Clinical features and functional significance of the P369S/R408Q variant in pyrin, the familial Mediterranean fever protein

Abstract

Objectives: Familial Mediterranean fever (FMF) is caused by mutations in MEFV, which encodes pyrin. The nature of substitutions P369S and R408Q in exon 3 remains unclear. Exon 3 encoding pyrin's B-box domain is necessary for interactions with proline serine threonine phosphatase interacting protein 1 (PSTPIP1). The aim was to characterise the phenotype of patients with these substitutions and to determine their functional significance.

Methods: A database of genetic tests undertaken at the US National Institutes of Health was interrogated. Symptoms and signs were classified according to Tel-Hashomer criteria. Coimmunoprecipitation techniques were employed to determine the variants' effects on pyrin/PSTPIP1 interactions.

Results: A total of 40 symptomatic and 4 asymptomatic family members with these substitutions were identified. P369S and R408Q were found in cis, and cosegregated in all patients sequenced. Clinical details were available on 22 patients. In all, 5 patients had symptoms and signs fulfilling a clinical diagnosis of FMF, and 15 received colchicine. In patients not achieving the criteria, trials of anti-tumour necrosis factor (TNF) agents resulted in partial or no benefit; resolution of symptoms was noted in those receiving anakinra. The carrier frequency was higher in the patient cohort than in controls but was not statistically significant. Coimmunoprecipitation studies demonstrated that these pyrin variants did not affect binding to PSTPIP1.

Conclusions: P369S/R408Q substitutions are associated with a highly variable phenotype, and are infrequently associated with typical FMF symptoms, however a trial of colchicine is warranted in all. Functional and modelling studies suggest that these substitutions do not significantly affect pyrin's interaction with PSTPIP1. This study highlights the need for caution in interpreting genetic tests in patients with atypical symptoms.

Figure 1

Modeling of B-Box domain. Substitutions at P369S, and R408Q highlighted, neither are predicted to have a significant effect on protein conformation.

Figure 2

P369S, R408Q and P369S/R408Q variants do not affect pyrin’s binding to wildtype or mutant PSTPIP1. GST tagged PSTIP1, wild type and variants known to affect binding to pyrin, were co-transfected with myc-tagged pyrin, wild types and variants of interest. Lanes 6 and 7 demonstrate that the A230T and to a lesser extent E250Q GST tagged PSTPIP1 mutants tightly bind to myc-tagged pyrin and act as a positive control. We also note a change in size of the immunoprecipitated pyrin band when co-expressed with the A230T PSTPIP1 mutation, a finding that was repeated in multiple experiments. This band of increased size is not observed in the protein lysate (Fig. 2C). None of the pyrin variants in exon 3 or the highly penetrant mutation in exon 10 (M694V) (lanes 3–5) bound PSTPIP1 with greater avidity than wild type (lane 2).