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Next‐generation sequencing reveals the presence of DDX41 mutations in acute lymphoblastic leukemia and aplastic anemia

Abstract Limited studies have been described DEAD‐box helicase 41 (DDX41) mutations in hematological diseases other than myeloid neoplasms. In this study, DDX41 mutations were identified in 0.8% of myeloid neoplasms, 0.9% of acute lymphoblastic leukemia (ALL), and 1.0% of aplastic anemia (AA). A total of 15 causal DDX41 variants in 14 patients were detected; seven of which have not been reported previously. In myeloid neoplasms, the median age of patients with germline missense was lower than that of germline nonsense mutations. In ALL, the characteristics of DDX41 mutation were distinct. This study first reported DDX41 mutations in ALL and AA, expanding its mutation and phenotypic spectrum.

firm criterion for causality. The fingernail specimens or blood samples in complete remission (CR) of acute leukemia were taken as a control to verify the possible germline origin.

Follow-up
The end of the follow-up period was December 30, 2020. CR was defined as morphologic CR. Overall survival (OS) is measured from the date of diagnosis to the date of death or the date of the last followup; relapse-free survival (RFS) is measured from the date of achievement of CR until the date of relapse, death, or to the date of the last follow-up.

Statistical analysis
Statistical analysis was performed using the SPSS 22.0 software, and chi-square or Fisher's exact tests calculated the significance between categorical data. A p-value of <0.05 was considered statistically significant.

The characteristics of DDX41 mutations
A total of 29 DDX41 variants were identified, including 22 germline variants and seven somatic variants. The germline variants were classified as causal (n = 8) and uncertain significance (n = 14) (Table S2); the latter were excluded in this assay. Seven causal variants (K102Rfs*32, S104F, L193P, Q210*, R282C, R323H, R471W) have not been reported previously. All of the germline variants were located on or upstream of the DEAD domain. Somatic DDX41 variants occurred throughout the whole coding region in ALL, but in the myeloid neoplasm, 80% of them were hotspot R525H ( Figure 1A).

DDX41 mutations in ALL and AA
DDX41 mutations were identified in 0.9% of the ALL cohort (1.0% of B-ALL (n = 6), 0.6% of T-ALL (n = 1)). The median age was 9 years (range, 4-27 years), and 56% were male. None of the patients had a family history of hematological malignancy and del 5/5q. Somatic (four missenses, one nonsense) and germline DDX41 mutations (two missenses) were respectively detected in five and two patients, which appeared to be mutually exclusive because no biallelic mutations were detected ( Figure 1B).
One case of AA in this cohort was observed with DDX41 muta-

Prognosis of patients with DDX41 mutations
Among six patients with AML, two received treatment, and the rest were abandoned after diagnosis. One relapsed after hematopoietic stem cell transplantation (HSCT) and received the second HSCT; the other one received 10 courses of chemotherapy and showed continuous no remission, then abandoned. Among five patients with ALL carried somatic DDX41 mutation, three relapsed and the median RFS was 21 months; four received HSCT (Table 1). Only one of ALL patients with germline DDX41 mutation was available to follow-up, which was continued to be minimal residual disease positive and then received HSCT. The patient with AA-PNH syndrome eventually received HSCT due to persistent, recurrent abdominal pain and dark-colored urine, and the need for platelet and red blood cell transfusion. At the time of this writing, the disease-free survival was 9 months after transplantation.

DISCUSSION
Previous studies have reported that DDX41 mutations were identified in 3% of families with suspected inherited HMs [2]. in 3.1% of Han Chinese patients with myeloid neoplasms [4], in 5.5% of Thai patients with myeloid neoplasms [3]. In this cohort, DDX41 mutations were found only in 0.8% of patients with myeloid neoplasms. It might be related to the composed of mainly primary AML patients in this cohort. The median age of this cohort is 28 years, which is lower than the mean age of onset (62 years) of HMs that have been reported [2]. However, our results also support that most AML cases who were carrying DDX41 mutations have an MDS history and are older.  There are a few reports of DDX41 mutations in lymphoid malignancies; it was speculated that dysregulation of the innate immune response might be linked to lymphoid malignancy [2]. However, it has not been reported in ALL. We identified DDX41 mutations in 0.9% of patients with ALL, and the characterize was distinct from myeloid tumors; with a young age of onset, somatic and germline DDX41 variants appeared to be mutually exclusive, the majority (80.0%) of the somatic variants were located on or upstream of the DEAD domain. It suggested that the pathogenesis of DDX41 mutations in ALL may differ from myeloid tumors, which merits further study.
Compared with other myeloid neoplasm predisposition syndromes, patients with germline DDX41 mutations are older at the time of presentation [10,11]. Loss of function (LOF) germline DDX41 mutations is by far most prevalent. However, the age of onset at different muta-  [2,5]. However, it is rare in East and Southeast Asian populations [3,4], neither was detected in this cohort. It is indicating that distinct ethnically associated with recurrent germline mutations. Approximately 50% of patients with germline DDX41 mutations also harbor somatic mutations in the other allele as a double hit event during progression to MDS or AML [5,12] with the missense mutation p.R525H being most common [13,14]. However, we and others have observed that individuals with single germline DDX41 mutations also progressed to hematological tumors [15]. In this cohort, we found one patient with AML concomitant with CSF3R, IDH2, RUNX1, and WT1 mutations, one patient with CMML concurrent with the MLL-ELL fusion gene. We speculate that these mutations or fusion genes may interact with germline DDX41 variants in the development of myeloid neoplasms.
In conclusion, we first reported the DDX41 mutations in ALL and AA, which have distinct characteristics, thereby expanding the mutation and phenotypic spectrum of DDX41 mutations. Moreover, the genotype-phenotype correlations regarding DDX41 mutations should be clarified more specifically in the future.

ACKNOWLEDGMENTS
We thank all the patients in this study. We acknowledge all the technicians working in the Laboratory Medicine Division of Hebei Yanda Lu Daopei Hospital.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS
Hongxing Liu designed the research and critically revised the paper.
Yang Zhang performed the research and wrote the paper. Fang Wang and Xue Chen supervised clinical and experimental findings. Hong Liu, Xiaoliang Wang, Jiaqi Chen, and Xiaoli Ma analyzed the data. Panxiang Cao performed bioinformatics analysis. All the authors read and approved the final version of the manuscript.

ETHICS APPROVAL
This study was approved by the Institutional Review Board and Ethical Committee of the Hebei Yanda Lu Daopei Hospital.

PATIENT CONSENT STATEMENT
Written informed consent was obtained from all the patients.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available upon request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.