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A common HLA nonsense mutation in aplastic anemia
(Figure 2A), clearly showing the presence of HLA-LL in patients for whom flow cytometry analysis of monocytes using anti-HLA-A2 antibodies produced unequivocal results regarding the presence of HLA-A2-lacking mono- cytes (Figure 2B).
Prevalence of Exon1mut in patients with aplastic anemia Using two different ddPCR mixtures for HLA-A and HLA-B, the presence of Exon1mut was evaluated in all of the 353 patients. Exon1mut was detected in 101 (28.6%) of the 353 patients, with the median frequency of 0.42% (range, 0.071% to 21.3%). The prevalence of Exon1mut was similar in HLA-A and HLA-B alleles in both treatment-naïve patients (n=291) and treated patients (n=62). Among the AA patients with Exon1mut, those who had been treated had a higher median frequency of Exon1mut than those who were untreated (0.96% vs. 0.33%, P=0.0079) (Figure 3A). Figure 3B shows the relationships between the presence of cells with Exon1mut, 6pLOH and a paroxysmal nocturnal hemoglobinuria (PNH) phenotype in the patients. Fifty-six (55.4%) and 67 (66.3%) of the 101 patients with Exon1mut had 6pLOH and PNH phenotype cells, respectively. Ten (2.8%) of the 353 patients had Exon1mut alone. The frequen- cy of Exon1mut was much lower than that of 6pLOH in 36
patients possessing both clones (P<0.001) (Figure 3C). A
Long-term persistence of Exon1mut-positive cells
Serial blood samples were available for longitudinal analyses of Exon1mut in 13 patients who responded to immunosuppressive therapy (cyclosporine [CsA] alone in 6 and rabbit antithymocyte globulin [rATG] + CsA in 5) or anabolic steroids (n=2). Exon1mut was persistently detected for 14-86 months in nine patients, including one patient (UPN 299) who had been off treatment for 7 years, sug- gesting that Exon1mut-positive leukocytes are derived from long-lasting HSPC (Figure 4A). The VAF of Exon1mut increased in two (15%, UPN 333 and UPN 339), remained stable in four (31%), and decreased in three (23%) patients. Exon1mut became undetectable at 7-33 months after the first detection of Exon1mut in the other four patients, all of whom were being treated with CsA. Figure 4B shows a gradual decline of the Exon1mut frequency over
3 years in one patient (UPN 213).
HLA-A and HLA-B alleles that acquire Exon1mut
Among the 101 patients with Exon1mut, HLA alleles that acquired Exon1mut could be determined by targeted deep sequencing with (n=21) or without (n=37) unique molec- ular identifiers, or deduced from alleles contained in the lost haplotype due to 6pLOH that was accompanied by Exon1mut (n=10) (Online Supplementary Figure S2). In the
BC
Figure 3. Prevalence and clone size of Exon1mut in patients with aplastic anemia. (A) Mutant allele frequency of Exon1mut in 291 and 62 Exon1mut positive untreated and treated patients with aplastic anemia, respective- ly. Red bars indicate median allele frequencies of Exon1mut. (B) The prevalence of Exon1mut and its relationship with paroxys- mal nocturnal hemoglobinuria phenotype (PNH) and copy num- ber neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH). Exon1mut -positive patients accounted for 58% of 6pLOH+PNH+ patients and for 67% of 6pLOH+ patients. (C)
Exon1mut and 6pLOH in individual patients who were positive for both mutant clones. The frequencies of both clones were determined by droplet digital polymerase chain reaction analysis. *P<0.001. VAF: variant allele frequency; AA: aplastic anemia; UPN: unique
Frequency of patient number.
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