P132 Complete nucleotide sequence characterization of DRB5 alleles indicate a homogeneous allele group which is distinct from other DRB genes

• Published in: Human immunology Vol. 77; p. 133
• Main Authors: Barsakis, Konstantinos, Babrzadeh, Farbod, Chi, Anjo, Mindrinos, Michael N., Fernandez Vinã, Marcelo A.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.09.2016
• Subjects: Allergy and Immunology
• ISSN: 0198-8859; 1879-1166
• DOI: 10.1016/j.humimm.2016.07.197

Next Generation Sequencing allows for testing and typing of entire genes of the HLA region. More complete sequence assessment can be achieved by the description of the entire common alleles at a given locus. The common alleles of DRB5 are under-characterized with the full exon-intron sequence of only one allele available. The haplotypes bearing DRB5 genes associate with susceptibility and resistance to many diseases; furthermore, the role of DRB5 mismatches in transplantation has not been utterly evaluated. In the present study, we cloned and sequenced DRB genes from 87 individuals including 18 subjects with DRB5 alleles. We developed robust and efficient methods for long range PCR and molecular cloning. Clones were sequenced by NGS and consensus was generated by a novel de novo assembly algorithm. The fragments generated overlapped and span exons 1–2 and 2–6 of DRB genes. We obtained DRB5 sequences spanning 12,638-12,681 of DRB5∗01:01, 01:02 and 02:01; partial information for the segment exon 2–6 was obtained for DRB5∗01:03, 01:08N and 02:03. Phylogenetic trees show that DRB5 alleles, group together and have distinctive differences with other DRB loci, with only 65, 73 and 83 percent homology with DRB1∗15, DRB4 and DRB3 alleles respectively. All DRB5 alleles are similar with at least 97.0 percent homology. Among 7 subjects we identified 4 intron variants of DRB5∗01:01, 3 differing in length in one intron2 STR and additional allele differing from the others by 1 SNP substitution. We identified 2 intron variants of DRB5∗01:02; with exception of the known exon substitutions one DRB5∗01:02 variant is identical to DRB5∗01:03 and DRB∗01:08N differs in introns from DRB5∗01:02 by length in one STR. Similarly, we identified two DRB5∗02:02 variants differing in length at the same STR while DRB5∗02:03 is identical in the intron sequences to one of these alleles. The DRB5 intron variants of each DRB5 allele were found in subject carrying distinct associations with alleles of DRB1, B and/or ethnicity. Our studies provide useful information that can be applied in NGS based typing. Unexpectedly, we identified intron variation that may shed light on understanding the evolution of the DRB region. Examination of DRB5 variants may also lead to identify the disease susceptibility factors of DRB5 containing haplotypes.