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209-P: Linkage disequilibrium between the HLA-G locus and neighbouring HLA loci
Abstracts
S117
208-P
EVALUATION OF HLA-DRB1*140101/ DRB1*1454 GENOTYPING USING PYROSEQUENCING METHODOLOGY. Melina Dinou, Vasiliki Vrani, Erasmia Stamathioudaki, Elissavet Kontou, Aliki Iniotaki, Maria Spyropoulou-Vlachou. Immunology Dp.-National Tissue Typing Laboratory, General Hospital of Athens “G. Gennimatas”, Athens, Greece. Aim: HLA DRB1*140101 and DRB1*1454 alleles are only distinguished by a single nucleotide at position 421 of exon 3. This exon is not covered by the RSSO kits routinely used for HLA typing in our laboratory. In order to differentiate between the two alleles, we developed an assay using the pyrosequencing technique. Methods: 128 adult transplant recipients and their donors who had an ambiguous DRB1*1401/1454 typing, using rSSO, were restrospectively tested. For distinguish ing DRB1*1401 and DRB1*1454 the nucleotide at position 421 of exon 3: a T in case of DRB1*1401 or a C for DRB1*1454 has to be defined.Three sets of primers were required in order to account for the sequence variations of the different alleles: one for the DRB1*1401/1454, DRB1*07 pairs, a second for the DRB1* 1401/1454, DRB1*09 or DRB1*10 pairs and a third for the rest of the cases. Results: Of the samples tested 42 were shown to have a T at position 421 and were defined as DRB1*1401 alleles. For the rest a DRB1*1454 assignment was made. Based on frequency occurrence the DRB1*1454 is much more common than DRB1*1401 allele in our population (68% vs 32% respectively) but the difference is not so significant as in other populations. These results were confirmed by PCR-SSP typing and no discrepancies were found. Conclusions: Pyrosequencing is an easy technique to use for SNP genotyping, allows the simultaneous analysis of numerous samples (up to 96) and the result is accurate and easy to interpret.Furthermore, it could be used for other similar cases where the usual methods fail to distinguish two alleles that are only different in one nucleotide position.
209-P
LINKAGE DISEQUILIBRIUM BETWEEN THE HLA-G LOCUS AND NEIGHBOURING HLA LOCI. Neifi H.S. Deghaide,1 Erick C. Castelli,1 Celso T. Mendes-Junior,2 Cassia Paula-Santos,1 Philippe Moreau,3 Eduardo A. Donadi.1 1Department of Medicine, University of Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil; 2 Departamento de Quı´mica, Universidade de Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil; 3Commissariat a` l’Energie Atomique, Hoˆpital Saint-Louis, Paris, France. Aim: Haplotypes and Linkage Disequilibrium (LD) observed in the HLA complex are frequently evaluated for population history inferences and association studies. In the present work, we used probabilistic models to computationally infer haplotypes and exact tests to study the pattern of LD of the HLA-G locus and several HLA loci. Methods: HLA-G was evaluated by sequence-based typing, the HLA-A, -B, -Cw, -DRB1 loci were evaluated by the Luminex® approach, and the class III TNFa-e microsatellites were evaluated by differential segregation pattern using PAGE. Results: The LD exact test revealed a highly significant LD only between HLA-G and the HLA-A locus (p ⫽ 0.0000 ⫾ 0.0000). In addition, statistically significant LDs between the HLA-G locus and HLA-Cw (0.0194 ⫾ 0.0111) and the TNFb (0.0334 ⫾ 0.0062) and TNFc (0.0126 ⫾ 0.0016) microsatellites were also observed. The absence of LD between HLA-G and other HLA loci may be due to the existence of multiple recombination hotspots in the MHC region. Among the associations observed between the HLA-G and HLA-A loci, it should be emphasized the following haplotypes: G*01010104/HLA-A29, G*01010105/HLA-A3 (which may indicate a European origin for the G*01010105 allele), G*010103/HLA-A11 (which may indicate a Eurasian origin for the G*010103 allele), the G*0105N/HLA-A30 (which may indicate an African origin for the G*0105N allele) and G*0106/HLA-A1. Conclusions: In conclusion, our data indicated a high LD only between HLA-G and HLA-A, spanning a region of approximately only 100 kb, without previously recognized recombination hotspots.
S117
208-P
EVALUATION OF HLA-DRB1*140101/ DRB1*1454 GENOTYPING USING PYROSEQUENCING METHODOLOGY. Melina Dinou, Vasiliki Vrani, Erasmia Stamathioudaki, Elissavet Kontou, Aliki Iniotaki, Maria Spyropoulou-Vlachou. Immunology Dp.-National Tissue Typing Laboratory, General Hospital of Athens “G. Gennimatas”, Athens, Greece. Aim: HLA DRB1*140101 and DRB1*1454 alleles are only distinguished by a single nucleotide at position 421 of exon 3. This exon is not covered by the RSSO kits routinely used for HLA typing in our laboratory. In order to differentiate between the two alleles, we developed an assay using the pyrosequencing technique. Methods: 128 adult transplant recipients and their donors who had an ambiguous DRB1*1401/1454 typing, using rSSO, were restrospectively tested. For distinguish ing DRB1*1401 and DRB1*1454 the nucleotide at position 421 of exon 3: a T in case of DRB1*1401 or a C for DRB1*1454 has to be defined.Three sets of primers were required in order to account for the sequence variations of the different alleles: one for the DRB1*1401/1454, DRB1*07 pairs, a second for the DRB1* 1401/1454, DRB1*09 or DRB1*10 pairs and a third for the rest of the cases. Results: Of the samples tested 42 were shown to have a T at position 421 and were defined as DRB1*1401 alleles. For the rest a DRB1*1454 assignment was made. Based on frequency occurrence the DRB1*1454 is much more common than DRB1*1401 allele in our population (68% vs 32% respectively) but the difference is not so significant as in other populations. These results were confirmed by PCR-SSP typing and no discrepancies were found. Conclusions: Pyrosequencing is an easy technique to use for SNP genotyping, allows the simultaneous analysis of numerous samples (up to 96) and the result is accurate and easy to interpret.Furthermore, it could be used for other similar cases where the usual methods fail to distinguish two alleles that are only different in one nucleotide position.
209-P
LINKAGE DISEQUILIBRIUM BETWEEN THE HLA-G LOCUS AND NEIGHBOURING HLA LOCI. Neifi H.S. Deghaide,1 Erick C. Castelli,1 Celso T. Mendes-Junior,2 Cassia Paula-Santos,1 Philippe Moreau,3 Eduardo A. Donadi.1 1Department of Medicine, University of Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil; 2 Departamento de Quı´mica, Universidade de Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil; 3Commissariat a` l’Energie Atomique, Hoˆpital Saint-Louis, Paris, France. Aim: Haplotypes and Linkage Disequilibrium (LD) observed in the HLA complex are frequently evaluated for population history inferences and association studies. In the present work, we used probabilistic models to computationally infer haplotypes and exact tests to study the pattern of LD of the HLA-G locus and several HLA loci. Methods: HLA-G was evaluated by sequence-based typing, the HLA-A, -B, -Cw, -DRB1 loci were evaluated by the Luminex® approach, and the class III TNFa-e microsatellites were evaluated by differential segregation pattern using PAGE. Results: The LD exact test revealed a highly significant LD only between HLA-G and the HLA-A locus (p ⫽ 0.0000 ⫾ 0.0000). In addition, statistically significant LDs between the HLA-G locus and HLA-Cw (0.0194 ⫾ 0.0111) and the TNFb (0.0334 ⫾ 0.0062) and TNFc (0.0126 ⫾ 0.0016) microsatellites were also observed. The absence of LD between HLA-G and other HLA loci may be due to the existence of multiple recombination hotspots in the MHC region. Among the associations observed between the HLA-G and HLA-A loci, it should be emphasized the following haplotypes: G*01010104/HLA-A29, G*01010105/HLA-A3 (which may indicate a European origin for the G*01010105 allele), G*010103/HLA-A11 (which may indicate a Eurasian origin for the G*010103 allele), the G*0105N/HLA-A30 (which may indicate an African origin for the G*0105N allele) and G*0106/HLA-A1. Conclusions: In conclusion, our data indicated a high LD only between HLA-G and HLA-A, spanning a region of approximately only 100 kb, without previously recognized recombination hotspots.