Transcriptome analysis of unmedicated heterozygous familial Mediterranean fever patients reveals a type I interferon signature driving increasing Pyrin expression

Abstract

Objectives: Familial Mediterranean fever (FMF) is traditionally viewed as an autosomal recessive autoinflammatory disorder. However, a significant subset of patients harbouring a single pathogenic MEFV mutation exhibit a clinical phenotype indistinguishable from that of homozygous patients. We aimed to compare the transcriptomic profiles of patients carrying a single pathogenic mutation who exhibit the classical FMF phenotype with those of healthy carriers (with 1 pathogenic mutation), as well as with homozygous or compound heterozygous patients (with 2 pathogenic mutations), to identify differential molecular signatures and potential diagnostic pathways.

Methods: Peripheral blood mononuclear cells (PBMCs) from 10 patients with FMF (phenotypic carriers/homozygotes) and 5 healthy MEFV mutation carriers were isolated during asymptomatic, treatment-naïve phases. Transcriptome profiling employed globin mRNA-depleted, strand-specific Qiaseq libraries sequenced on Illumina NextSeq 500/550 (paired-end). Differential expression analysis applied TMM (trimmed mean of M values) based-normalised negative binomial models (|log₂FC| > 1, adjusted *P* < .01), with Reactome pathway enrichment. For immunoblotting, interferon (IFN)-α-stimulated monocytes/PBMCs of healthy individuals were lysed, denatured, and probed with antibodies targeting key proteins (IRF-3, IFN-stimulated gene 15 [ISG15], Pyrin, STAT1, AIM2, caspase-5, β-actin). CXCL10 levels were quantified using Luminex.

Results: PBMC profiling revealed 147 differentially expressed genes. Pathway analyses highlighted enrichment in type I IFN signalling and inflammasome-related pathways, with marked upregulation of type I ISGs: ISG15, IFIT2, STAT1, and the inflammasome sensor Pyrin encoded by the MEFV gene, mutated in FMF. Moreover, functional assays demonstrated that type I IFN stimulation increases Pyrin protein levels in PBMCs and isolated monocytes, revealing cross-talk between IFN responses and inflammasome signalling.

Conclusions: These findings suggest that type I IFN signalling acts as a critical 'second hit', amplifying Pyrin expression in heterozygous individuals and enabling disease manifestation despite a single MEFV mutation. This study offers an explanation for the much-debated issue of the carriers expressing disease phenotypes in diseases such as FMF and presents novel insights for precision diagnosis and therapeutic intervention.