Identification of the Novel HLA-A*24:518N Null Allele and Evaluation of its Cell Surface Expression on Lymphocytes.

Abstract

Here we describe the identification of a new HLA-A allele, A*24:518N by next-generation sequencing (NGS) and the confirmation of a lack of expression as measured by flow cytometry. A patient of Filipino ancestry was a candidate for allogeneic hematopoietic cell transplantation because of polycythemia vera. HLA typing was performed by NGS (ScisGo HLA v.5 Kit, Scisco Genetics), and the HLA assignments were as follow: A*24:518N(NEW), A*11:01; B*15:35, B*40:01; C*07:02, C*04:82; DRB1*15:02, DRB1*09:01; DQB1*05:02, DQB1*03:03; DPB1*05:01, DPB1*01:01. NGS revealed an insertion of guanine at either codon 45 or 46 (positions 207–209) in exon 2 of HLA-A*24:02:01:01 (Figure 1A), with codon 46 changing from GAG to GGA. Zero reads aligned to A*24:02:01:01, whereas 700 reads aligned to A*24:02:01:01 with a “G” insertion. This insertion was confirmed by sequence-based typing. The insertion resulted in a frameshift, with codon 74 changing from aspartic acid (GAC) to a stop codon (TGA). This was predicted to lead to a truncated HLA-A24 protein. The new allele was named A*24:518N (accession# HLA29436)1 by the World Health Organization Nomenclature Committee for Factors of the HLA System in December 2020.2 This insertion was absent in all HLA-A alleles, but the newly discovered A*24:529N (insertion in codon 30) has a similar truncated protein sequence. Nonfunctional HLA is a plausible mechanism of immune escape in blood cancers.3 Inheritance of this new null allele was confirmed by HLA typing of the father and DNA isolated from the patient’s buccal brush sample.FIGURE 1.: A, A representative alignment comparing exon 2 of HLA-A*24:02:01:01 to A*11:01:01:01 and A*24:518N. Highlighted in blue is the guanine insertion at codon 45/46, unique to HLA-A*24:518N. B, Depiction of the cellular expression of HLA-A24 (blue line) on the surface of CD3+ T cells (B lymphocytes not shown) as measured by flow cytometry. The HLA-A11 antibody (red line) was included to establish normal expression of HLA for this patient (patient #1). Background fluorescence was assessed with mouse IgG1 (black dashed line). Reference expression levels of A11 and A24 were obtained using cells from donor #1 (A*24:02 heterozygous) and donor #2 (A*11:01 heterozygous). C, Lists of the patient’s genotype frequencies relative to the NMDP donor populations: API, Filipino, and HAWI. “A*24 null genotype” depicts the patient’s genotype when considering A*11 to be homozygous. API, Asian-Pacific Islander; HAWI, Hawaiian; IgG1, immunoglobulin G1; NMDP, National Marrow Donor Program.In HLA alleles, premature termination codons from exons 1 to 4 are well known to be associated with reduced mRNA transcripts.4 Therefore, to confirm the absence of cell surface expression of A*24:518N, we tested the patient’s lymphocytes by flow cytometry using a monoclonal antibody specific for A23/24 (One Lambda). Antibodies against CD3 and CD19 (BD Biosciences) were included to distinguish T and B lymphocytes. This study was reviewed by an institutional board. Flow cytometric testing revealed that HLA-A*24:518N was not expressed on the surface of the patient’s lymphocytes above that of background controls (mouse immunoglobulin G1; Abcam) (Figure 1B). However, the expression level of HLA-A11 (anti-HLA-A11; One Lambda) on patient’s lymphocytes was comparable with that on the donor’s lymphocytes (donor 2). The impact of a null allele in a hematopoietic cell transplantation setting is immediate as this patient would now be considered A*11 homozygous in an unrelated donor search. As a result, the number of potential 10 of 10 National Marrow Donor Program donors (with 75% probability) drops from 4 to 0. Haplostat (www.haplostats.org) also shows a considerable decrease in the genotype frequency (Figure 1C).5 This is most evident in the Filipino donor population as the patient’s genotype frequency with A*24:02 (original genotype) is 0.013% but drops to 0.0003% when A*11 homozygosity is factored in the A*24 null genotype. Even from a bigger pool of 100 000 Filipino donors, the calculated number of 10 of 10 matched donors would change from 13 to 0 using a binomial distribution formula. In summary, we identified a novel null A*24 allele (A*24:518N) caused by an insertion in exon 2. Analysis by flow cytometry confirmed the lack of cellular A*24:518N expression. This case report emphasizes the importance of confirming the inheritance of new HLA mutations in donor selection. ACKNOWLEDGMENTS The authors would like to thank Amanda Fulton for her assistance in the NMDP donor search and Dr Arnab Chowdhury for his assistance with binomial distribution calculation.