Overall cancer risk in people with deleterious germline DDX41 variants

Abstract

Germline loss-of-function (LoF) DDX41 variants predispose to late-onset hematopoietic malignancies (HM), predominantly of myeloid lineage. Among 43 families with germline DDX41LoF variants, bone marrow (BM) biopsies in those without (N=8) or with malignancies (N=21) revealed mild dysplasia in peripheral blood (57%) and BM (88%), long before the average age of DDX41-related HM onset. Therefore, we recommend baseline BM biopsies in people with germline DDX41LoF alleles to avoid over-diagnosis of myelodysplastic syndromes. A variety of solid tumors were also observed in our cohort, with 24% penetrance by age 75. Although acquired DDX41 mutations are common in HM, we failed to identify such alleles in solid tumors arising in those with germline DDX41LoF variants (N=15), suggesting an alternative mechanism driving solid tumor development. Furthermore, 33% of pedigrees in which ≥15% of first-degree relatives including the proband were diagnosed with a solid tumor had second germline deleterious variants in other cancer-predisposition genes, likely serving as primary cancer drivers. Finally, both lymphoblastoid cell lines and primary peripheral blood from individuals with germline DDX41LoF variants exhibited differential levels of inflammation-associated proteins. These data provide evidence of inflammatory dysfunction mediated by germline DDX41LoF alleles that may contribute to solid tumor growth in the context of additional germline cancer- associated variants. For those with HM and personal/family histories of solid tumors, we recommend broad germline testing. DDX41 may be an indirect modifier of solid tumor pathogenesis compared to its tumor suppressor function within hematopoietic tissues, a hypothesis that can be addressed in future work.

Figure 1.

Family-associated germline DDX41 variants and morphologic features of baseline and malignant peripheral blood and bone marrow in individuals with deleterious germline DDX41 variants at different ages. (A) Deleterious germline DDX41 variants identified in patients and families with hematopoietic malignancies (HM) are shown above the protein schematic, and variants of uncertain significance (VUS; blue circles) are shown below. Pathogenic variants are indicated by red diamonds, and likely pathogenic variants by orange diamonds. Non-protein coding variants are listed in the bottom right. The likely pathogenic copy number variant (CNV) is indicated by an orange line. Novel variants are shown with glow and previously identified variants are shown without glow. DDX41 protein domains are indicated by color: RecA-like domain 1 (light blue), RecA-like domain 2 (lilac), and zinc finger (ZnF, light green). (B-Q) Images shown include (B, F, J, N) peripheral blood, (C, G, K, O) bone marrow (BM) aspirate, (D, H, L, P) BM trephine, and (E, I, M, Q) immunohistochemistry on trephine. (B-E) A 17-year-old (yo) female with a pathogenic DDX41 variant (p.M1?), mild dysplastic changes in erythroid lineage (red arrow) and megakaryocytic lineage (yellow arrow), but insufficient for diagnosis of myelodysplastic syndromes (MDS). (F-I) A 46yo female with a likely pathogenic DDX41 deletion of exons 12-17, significant (>10%) dysplastic changes in both erythroid and megakaryocytic lineages, but no granulocytic dysplasia. (J-M) A 73yo male (father of (F-I)) with a likely pathogenic DDX41 deletion of exons 12-17, 4.6% blasts and significant dyserythropoiesis and dysmegakaryopoiesis as well as dysgranulopoiesis manifested mainly by abnormal nuclear morphology including hyposegmentation, dense chromatin and nuclear membrane projections (orange arrow), but not cytoplasmic hypogranulation, diagnosed with MDS with multilineage dysplasia. (N-Q) A 61yo male with a likely pathogenic DDX41 variant (p.P258L), 18% blasts, and multilineage dysplasia (particularly prominent in granulocytes), diagnosed MDS with excess blasts-2 progressing toward acute myeloid leukemia.

Figure 2.

Disease breakdown by DDX41 variant and timelines of solid tumors and hematopoietic malignancies in patients with multiple malignancies. (A) DDX41 protein schematic showing all malignancies identified in individuals with loss-of-function (LoF) DDX41 alleles plotted by corresponding variant. Diseases represented include hematopoietic malignancies (HM) (red), and solid tumors such as breast (pink), prostate (orange), melanoma (yellow), colon (light green), gastric (black), endometrial (light blue), lung (dark blue), tonsillar (magenta), ovarian/vulvar (purple), renal (brown), neuroendocrine carcinoma (peach), mastocytosis (dark green), head and neck (green), small bowel (teal), basal cell carcinoma (gray), fallopian tube (fuchsia), mesothelioma (light pink), and kidney (salmon) cancers. DDX41 protein domains are indicated by color: RecA-like domain 1 (light blue), RecA-like domain 2 (lilac), and zinc finger (ZnF) (light green). (B) Age at cancer diagnoses and treatments in individuals with DDX41^LoF alleles who were diagnosed with more than 1 cancer. HM are shown in red. Solid tumors represented are breast (pink), prostate (orange), colon (light green), lung (dark blue), renal (brown), basal cell carcinoma (grey), gastric (black), ovarian/vulvar (purple), tonsillar (magenta), and neuroendocrine carcinoma (peach). Solid tumor cancer therapies are indicated by: radiation therapy (R); chemotherapy (C); hormonal therapy (H); surgery (S); and unknown (?). Mean latency refers to the average years between the onsets of solid tumors and HM, whereas the range refers to the minimum and maximum latencies present.

Figure 3.

RNA sequencing, cytokine arrays, and Luminex assays reveal inflammatory dysregulation in DDX41^{var/ +} patient-derived lymphoblastoid cell lines. (A) Principal component analysis (PCA) plot of RNA-sequencing data for DDX41^var/+ (N=5, purple) and DDX41^WT (N=3, green) lymphoblastoid cell lines (LCL). Noted clustering in gene expression is demonstrated between DDX41^P258L/+ and DDX41^A500Cfs*9/+, DDX41^A492Gfs*17/+ and DDX41^{del ex12-17/+}, and of DDX41^WT LCL. (B) Volcano plot showing significantly (CI=95%) upregulated (red) and downregulated (blue) genes in DDX41^var/+ LCL (N=5) compared to DDX41^WT LCL (N=3). Genes with no statistically significant change are in grey. (C) Normalized enrichment plot of genes from 24 hallmark signaling pathways. Increases in overall gene expression in DDX41^var/+ LCL from wild-type (WT) are in red, while decreases from WT are in blue. P values were determined by Pearson’s correlation. (D) Heat map of cytokine array data showing fold changes in pixel densities of increased cytokines in patient-derived DDX41^var/+ (purple) LCL-conditioned media compared to DDX41^WT. Fold changes range from 0.1 (blue) to >10 (red). (E) Heat map of commercial Luminex data showing fold changes in pixel densities of increased cytokines in patient-derived DDX41^var/+ (purple) LCL-conditioned media compared to DDX41^WT. Fold changes range from 0.1 (turquoise) to >2 (magenta). (F-I) Bar graphs showing data from a custom Luminex panel. P values were determined using two-tailed t tests with Welch’s correction and confirm higher levels of (F) ANG, (G) CXCL13, (H) CXCL8, and (I) IL-9 in DDX41^var/+ (purple) LCL-conditioned media compared to DDX41^WT (green).

Figure 4.

Testing the proposed mechanism of inflammatory dysregulation in germline DDX41^var/+ lymphoblastoid cell lines. (A) Western blots to quantify NF-kB (p65 subunit) in nuclear and cytoplasmic protein fractions from patient-derived DDX41^var/+ (purple) and DDX41^WT (green) lymphoblastoid cell lines (LCL). Histone H3 was used as a nuclear marker and loading control while GAPDH was used as a cytoplasmic marker and loading control. (B) Average NF-kB pixel densities in nuclear protein fractions from patient-derived DDX41^var/+ (purple) and DDX41^WT (green) LCL normalized to histone H3. Higher levels of NF-kB were detected in DDX41^var/+ LCL compared to DDX41^WT (P=0.008) according to a two-tailed t test with Welch’s correction. (C) Average NF-kB pixel densities in cytoplasmic protein fractions from patient-derived DDX41^var/+ (purple) and DDX41^WT (green) LCL normalized to GAPDH. No significant change in NF-kB was detected according to a two-tailed t test with Welch’s correction. (D) Visual summary of cytokine array, Luminex, and western blot data. Cytokines whose levels were higher in DDX41^var/+ than in DDX-41^WT LCL-conditioned media according to cytokine array and Luminex are shown in red. Increased activation and translocation of NF-kB (gold) is indicated by red upward arrows. Direct protein interactions are indicated by solid black arrows. Indirect activation of NF-kB by inflammatory cytokine signaling is indicated by a dotted red arrow. Proteins/receptors that were identified in literature but were not quantified are shown in grey. Created in https://BioRender.com.

Figure 5.

Inflammatory dysregulation in UK Biobank participants with likely germline DDX41^LoF variants. (A) Volcano plot showing proteins that are increased (red; 95% confidence interval [CI]) or decreased (blue) in individuals with likely germline loss-of-function (LoF) DDX41 variants compared to wild-type (WT) controls. (B) Protein-protein interaction network showing proteins found to decrease in individuals with likely germline DDX41^LoF variants compared to WT controls. Only proteins with “high confidence” (0.700) or “highest confidence” (0.900) interactions (N=30) according to the STING database are shown. The level of significance with which proteins were found to decrease are indicated by color: P<0.001 (green), P<0.01 (light blue), or P<0.05 (dark blue). (C) Enrichment plot showing pathways enriched among the 114 proteins found to decrease in individuals with likely germline DDX41^LoF variants compared to WT controls according to the STRING database.