Functional Characterization of a Novel Intronic Complement Factor I Variant in Factor I Deficiency and Atypical...

The role of noncanonical intronic splice site variants in atypical hemolytic uremic syndrome (aHUS) cases without identified pathogenic variants has long been postulated. We previously identified a novel intronic variant (c.328+42G>A) in the complement factor I (CFI) gene in a patient with factor I (FI) deficiency. This study aimed to identify additional carriers and functionally characterize this variant and its impact on the expression of FI, a crucial complement regulator with a well-established role in aHUS.

After screening our genetic registry for patients with potential FI-driven pathologies, the identified c.328+42G>A carriers and their family members underwent comprehensive complement profiling and genetic analysis. To investigate mRNA isoforms with real-time polymerase chain reaction (PCR), we cultured monocyte-derived dendritic cells (mo-DCs) from carriers and used geneticin to assess the role of nonsense-mediated decay (NMD).

We identified 3 additional carriers, demonstrating marked enrichment within our aHUS cohort. All carriers exhibited consistently reduced FI levels. Based on the mo-DC experiments, the variant creates a strong noncanonical splice site, leading to the predominant production of a splice variant with a 43-bp extension of exon 2, which generates a premature stop codon. Geneticin treatment increased the aberrant transcript level 4- to 7-fold, supporting its degradation via NMD.

The CFI c.328+42G>A variant causes aberrant splicing and nearly complete loss of FI protein production. Besides causing FI deficiency in compound heterozygous form, this variant may contribute to the development of aHUS in heterozygotes. Our findings underscore the importance of analyzing intronic regions in complement-mediated diseases with unexplained genetic predisposition.