A rare missense p.C125Y mutation in the TNFRSF1A gene identified in a Chinese family with tumor necrosis factor receptor-associated periodic fever syndrome

Abstract

Background: Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is a rare autosomal dominant disorder with a low incidence in Asia. The most frequent clinical manifestations include fever, rash, myalgia, joint pain and abdominal pain. Misdiagnosis rates are high because of the clinical and genetic variability of the disease. The pathogenesis of TRAPS is complex and yet to be fully defined. Early genetic diagnosis is the key to precise treatment.

Methods: In this study, a Chinese family with suspected TRAPS were analyzed by genome-wide SNP genotyping, linkage analysis and targeted sequencing for identification of mutations in causative genes. To study the pathogenicity of the identified gene mutation, we performed a conservation analysis of the mutation site and protein structure analysis. Flow cytometry was used to detect TNFRSF1A shedding and quantitative real-time PCR were used to assess the activation of unfolded protein response (UPR) in the mutation carriers and healthy individuals.

Results: A typical TRAPS family history, with a pattern of autosomal dominant inheritance, led to the identification of a rare mutation in the TNFRSF1A gene (c.G374A [p.Cys125Tyr]) with unknown significance. The patient responded well to corticosteroids, and long-term therapy with colchicine effectively reduced the inflammatory attacks. No amyloid complications occurred during the 6-year follow-up. In silico protein analysis showed that the mutation site is highly conversed and the mutation prevents the formation of intrachain disulfide bonds in the protein. Despite a normal shedding of the TNFRSF1A protein from stimulated monocytes in the TRAPS patients with p.C125Y mutation, the expression of CHOP and the splicing of XBP1 was significantly higher than healthy controls, suggesting the presence of an activation UPR.

Conclusion: This is the first report of a Chinese family with the rare p.C125Y mutation in TNFRSF1A. The p.C125Y mutation does not result in aberrant receptor shedding, but instead is associated with an activated UPR in these TRAPS patients, which may provide new insights into the pathogenesis of this rare mutation in TRAPS.

Keywords: TNFRSF1A summary; autoinflammatory disease; fever; primary immunodeficiency disease; tumor necrosis factor receptor-associated periodic syndrome.

FIGURE 1

Identification of a germline missense mutation in TNFRSF1A. (A) Family Pedigree of the patient. The proband is indicated by a filled square with a black arrow. (B) Chromosome linkage analysis. A strongly linked segment was found on chromosome 12, marked with a red circle. (C) Sanger sequencing. The mutated TNFRSF1A gene (c.374G>A [p.Cys125Tyr]) was verified by Sanger sequencing in eight family members. (D) Conservation analysis. This mutation site was highly conserved across species. (E) Protein structure simulation. Structural alignment between the wild type protein structure and mutant protein. The left-hand box shows the normal structure surrounding the site of the wild type amino acid, with disulfide bonds present. The structure of the mutant protein is shown in the right-hand box, with no disulfide bonds.

FIGURE 2

TNFRSF1A shedding and the activation of unfolded protein response (UPR) in patients with tumor necrosis factor receptor-associated periodic syndrome (TRAPS). (A) Gating strategy and representative histograms of TNFRSF1A level in a patient with the mutation. (B) Change in TNFRSF1A median fluorescence intensity (MFI) in peripheral CD14⁺ monocytes during 20 ng/mL phorbol 12-myristate 13-acetate (PMA) stimulation in patients with the mutation (red lines, n = 4), a family member with wild type (WT) TNFRSF1A (square icon, blue line, n = 1) and healthy controls (circle icon, blue line, n = 3). (C) Change in TNFRSF1A level in peripheral CD14⁺ monocytes after 10-min and 60-min PMA stimulation (MFI of PMA-stimulated cells/unstimulated cells ratio), in patients with the mutation (n = 4) and individuals with WT TNFRSF1A (n = 4). (D) The relative mRNA expression of spliced XBP1 (sXBP1, left), CHOP (middle) and BIP (right) in TRAPS patients with the p.C125Y mutation (n = 4) and individuals with WT TNFRSF1A (n = 6) were assessed by quantitative real-time PCR. Results were analyzed using the ΔΔC_t method, with GAPDH serving as an internal control and unrelated individuals with WT TNFRSF1A serving as the control group. Data are shown as mean ± SD and compared using unpaired t-test. *, p < 0.05. **, p < 0.01.ns, not significant.