Are DDX41 variants of unknown significance and pathogenic variants created equal?

A variant of unknown significance (VUS) is a type of genetic mutation whose impact on an individual’s health remains undetermined. It falls between benign and pathogenic variants, and we can reclassify it as we gather more evidence. In this issue of Hematologica, Bader et al. describe a comprehensive study to uncover the clinical impact of VUS for DDX41 (DDX41^VUS) and compared it to that of the pathogenic variants of DDX41 (DDX41^path) and the associated cytopenia, myelodysplasia syndromes and acute myeloid leukemia.¹

The DDX41 gene, also known as DEAD/H-box RNA helicase, is located on chromosome 5q35 and belongs to a class of tumor suppressor genes. It plays a crucial role in hematopoiesis by regulating the processing of small nucleolar RNA, assembling ribosomes, and synthesizing proteins.^2,3 When the function of DDX41 is reduced, it can lead to defects in hematopoietic cells and increase the risk of developing hematologic malignancies, such as certain types of blood cancer. It is important to distinguish precisely between germline and somatic variants of DDX41, as well as between DDX41^VUS and DDX41^path variants, in order to make accurate prognoses and manage myeloid neoplasms associated with DDX41 mutations. However, despite the availability of advanced sequencing techniques for diagnosing inherited myeloid neoplasmas,⁴ we still face challenges in differentiating between causal variants and those classified as VUS. This difficulty arises from our incomplete understanding of the function of DDX41 protein. As a result, we have yet to fully define the landscape of causal DDX41 variants.

DDX41^path-associated familial myelodysplastic syndrome/ acute myeloid leukemia has distinctive features. It typically occurs later in life, at a median age of 65-70 years, and affects males more often than females (ratio of 3:1). There is also variability in the family history of hematologic malignancies.^5-7 Many of these patients (46%) have pre-existing cytopenia and a long latency (5.2 years) before the diagnosis of the myeloid neoplasms.⁵ Patients with DDX41-mutant myeloid neoplasms typically present with hypocellularity on the bone marrow sample and with normal cytogenetics.⁸ Fortunately, patients with DDX41^path-associated myeloid neoplasms generally respond well to treatment. In fact, a study demonstrated that they had a 100% overall response rate and nearly 90% overall survival at the 2-year mark when treated with intensive chemotherapy or hypomethylating-based agents.⁹

To investigate the potential pathogenicity of DDX41^VUS, Bader et al. screened DDX41 mutations from 4,524 patients treated at the Mayo Clinic who underwent targeted sequencing for suspected or known myeloid neoplasms. They classified the mutations into DDX41 causal variants and VUS based on established guidelines from the American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP). The researchers carefully categorized the DDX41 variants as either purely DDX41^VUS (63 cases) or DDX41^path (44 cases, with 11 having both pathogenic and VUS variants). The authors described the clinical features and outcomes of this cohort and found comparable features, including molecular profiles, with no differences between patients in variant allele fraction, co-mutation patterns, and cytogenetics. Family history of hematologic malignancies, and the outcomes, including time to initiate treatment, progression-free survival, and overall survival were also comparable between patients with DDX41^vus and DDX41^path (Figure 1).

It is worth noting that some specific variants of DDX41^VUS, such as p.P258L, p.G173R, and M155L, were associated with a remarkably high frequency of myeloid neoplasms. For instance, the occurrence of myeloid neoplasms was 86%, 75%, and 40% among patients with these variants, respectively. In addition, 28% (5/18) of patients had cytopenia. Similarly to a previous study led by Li et al.,⁷ only two patients with DDX41^vus had concurrent somatic DDX41^path (R5252H) mutations, which suggests that these DDX41^VUS alone could be oncogenic. On the other hand, a study by Chlon et al. using mouse models demonstrated that a single DDX41 mutation, known as a monoallelic mutation, is associated with age-dependent hematopoietic defects.³ However, the acquisition of a "second-hit" mutation, often R525H, has a disease-modifying effect that accelerates hematopoietic defects and leads to hematologic malignancies.³ Clinical studies led by Li et al.⁷ and Duployez et al.⁸ also demonstrated that 70-80% of patients with DDX41^path mutations develop acute myeloid leukemia after the acquisition of the “second-hit” DDX41 somatic mutation. The development of myeloid neoplasms without an additional somatic hit in these variants suggests that these DDX41^vus might represent bona fide risk factors for the progression of myeloid neoplasms rather than trivial or inconsequential findings.

Previous studies showed that the enrichment of somatic mutations in ASXL1, EZH2, and SRSF2 is associated with acute myeloid leukemia progression, and patients with DDX41 variants that result in truncation of the protein experience a rapid progression to acute myeloid leukemia compared to those with non-truncating variants.¹⁰ However, within the current study, the authors did not find significant differences in clinical features or outcomes when comparing patients with isolated DDX41 variants and those with co-mutations nor between patients with proteintruncating variants versus non-protein-truncating variants.

Figure 1.Comparable features between DDX41^vus and DDX41^path variants. VAF: variant allele frequency; AML: acute myeloid leukemia; MDS: myelodysplastic syndromes/myeloid neoplasms; DDX41^vus: variants of DDX41 of unknown significance; DDX41^path: pathogenic variants of DDX41.

It is important to note that this lack of significance may be due to the small number of cases included in the analysis.

Collectively, the high frequency of myeloid neoplasms observed in patients with DDX41^VUS, along with the comparable clinical features and outcomes between those with DDX41^VUS and DDX41^path, suggest that these VUS might actually be oncogenic. This study underscores the importance of accurately classifying variants as VUS or pathogenic and understanding the causal landscape of DDX41 variants. It is crucial to establish a standardized classification specifically for DDX41 variants. For hematologists, the identification of DDX41^VUS should prompt increased vigilance in terms of genetic counseling referrals, monitoring blood counts, and tailoring management accordingly. Future studies should focus on performing functional analyses of DDX41^VUS to overcome the challenges associated with interpreting these variants of uncertain relevance.

Footnotes

• Received May 25, 2023
• Accepted June 6, 2023

Correspondence

Z. Xie zhuoer.xie@moffitt.org

Disclosures

DTS serves on the scientific advisory board at Kurome Therapeutics, is a consultant for and/or received funding from Kurome Therapeutics, Captor Therapeutics, Treeline Biosciences, and Tolero Therapeutics, and has equity in Kurome Therapeutics. ZX has received speaker bureau fees from Novatis.

Contributions

ZX wrote the initial draft; both authors reviewed, provided edits to subsequent manuscript versions, and approved the final manuscript for submission.