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Ethnic features of DDX41 mutations in ICUS/MDS/AML
most other cases of cancers with germline predisposing mutations typically develop in adolescence or early adult- hood.5-11
The DDX41 gene is located at 5q35.3 and encodes a DEAD-box RNA helicase, which is involved in pre-mRNA splicing, RNA processing, and ribosome biogenesis.12 Several mechanisms have been proposed to explain the contributions of DDX41 mutations to the development of hematologic malignancies. DDX41 mutations can: (i) cause aberrant mRNA splicing leading to exon retention or exon skipping, (ii) disrupt the STING-interferon pathway; and (iii) induce aberrant pre-rRNA trimming and ribosome biogenesis.3 DDX41 mutations include both germline and somatic mutations, with the latter being found in over half of the patients with germline mutations in the other allele of DDX41.5 In recent studies, germline DDX41 variants were found in 2.4% of 1,385 patients with MDS or AML,11 and germline or somatic DDX41 variants were found in 3.4% of 1,002 patients with myeloid neo- plasms.10
Following advances in genetic testing, clinical next-gen- eration sequencing (NGS)-based leukemia panels are being increasingly used to identify somatic mutations to facilitate the diagnosis, improve prognostication, and select optimal treatment strategies in patients with hema- tologic malignancies. Some variants found in the panels can also be germline mutations in genes associated with hereditary hematopoietic malignancies.13-15 The myeloid leukemia panel used at our institute includes the DDX41 gene, and the frequencies of DDX41 mutations in Korean patients with MDS or AML appeared to be higher than the previously reported incidences. Importantly, DDX41 mutations have not been evaluated in patients with idio- pathic cytopenia of undetermined significance (ICUS), which is a known precursor lesion of MDS. In this study, we investigated the incidence, genetic characteristics, and clinical features of DDX41 mutations in Korean patients with ICUS, MDS, or AML.
Methods
Patients
We included patients with ICUS, MDS, or AML whose bone marrow samples were collected between 2009 and 2019 at Asan Medical Center (Seoul, Korea). Patients with ICUS or lower-risk MDS were either prospectively enrolled (since January 2018) or retrospectively analyzed, while those with higher-risk MDS or AML were retrospectively analyzed. All patients in the study cohort were unrelated individuals, not including an index case and his or her family members. Diagnoses of MDS and AML were based on the WHO 2016 Classification.2 ICUS was defined by the proposed criteria of the 2007 Consensus Group:16 cytopenia in one or more of cell lineages for ≥6 months (hemoglobin <11 g/dL, neu- trophils <1.5x109/L, and platelets <100x109/L) while excluding other causes of cytopenia such as a history of pelvic irradiation or cytotoxic chemotherapy, splenomegaly, heart failure or liver cir- rhosis with portal hypertension, active viral infections, and a his- tory of blood or bone marrow diseases. Clonal cytopenia of unde- termined significance was defined as ICUS with myeloid neo- plasm-related somatic mutations with a variant allele frequency ≥ 2%, or clonal karyotypic abnormalities. Myeloid neoplasm-relat- ed somatic mutations were based on those specified in the updat- ed National Comprehensive Cancer Network guideline for MDS.17
The Institutional Review Board of Asan Medical Center approved the protocols of this study (2018-0042 and 2018-0048 [for prospective and retrospective analysis of patients with lower- risk MDS or ICUS], 2019-0794 [for sequencing the DDX41 gene in DDX41-mutated patients and their family members], and 2020- 0131 [for retrospective analysis of data from patients with higher- risk MDS or AML]), which was carried out in accordance with the 2008 Declaration of Helsinki.
Mutational and cytogenetic analysis
For rhe NGS assay, we prepared the sequencing libraries from genomic DNA using customized probes (Integrated DNA Technologies, Inc., Coralville, IA, USA) to capture and enrich the entire coding regions of 61 target genes (HEMEaccuTest DNA Target Enrichment kit; NGeneBio, Seoul, Korea) (Online Supplementary Table S1). We carried out sequencing on the MiSeqDx (Illumina, San Diego, CA, USA) with 2×150 bp, paired- end reads according to the manufacturer’s instructions. Initial read mapping was carried out against the human reference genome (hg19/GRCh37). We subsequently analyzed the sequencing data for variant calling using commercial software (CLC Genomics Workbench; QIAGEN Bioinformatics, Redwood City, CA, USA). We retained the potentially pathogenic variants by filtering out common polymorphisms (minor allele frequency in the popula- tion ≥1%) and sequencing/mapping errors, and by filtering in the known oncogenic variants based on the available population or cancer mutation-specific databases. We set the minimum cutoff of variant allele frequency at 2.0% for reporting. We performed the cytogenetic analysis using conventional G-banding techniques based on the analysis of 20 or more metaphase cells.
Germline variant confirmation and determination of causality
Variants with allele frequencies between 40% and 60% were considered to be probable germline mutations. We performed germline-based testing in 11 of 34 patients with probable germline DDX41 mutations using sorted blood T cells. This strategy of using sorted T cells was based on recent work confirming that T cells yield sufficient DNA and high rates of somatic variant calls in MDS. It was suggested that, given the challenge of obtaining skin biopsies, T cells would be preferential germline tissues for MDS genomic studies.18 Peripheral blood mononuclear cells were harvested by standard Ficoll (GE Healthcare, Sweden) density gra- dient centrifugation, and T cells were isolated using the Pan T Cell Isolation Kit, human (MACS Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions. The isolated T cells were analyzed with CD3-FITC using a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA), and genomic DNA was puri- fied by the QIAamp DNA mini kit (Qiagen, QIAGEN GmbH, Germany). The pathogenicity of probable germline DDX41 mutation was determined according to the guideline from the American College of Medical Genetics and Genomics (ACMG).19 The concurrence of a somatic DDX41 muta- tion was considered as strong evidence for causality. Thus, we classified germline DDX41 variants as “causal” if they were either pathogenic (or likely pathogenic) by the ACMG guideline or accompanied by a somatic DDX41 mutation regardless of the ACMG interpretation.
Statistical analysis
Categorical variables were compared using the χ2 test or Fisher exact test, and continuous variables were compared using the Mann-Whitney U-test or the Student t-test, as appropriate. Survival was calculated by the Kaplan-Meier method and the resulting survival curves were compared using the log-rank test
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