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A common HLA nonsense mutation in aplastic anemia
alleles contained in the lost haplotype due to 6pLOH that was accompanied by Exon1mut (Online Supplementary Figure S3). The low VAF of Exon1mut (VAF <1%) was confirmed by deep sequencing with unique molecular identifiers (xGen® Dual Index UMI Adapters: Integrated DNA Technologies, IA, USA).19 The correla- tion between Exon1mut’ VAF determined by deep sequencing with unique molecular identifiers and those determined by the ddPCR assay was examined using 24 different samples (Online Supplementary Figure S4). HLA class I alleles acquiring Exon1mut were determined using the nearest allele-specific SNP. Details on deep
A
sequencing with unique molecular identifiers are provided in the Online Supplementary Methods.
Statistical analysis
Comparisons were performed using the Fisher exact test for cat- egorical variables and Mann-Whitney U test for continuous vari- ables with a two-tailed significance level of 0.05. Statistical analy- ses were performed using the EZR software program.20 Graphs were generated using GraphPad PRISM7.0 (GraphPad Software Inc, CA, USA).
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Figure 1. Identification of Exon1mut in patients with aplastic anemia. (A) Exon1mut [p.R7*(c.19C>T)] was detected by targeted deep sequencing of sorted HLA-A2– gran- ulocytes (UPN 262) and HLA-A2+B60– granulocytes (UPN 211) in two patients with aplastic anemia. Sequencing results of sorted HLA-allele lacking leukocytes from these two patients and germline controls and flow cytometry results of granulocytes are shown. (B) Loss-of-function mutations detected in 14 patients by targeted deep sequencing. Exon1mut was detected in HLA-A alleles of eight patients and HLA-B alleles of four patients. UPN: unique patient number.
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