtx metalloproteinases. The resi-I promoter contains a TATA-box at nucleotkte -29 relative to the transcription start site. We have detected a consensus sequence for the transcription factor AP-1 at position -73 and a putative PEA-3 element at position -155. This arrangement is typical for many MMPs (4). Furthermore potential binding sites for the transctiption factors NF-kappa& AP-2, SP-1 and elements which show similarity to the TGF-beta inhibitory element (TIE) and a retinoic acid response element (RARE) have been detected. In addition we recognized some other binding sites that can not be found in other MMPs. Conclusion: Taken together these data suggest that msi-I is a tightly controlled gene under physiological conditions resembling many of the regulatory characteristics of most other MMPs. Further experiments such as DNA-footprinting and promoter/reporter-gene assays have to proof the regulatory effect of the above mentioned transcription factors. Because MMPs play a crucial role in the pathogenesis of systemic autoimmune diseases, obtaining insights into their transcriptional regulation is of particular interest and may lead to the development of new therapy concepts to treat such disorders. [l] Sedlacek, R., Mauch, S., Eibel, H., Peter, H.-H. and Krawinkel, U. (1995) Immunobiol., 194,153 121 _ _ Bid&al-Hansen, H., Moore, W., Bodden, M.. Windsor, L., Birkedal-Hansen, B., DeCado, A. and Angler, J.(1993) Crit. Rev. Oral Biol. Med., 4 (2), 197-250 [3] Murphy, G. and Hembry, R. (1992) J. Rheumatol. Suppl., 32,61-84 [4] Mattisian, L. (1994) Ann. N. Y. Acad. Sci., 732, 42-50
P.5.04.04
Frequencies of HLA-ORB1 and HLA-DQBl alleles In patients buffering from vltlllgo
M. But’, H. FazekaSovB’, E. Hegyi 2, K. Kolib&Bov62, S. FerenEik3. ’ Department of Immunology, Faculty of Medicine, Comanius Univatsi~, Bmtislava, Slovakia, ‘Depamant of Darma~ology,Hospital R&noy Bratislava, Slovakia, 31nstkuteof Immunoiog~ University Hospital, Essan, Germany Introduction: Our previous studies on the associations between vitiligo and HLA an&ens disclosed the increased frequency of HLA-Dw7 antigen (But et. al., Foliabiol. 42: 23-25, 1998). To confirmthe &lular typing results we retyped the studied group of patients for the frequencies of HIA-DRBl and HlA-DQBl alleles, resp&ti;ely. Matertal and Methods: 21 patients were investigated. 13 alleles of the HLA-DRBl locus and 17 alleles of the HLA-DQBl locus were determined by PCR-SSP method according to Qleriup et al. mssue Antigens, 39: 225-235, 1992). Results: The statistically significant deviations in frequencies of the following alleles were found: DRB1’0701 (28.5% compared to 15.3% in the healthy population, p < 0.001) and WB1’0201 (38.0% compared to 21.1% in the control group, p < 0.0001). No other statistically significant differences were obsenred. Conctuslon: The allele DRB1’0701 encodes the antigen HLA-DR7 and the allele DQBl”O201 encodes HlA-Dw7, an epttope present on B-chain of the antigen HLA-DQ2. So DNA typing results confirm the previously recorded associations of HLA-DR7 and HLA-Dw7 antigens with vitiligo and indicate that HLA antigens do play a role in the immunopathogenesis of vitiligo.
Genetics of autoimmuni~
P.5.04.05
167
Study of the HLA-BZ;Nankyloslng spondylltls association about 34 cases
H. Makni 1.4,N. Lahlani-Mahfoudh ‘, S. Baktouti 2, 2. Bahloul 3, H. Ayadi 4.
’ Laboratoire d’hishxwmpatibili~6, EfS H. Chakac S&x, Tunisia,2San&a da Rhumatolwia, EPS H. Chakar, S&x, Tunisia,3Sawim da Medacina lnfeme. EPS H. Chakac Sfax, Tunisia,4La~tatoira d’lmmunobgia at da Biokqia Mol&u/aire, Faculte da hMdecine, Sfax, Tunisia Thirty four patients from south Tunisia fulfilled the new York criteria of ankylosing spondylitis (AS) and were phenotyped for HLA-A and -B loci. A complement dependent micrccytotoxictty assay was perfom?ed using a battery of local sera screened from multiparous women: A sample of 105 healthy and unrelated individuals was used as controls. Nineteen out of 34 patients (55.85%) were HLA-B27 positive whereas the HLA-B27 frequency was of 1.9% in controls. The relative Risk value (RR) was of 85.23: The most frequent haplotype associated to AS was A2 827. As for most populations worldwide, we found a significant increased frequency of HLA-B27 in patients with AS. Nevertheless, HLA-B27 was associated to AS at a lower frequency than in Caucasians. Moreover, the RR value found was lower than those reported in Caucasians. Our results showed an equal distribution of HLA-827 in the population of south Tunisia, in Kabyles and in Saudi Arabs. In addition, 15 AS patients of 34 (44.2%) lacked the HlA-B27 antigen. Altogether, these data suggest that besides the HLA-B27 antigen, additional genes might also be involved in the susceptibility to develop AS.
P-5.04.06
Polymorphism of the human lmmunoglobulln heavy chain locus In rheumatoid arthritis
F. Fakhfakh ‘, A. Maalej I, 2. Bahloul ’ , A. Jarraya ‘, H. Ayadi ‘, M.Zouali 2. ’ LaborMoire d’lmmunokgia at da Biologic Mol&ulaira, FaculM da M6decina. St&x, Tunisia,2LMpartement d’lmmunolcgia, lnsritot Pastaq 28, rue du Dr Roux, Paris, France The genetic origin of Rheumatoid Arthritis (RA) is largely unknown. The purpose of this investigation was to assess the potential genetically determined involvement of the immunoglobulin (lg) heavy chain variable region (VH) locus in the pathogenesis of RA. We tested the hypothesis of whether there is a genetic linkage between a structural abnormality of the VH gene complex and autoantibody hyperproduction in RA. We used restfiction endonuclease generated polymorphism in human VH gene-family specific probes to examine genomic DNA from a RA family and from unrelated RA patients from both the Tunisian and the European populations. The use of DNA samples from these ethnic origins permitted a further evaluation of the polymorphism of the humanVH locus. While we found that the polymorphism of the VH locus was lower in the Tunisian population, we could not detect a restriction site polymorphism pattern restricted to RA. Our results argue against a genetic linkage between RA and polymorphism in the VH locus. However it is possible that the method of restriction fragment length polymorphism was not able to detect subtle variations in DNAs of RA patients and, hence, reveal minor abnormalities in the VH locus of the RA patients. Likewise, genomic changes or somatic variation in a B-cell subset, both undetectable by our restriction fragment analysis, would need to be further investigated.
1P.5.04.07 ] Molecular analysis of Class II sequences Involved In autolmmune hepatitis In Mexicans M.N. Vtiquez-Garcia 1.3,C. Alaez ‘, A. Olivo ‘, H. Debaz I, E. P&z-Luque ‘, G. De La Rosa ‘, S. Cano ‘, A. Burguete ‘, J. Bandera 2, D. Kershenobich 2, C. Gorodezky ’ ’ Department of Immunogenetics, INDRE, SSA, Mexico Ci& Mexico, 2Gastroentarol~y Sarvica, lnsikuto National da la Nutriciidn,SSA, Mexico Ciw Mexiw, 3Prvgrama da Biomedicina Molecular CINVESTAV-IPN, Mexico Ci& Maxkw Introduction: Autoimmune hepatitis (AH) is a progressive condition of the liver characterized by piecemeal necrosis, the presence of diierent autoantibodies and by its prevalence in females. DRBl’O301 and DRBl l0401 are associated in Caucasians and DRB1.0405 is a susceptibility gene in Japanese. This is the first study done looking for HLA class I antigens and class II DNA alleles in Mexicans with the disease. Materials and Methods: We analyzed 30 Mexican Mestizo patients with type I AH compared to 175 healthy controls. The following autoantibodies were determined: (r-DNA, smooth muscle, ANA, mitocondrtal, thyroglobulin and LKMl (microsomal). Serotogical class I typing was done using the microcytotoxicity technique with magnetic beads and double fluorescence staining. DNA typing for DRBI, DQAl, and DQBl loci was performed with the protocols, primers and probes developed for the 12th International Wokshop. PCR-SSP was done to confirm certain DRBl, DQAl or WBI alleles. Frequencies were calculated and the most probable combinations were deduced.
P.5.04.03
promoter analysis of the matrix metalloprotelnase RASI-1, a novel autoantlgen In rheumatold arthritis
M. Mueller. S. Mauch, R. Sedlacek, U. Krawinkel. Departmentof fmmunofog~! University of Konstanz, Konstanz, Germany introduction: The matrix metalloproteinase (MMP) RASI-1 was detected as a novel autoantigen in Rheumatoid Arthritis (RA) (1). Many MMPs seem to play a critical role in the pathology of systemic autoimmune diseases such as FiA (2, 3). In order to study the transcriptional regulation of RASI-1 we have isolated the complete rasi-7 gene and analyzed its 5’8anking sequence. This study allows first insights into possible regulatory mechanisms controlling the expression of RASI-1. Materials and Methods: We have isolated the complete rasi-I gene from a human genomic DNA library. The promoter/enhancer region has been sequenced and the transcription start site was identified by primer extension analysis. Potential DNA-binding sites for transcription factors were detected by database analysis. Fteaulta: We have found many potential DNA-binding sites for transcription factors, several of which are typical for matrtx metalloproteinases. The resi-I promoter contains a TATA-box at nucleotkte -29 relative to the transcription start site. We have detected a consensus sequence for the transcription factor AP-1 at position -73 and a putative PEA-3 element at position -155. This arrangement is typical for many MMPs (4). Furthermore potential binding sites for the transctiption factors NF-kappa& AP-2, SP-1 and elements which show similarity to the TGF-beta inhibitory element (TIE) and a retinoic acid response element (RARE) have been detected. In addition we recognized some other binding sites that can not be found in other MMPs. Conclusion: Taken together these data suggest that msi-I is a tightly controlled gene under physiological conditions resembling many of the regulatory characteristics of most other MMPs. Further experiments such as DNA-footprinting and promoter/reporter-gene assays have to proof the regulatory effect of the above mentioned transcription factors. Because MMPs play a crucial role in the pathogenesis of systemic autoimmune diseases, obtaining insights into their transcriptional regulation is of particular interest and may lead to the development of new therapy concepts to treat such disorders. [l] Sedlacek, R., Mauch, S., Eibel, H., Peter, H.-H. and Krawinkel, U. (1995) Immunobiol., 194,153 121 _ _ Bid&al-Hansen, H., Moore, W., Bodden, M.. Windsor, L., Birkedal-Hansen, B., DeCado, A. and Angler, J.(1993) Crit. Rev. Oral Biol. Med., 4 (2), 197-250 [3] Murphy, G. and Hembry, R. (1992) J. Rheumatol. Suppl., 32,61-84 [4] Mattisian, L. (1994) Ann. N. Y. Acad. Sci., 732, 42-50
P.5.04.04
Frequencies of HLA-ORB1 and HLA-DQBl alleles In patients buffering from vltlllgo
M. But’, H. FazekaSovB’, E. Hegyi 2, K. Kolib&Bov62, S. FerenEik3. ’ Department of Immunology, Faculty of Medicine, Comanius Univatsi~, Bmtislava, Slovakia, ‘Depamant of Darma~ology,Hospital R&noy Bratislava, Slovakia, 31nstkuteof Immunoiog~ University Hospital, Essan, Germany Introduction: Our previous studies on the associations between vitiligo and HLA an&ens disclosed the increased frequency of HLA-Dw7 antigen (But et. al., Foliabiol. 42: 23-25, 1998). To confirmthe &lular typing results we retyped the studied group of patients for the frequencies of HIA-DRBl and HlA-DQBl alleles, resp&ti;ely. Matertal and Methods: 21 patients were investigated. 13 alleles of the HLA-DRBl locus and 17 alleles of the HLA-DQBl locus were determined by PCR-SSP method according to Qleriup et al. mssue Antigens, 39: 225-235, 1992). Results: The statistically significant deviations in frequencies of the following alleles were found: DRB1’0701 (28.5% compared to 15.3% in the healthy population, p < 0.001) and WB1’0201 (38.0% compared to 21.1% in the control group, p < 0.0001). No other statistically significant differences were obsenred. Conctuslon: The allele DRB1’0701 encodes the antigen HLA-DR7 and the allele DQBl”O201 encodes HlA-Dw7, an epttope present on B-chain of the antigen HLA-DQ2. So DNA typing results confirm the previously recorded associations of HLA-DR7 and HLA-Dw7 antigens with vitiligo and indicate that HLA antigens do play a role in the immunopathogenesis of vitiligo.
Genetics of autoimmuni~
P.5.04.05
167
Study of the HLA-BZ;Nankyloslng spondylltls association about 34 cases
H. Makni 1.4,N. Lahlani-Mahfoudh ‘, S. Baktouti 2, 2. Bahloul 3, H. Ayadi 4.
’ Laboratoire d’hishxwmpatibili~6, EfS H. Chakac S&x, Tunisia,2San&a da Rhumatolwia, EPS H. Chakar, S&x, Tunisia,3Sawim da Medacina lnfeme. EPS H. Chakac Sfax, Tunisia,4La~tatoira d’lmmunobgia at da Biokqia Mol&u/aire, Faculte da hMdecine, Sfax, Tunisia Thirty four patients from south Tunisia fulfilled the new York criteria of ankylosing spondylitis (AS) and were phenotyped for HLA-A and -B loci. A complement dependent micrccytotoxictty assay was perfom?ed using a battery of local sera screened from multiparous women: A sample of 105 healthy and unrelated individuals was used as controls. Nineteen out of 34 patients (55.85%) were HLA-B27 positive whereas the HLA-B27 frequency was of 1.9% in controls. The relative Risk value (RR) was of 85.23: The most frequent haplotype associated to AS was A2 827. As for most populations worldwide, we found a significant increased frequency of HLA-B27 in patients with AS. Nevertheless, HLA-B27 was associated to AS at a lower frequency than in Caucasians. Moreover, the RR value found was lower than those reported in Caucasians. Our results showed an equal distribution of HLA-827 in the population of south Tunisia, in Kabyles and in Saudi Arabs. In addition, 15 AS patients of 34 (44.2%) lacked the HlA-B27 antigen. Altogether, these data suggest that besides the HLA-B27 antigen, additional genes might also be involved in the susceptibility to develop AS.
P-5.04.06
Polymorphism of the human lmmunoglobulln heavy chain locus In rheumatoid arthritis
F. Fakhfakh ‘, A. Maalej I, 2. Bahloul ’ , A. Jarraya ‘, H. Ayadi ‘, M.Zouali 2. ’ LaborMoire d’lmmunokgia at da Biologic Mol&ulaira, FaculM da M6decina. St&x, Tunisia,2LMpartement d’lmmunolcgia, lnsritot Pastaq 28, rue du Dr Roux, Paris, France The genetic origin of Rheumatoid Arthritis (RA) is largely unknown. The purpose of this investigation was to assess the potential genetically determined involvement of the immunoglobulin (lg) heavy chain variable region (VH) locus in the pathogenesis of RA. We tested the hypothesis of whether there is a genetic linkage between a structural abnormality of the VH gene complex and autoantibody hyperproduction in RA. We used restfiction endonuclease generated polymorphism in human VH gene-family specific probes to examine genomic DNA from a RA family and from unrelated RA patients from both the Tunisian and the European populations. The use of DNA samples from these ethnic origins permitted a further evaluation of the polymorphism of the humanVH locus. While we found that the polymorphism of the VH locus was lower in the Tunisian population, we could not detect a restriction site polymorphism pattern restricted to RA. Our results argue against a genetic linkage between RA and polymorphism in the VH locus. However it is possible that the method of restriction fragment length polymorphism was not able to detect subtle variations in DNAs of RA patients and, hence, reveal minor abnormalities in the VH locus of the RA patients. Likewise, genomic changes or somatic variation in a B-cell subset, both undetectable by our restriction fragment analysis, would need to be further investigated.
1P.5.04.07 ] Molecular analysis of Class II sequences Involved In autolmmune hepatitis In Mexicans M.N. Vtiquez-Garcia 1.3,C. Alaez ‘, A. Olivo ‘, H. Debaz I, E. P&z-Luque ‘, G. De La Rosa ‘, S. Cano ‘, A. Burguete ‘, J. Bandera 2, D. Kershenobich 2, C. Gorodezky ’ ’ Department of Immunogenetics, INDRE, SSA, Mexico Ci& Mexico, 2Gastroentarol~y Sarvica, lnsikuto National da la Nutriciidn,SSA, Mexico Ciw Mexiw, 3Prvgrama da Biomedicina Molecular CINVESTAV-IPN, Mexico Ci& Maxkw Introduction: Autoimmune hepatitis (AH) is a progressive condition of the liver characterized by piecemeal necrosis, the presence of diierent autoantibodies and by its prevalence in females. DRBl’O301 and DRBl l0401 are associated in Caucasians and DRB1.0405 is a susceptibility gene in Japanese. This is the first study done looking for HLA class I antigens and class II DNA alleles in Mexicans with the disease. Materials and Methods: We analyzed 30 Mexican Mestizo patients with type I AH compared to 175 healthy controls. The following autoantibodies were determined: (r-DNA, smooth muscle, ANA, mitocondrtal, thyroglobulin and LKMl (microsomal). Serotogical class I typing was done using the microcytotoxicity technique with magnetic beads and double fluorescence staining. DNA typing for DRBI, DQAl, and DQBl loci was performed with the protocols, primers and probes developed for the 12th International Wokshop. PCR-SSP was done to confirm certain DRBl, DQAl or WBI alleles. Frequencies were calculated and the most probable combinations were deduced.