Complete nucleotide sequence characterization of DRB5 alleles reveals a homogeneous allele group that is distinct from other DRB genes

• Published in: Human immunology 2019-07, Vol.80 (7), p.437-448
• Main Authors: Barsakis, Konstantinos, Babrzadeh, Farbod, Chi, Anjo, Mallempati, Kalyan, Pickle, William, Mindrinos, Michael, Fernández-Viña, Marcelo A.
• Format: Article
• Language: English
• Subjects: 5' Untranslated Regions - genetics; Alleles; Animals; Base Sequence - genetics; Cell Line; Cercopithecidae - genetics; Cloning, Molecular; De novo assembly; Exons - genetics; Gene conversion; Genetic Loci; Genotype; Haplotypes - genetics; High-Throughput Nucleotide Sequencing; Histocompatibility Testing; HLA-DRB5; HLA-DRB5 Chains - genetics; Humans; Introns - genetics; Next Generation Sequencing; Pan troglodytes - genetics; Phylogeny; Polymerase Chain Reaction; Sequence Analysis, DNA; Single tandem repeats
• ISSN: 0198-8859; 1879-1166
• DOI: 10.1016/j.humimm.2019.04.001

Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. A better and comprehensive sequence assessment can be achieved by the inclusion of full gene sequences of all the common alleles at a given locus. The common alleles of DRB5 are under-characterized with the full exon-intron sequence of two alleles available. In the present study the DRB5 genes from 18 subjects alleles were cloned and sequenced; haplotype analysis showed that 17 of them had a single copy of DRB5 and one consanguineous subject was homozygous at all HLA loci. Methodological approaches including robust and efficient long-range PCR amplification, molecular cloning, nucleotide sequencing and de novo sequence assembly were combined to characterize DRB5 alleles. DRB5 sequences covering from 5′UTR to the end of intron 5 were obtained for DRB5*01:01, 01:02 and 02:02; partial coverage including a segment spanning exon 2 to exon 6 was obtained for DRB5*01:03, 01:08N and 02:03. Phylogenetic analysis of the generated sequences showed that the DRB5 alleles group together and have distinctive differences with other DRB loci. Novel intron variants of DRB5*01:01:01, 01:02 and 02:02 were identified. The newly characterized DRB5 intron variants of each DRB5 allele were found in subjects harboring distinct associations with alleles of DRB1, B and/or ethnicity. The new information provided by this study provides reference sequences for HLA typing methodologies. Extending sequence coverage may lead to identify the disease susceptibility factors of DRB5 containing haplotypes while the unexpected intron variations may shed light on understanding of the evolution of the DRB region.