- Email: [email protected]
Differential transcription of the HLA-DR genes
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Abstracts Abstracts 65-73
Group discussion VI: N e w alleles and new genes
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POPULATION AND FUNCTIONAL ANALYSIS OF A NEW TNF ALPHA PROMOTER POLYMORPHISM. S.D'Alfonso1, F.Pociot2, C.Carcassi 3, P. Momigliano Richiardi 1. 1 Centro CNR lmmunogenetica e Istoeompatibilit/t, Torino and Dip. Scienze Mediche, Novara, Universith di Torino, Italy 2 Steno Diabetes Center, Gentofle, Denmark. 3 Cattedra di Genetica Medica, Universitb. di Cagliari, Italy A new polymorphism was identified in the TNFct promoter gene consisting ofa G to A transition at position -238 in a putative regulatory sequence (Y box). This nucleotide substitution induced a differential electrophoretic migration on po/yanrilamide gel of the amplified fragments, probably due to a modification in DNA bending. The gene frequencies of the two alleles, named TNFA-G and TNFA-A, were 0.93 and 0.07 in the North Italian population. The TNFA-A allele was significantly associated with alleles belonging to the two extended haplotypes [B18, F1C30, DR3] and [B57, SC61, DR7]. Notably, linkage disequilibria between TNFA-A and the extended haplotypes were higher than with any single haplotype marker. The association with the [B18, F1C30, DR3] hapiotype was confirmed both in normal individuals and in diabetic patients in the Sardinian population, where this haplotype is particularly frequent. The location of the G/A polymorphism in a regulatory box raises the question of its relevance for the control of TNFa expression. We therefore characterized for this polymorphism 49 individuals tested for TNF~ production upon in vitro stimulation with LPS. No significant correlation was detected between TNFA-G/A alleles and the amount of cytokine. A possible association with baseline TNF production or with its induction by different stimuli will be searched.
DIFFERENTIAL TRANSCRIPTION OF THE HLA-DR GENES [~, Vincent. I. Papa, J Clot, J-F Eliaou. Laboratoire dqmmunologie, CHRU Montpellier, France The MHC class II molecules participate in the immune response mediated by the activation of T-CD4 cells both by the allelic polymorphism of antigen presentation and by the density of peptide-binding class II molecules expressed on the cell surface. Differential expression of the different DRB genes has been reported. Moreover, it has been shown that abnormal expression of those geee-encoded molecales occurs in autoimmune diseases such as Rheumatoid Arthritis (RA). Regulation of I-ILA class II gene expression will then have fundamental as well as pathological implications. A structural as well as a functional polymorphism of the proximal promoters of the DRB genes have been described in the Laboratory. These results permitted us to assume that a differential transcriptional regulation could exist among the DRB genes. In order to confirm this hypothesis, we performed a quantification of the corresponding messengers RNAs using a quantitative PCR technology, based on the competition between an internal control and the target. This competitive PCR allowed us to compare the amount of messengers RNAs transcribed from the different DRB genes. We found quantitative differences between the transcripts of the DRB genes, that are correlated with the polymorphism of the DRB geee regulatory regions. In a second time, using the same technology approach, we compared the DRA gene transcripts with the sum of the DRB gene transcripts. At this time we found an invariant ~!3 DR RNA ratio. From a clinical point of view those results will offer a greater understanding of the mechanisms of the association between autoimraune diseases and HLA by lookizg for differential expressions of the DRB 1 alleles associated with RA compared with nonassociated alleles.
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DEFINING FOUR CIS-LOCATED SEQUENCE MOTIFS BY 2ND GENERATION PCR AMPLIFICATION WITH SEQUENCE-SPECIFIC PRIMERS - DROP-IN PCRSSP - FOR HIGH RESOLUTION HLA CLASS I AND II TYPING. A, Al~Jcnfr, A. Fogdell and O. Olerup. Center tbr BioTechnology, Karolinska Institute, NOVUM and Dept of Clinical Immunology, Karolinska Institute at Huddinge Hospital, Huddinge, Sweden For high resolution HLA class I and class II typing it is, in many cases, not enough to merely determine the presence or absence of sequence polymorphisms. It may be necessary to define that two to fimr sequence motifs are located on the same chromosome, i.e. in cis. This degree of resolution may be obtained by e.g. PCR amplification with sequencespecific primers (PCR-SSP) using an outer and an inner primer pair, i.e. a nested approach. However, the handling of the amplified product of the 1st PCR reaction in conventional nested PCR carries a substantial risk of contamination and is also relatively labor-intensive. In order to avoid these drawbacks, we have developed a simple technique whereby the second primer pair merely is added to - dropped-in - and concomitantly diluting the product of the Ist round of amplification. Thus, the pipettthg of PCR products is eliminated. First, 16 PCR cycles in 2 ~1 reaction volume, containing the 1st primer pair (0. l .aM), were performed. Then, 18/zl were added containing the 2nd primer pair (1.0 ~tM), buffer, dNTPs and water (but no additional Taq polymeras), diluting the 1st PCR product 1 to 10, and a 2nd round of 18 PCR cycles were performed. Each PCR reaction also contained a non-allelic primer pair (0.1 .uM). So far the drop-in FCR-SSP approach has been successfully applied for defining alleles of the AI9, DRBI*08 and DRBI*II groups. HLA class I and class II typing by the drop-in PCR-SSP technique offers an extremely high resolution, since the chromosomal locations of four sequence motifs are detected by the two primer pairs of each PCR reaction, combined with being fast, technically simple and carrying a low risk of contamination.
IRREGULAR PCR SSO TYPINGS IDENTIFY NEW HL~-DRBI ALLELES. J.DH. Anholts, W. Verduyn, J. D'Amaro, I.I.N. Doxiadis. M.J. Giphart, G.G. Persijn, G.M.Th. Schreuder. Dept of Immunohematology and Bloodbank, University Hospital Leiden, and Eurotransplant Foundation, Leiden. The Netherlands The PCR-SSO technique is a reliable method to study MHC class II polymorphism. Since three years this method is applied in our laboratory using a non-radioactive labelling system. In this period over 7.000 patients, blood and organ donors have been typed. The results were compared to conventional serology. Using a locally developed computer program for interpretation of the DRgl PCR-SSO readings a number of samples were detected with irregular hybridisation patterns. Several of such samples were sequenced and appeared to identify the following new DRBI alleles: -- DRBl*O301-1ike. that was observed in three unrelated caucasoid individuals; - DRBI*I603 variant; -- DRgl*13Ol-like, found in a Surinam family; -- DRBI*I3 with sequences related to DRgl*1305 and "1307: - DRBI*1401 with a HV3 derived from *1301: -- DRBI*I401 including some DR11 sequences: - DRBI*0801 including some DRBI*I401 sequences. These new alleles lead also to complications when typing by sequence specific priming (SSP). Some would have been missed whereas others would occur as triplet.
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A S T U D Y OF I t I ~ - D R 2 - L U M HAPLOTYPE G E N E R A T I O N A T T H E M O L E C U L A R LEVEL. H I ~ - D R B 6 ALLELE D I S T R I B U T I O N . E. Paz-Artai, A. Corell, J. Martinez-Laso, P. Morales, M. Alvarez, P. Varela, J.M. Marffn-Villa, E. du Toit* and A. Arnaiz-Vigena. INMUNOLOGIA, Hcapital Universitario '12 de Octubre'. Universidad Complutense. Ctra. Andaluc~a, 28041 MADRID (SPAIN) and *Provincial Laboratory for Tissue Immunology, Cape Town, SOUTH AFRICA A new class 1] locus has been recently characterized within the DR subregion of the HLA system: HLA-DRB6. This locus is found in DR1, DR2 and DR10 haplotypes and, to date, three DRB6 alleles have been identified: DRB6*0101 in DR1 and DR10 haplotypes, DRB6*0201 in DRB 1*15 haplotypes and DRB6*0202 in DRB I *16 ones. On the other hand, two hybrid DR1-DR2, haplotypes have been described: one of them (designed DR1/2) has been characterized by means of RFLP and sequencing studies, and the other one (DR2"LUM") has been def'med at the serological and RFLP levels. In the present work, the genetic structure and recombinational origin of these two haplotypes has been studied based on the DRB6 allele carried by tho~e haplotypes. In addition, the distn'bution of the DRB6 alleles in a panel of 27 HLA-DR2 ceils has been analyzed. Specific HLA-DRB6 amplification and direct DNA sequence analysis with dye-labe,iled didcx3~-terminators was carried out. All eighteen DR15 cells had the DRB6*~201 allele, while all eight DR16 had the DRB6*ff202 allele. Thus, there was a 100% association between each DR2 subtype and the corresponding DRB6 allele in the non-recombinant haplotypes. DRB gen ~ e n 2 sequences obtained from a DR2"LUM" heterozygous individual showed only two different scx]uenees, both belenglng to the DR2 chromesome: 1) DRB1 *1501 and 2) DRB6*0201. Of 50 clones sequenced, none were isolated that corresponded to any DRB5 allele. Th~s suggests that the DRB5 gene is absent in the DR2"LUM" haplotype, a notion further supported by the concordance with the number of RFLPs obtained in a homozygous DR2LUM individual.The putative recombinational point generating DR1/2 and D1L2"LUM" recombinant haplotypes will be discussed. Also the DRB6 pers/stence in DR1, DR2 and DR10 haplotypes may be favouring intra-DRB6-grenp, specifie crossing-over events.
A N O V E L DRB1 A L L E L E (DRBI*1415): F O R M E D BY I N T E R A L L E L I C C R O S S I N G O V E R B E T W E E N T H E DRBI*1404 A N D DRBI*0804 A L L E L E S . A. Foedell and 0. Olerup. Center for BioTechnology, Karolinska Institute, NOVUM, Huddinge and Department of Clinical Immeno]ogy, Karolinska Institute at Huddinge Hospital, Hnddinge, Sweden The extensive polymorphism of the human leukocyte antigens is mainly encoded by the second exon of the different class I and class II genes. Of the class II loci, DRBI is the most polymorphio with over 70 recognised alleles. Here, we report a novel DRB1 allele found in a heterozygoas patient of East Asian origin. The deserepant typing results obtained by serology (DR2,8;DQ1), TaqI RFLP analysis (DR2(15),6(14), DR51,52;DQ5,5) and PCR-SSP typing (DR2(15),8;DR51,52;DQ5,5), indicated the presence of a new allele. Sequencing was performed in both directions using biotinylated primers and magnetic beads in the first PCR amplification and fluorescent didenxynocleotides in the cycling sequencing reaction. Both the second exon of the DRB1 and DRB3 genes where sequenced at least three times using separate PCR products. The result revealed that the novel DRB1 allele was a combination of sequence motifs from the DRBI*I404 and DRBI*0804 alleles and that the DRB3 allele was D R B 3 * 0 2 0 2 . The deduced haplotype of the novel allele was the same as for DRB1 "1401, namely DRBl*1415, DRB3 *0202, DQA1 "0104, DQB1 *0503. The novel allele probably arose by intraexvnic double crossing over, where the orhelical encoding part was donated from DRBI *0804 to the recipient allele DRB1 "1404. The Y-recombination site is somewhere between codon 38 to 56, but probably at codon 51 to 55, which has been suggested to be a putative recombination signal. The Trecombination site could not be delineated due to sequence homology in this region between DRB1 "1404 and DRB1 *0804, but is located 3' of codon 78. The novel allele was also an additional example of a haplotype carrying the DQAI*OI04 allele previously observed on DRI0, some DRI2 and the DRB1 "1401 haplotypes.
Abstracts Abstracts 65-73
Group discussion VI: N e w alleles and new genes
65
66
POPULATION AND FUNCTIONAL ANALYSIS OF A NEW TNF ALPHA PROMOTER POLYMORPHISM. S.D'Alfonso1, F.Pociot2, C.Carcassi 3, P. Momigliano Richiardi 1. 1 Centro CNR lmmunogenetica e Istoeompatibilit/t, Torino and Dip. Scienze Mediche, Novara, Universith di Torino, Italy 2 Steno Diabetes Center, Gentofle, Denmark. 3 Cattedra di Genetica Medica, Universitb. di Cagliari, Italy A new polymorphism was identified in the TNFct promoter gene consisting ofa G to A transition at position -238 in a putative regulatory sequence (Y box). This nucleotide substitution induced a differential electrophoretic migration on po/yanrilamide gel of the amplified fragments, probably due to a modification in DNA bending. The gene frequencies of the two alleles, named TNFA-G and TNFA-A, were 0.93 and 0.07 in the North Italian population. The TNFA-A allele was significantly associated with alleles belonging to the two extended haplotypes [B18, F1C30, DR3] and [B57, SC61, DR7]. Notably, linkage disequilibria between TNFA-A and the extended haplotypes were higher than with any single haplotype marker. The association with the [B18, F1C30, DR3] hapiotype was confirmed both in normal individuals and in diabetic patients in the Sardinian population, where this haplotype is particularly frequent. The location of the G/A polymorphism in a regulatory box raises the question of its relevance for the control of TNFa expression. We therefore characterized for this polymorphism 49 individuals tested for TNF~ production upon in vitro stimulation with LPS. No significant correlation was detected between TNFA-G/A alleles and the amount of cytokine. A possible association with baseline TNF production or with its induction by different stimuli will be searched.
DIFFERENTIAL TRANSCRIPTION OF THE HLA-DR GENES [~, Vincent. I. Papa, J Clot, J-F Eliaou. Laboratoire dqmmunologie, CHRU Montpellier, France The MHC class II molecules participate in the immune response mediated by the activation of T-CD4 cells both by the allelic polymorphism of antigen presentation and by the density of peptide-binding class II molecules expressed on the cell surface. Differential expression of the different DRB genes has been reported. Moreover, it has been shown that abnormal expression of those geee-encoded molecales occurs in autoimmune diseases such as Rheumatoid Arthritis (RA). Regulation of I-ILA class II gene expression will then have fundamental as well as pathological implications. A structural as well as a functional polymorphism of the proximal promoters of the DRB genes have been described in the Laboratory. These results permitted us to assume that a differential transcriptional regulation could exist among the DRB genes. In order to confirm this hypothesis, we performed a quantification of the corresponding messengers RNAs using a quantitative PCR technology, based on the competition between an internal control and the target. This competitive PCR allowed us to compare the amount of messengers RNAs transcribed from the different DRB genes. We found quantitative differences between the transcripts of the DRB genes, that are correlated with the polymorphism of the DRB geee regulatory regions. In a second time, using the same technology approach, we compared the DRA gene transcripts with the sum of the DRB gene transcripts. At this time we found an invariant ~!3 DR RNA ratio. From a clinical point of view those results will offer a greater understanding of the mechanisms of the association between autoimraune diseases and HLA by lookizg for differential expressions of the DRB 1 alleles associated with RA compared with nonassociated alleles.
67
68
DEFINING FOUR CIS-LOCATED SEQUENCE MOTIFS BY 2ND GENERATION PCR AMPLIFICATION WITH SEQUENCE-SPECIFIC PRIMERS - DROP-IN PCRSSP - FOR HIGH RESOLUTION HLA CLASS I AND II TYPING. A, Al~Jcnfr, A. Fogdell and O. Olerup. Center tbr BioTechnology, Karolinska Institute, NOVUM and Dept of Clinical Immunology, Karolinska Institute at Huddinge Hospital, Huddinge, Sweden For high resolution HLA class I and class II typing it is, in many cases, not enough to merely determine the presence or absence of sequence polymorphisms. It may be necessary to define that two to fimr sequence motifs are located on the same chromosome, i.e. in cis. This degree of resolution may be obtained by e.g. PCR amplification with sequencespecific primers (PCR-SSP) using an outer and an inner primer pair, i.e. a nested approach. However, the handling of the amplified product of the 1st PCR reaction in conventional nested PCR carries a substantial risk of contamination and is also relatively labor-intensive. In order to avoid these drawbacks, we have developed a simple technique whereby the second primer pair merely is added to - dropped-in - and concomitantly diluting the product of the Ist round of amplification. Thus, the pipettthg of PCR products is eliminated. First, 16 PCR cycles in 2 ~1 reaction volume, containing the 1st primer pair (0. l .aM), were performed. Then, 18/zl were added containing the 2nd primer pair (1.0 ~tM), buffer, dNTPs and water (but no additional Taq polymeras), diluting the 1st PCR product 1 to 10, and a 2nd round of 18 PCR cycles were performed. Each PCR reaction also contained a non-allelic primer pair (0.1 .uM). So far the drop-in FCR-SSP approach has been successfully applied for defining alleles of the AI9, DRBI*08 and DRBI*II groups. HLA class I and class II typing by the drop-in PCR-SSP technique offers an extremely high resolution, since the chromosomal locations of four sequence motifs are detected by the two primer pairs of each PCR reaction, combined with being fast, technically simple and carrying a low risk of contamination.
IRREGULAR PCR SSO TYPINGS IDENTIFY NEW HL~-DRBI ALLELES. J.DH. Anholts, W. Verduyn, J. D'Amaro, I.I.N. Doxiadis. M.J. Giphart, G.G. Persijn, G.M.Th. Schreuder. Dept of Immunohematology and Bloodbank, University Hospital Leiden, and Eurotransplant Foundation, Leiden. The Netherlands The PCR-SSO technique is a reliable method to study MHC class II polymorphism. Since three years this method is applied in our laboratory using a non-radioactive labelling system. In this period over 7.000 patients, blood and organ donors have been typed. The results were compared to conventional serology. Using a locally developed computer program for interpretation of the DRgl PCR-SSO readings a number of samples were detected with irregular hybridisation patterns. Several of such samples were sequenced and appeared to identify the following new DRBI alleles: -- DRBl*O301-1ike. that was observed in three unrelated caucasoid individuals; - DRBI*I603 variant; -- DRgl*13Ol-like, found in a Surinam family; -- DRBI*I3 with sequences related to DRgl*1305 and "1307: - DRBI*1401 with a HV3 derived from *1301: -- DRBI*I401 including some DR11 sequences: - DRBI*0801 including some DRBI*I401 sequences. These new alleles lead also to complications when typing by sequence specific priming (SSP). Some would have been missed whereas others would occur as triplet.
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A S T U D Y OF I t I ~ - D R 2 - L U M HAPLOTYPE G E N E R A T I O N A T T H E M O L E C U L A R LEVEL. H I ~ - D R B 6 ALLELE D I S T R I B U T I O N . E. Paz-Artai, A. Corell, J. Martinez-Laso, P. Morales, M. Alvarez, P. Varela, J.M. Marffn-Villa, E. du Toit* and A. Arnaiz-Vigena. INMUNOLOGIA, Hcapital Universitario '12 de Octubre'. Universidad Complutense. Ctra. Andaluc~a, 28041 MADRID (SPAIN) and *Provincial Laboratory for Tissue Immunology, Cape Town, SOUTH AFRICA A new class 1] locus has been recently characterized within the DR subregion of the HLA system: HLA-DRB6. This locus is found in DR1, DR2 and DR10 haplotypes and, to date, three DRB6 alleles have been identified: DRB6*0101 in DR1 and DR10 haplotypes, DRB6*0201 in DRB 1*15 haplotypes and DRB6*0202 in DRB I *16 ones. On the other hand, two hybrid DR1-DR2, haplotypes have been described: one of them (designed DR1/2) has been characterized by means of RFLP and sequencing studies, and the other one (DR2"LUM") has been def'med at the serological and RFLP levels. In the present work, the genetic structure and recombinational origin of these two haplotypes has been studied based on the DRB6 allele carried by tho~e haplotypes. In addition, the distn'bution of the DRB6 alleles in a panel of 27 HLA-DR2 ceils has been analyzed. Specific HLA-DRB6 amplification and direct DNA sequence analysis with dye-labe,iled didcx3~-terminators was carried out. All eighteen DR15 cells had the DRB6*~201 allele, while all eight DR16 had the DRB6*ff202 allele. Thus, there was a 100% association between each DR2 subtype and the corresponding DRB6 allele in the non-recombinant haplotypes. DRB gen ~ e n 2 sequences obtained from a DR2"LUM" heterozygous individual showed only two different scx]uenees, both belenglng to the DR2 chromesome: 1) DRB1 *1501 and 2) DRB6*0201. Of 50 clones sequenced, none were isolated that corresponded to any DRB5 allele. Th~s suggests that the DRB5 gene is absent in the DR2"LUM" haplotype, a notion further supported by the concordance with the number of RFLPs obtained in a homozygous DR2LUM individual.The putative recombinational point generating DR1/2 and D1L2"LUM" recombinant haplotypes will be discussed. Also the DRB6 pers/stence in DR1, DR2 and DR10 haplotypes may be favouring intra-DRB6-grenp, specifie crossing-over events.
A N O V E L DRB1 A L L E L E (DRBI*1415): F O R M E D BY I N T E R A L L E L I C C R O S S I N G O V E R B E T W E E N T H E DRBI*1404 A N D DRBI*0804 A L L E L E S . A. Foedell and 0. Olerup. Center for BioTechnology, Karolinska Institute, NOVUM, Huddinge and Department of Clinical Immeno]ogy, Karolinska Institute at Huddinge Hospital, Hnddinge, Sweden The extensive polymorphism of the human leukocyte antigens is mainly encoded by the second exon of the different class I and class II genes. Of the class II loci, DRBI is the most polymorphio with over 70 recognised alleles. Here, we report a novel DRB1 allele found in a heterozygoas patient of East Asian origin. The deserepant typing results obtained by serology (DR2,8;DQ1), TaqI RFLP analysis (DR2(15),6(14), DR51,52;DQ5,5) and PCR-SSP typing (DR2(15),8;DR51,52;DQ5,5), indicated the presence of a new allele. Sequencing was performed in both directions using biotinylated primers and magnetic beads in the first PCR amplification and fluorescent didenxynocleotides in the cycling sequencing reaction. Both the second exon of the DRB1 and DRB3 genes where sequenced at least three times using separate PCR products. The result revealed that the novel DRB1 allele was a combination of sequence motifs from the DRBI*I404 and DRBI*0804 alleles and that the DRB3 allele was D R B 3 * 0 2 0 2 . The deduced haplotype of the novel allele was the same as for DRB1 "1401, namely DRBl*1415, DRB3 *0202, DQA1 "0104, DQB1 *0503. The novel allele probably arose by intraexvnic double crossing over, where the orhelical encoding part was donated from DRBI *0804 to the recipient allele DRB1 "1404. The Y-recombination site is somewhere between codon 38 to 56, but probably at codon 51 to 55, which has been suggested to be a putative recombination signal. The Trecombination site could not be delineated due to sequence homology in this region between DRB1 "1404 and DRB1 *0804, but is located 3' of codon 78. The novel allele was also an additional example of a haplotype carrying the DQAI*OI04 allele previously observed on DRI0, some DRI2 and the DRB1 "1401 haplotypes.