HLA sequencing based typing (SBT) methods for resolving ambiguities of heterozygous allele combinations may utilize new tools able to separate PCR sequences. In this study we evaluated the resolution level achieved by the Heterozygous Ambiguity Resolution Primers (HARPs) method in patients who underwent haemopoietic stem cell transplantation (HSCT) and relative donors. 664 SBT typed individuals (373 for HLA-A,B,DRB1 – year 2004 - and 291 for HLA-C - 2005) were reanalyzed by the light of the 2006 database (Assign SBTTM 3.5 software, Conexio Genomics, Applecross, Western Australia) in order to define ambiguous typings and relative frequencies: 67.8% of samples resulted ambiguous for locus HLA-A, 61.9% for HLA-B, 81.8% for HLA-C and 49.9% for HLA-DRB1. We thus evaluated the hypothetical resolution percentages of HARPs. Finally we performed analyses using specific HARPs for 90 ambiguous typings (23 HLA-A, 26 HLA-B, 27 HLA-C, 14 HLA-DRB1). We obtained different levels of resolution: 1) 28.9% ambiguities were completely resolved (11/23 for HLA-A, 8/26 for HLA-B, 3/27 for HLA-C, 4/14 for HLA-DRB1); 2) 33.3% were resolved with the HARPs, although ambiguities due to polymorphisms located out of exons 2 (class I-II) and 3 (class I) still remain (7/26 HLA-B, 13/27 HLA-C, 10/14 HLADRB1); 3) 22.2% were resolved, but null alleles still remain (10/ 23 HLA-A, 5/26 HLA-B, 5/27 HLA-C); 4) 8.9% were partially resolved (1/23HLA-A, 2/26HLA-B, 5/27HLA-C); 5) 6.7%were not resolved at all (1/23 HLA-A, 4/26 HLA-B, 1/27 HLA-C). In our preliminary results, this strategy seems to be useful to resolve most of the ambiguities of HLA class I-II, although a new Assign software - able to indicate theHARPsrequired for HR typing of the sample - should be implemented.
Heterozygous ambiguities in Hla sequencing: evaluation of a new typing strategy
BERTINETTO, FRANCESCA;GARINO, Elena;AMOROSO, Antonio
2007-01-01
Abstract
HLA sequencing based typing (SBT) methods for resolving ambiguities of heterozygous allele combinations may utilize new tools able to separate PCR sequences. In this study we evaluated the resolution level achieved by the Heterozygous Ambiguity Resolution Primers (HARPs) method in patients who underwent haemopoietic stem cell transplantation (HSCT) and relative donors. 664 SBT typed individuals (373 for HLA-A,B,DRB1 – year 2004 - and 291 for HLA-C - 2005) were reanalyzed by the light of the 2006 database (Assign SBTTM 3.5 software, Conexio Genomics, Applecross, Western Australia) in order to define ambiguous typings and relative frequencies: 67.8% of samples resulted ambiguous for locus HLA-A, 61.9% for HLA-B, 81.8% for HLA-C and 49.9% for HLA-DRB1. We thus evaluated the hypothetical resolution percentages of HARPs. Finally we performed analyses using specific HARPs for 90 ambiguous typings (23 HLA-A, 26 HLA-B, 27 HLA-C, 14 HLA-DRB1). We obtained different levels of resolution: 1) 28.9% ambiguities were completely resolved (11/23 for HLA-A, 8/26 for HLA-B, 3/27 for HLA-C, 4/14 for HLA-DRB1); 2) 33.3% were resolved with the HARPs, although ambiguities due to polymorphisms located out of exons 2 (class I-II) and 3 (class I) still remain (7/26 HLA-B, 13/27 HLA-C, 10/14 HLADRB1); 3) 22.2% were resolved, but null alleles still remain (10/ 23 HLA-A, 5/26 HLA-B, 5/27 HLA-C); 4) 8.9% were partially resolved (1/23HLA-A, 2/26HLA-B, 5/27HLA-C); 5) 6.7%were not resolved at all (1/23 HLA-A, 4/26 HLA-B, 1/27 HLA-C). In our preliminary results, this strategy seems to be useful to resolve most of the ambiguities of HLA class I-II, although a new Assign software - able to indicate theHARPsrequired for HR typing of the sample - should be implemented.
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