Clinical and functional characterization of a novel TNFRSF9 variant causing immune dysregulation with predisposition to EBV-driven lymphomagenesis

Abstract

Introduction: The TNFRSF9 gene encodes the costimulatory receptor CD137, also known as 4-1BB, which plays a critical role in sustaining effective cytotoxic T-cell responses. Variants in the TNFRSF9 gene are associated with an extremely rare autosomal recessive primary immunodeficiency disorder characterized by recurrent sinopulmonary infections and EBV-induced lymphoproliferation.

Methods: We report a case siblings exhibiting EBV viremia, recurrent respiratory infections, and Burkitt lymphoma. Whole-exome sequencing (WES) was performed. Sanger sequencing was used to validate the variants. In vitro functional study was performed by western blot, flow cytometry assays and luciferase assays.

Results: Genetic analysis identified a novel missense variant in the TNFRSF9 gene (NM_001561.5: c.359G>C, p.C120S). Functional analysis in vitro demonstrated that this variant decreased the expression of TNFRSF9 both mRNA and protein levels. Western blot analysis revealed a significant decrease in phosphorylated-AKT. Luciferase assays showed that the p.C120S variant diminished the activity of the NF-κB pathway. Immunophenotyping of the patient's peripheral blood revealed a significant reduction in CD27+ memory B cells, which are critical for long-term humoral immunity. Additionally, there was a notable decrease in IFN-γ secretion in CD8+ T cells, suggesting impaired cytotoxic T-cell function. These findings align with the clinical presentation of immunodeficiency and lymphoproliferation observed in the patients. We also reviewed 9 previously reported patients with homozygous or compound heterozygous TNFRSF9 variants. The clinical manifestations among these patients were highly heterogeneous, ranging from asymptomatic to malignancies.

Discussion: In summary, we identified a novel TNFRSF9 variant associated with immunodeficiency and lymphoproliferation, supported by functional evidence demonstrating its impact on gene expression, AKT and NF-κB signaling pathways, and immune cell function. Our findings expand the mutation spectrum of the TNFRSF9 gene and provide new insights into the molecular mechanisms underlying this rare immunodeficiency disorder.

Keywords: AKT; EBV viremia; NF-κB; TNFRSF9; immunodeficiency; lymphoproliferation.

Figure 1

Clinical presentation and timeline of key clinical events. (A) Pathological and immunohistochemical characteristics at initial diagnosis of Burkitt’s lymphoma. CD20 positivity confirms B-cell origin, while CD3 negativity excludes T-cell origin. High Ki67 expression (near 100%) indicates a high proliferation rate, typical of Burkitt’s lymphoma. EBER positivity suggests Epstein-Barr virus (EBV) infection. (B) EBV sorting PCR to identify EBV-infected cell types and EBV load over time. EBV primarily infects B cells but can also infect T cells and NK cells, particularly in immunocompromised individuals. EBV DNA load (copies/mL) serves as an indicator of viral activity, disease progression, and treatment response. The graph illustrates changes in EBV load and cell type distribution throughout the disease course. (C) Timeline of key clinical events and laboratory test results, spanning from 2020 to 2025. Key events include symptoms (e.g., abdominal pain, abdominal mass, recurrent infections, EBV viremia, lymphadenopathy), complications (e.g., sepsis, pneumonia, conjunctivitis), and treatment milestones (e.g., chemotherapy completion, administration of cyclophosphamide, vincristine, cytarabine, methotrexate, doxorubicin, levofloxacin, IVIG, caspofungin, ganciclovir). Notable events such as TNFRSF variant and respiratory tract infections are highlighted.

Figure 2

Genetic and molecular characterization of the patient with TNFRSF9 variants. (A) Pedigrees of this family showing the inheritance pattern of the TNFRSF9 c.359G>C variant. (B) Sanger sequencing of the TNFRSF9 c.359G>C variant in this family, confirming the homozygous status of the c.359G>C variant in the patient and the heterozygous status in the parents. (C) Amino acid sequence alignment across species. The black bar indicated by red arrow highlights the highly conserved position of the 120^th residue in human emphasizing its evolutionary significance. (D) Structural impact of the C120S variant on protein stability. The variant introduces additional hydrogen bonds with neighboring residues (e.g., LYS-152 and THR-124), potentially affecting the protein’s conformation and function. (E) Distribution of TNFRSF9 variants and protein domain structure. The variant studied in this work (C120S) is highlighted in red. The domain structure of the protein includes the signal peptide (amino acids 1-24), four extracellular cysteine-rich domains (25-186), including the ligand binding sites located in CRD2 and CRD3, transmembrane domain (187-213), and cytoplasmic region (214-255).

Figure 3

Immunological phenotypes of the patient. (A) Fluorescence-activated quantitative analysis of IFN-γ in CD8+T cells after stimulation with PMA/Ion, demonstrating a reduced release of IFN-γ in CD8+ T cells of the patient compared to the mother. (B) Peripheral blood B-cell immunophenotyping: Memory (CD19+ CD27+) frequencies in the patient and his mother, as measured by flow cytometry. The patient showed a lower frequency of CD27+ memory B cells compared to the mother, both under non-stimulated and stimulated conditions. These experiments were performed once and not repeated.

Figure 4

Functional analysis of the C120S variant in CD137 gene expression and signaling pathways. (A, B) Fluorescence intensity was analyzed by flow cytometry to evaluate cell transfection efficiency. (C) Expression levels of wild-type and mutant TNFRSF9 in cells by real-time PCR. The WT group showed the highest expression level, while the C120S and G109S mutants exhibited slightly lower expression levels. (D, E) Western blot analysis of AKT and phosphorylated AKT (p-AKT) in cells transfected with wild-type or mutant TNFRSF9 (including G109S as a positive control) and GAPDH was used as a loading control, and quantified by gray value using Image J software. The data indicate potential alterations in AKT signaling pathways due to the C120S mutation, as reflected by changes in the levels of total AKT and its phosphorylated form (p-AKT). (F) NF-kB-dependent luciferase activity in cell extracts from each sample, normalized to control cells transfected with WT-TNFRSF9 plasmid. All experiments were repeated three times independently. One-way analysis of variance (ANOVA) was used when comparing three or more groups and statistical significance is indicated by *P<0.05; **P<0.01, ***P<0.001.