Full‐length HLA‐DRB1 coding sequences generated by a hemizygous RNA‐SBT approach

Abstract

Currently 1582 HLA-DRB1 alleles have been identified in the IMGT/HLA database (v3.18). Among those alleles, more than 90% have incomplete allele sequences, which complicates the analysis of the functional relevance of polymorphism beyond exon 2. The polymorphic index of each individual exon of the currently known allele sequences, shows that polymorphism is present in all exons, albeit not equally abundant. Full-length HLA-DRB1 RNA sequencing identifies polymorphism of the complete coding region. Here we describe a hemizygous full-length RNA sequence-based typing (SBT) approach based on group-specific HLA-DRB1 amplification and subsequent sequencing. RNA full-length sequences can easily be accessed because of the short amplicon length (801 bp). The RNA-SBT approach was successfully validated on a panel of DRB1 alleles having fully known coding sequences according to the IMGT/HLA database, and cover all serological equivalents. Subsequently, the approach was applied on a panel of 54 alleles with incomplete allele sequences, resulting in full-length coding sequences and the identification of one new and one corrected allele. This study shows the universal applicability of the RNA-based sequencing approach to identify full-length coding sequences and to define the polymorphic content of HLA-DRB1 alleles.

The role of gene polymorphism in HLA class I splicing

“…One of the risk factors in this matter is that a synonymous nucleotide substitution is accepted for bone marrow transplantation, with no knowledge about the effect of this substitution on splicing and/or on expression levels. Routinely applicable RNA sequencing approaches as previously published by our group for HLA class I, DRB1 and DPB1 (Lauterbach et al ., ; Gerritsen et al ., , ) might be a first start to determine transcript sequences and to detect transcriptional aberrations.…”