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Genome analysis for MDS among A-bomb survivors
tions are sequentially acquired de novo with age, and lead to the development of MDS through the aging-related hematopoietic condition called clonal hematopoiesis of indeterminate potential (CHIP).5-8
Chemotherapy and radiotherapy are well-known risk factors for the development of myeloid neoplasms (thera- py-related myeloid neoplasms, t-MN) including MDS (t-MDS), and the clinical and genetic features of t-MDS are different from those of de novo MDS with some overlap.9 For example, response to treatment and survival rates are very poor for t-MDS, and the karyotypes of t-MDS fre- quently show deletions of the long arms or the whole of chromosomes 5 and 7, often associated with complex karyotypes.10 The most frequently mutated gene in t-MDS is TP53 followed by RUNX1. However, TET2 mutations are less frequent in t-MDS than in de novo cases. Several reports have demonstrated that chemotherapy/radiotherapy provides an opportunity for the proliferation of pre-existing hematopoietic stem cells carrying mutations in genes such as TP53 and RUNX1, which eventually progresses to overt t-MN.11
Atomic bomb (A-bomb) radiation has also been report- ed to be a risk factor for developing MDS, with the degree of risk being associated with radiation dose exposure and distance from the hypocenter.12 Our previous reports showed an increase in chromosomal aberrations and com- plex karyotypes in MDS among the proximally exposed survivors. However, the median survival time and time to progression to leukemia did not differ between the proxi- mally and distantly exposed groups.13 Detailed compar- isons of chromosomal aberrations between A-bomb sur- vivors and unexposed patients with MDS demonstrated that structural alterations in chromosomes 3, 8, and 11 were significantly increased in MDS among survivors, while alterations in chromosomes 5 and 7 were equally frequent in both groups.14
These observations suggest that MDS among A-bomb survivors may have a different pathogenesis compared with de novo and t-MDS cases, which may be reflected by their different patterns of genome alterations. To address these issues, we analyzed MDS among A-bomb survivors using next generation sequencing technologies. We found different profiles of driver mutations among proximally exposed patients, as well as frequent deletion of the long arm of chromosome 11 associated with aberrations of ATM.
Methods
We analyzed 32 patients diagnosed as having MDS, and three patients as idiopathic cytopenia of undetermined significance among A-bomb survivors (Online Supplementary Table S1), and we divided them into two groups: patients exposed within 2.7 km of the hypocenter were categorized as the proximally exposed (PE) group, and the others as the distally exposed (DE) group according to the approach adopted by the Radiation Effect Research Foundation15 (Online Supplementary Methods). In this study, we compared clinical/genome data between PE and DE groups because these two groups would have lived in similar circum- stances (stayed in Nagasaki after A-bomb under similar environ- mental circumstances including medical access) except for the dose of A-bomb radiation, more than 5mGy (at 2.7 km) or less, which suggested DE as an appropriate control for PE. Based on our previous epidemiological analysis, excess relative risk (ERR) of
MDS among survivors that had been exposed to 5mGy would be 0.02 (ERR, 4.3 / Gy).12 Several different sequencing methods were applied in this study depending on the amount and the quality of DNA samples (Online Supplementary Tables S1 and S2).
First, we performed whole exome sequencing (WES) with matched germline controls for five patients in the PE group, coded as unbiased-WES (U-WES), then WES without matched germline controls (B-WES-T) for three and eight patients in the PE and DE groups, respectively. Limited number of genes (356 genes) were validated for B-WES-T, which were putative drivers of hematolog- ic malignancies3,4,16,17 or candidate genes identified through U- WES (Online Supplementary Table S3). Targeted capture sequencing (T-S) of 154 genes was performed for another ten and nine patients in the PE and DE groups, respectively, without matched controls. Target genes were selected based on published data3,4,16,17 and results of the U-WES cases (Online Supplementary Table S4).
We also investigated three cases (U-WES-3, 4, and 7) using whole genome sequencing (WGS) with matched germline con- trols (Online Supplementary Methods and Online Supplementary Table S5).
The DNA copy number alterations (CNA) were analyzed with a SNP array (CytoScan HD Array, Affymetrix, Santa Clara, CA, USA), and CNA in T-S cases were identified in the sequencing data using the CNACS pipeline,18 because of the insufficient qual- ity and quantity of DNA for SNP array. Although copy number states of whole chromosomes could not be evaluated, frequently affected regions in MDS, such as the long arms of chromosome 5 (5q), 7q, and 20q, were included among the targets. We also tar- geted the genes affected by 11q deletion to evaluate the whole arm of 11q because this region was of interest in this study. This study was approved by the ethics committee of Nagasaki University.
Further details of the methods used for this study are available in the Online Supplementary Methods.
Results
Clinical features of patients in this study
The major clinical characteristics of the 35 patients who took part in this study are listed in Table 1 with further details provided in Online Supplementary Table S1. The median exposure distance from the hypocenter was 1.1 km in the PE group and 3.4 km in the DE group (P<0.001). There were no significant differences in the sex, subtype of MDS, age at diagnosis, or age at the time of the bomb- ing between the two groups. The frequencies of abnormal karyotype and complex karyotype were higher in PE but without statistical significance (Table 1). There was no dif- ference in survival time after diagnosis between two groups (P=0.652) (Online Supplementary Figure S7).
Somatic mutations, mutational spectrum, and clonal architecture of myelodysplastic syndromes in the proximally exposed group
Among the five patients (U-WES-3, 4, 5, 7, 8) in the PE group who were analyzed using WGS and/or WES, we identified 5-15 somatic missense and nonsense SNV (mean 9.2 per sample), and 0-2 somatic INDEL (insertions or deletions; mean 1 per sample) on coding exons (Figure 1 and Online Supplementary Table S6) per patient. The number of somatic SNV identified using WGS of three patients (U-WES-3, 4 and 7) in the PE group was 1,695, 573, and 756, respectively (Online Supplementary Tables S7- 1, -2, and -3). The most frequent pattern of nucleotide sub-
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