Abstract
Sequence-based typing (SBT) is one of the most comprehensive methods utilized for HLA typing. However, one of the inherent problems with this typing method is the interpretation of ambiguous allele combinations which occur when two or more different allele combinations produce identical sequences. The purpose of this study is to investigate the probability of this occurrence. We performed HLA-A,-B SBT for Exons 2 and 3 on 676 donors. Samples were analyzed with a capillary sequencer. The racial distribution of the donors was as follows: 615-Caucasian, 13-Asian, 23-African American, 17-Hispanic and 8-Unknown. 672 donors were analyzed for HLA-A locus ambiguities and 666 donors were analyzed for HLA-B locus ambiguities. At the HLA-A locus a total of 548 total ambiguous allele combinations were identified (548/1344 = 41%). Most (278/548 = 51%) of these ambiguities were due to the fact that Exon 4 analysis was not performed. At the HLA-B locus 322 total ambiguous allele combinations were found (322/1332 = 24%). The HLA-B*07/08/15/27/35/44 antigens, common in Caucasians, produced a large portion of the ambiguities (279/322 = 87%). A large portion of HLA-A and B ambiguous allele combinations can be addressed by utilizing a group-specific primary amplification approach to produce an unambiguous homozygous sequence. Therefore, although the prevalence of ambiguous allele combinations is high, if the resolution of these ambiguities is clinically warranted, methods exist to compensate for this problem.
Highlights
The precise identification of HLA Class I and Class II alle- transplants, the development of peptide based viral and
The prevalence of ambiguities in HLA typing relates to the nature of polymorphisms which exists in the sequence of the major histocompatibility complex (MHC) Class I and Class II genes
Sequence based typing analysis of HLA-A and B alleles was performed on a population of 676 normal donors
Summary
The precise identification of HLA Class I and Class II alle- transplants, the development of peptide based viral and (page number not for citation purposes). Sequence-based typing (SBT) involves PCR amplification of specific coding regions of HLA genes and sequencing of the amplicons [3,4]. SBT allows for a detailed interpretation of HLA alleles by comparing nucleotide sequences of the polymorphic, and sometimes conserved, regions of the HLA gene to a database of possible allelic combinations. Sequence-based typing involves PCR amplification and sequencing of specific HLA exons, which are known to be polymorphic, from genomic DNA. In the Class I region, HLA-B*070201, 3503 would have the same nucleotide sequence as HLAB*0724, 3533 in positions 559 and 560 (Figure 1) In this example, the SBT produces a heterozygous base pair combination at positions 559 and 560 with an international union of biochemists (IUB) designation of K(G+C)W(A+T). The interpretation to the high resolution level can not be made because it is not known which allele combination is correct
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