A patient with myelodysplastic/myeloproliferative neoplasm with neutrophilia (MDS/MPN-N) harboring a DDX41 germline mutation: a case report

Abstract

We report a 39-year-old male with MDS/MPN with neutrophilia​ harboring a germline DDX41mutation. Next sequencing revealed mutational redundancy, wherein high-risk somatic lesions (ASXL1, SRSF2) may contribute to disease evolution in lieu of​ a detectable DDX41second hit. Despite a favorable karyotype, the patient relapsed early after the first allogeneic hematopoietic stem cell transplantation (allo-HSCT). He achieved remission via venetoclax/azacitidine(VA) bridging followed by a second allo-HSCT. This case expands the phenotypic spectrum of DDX41and underscores that allo-HSCT remains the definitive curative strategy, irrespective of non-transplant response rates or conventional risk models.

1. Introduction

Over the past century, our comprehension of heritable cancer susceptibility has undergone a profound transformation. Initially rooted in the recognition of familial aggregation patterns, the field has progressed to the definitive characterization of specific germline pathogenic variants, propelled by seminal advancements in molecular biology and next-generation sequencing (NGS) (1). Within the spectrum of myeloid neoplasms, more than ten germline predisposition genes have now been identified (2). Among these, germline DDX41(DEAD-box RNA helicase 41)​ mutations are predominantly reported in adult patients with myelodysplastic neoplasms (MDS)​ and acute myeloid leukemia (AML), with a reported detection rate ranging from 1.6% to 6.0% (3). While DDX41is increasingly recognized as a key genetic culprit, its association with myelodysplastic syndromes/myeloproliferative neoplasms with neutrophilia (MDS/MPN-N), encompassing atypical chronic myeloid leukemia (aCML), remains exceedingly rare. These disorders are characterized by persistent neutrophilia and dysgranulopoiesis but lack targetable fusions like BCR::ABL1, presenting significant therapeutic challenges.

Herein, we report a unique case of a 39-year-old male who presented with MDS/MPN-N harboring a germline DDX41mutation. This case is notable for the comprehensive documentation of its molecular architecture, featuring the synergistic co-occurrence of the germline DDX41variant with high-risk somatic mutations in splicing factors (SRSF2) and epigenetic regulators (ASXL1, EZH2, TET2, RUNX1). We detail the clinical course, including the failure of the first allogeneic hematopoietic stem cell transplantation (allo-HSCT) leading to early relapse, and the successful salvage therapy utilizing a VA bridge followed by a second allo-HSCT. This report aims to expand the phenotypic spectrum of germline DDX41mutations and underscore the critical importance of germline genetic testing in young patients with MDS/MPN-N, even in the presence of complex somatic mutational profiles.

2. Case report

We discuss a 39-year-old male patient with an unremarkable previous medical history who presented to the hospital due to a persistent fever lasting one week.

Complete blood count(CBC) showed white blood cells (WBC) of 56.65 × 10⁹/L with absolute neutrophil count (ANC) of 48.3 × 10⁹/L, absolute monocyte count of 0.45× 10⁹/L (0.8%), hemoglobin (Hb) of 72 g/L, and platelets of 70 ×10⁹/L. Manual differential identified that the proportion of neutrophils is increased with a left shift (4% band forms, 2% segment forms, 2% myelocytes, 12% metamyelocytes, 2% promyelocytes, and 1% blasts) and 65% dysgranulopoiesis (Figure 1A). Physical examination revealed no hepatosplenomegaly. Both bone marrow (BM) aspirate and trephine biopsy showed hypercellularity with myeloid hyperplasia to 90%, 3% blasts, and15% dysgranulopoiesis (Figure 1B). Flow cytometry immunophenotyping of the bone marrow aspirate revealed an abnormal population of myeloid progenitor cells, characterized by the phenotype CD34+CD117+CD38+HLA-DR+CD13+CD33+CD11c+, with partial expression of CD64 and absence of CD5 and CD7, representing 1.4% of all nucleated cells.

Figure 1.

Peripheral blood and bone marrow morphology, with bone marrow biopsy images. (A) The peripheral blood smear reveals neutrophilia and evidence of dysgranulopoiesis, characterized by hyposegmented nuclei and hypogranular cytoplasm. Additionally, a neutrophilic precursor is present, and occasional cells display abnormal chromatin clumping(Wright-Giemsa staining, ×1000). (B) The bone marrow aspirate smear is notable for a marked predominance of granulocytic cells, which display dysplastic features(Wright-Giemsa staining, ×1000).

Karyotype was 46, XY, and molecular testing for the BCR::ABL1 fusion gene, as well as CSF3R, SF3B1, JAK2V617F, CALR, and MPL gene mutations, all showed no abnormalities. Genomic DNA was extracted from bone marrow or peripheral blood samples. Targeted next-generation sequencing (NGS) was performed using a customized 56-gene myeloid panel on the Illumina high-throughput sequencing platform, which captures the protein-coding regions and flanking intronic regions of genes closely associated with myeloid malignancies. The library was prepared with unique dual indices and sequenced to a mean depth of >500×, with >95% of target regions covered at ≥100×.​ Variants were called using standard pipelines for single-nucleotide variants (SNVs) and small insertions/deletions (indels). Copy-number variation (CNV) and loss of heterozygosity (LOH) analysis were not performed as part of this assay. The results revealed the presence of pathogenic variants, including SRSF2, ASXL1, RUNX1, TET2, EZH2, and DDX41(Table 1). No additional DDX41 coding mutations were detected. The diagnosis of MDS/MPN with neutrophilia (MDS/MPN-N) was established according to the WHO Classification of Tumours: Haematolymphoid Tumours (5th edition, 2022). The diagnostic criteria included persistent neutrophilic leukocytosis, prominent granulocytic dysplasia, and the absence of BCR::ABL1 fusion or PDGFRA/B/FGFR1rearrangements. Mutations in classic MPN driver genes (JAK2, CALR, MPL) and the CSF3R gene were absent. Importantly, peripheral blood examination revealed an absolute monocyte count of 0.45 × 10₉/L (representing 0.8% of total white blood cells), effectively ruling out chronic myelomonocytic leukemia (CMML).

Table 1.

Clinical, morphologic, cytogenetic, and molecular characteristics at diagnosis.

Parameter	Patient value (reference range)
Age (years)	39
Gender	Male
HSCT	Yes
Follow-up time(months after HSCT) (months)	26
WBC (×109/L)	56.65 (3.5-9.5)
ANC (×109/L)	48.3 (1.8-6.3)
absolute monocyte count	0.45 (0.1-0.6)
Hemoglobin (g/L)	68 (121-169)
Platelets (×109/L)	47 (125-350)
Manual differential (PB)	Segmented Neutrophils 2%, Band Neutrophils 4% Promyelocytes 2%, Myelocytes 2% Metamyelocytes 12%, Blasts 1% Dysgranulopoiesis 65%
BM blasts(%)	3
Karyotype	46, XY (20)
BCR::ABL fusion gene	Negative
Mutations in CSF3R, SF3B1, JAK2, CALR, and MPL	Negative
Gene mutations identified by the NGS panel	SRSF2 (c.284C >A, p.P95H, VAF48.70%) ASXL1 (c.2468delT, p.L823*, VAF48.20%) RUNX1 (c.934dupA, p.T312Nfs*288, VAF46.7%) TET2 (c.3058C >T, p.Q1020*, VAF48.6%) EZH2 (c.1355dupA, p.Y452*, VAF1.6%) DDX41 (c.22C>T, p.R8W, VAF49.1%)

Notably, the DDX41 mutation allele frequency of 49.8% strongly suggests a germline origin. Nail, hair, and oral mucosa samples were collected from the patient, his parents, and his daughter. Sanger sequencing confirmed the presence of the DDX41variant c.22C>T (p.R8W) in the patient, father, and daughter (Figure 2). According to the ACMG/AMP guidelines, this variant is currently classified as Variant of Uncertain Significance (VUS). It is extremely rare in population databases (gnomAD: not observed/very low frequency) and is located in the critical DEXDc helicase domain. The familial cosegregation observed in this pedigree provides strong supportive evidence (PP1)​ for its clinical relevance. This missense variant results in an arginine-to-tryptophan substitution at position 8 of the DDX41 protein. Based on the identification of this germline alteration, a final diagnosis of MDS/MPN-N associated with a DDX41 germline mutation was established.

Figure 2.

Pedigree and Sanger sequencing. Panels A–D show Sanger sequencing results of the DDX41 gene in the patient (A), father (B), daughter (C), and mother (D), respectively, from the pedigree of a patient with MDS/MPN-N harboring a germline DDX41 mutation. The red arrow indicates the mutation site (c.22C>T, p.R8W). No DDX41 mutation was detected in the patient's mother.

The patient was initially diagnosed with MDS/MPN-N and commenced first-line therapy with a combination of azacitidine (140 mg daily, days 1–7) and venetoclax. Venetoclax was administered via a dose-escalation regimen, starting at 100 mg daily and titrated to a target dose of 400 mg daily (days 1–7). After one cycle, the patient achieved a morphologic complete remission (mCR). To mitigate the risk of donor cell leukemia (DCL), a fully HLA-matched unrelated donor was identified through the China Marrow Donor Program. Conditioning was myeloablative: fludarabine 50 mg qd (days 8 to 3), busulfan 60 mg q6h (days 7 to 5), and melphalan 300 mg qd (day 3). GvHD prophylaxis was quadruple: ATG (25 mg on day 3, 150 mg on day 2, 200 mg on day 1), cyclosporine A 150 mg starting day 3, mycophenolate mofetil 1 g q12h starting day 3, and methotrexate 30 mg on day 1, 20 mg on days 3 and 6.​ The transplant proceeded without major complications. Neutrophil engraftment occurred on day 11 and megakaryocyte engraftment on day 13.

During routine monitoring, donor chimerism fell from 79.91% at month 7 to 16.35% at month 9. NGS detected re-emergence of somatic mutations (SRSF2, TET2, EZH2, ASXL1, RUNX1, CREBBP) without a second DDX41 hit. Bone marrow morphology and immunophenotyping showed 2% and 4% myeloid blasts, establishing relapsed MDS/MPN-N post-allo-HSCT. After family counselling, a second allo-HSCT was planned. Two re-induction cycles of VA were given; post-treatment marrow blasts were 2%, and donor chimerism improved to 69.66%. Due to inadequate hematopoietic recovery, the regimen was switched to azacitidine 160 mg (days 1–7) + rizotoclax, escalated to 200 mg (day 6) → 400 mg (day 7) → 600 mg daily thereafter. The choice of rizotoclax was based on the synthetic-lethal dependency of DDX41-deficient myeloid cells on BCL-2 inhibition, aiming to deepen remission before the second transplant.​ Two cycles were delivered.

One month after completing AZA–rizotoclax, the patient received a second myeloablative allo-HSCT from a different fully HLA-matched unrelated donor.​ GvHD prophylaxis followed the same quadruple regimen (ATG + CSA + MMF + MTX). Follow-up at one month post-second transplant showed 100% donor chimerism and sustained morphologic remission (Figure 3).

Figure 3.

Trends of donor chimerism after allo-HSCT. The blue line represents the period following the first allo-HSCT, during which donor chimerism initially reached 100% but subsequently declined, hitting a nadir of 16.35% on July 30, 2025 (marked as “Relapse”). The red line indicates the second allo-HSCT performed on March 9, 2026, after which complete donor chimerism was successfully restored and maintained.

3. Discussion

This case report describes a 39-year-old male with MDS/MPN with neutrophilia (MDS/MPN-N) harboring a germline DDX41variant c.22C>T (p.R8W). The p.R8W substitution occurs within the N-terminal DEXDc helicase domain of DDX41, a region frequently implicated in germline predisposition to myeloid malignancies.

DDX41 plays essential roles in RNA metabolism, particularly in pre-mRNA splicing and ribosomal biogenesis. As a component of the spliceosome C complex, its loss can induce splicing aberrations—such as exon skipping and intron retention—in hematopoietic cells (4, 5). Furthermore, recent studies suggest that impaired R-loop resolution and subsequent innate immune activation are involved in the pathogenesis of DDX41-related myeloid neoplasms (6, 7). However, the precise contribution of these specific pathways to individual clinical phenotypes requires further functional validation. In our patient, the germline DDX41variant c.22C>T, resulting in a p.R8W substitution, likely initiated this cascade by disrupting helicase function, thereby creating a pro-inflammatory microenvironment and disrupting protein homeostasis. This state of genomic instability created a permissive environment for the acquisition of high-risk somatic mutations, ultimately driving the phenotypic progression to MDS/MPN-N. Consistent with our observation, a recent Chinese single-center study also listed p.R8W among the prevalent DDX41variants of uncertain significance or probable pathogenicity, although that cohort lacked formal family cosegregation data (8). Our identification of the same c.22C>T (p.R8W) variant in the patient’s father and daughter provides the germline inheritance evidence that was otherwise missing from the population-level data, reinforcing p.R8W as a clinically relevant DDX41 allele in Chinese kindreds with MDS/MPN.

While the germline DDX41 variant likely establishes the predisposing background, the acquisition of high-risk somatic mutations (ASXL1, SRSF2) appears to drive malignant transformation. This case suggests that cooperating high-risk somatic lesions may contribute to disease evolution even in the absence of a detectable somatic DDX41 second hit, aligning with the concept of genetic cooperation in myeloid neoplasms.

An examination of East Asian cohorts reveals that four independent studies have delineated germline DDX41variants linked to myeloid malignancies across Japan, China, and Korea. A striking geographic specificity exists in the dominant mutations: p.A500fs prevails in Japan, p.Y259C in Korea, and the splicing variant c.935 + 4A>T in China (9–12). Interestingly, our patient carried the c.22C>T​ variant, differing from the predominant Chinese founder mutation. This suggests broader genetic diversity within the Chinese population than previously appreciated based solely on hotspot testing. Clinicians should be wary that ACMG guidelines can result in the overclassification of DDX41variants as variants of uncertain significance (VUS); however, familial segregation supports the germline origin.

It is noteworthy that the patient presented at the age of 39. Although the penetrance of germline DDX41variants remains negligible before the age of 40 (<5%), it undergoes a steep escalation to 49–54% by the age of 90 (13).​ This explains why early-onset cases like ours are rare but highlights the cumulative risk over time. The predominance of a normal karyotype, coupled with the unique molecularfingerprint​ of DDX41variants, has prompted the realization that conventional cytogenetic risk stratification models fail to reliably prognosticate outcomes​ in this specific patient cohort (14).

This highlights that even amid promising responses to non-transplant therapies, patients harboring DDX41-mutated MDS or AML retain a considerable propensity for disease recurrence unless allo-HSCT is performed (15).​ Our patient, despite achieving a complete remission with VA therapy, experienced an early relapse following the first transplant attempt. This case also highlights the risk of donor cell leukemia (DCL), emphasizing the need for rigorous donor screening. The successful salvage using a sequential strategy involving Venetoclax + Azacitidine (VA) induction followed by a second allo-HSCT provides valuable clinical insight. In conclusion, this case expands the phenotypic spectrum of germline DDX41 mutations and underscores that while ELN 2024 guides initial therapy, allo-HSCT remains the definitive curative option for fit patients with germline DDX41variants.

4. Conclusion

We report a rare case of MDS/MPN-N​ driven by a germline DDX41mutation in a 39-year-old male. Molecular profiling revealed mutational redundancy, wherein high-risk ASXL1 and SRSF2 mutations substituted for the canonical somatic DDX41 “second hit.” Clinically, despite a favorable karyotype, the patient experienced early relapse after the first transplant. This case underscores that conventional risk models may be inadequate for DDX41carriers. Successful salvage with VA bridging followed by a second allo-HSCT​ highlights the necessity of allo-HSCT as the definitive curative strategy, regardless of non-transplant response rates or apparent low-risk stratification.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This study was supported by Shenzhen Science and Technology Program (JCYJ20230807142204008).

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

Author contributions

XiC: Writing – original draft. CF: Writing – original draft, Writing – review & editing. JZ: Writing – review & editing, Writing – original draft. XuC: Writing – original draft, Writing – review & editing.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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