The tumor-necrosis-factor receptor-associated periodic syndrome: new mutations in TNFRSF1A, ancestral origins, genotype-phenotype studies, and evidence for further genetic heterogeneity of periodic fevers

Abstract

Mutations in the extracellular domain of the 55-kD tumor-necrosis factor (TNF) receptor (TNFRSF1A), a key regulator of inflammation, define a periodic-fever syndrome, TRAPS (TNF receptor-associated periodic syndrome [MIM 142680]), which is characterized by attacks of fever, sterile peritonitis, arthralgia, myalgia, skin rash, and/or conjunctivitis; some patients also develop systemic amyloidosis. Elsewhere we have described six disease-associated TNFRSF1A mutations, five of which disrupt extracellular cysteines involved in disulfide bonds; four other mutations have subsequently been reported. Among 150 additional patients with unexplained periodic fevers, we have identified four novel TNFRSF1A mutations (H22Y, C33G, S86P, and c.193-14 G-->A), one mutation (C30S) described by another group, and two substitutions (P46L and R92Q) present in approximately 1% of control chromosomes. The increased frequency of P46L and R92Q among patients with periodic fever, as well as functional studies of TNFRSF1A, argue that these are low-penetrance mutations rather than benign polymorphisms. The c.193-14 G-->A mutation creates a splice-acceptor site upstream of exon 3, resulting in a transcript encoding four additional extracellular amino acids. T50M and c.193-14 G-->A occur at CpG hotspots, and haplotype analysis is consistent with recurrent mutations at these sites. In contrast, although R92Q also arises at a CpG motif, we identified a common founder chromosome in unrelated individuals with this substitution. Genotype-phenotype studies identified, as carriers of cysteine mutations, 13 of 14 patients with TRAPS and amyloidosis and indicated a lower penetrance of TRAPS symptoms in individuals with noncysteine mutations. In two families with dominantly inherited disease and in 90 sporadic cases that presented with a compatible clinical history, we have not identified any TNFRSF1A mutation, despite comprehensive genomic sequencing of all of the exons, therefore suggesting further genetic heterogeneity of the periodic-fever syndromes.

Figure 1

TRAPS pedigrees with novel TNFRSF1A mutations. Affected individuals are indicated by blackened symbols; individuals who are mutation positive but asymptomatic are represented by gray-shaded symbols. For the H22Y, R92Q, and c.193−14 G→A mutations, only representative families are shown.

Figure 2

DNA sequence electropherograms for the seven TNFRSF1A mutations identified in the present study. For each sequence, the upper tracing is from a patient, and the lower tracing is from a normal control.

Figure 3

A, Diagram of normal and aberrantly spliced TNFRSF1A transcript. The conventional splice-donor site, splice-acceptor site, and mutated nucleotide in intron 3 are in boldface. The c.193−14 G→A mutation creates a new splice-acceptor site, as shown. Single-letter designations for amino acids are shown immediately above the first nucleotide in each codon. Four additional amino acids that are present in the alternatively spliced transcript are encoded by the nucleotides that are underlined. B, DNA-sequence electropherograms showing in vitro splicing of c.193−14 G→A transcripts (top) and normal transcripts (bottom) in Cos-7 cells.

Figure 4

Defective clearance of TNFRSF1A in patients with TRAPS. Monocytes were analyzed for TNFRSF1A expression (thicker line in each panel), before (left-hand panels) or after (right-hand panels) PMA activation (100 ng/ml) for 10 min. Single-cell suspensions (10⁶ cells) were incubated with either anti–TNFRSF1A-PE or isotype-matched IgG-PE as control (thinner line in each panel), were washed, and were analyzed by flow cytometry. Fluorescence histograms are shown for controls and for patients with the C52F, T50M, P46L, and R92Q TRAPS mutations.

Figure 5

Pedigree of family affected with dominantly inherited periodic-fever syndrome. One affected member of the family was screened for the TNFRSF1A mutation, throughout the coding and promoter regions; no mutation was identified. Linkage analysis was done to rule out other mutations in TNFRSF1A. Genotypes for six DNA markers from chromosome 12p are shown. Affected sibs in the second generation inherited opposite haplotypes across the interval.

Figure 6

Genomic structure of TNFRSF1A (adapted from Fuchs et al. 1992). All 16 known TRAPS-causing mutations are clustered in exons 2–4, which encode the TNFRSF1A extracellular domain. The c.218−9 T→C polymorphism is shown in intron 8 of TNFRSF1A.

Figure 7

Crystallographically determined structure of the TNFRSF1A extracellular domains 1 and 2 (Banner et al. 1993). TRAPS mutations are shown as circled amino acids. The three disulfide bonds of CRD1 and CRD2 are depicted by thick black bars. Structurally conserved regions of the CRDs are represented by thicker lines. The β-turn positions are indicated by “β.” Loop domains are denoted as “L1”–“L3.”