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44 hybrid allele
S4
Abstracts
Tuesday, November 3, 2009 2:00 PM - 3:30 PM Workshop 2: Case Studies in Stem Cell Transplantation 8-W
CHIMERISM AFTER HSCT DETERMINED BY SHORT TANDEM REPEATS – COMPARISON OF EUROCHIMERISM AND IDENTIFILER APPROACH. Anna Adamusiak, Christina E.M. Voorter, Marcel G.J. Tilanus. Transplantation Immunology, Tissue Typing Laboratory, Maastricht University Medical Centre, Maastricht, Netherlands. Aim: Patient-donor chimerism analysis after HSCT is used to monitor engraftment and enables identification of relapse. In this study STR markers defined by the Eurochimerism Consortium were compared with the Identifiler approaches, originally developed for forensic purposes, with regard to sensitivity and specificity. Methods: Artificial DNA mixtures containing 10%, 5%, 3%, 1% and 0.5% of minor component for five patient/donor pairs were prepared. The Eurochimerism panel consisted of 13 STR markers. Multiplex reactions were used to determine the informative markers, singleplex reactions for screening and quantification of chimeric samples. In the Identifiler panel 15 STR markers were amplified in one multiplex reaction for both purposes. Peak heights/areas were taken for calculation (3730 DNA Analyser, GeneMapper Software). Sensitivity was defined as the ability to detect minor DNA in the mixture at the lowest concentration. The linearity of results within the range from 10% to 0.5% was calculated. Results: The Eurochimerism markers showed higher sensitivity than the Identifiler markers. For 9/13 Eurochimerism markers the sensitivity was 0.5%, 3 had a sensitivity of 1% and 1 of 3%. Of the 15 Identifiler markers only 2 had a sensitivity of 0.5%, 7 showed a sensitivity of 1%, 5 of 3% and 1 had a 5% sensitivity. The Eurochimerism markers showed a better linearity of standard curves plotted from DNA mixtures than Identifiler markers (mean R2 0.9754 ⫾ 0.0141 vs. 0.8624 ⫾ 0.0942). Conclusions: The Eurochimerism approach is not only a more sensitive method than Identifiler, but it offers also more reliable data for sequential quantification of mixed chimerism after HSCT when the presence of minor component is below 10%.
9-W
IDENTIFICATION OF A NOVEL HLA-B*27/44 HYBRID ALLELE. M. Sprague,1 A. Smith,1 L. Regen,1 R. Williams,1 S. McKinney,1 P. Peterson,1 C. Kellum,1 S. Pereira.1,2,3 1Seattle Cancer Care Alliance; 2 FHCRC; 3Dept.of Lab Medicine, U. of Washington. Aim: Sequence based typing was performed on a potential URD for a HSCT patient. Initial results indicated B*2710, 4418 with 2 nucleotide mismatches at N559, 60. Additional testing revealed a novel B*27/44 hybrid allele and B*4901. Methods: Initial sequencing was performed with Atria B-locus reagents for exons 2, 3, and 4, and with Heterozygous Ambiguity Resolution sequencing primers (HARPs) in exons 2 and 3. When HLA-B group specific primers (BG) were used to isolate and sequence the individual alleles at exons 2 and 3, results indicated a B*4901 and a novel B*27 or 44. Primers were designed to amplify and sequence exons 1, 4, 5, 6, and 7. Data was collected on the ABI 3130xl and analyzed with Conexio Genomics Assign software. Intron data was read manually from the raw data output files in ABI’s Sequencing Analysis software. The sample was also tested by rSSOP and serology. Results: rSSOP typing suggested B*2705/38, 4418, B*2738, 4901, or B*2710, 4418 with several false positive and/or false negative reactions. Serological typing was most consistent with B49, B27 and a short B12. HARPs data appeared to confirm B*4418, but BG results indicated B*4901 and a novel B*27. Hemizygous sequencing of exons 1-7 for each allele confirmed B*4901 and determined that the novel B*27 allele was identical to B*270502 until N538 in exon 3, at which point it switched to match B*44020101 through the end of exon 7. The suspected crossover was confirmed by examining available intron data. The hybrid allele matched B*270502 in introns 1 and 2, and B*44020101 in introns 5 and 6. Introns 3 and 4 are indistinguishable between the two alleles. Conclusions: We have identified a novel hybrid allele of B*2705/B*4402, with the crossover occurring at N538 in exon 3.
Abstracts
Tuesday, November 3, 2009 2:00 PM - 3:30 PM Workshop 2: Case Studies in Stem Cell Transplantation 8-W
CHIMERISM AFTER HSCT DETERMINED BY SHORT TANDEM REPEATS – COMPARISON OF EUROCHIMERISM AND IDENTIFILER APPROACH. Anna Adamusiak, Christina E.M. Voorter, Marcel G.J. Tilanus. Transplantation Immunology, Tissue Typing Laboratory, Maastricht University Medical Centre, Maastricht, Netherlands. Aim: Patient-donor chimerism analysis after HSCT is used to monitor engraftment and enables identification of relapse. In this study STR markers defined by the Eurochimerism Consortium were compared with the Identifiler approaches, originally developed for forensic purposes, with regard to sensitivity and specificity. Methods: Artificial DNA mixtures containing 10%, 5%, 3%, 1% and 0.5% of minor component for five patient/donor pairs were prepared. The Eurochimerism panel consisted of 13 STR markers. Multiplex reactions were used to determine the informative markers, singleplex reactions for screening and quantification of chimeric samples. In the Identifiler panel 15 STR markers were amplified in one multiplex reaction for both purposes. Peak heights/areas were taken for calculation (3730 DNA Analyser, GeneMapper Software). Sensitivity was defined as the ability to detect minor DNA in the mixture at the lowest concentration. The linearity of results within the range from 10% to 0.5% was calculated. Results: The Eurochimerism markers showed higher sensitivity than the Identifiler markers. For 9/13 Eurochimerism markers the sensitivity was 0.5%, 3 had a sensitivity of 1% and 1 of 3%. Of the 15 Identifiler markers only 2 had a sensitivity of 0.5%, 7 showed a sensitivity of 1%, 5 of 3% and 1 had a 5% sensitivity. The Eurochimerism markers showed a better linearity of standard curves plotted from DNA mixtures than Identifiler markers (mean R2 0.9754 ⫾ 0.0141 vs. 0.8624 ⫾ 0.0942). Conclusions: The Eurochimerism approach is not only a more sensitive method than Identifiler, but it offers also more reliable data for sequential quantification of mixed chimerism after HSCT when the presence of minor component is below 10%.
9-W
IDENTIFICATION OF A NOVEL HLA-B*27/44 HYBRID ALLELE. M. Sprague,1 A. Smith,1 L. Regen,1 R. Williams,1 S. McKinney,1 P. Peterson,1 C. Kellum,1 S. Pereira.1,2,3 1Seattle Cancer Care Alliance; 2 FHCRC; 3Dept.of Lab Medicine, U. of Washington. Aim: Sequence based typing was performed on a potential URD for a HSCT patient. Initial results indicated B*2710, 4418 with 2 nucleotide mismatches at N559, 60. Additional testing revealed a novel B*27/44 hybrid allele and B*4901. Methods: Initial sequencing was performed with Atria B-locus reagents for exons 2, 3, and 4, and with Heterozygous Ambiguity Resolution sequencing primers (HARPs) in exons 2 and 3. When HLA-B group specific primers (BG) were used to isolate and sequence the individual alleles at exons 2 and 3, results indicated a B*4901 and a novel B*27 or 44. Primers were designed to amplify and sequence exons 1, 4, 5, 6, and 7. Data was collected on the ABI 3130xl and analyzed with Conexio Genomics Assign software. Intron data was read manually from the raw data output files in ABI’s Sequencing Analysis software. The sample was also tested by rSSOP and serology. Results: rSSOP typing suggested B*2705/38, 4418, B*2738, 4901, or B*2710, 4418 with several false positive and/or false negative reactions. Serological typing was most consistent with B49, B27 and a short B12. HARPs data appeared to confirm B*4418, but BG results indicated B*4901 and a novel B*27. Hemizygous sequencing of exons 1-7 for each allele confirmed B*4901 and determined that the novel B*27 allele was identical to B*270502 until N538 in exon 3, at which point it switched to match B*44020101 through the end of exon 7. The suspected crossover was confirmed by examining available intron data. The hybrid allele matched B*270502 in introns 1 and 2, and B*44020101 in introns 5 and 6. Introns 3 and 4 are indistinguishable between the two alleles. Conclusions: We have identified a novel hybrid allele of B*2705/B*4402, with the crossover occurring at N538 in exon 3.