PS1110 THE IDENTIFICATION OF A COMMON LOSS‐OF‐FUNCTION MUTATION ACROSS DIFFERENT HLA CLASS ALLELES FREQUENTLY DETECTED IN PATIENTS WITH ACQUIRED APLASTIC ANEMIA
Authors: H. Mizumaki, K. Hosomichi, M. Tanabe, T. Yoroidaka, T. Imi, K. Hosokawa, T. Katagiri, H. Takamatsu, T. Ozawa, F. Azuma, H. Kishi, A. Tajima, S. nakao
Journal: HemaSphere (2019)

Background: The presence of HLA class I allele‐lacking leukocytes due to the copy‐number neutral loss of heterozygosity in the short arm of chromosome 6 (6pLOH) or HLA allelic mutations is compelling evidence supporting the notion that cytotoxic‐T lymphocyte attack on hematopoietic stem and progenitor cells (HSPCs) underlies the development of acquired aplastic anemia (AA). We previously reported that deep sequencing of HLA class I genes of HLA‐class I allele‐lacking granulocytes revealed various loss‐of‐function mutations in alleles such as HLA‐B ∗ 40:02 , A ∗ 02:06 , and A ∗ 31:01 , suggesting that limited HLA‐class I alleles are involved in the autoantigen presentation of AA (Zaimoku, et al. Blood . 2017;129:2908–16; Mizumaki, et al. Blood . 2018;132:2584). Of interest, our previous studies have revealed a common nonsense mutation in exon1 of the HLA‐A and B genes ( Exon1 mut ) leading to the loss of specific HLA alleles across different class I alleles in AA patients harboring 6pLOH. If the Exon1 mut is frequently detected with a sensitive assay in AA patients regardless of the presence of 6pLOH, it would serve as a useful indicator of the presence of an immune pathophysiology of bone marrow failure. Aims: To determine the prevalence and clinical significance of Exon1 mut in AA, we established a novel droplet digital polymerase chain reaction (ddPCR) assay for precisely detecting Exon1 mut in the peripheral blood of AA patients and investigated the correlation of the mutated sequence with the response to immunosuppressive therapy (IST). Methods: Exon1 regions of HLA‐A and HLA‐B alleles were amplified using two different sets of primer pairs that are complementary to the consensus sequences of the HLA class I alleles. The amplicons were subjected to a ddPCR assay using TaqMan probes with substituted locked nucleic acid bases complementary to wild‐type (WT) and Exon1 mut sequences, which were labeled with different fluorochromes (FAM and HEX). The fractional abundance of the mutant allele was obtained by dividing the number of copies per microliter of the mutant allele (FAM) by the total copies per microliter of the WT allele (HEX) plus the mutant allele (FAM). Peripheral blood leukocytes from 248 patients with AA who were 14–93 years of age (97 with severe AA and 151 with non‐severe AA; 115 males and 133 females; 80 6pLOH[+] and 168 6pLOH[‐]) were subjected to the ddPCR assay, and the correlation between the presence of Exon1 mut and response to immunosuppressive therapy in newly diagnosed AA patients was examined. Results: Mixing experiments showed the ddPCR assay was able to detect as few as 0.07% leukocytes with Exon1 mut in the background WT leukocytes. Exon1 mut was detected in 51 (64%) of the 80 6pLOH(+) patients; their lost alleles were A ∗ 02:06 (n = 11), A ∗ 31:01 (n = 3), B ∗ 40:02 (n = 23), B ∗ 54:01 (n = 6), and other alleles (n = 8). Exon1 mut was also detected in 35 (21%) of the 168 6pLOH(‐) patients. The median variant allele frequencies of Exon1 mut was 0.59% (range, 0.074% to 8.70%). Overall, 86 (35%) of the 248 patients with or without 6pLOH were positive for Exon1 mut . Fifty Exon1 mut (+) patients were treated with IST (antithymocyte globulin+cyclosporine [CsA], n = 30 or CsA alone, n = 20), and 43 (86%) showed a response. Summary/Conclusion: HLA allelic loss due to Exon1 mut is common in AA patients. The presence or absence of this mutated allele can be determined using the ddPCR assay within 8 h without any information about patients’ HLA genotypes. This assay is therefore thought to be a powerful tool for diagnosing an immune pathophysiology of bone marrow failure.