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Related Concept Videos

Complementation Tests00:49

Complementation Tests

A complementation test is a simple cross to identify whether the two mutations are located on the same gene or different genes. It was first performed by Edward Lewis in the 1940s while working on fruit flies. He developed the test to identify the location and arrangement of different mutations on chromosomes.
Organisms heterozygous for different mutations are crossed pairwise in all combinations. If present on different genes, the mutations can complement each other by providing the missing...

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Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA
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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.

Frontiers in Immunology
|September 6, 2024
PubMed
Summary

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.

Keywords:
Cis/trans-acting elementsRNA splicingalternative pathwaycomplementcomplement factor Icomplement-mediated diseaseshaploinsufficiency

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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.