P128 Identification of novel A24 allele without subcloning

Abstract

Aim To identify the mutation in one of the alleles at HLA-A locus in African American kidney transplant recipient. Methods Initial evaluation was done using OneLambda’s PCR-SSOP. Sequencing kits from GenDx’s AlleleSEQR (HLA-A) and Protrans’ Domino Stones 2.0 (HLA-A4) were used to sequence the DNA. GenDx SBT Engine Software was used for sequencing data analysis. BLAST search was performed using IMGT database. Allele specific re-sequencing was done using primers tailed with M13 sequence. The 5′ primer was located in the second exon (240–257) and 3′ primer (540–557) was located in the 2nd intron. Results PCR-SSOP listed A∗23:01 allele in the possible genotypes, but final typing was inconclusive (“No Match”). Exclusion of false positive reacting beads resulted in A∗23:01 homozygous typing. Olerup’s PCR-SSP confirmed presence of A∗23:01 allele. However, SBT Engine indicated presence of potentially new allele. Knowledge of A∗23:01 sequence helped in discriminating the known (A∗23:01) from the unknown A∗New heterozygote at exons 1–4. A∗23:01 and A∗ New were identical at exons 1, 3 and 4. BLAST search of A∗New allele gave lowest score/highest similarity with A∗24:24. Data from direct sequencing of A∗24:New allele (M13 primers) identified a base substitution at 282 position (codon 70), G->C. This mutation leads to amino acid change from Glutamine (CAG) to Histidine (CAC). The sequence of novel HLA-A∗24 allele was submitted to IMGT/HLA database and designated HLA-A∗24:392 by the WHO Nomenclature Committee in December, 2017. GenBank Database assigned Accession Number BankIt 2058784 RBHLA MG463105. Thus, specimen’s HLA typing is HLA-A∗23:01, A∗24:392; B∗07:02, 50:01 and HLA-DRB1∗08:06, 09:01. Conclusions Three-dimensional structure analysis of the HLA protein (Fig. 1) encoded by A∗24:392 showed that it is likely that mutation at codon 70 (α-helical coil) is involved in peptide binding, and mismatched 70HT epitope can elicit antibody production.