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A common HLA nonsense mutation in aplastic anemia
We previously used targeted deep sequencing to identify frequent loss-of-function mutations in three HLA class I alleles, B*40:02, A*02:06, and B*54:01.9,16,17 The highly sensi- tive ddPCR assay described herein that was capable of detecting Exon1mut newly identified three HLA-A (A*02:01, A*02:07, A*31:01) and six HLA-B alleles (B*13:01, B*40:01, B*40:03, B*44:03, B*55:02, B*56:01) as HLA alleles that are susceptible to allelic loss. Compared with their frequency in the general Japanese population, these HLA alleles were found to be highly enriched in AA patients. Among the 14 HLA class I supertypes that are defined based on similarities in the antigen-presenting peptide motif, the 12 alleles men- tioned above belong to only four of the supertypes.21 These findings suggest that autoantigens of AA may be presented to T cells by these specific HLA alleles on HSPC.
Like HSPC positive for 6pLOH, those positive for Exon1mut are thought to escape the attack of cytotoxic T lymphocytes specific to autoantigens presented by the missing HLA-A or HLA-B allele and contribute to hematopoiesis over the long-term. However, it is unclear why Exon1mut occurs more frequently in HSPC than loss-of- function mutations in other positions of HLA class I alle- les. Shukla et al. reported different hotspots of mutations in class I HLA genes according to cancer type, and identi- fied Exon1mut only in head and neck squamous cell can- cers.24 HSPC may thus share a common property in that Exon1mut is likely to occur in class I HLA genes in head and neck squamous cell cancers.
The median VAF of Exon1mut in patients with Exon1mut patients was only 0.42%, a level that cannot be detected by targeted deep sequencing. This low VAF was in sharp contrast to the high proportion of concomitant 6pLOH in individual patients (Figure 3C). We previously reported that 6pLOH+ leukocytes were often polyclonal, consisting of leukocytes having different breakpoints of uniparental disomy in the short arm of chromosome 6.10 This poly- clonality may account for the high proportion of 6pLOH. Although the leukocytes with Exon1mut represent a minor leukocyte population, the long-term (1-7 years) persist- ence of these mutated leukocytes indicates that they are derived from HSPC with self-renewal capacity. Arends et al. showed that clone size of cells with somatic mutations of epigenetic regulation genes expanded from most imma- ture hematopoietic stem cells to mature peripheral blood cells in patients with clonal hematopoiesis of indetermi- nate potential.26 Leukocyte positive for Exon1mut may also be derived from most immature hematopoietic stem cells. The persistence of similarly minor clones in peripheral blood has been reported for GPI– granulocytes in AA, the median frequency of which was 0.25%.18,27 In contrast to PIGA-mutated or 6pLOH+ leukocytes, which can be oligo- clonal and dysfunctional due to the lack of all GPI- anchored proteins or a large segment of 6p, Exon1mut-posi- tive leukocytes are derived from a single HSPC that is phe- notypically normal except for the lack of one HLA allele. According to Dingli’s hypothesis, approximately 400 HSPC are actively involved in human hematopoiesis.28 Thus, the small proportion of Exon1mut-positive leukocytes among the entire leukocyte population may reflect an average clone size of individual HSPC in the bone mar- row.
HLA-LL are useful markers that indicate the presence of an immune pathophysiology in patients with bone mar-
row failure. Here we showed a high response rate to immunosuppressive therapy in patients with Exon1mut, although patients without Exon1mut also had a high response rate likely due to the high prevalence of GPI– cells.29-31 Several methods can be used to detect HLA-LL, including flow cytometry assays with monoclonal anti- bodies specific to HLA-A or HLA-B alleles, ddPCR or SNP arrays for detecting 6pLOH, and targeted deep sequenc- ing.8-10,12 However, these methods require HLA typing of patients, take a long time to produce results, and are unable to detect HLA-LL that account for less than 1% of total leukocytes. The ddPCR assay used in the present study to detect Exon1mut enables the detection of HLA-LL accounting for as few as 0.07% of the total leukocyte pop- ulation within 6 h of blood collection, highlighting the powerful nature of this assay for diagnosing immune pathophysiology in patients with bone marrow failure.
Disclosures
No conflicts of interest to disclose.
Contributions
HM, TI, KT, YZ and SN collected clinical data and blood samples. FA performed HLA genotyping. YF and SO conducted the SNP array analyses. YZ, HT, TO, HK and AM generated an original monoclonal antibody specific to HLA-B13, B60 and B61. HM and TY performed cell sorting. HM, KHosomichi, TI, YZ and AT performed deep sequencing. HM, YZ, NMA and TCD performed the droplet digital polymerase chain reaction. KC and YY generated the induced pluripotent stem cells. MIE performed the in vitro experiments. HM, KHosokawa and SN designed the research and wrote the manuscript. All authors crit- ically reviewed the manuscript and checked the final version.
Acknowledgements
The authors thank the patients and donors and their physi- cians, including M. Yamaguchi of Ishikawa Prefectural Central Hospital of Kanazawa, Ishikawa, T. Takaku of Juntendo University Hospital of Bunkyo-ku, Tokyo, H. Yano of Kainan Hospital of Yatomi, Aichi, K. Watamoto of Komaki City Hospital of Komaki, Aichi, M. Mizutani of Matsusaka Central General Hospital of Mastusaka, Mie, S. Morishima of University of Ryukyu Hospital of Nishihara, Okinawa, and Mikko Ker¬änen, Sofie Lun¬d¬gren and Satu Mustjoki of the University of Helsinki, Helsinki, Finland for sending their patients’ samples for screening for Exon1mut, and the Advanced Preventive Medical Sciences Research Center, Kanazawa University for the use of facilities. HM is a PhD candidate at Kanazawa University and this work is submitted in partial ful- fillment of the requirements for the PhD.
Funding
This work was supported by MEXT KAKENHI (Grant-in- Aid for Scientific Research [B], grant number: 16H05335 and 19H03686) to SN, MEXT KAKENHI (Grant-in-Aid for Young Scientists [B], grant number: 17K16184) to K.Hosokawa, MEXT KAKENHI (Grant-in-Aid for Scientific Research [C], grant number: 17K09007) to TK, MEXT KAK- ENHI (Grant-in-Aid for Scientific Research on Innovative Areas, grant number: 16H06502 and 19H05344) to K. Hosomichi., Hokuriku Bank Research Grant for Young Scientists to TK and Hokkoku Foundation for Cancer Research to TK and KHosokawa.
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