Unambiguous HLA-DQB1 typing through a group-specific sequencing strategy

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Abstracts

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UNAMBIGUOUS HLA-DQB1 TYPING THROUGH A GROUP-SPECIFIC SEQUENCING STRATEGY Sarah Grimpe,1 Marta Albis-Camps,1 Holger-Andreas Elsner,2 Rainer Blasczyk1, 1Transfusion Medicine, Hannover Medical School, Hannover, Germany; 2MMD Medical Molecular Diagnostics GmbH, Dresden, Germany The HLA-DQB1 gene encodes the b-chain of the class II antigen HLA-DQ, and thus represents an important matching criterium in hematopoietic stem cell transplantation. Therefore, a PCR-SBT sequencing strategy for DQB1 should enable complete sequence analysis of exon 2, which is the most polymorphic part of the gene. Furthermore, to avoid ambiguous typing results, the method should allow for the definition of the cis/trans linkage of sequence motifs by a group-specific amplification step. Previous approaches in this field were limited since none of them fulfilled both criteria. In this study we have developed a PCR-SBT strategy enabling haplotype-specific sequencing of exon 2 of DQB1. This new approach is based on a PCR amplification step that utilizes group-specific polymorphisms in introns 1 and 2. The variations in these noncoding regions made it possible to separate all allelic groups by 6 primer mixes specific for DQB102, 03 group 1, 03 group 2, 04, 05, and 06. Compared to other methods, group-specific PCR-SBT of exon 2 of DQB1 provides the most precise sequence information and has a high potential for automation in highthroughput HLA typing formats.

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AMBIGUITIES OF HLA-B RESOLVED BY HAPLOTYPE-SPECIFIC SEPARATION AND SEQUENCE BASED TYPING Marion Nagy,1 Petra Otremba,1 Constanze Schoenemann,2 Johannes Dapprich,3 Nancy Murphy4, 1Institute of Legal Medicine, Humboldt-University, Charite, Berlin, Berlin, Germany; 2Institute for Transfusion Medicine, Humboldt-University, Charite, Berlin, Berlin, Germany; 3Generation Biotech, Lawrenceville, NJ, USA; 4GenoVision, Inc., West Chester, PA, USA Haplotype specific extraction, HSE, allows the specific collection of individual alleles by separating diploid samples into their haploid components without PCR. The separation step is performed with magnetic beads based on allele-group specific sequence binding sites. The separated DNA samples can be directly typed with downstream applications like sequence based typing. We have used the HLA-B Ampl Seq Module (ABBOTT, Wiesbaden). The haplotype specific extraction was carried out in a six sample parallel format on the GenoM™-6 robot (Genovision, Wien). Haplotype specific separations of HLA-B locus specific ambiguities are shown. The influence of DNAconcentration and DNA quality on the separation step was studied and the sequence based typing procedure adapted and optimized. For a HLA-B*47, 56? sample, false positive and negative reactions were present after sequence specific PCR and sequence specific oligonucleotide typing and could not be solved by generic sequencing. Only sequence based typing after haplotype specific separation could with certainty detect base changes in the HLA-B*5601 allele besides the HLA-B*4701 allele. The applied method allows direct sequencing of an individual allele. Failed resolution of diploid HLA-B allele pair combinations by sequence specific PCR (SSP), sequence specific oligonucleotides (SSO) or sequence based typing (SBT) can be unambiguously typed after separation by HSE. New alleles can be easily detected without cloning. Research was supported in part by GenoVision GmbH, Vienna, Austria.