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P201 Erroneous HLA-B allele typing by sequencing-based typing method that utilizes combinations of Sanger sequencing and rSSO typing

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Allele-level HLA matching reduces graft-versus-host disease and improves overall patient survival after hematopoietic stem cell transplantation (HSCT). Sanger sequencing has been the gold standard for allele-level HLA typing since 1996.… Click to show full abstract

Allele-level HLA matching reduces graft-versus-host disease and improves overall patient survival after hematopoietic stem cell transplantation (HSCT). Sanger sequencing has been the gold standard for allele-level HLA typing since 1996. The typing strategy typically employs the sequencing of exons 2 and 3 for HLA Class I and exon 2 for HLA Class II. These exons encode the antigen recognition domains and sequencing only these exons are considered to be clinically relevant for HSCT. Sanger sequencing results significant number of ambiguities in HLA typing due to partial sequencing of limited exons, inability to phase heterozygous alleles, and growing number of new HLA alleles. Additional typing methods are required to resolve phasing and ambiguity problems. Next-generation sequencing (NGS) permits sequencing of the entire HLA gene and generates unambiguous and phased-resolved HLA typing results up to four field levels. Here we describe an erroneous HLA-B allele typing by Sanger sequencing. Sanger sequencing (Genome Diagnostics B.V.) of an unrelated NMDP donor showed the following possible HLA types: B ∗ 15:01:01,18:14 or B ∗ 15:07:01,18:01:01 or B ∗ 15:07:01,18:17 N or B ∗ 15:07:03,18:01:10 . The B ∗ 15:01:01, B ∗ 15:07:01, B ∗ 18:01:01 , and B ∗ 18:14 are common and well-documented alleles. The high-definition reverse sequence-specific oligonucleotide (rSSO) typing (One Lambda) ruled out all possible types except B ∗ 15:01:01,18:14 . The rSSO bead for B ∗ 15:07:01 showed reactivity below the specified cutoff. We performed NGS typing on this sample, which enabled phasing of polymorphic positions 363 and 485 in exon 3 resulted unambiguous B ∗ 15:07:01,18:01:01 typing. In summary, the strategy of Sanger sequencing plus rSSO typing resulted in incorrect HLA-B assignment for the NMDP donor, which in turn caused the exclusion of matched donor. The incorrect HLA allele assignment can be detrimental to HSCT recipient survival. NGS provides the capability to phase polymorphisms thereby eliminating most ambiguities and provides high-resolution HLA typing without reflexive, time-consuming and expensive additional testing, and therefore NGS is the new gold standard for allele-level HLA typing.

Keywords: rsso typing; erroneous hla; hla typing; hla; hla allele; sanger sequencing

Journal Title: Human Immunology
Year Published: 2017

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