Functional evaluation of rare variants in complement factor I using a minigene assay
Cobey J H Donelson1, Nicolo Ghiringhelli Borsa1, Amanda O Taylor1
1Molecular Otolaryngology and Renal Research Laboratory, Carver College of Medicine, University of Iowa, Iowa City, IA, United States.
Insights
Functional assays reclassified four ultra-rare complement factor I (CFI) gene variants from uncertain significance to pathogenic. This study highlights the importance of minigene assays for understanding rare CFI variants and their clinical impact.
Area of Science:
- Immunology and Genetics
- Molecular Biology
Background:
- Complement factor I (FI) is a regulatory serine protease crucial for controlling complement system activity by inactivating C3b and C4b.
- Missense variants in the CFI gene, especially ultra-rare ones (0.01%-0.1% MAF), are often classified as variants of uncertain significance (VUS) due to limited functional studies.
- Accurate classification of these variants is essential for clinical genetic testing and patient diagnosis.
Purpose of the Study:
- To functionally assess the impact of 36 ultra-rare variants in the complement factor I (CFI) gene using a minigene splicing assay.
- To reclassify variants of uncertain significance (VUS) based on their functional consequences.
- To evaluate the utility of minigene assays in determining the clinical relevance of rare genetic variants.
Main Methods:
- A minigene splicing assay was employed to investigate the splicing effects of 36 selected ultra-rare CFI variants.
- Variants were chosen based on minor allele frequencies (MAF) and association with low-normal FI levels.
- Standard ACMG (American College of Medical Genetics and Genomics) guidelines were used for variant reclassification.
Main Results:
- Four CFI variants demonstrated aberrant splicing: one at a 5' consensus splice site (c.1429G>C, p.Asp477His) and three exonic changes (c.355G>A, p.Gly119Arg; c.472G>A, p.Gly158Arg; and c.950G>A, p.Arg317Gln).
- These four variants were successfully reclassified as likely pathogenic (LP) or pathogenic (P) according to ACMG criteria.
- The study identified specific variants impacting splicing, providing concrete evidence for their pathogenicity.
Conclusions:
- Functional assessment using minigene splicing assays is critical for accurately interpreting the clinical significance of rare CFI variants.
- This study successfully reclassified four VUS in the CFI gene to pathogenic, improving diagnostic clarity.
- The findings emphasize the importance of integrating functional data into genetic variant interpretation workflows for complement-related disorders.
Abstract:
The regulatory serine protease, complement factor I (FI), in conjunction with one of its cofactors (FH, C4BP, MCP, or CR1), plays an essential role in controlling complement activity through inactivation of C3b and C4b. The functional impact by missense variants in the CFI gene, particularly those with minor allele frequencies of 0.01% to 0.1%, is infrequently studied. As such, these variants are typically classified as variants of uncertain significance (VUS) when they are identified by clinical testing. Herein, we utilized a minigene splicing assay to assess the functional impact of 36 ultra-rare variants of CFI. These variants were selected based on their minor allele frequencies (MAF) and their association with low-normal FI levels. Four variants lead to aberrant splicing-one 5' consensus splice site (NM_000204.5: c.1429G>C, p.Asp477His) and three exonic changes (c.355G>A, p.Gly119Arg; c.472G>A, p.Gly158Arg; and c.950G>A, p.Arg317Gln)-enabling their reclassification to likely pathogenic (LP) or pathogenic (P) based on ACMG guidelines. These findings underscore the value of functional assays, such as the minigene assay, in assessing the clinical relevance of rare variants in CFI.
