B-252 Functional Validation of Novel FAS Intronic and Splice Region VUS Through Minigene Assays

Abstract

Background Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder characterized by chronic lymphadenopathy, splenomegaly, cytopenias, and an increased risk of lymphoma. Molecular genetic diagnosis is essential for the accurate diagnosis and management of ALPS, particularly due to its overlapping clinical features with autoimmune lymphoproliferative immunodeficiency (ALPID). About 80% of ALPS cases result from germline or somatic pathogenic variants in FAS (ALPS-FAS, ALPS-sFAS), primarily through dominant-negative interference or haploinsufficiency. The FAS mutation spectrum includes nonsense, frameshift, missense, splicing defect variants, and copy number variants. Notably, around 13% ALPS-FAS cases are attributed to splice site/region mutations in FAS that disrupt splicing. However, novel splice region or deep intronic FAS variants are often classified as variants of unknown significance (VUS) due to the lack of functional validation, making their precise classification difficult to determine. This study aims to identify and characterize novel intronic or splice region FAS VUS found in a cohort of 1,488 ALPS cases at a pediatric center, as well as those reported in the ClinVar database. Methods We retrospectively reviewed the records of 1,488 patients with suspected ALPS referred to our institution for either the FAS gene sequencing or ALPS next-generation sequencing (NGS) panel between 2005 and 2023. Previously unreported variants with potential splicing effect were identified. Additionally, we reviewed the ClinVar database for rare FAS variants with predicted effects on splicing. To functionally assess these variants, we plan to use a minigene assay, which involves constructing a simplified version of the gene containing the exon of interest and its flanking introns into a plasmid. This plasmid is then transfected into cells to analyze how the variant affects the resulting mRNA transcript. Results We identified 19 novel FAS variants with predicted splicing effect in 30 independent probands from our cohort. These variants were distributed across eight FAS intronic splicing regions, including one synonymous change in a deep exonic region predicted to activate a nearby cryptic splice donor. Additionally, a review of the ClinVar database revealed 19 previously unreported rare splice region variants in FAS, of which, 13 were predicted to affect splicing. We will use the minigene system to experimentally validate the functional consequences of these variants. Conclusion FAS pathogenic variants affecting splicing represent a significant contributor to ALP-FAS and ALP-sFAS. The minigene assay will provide crucial functional evidence to further characterize the pathogenicity of rare VUS with predicted splicing effects, thereby improving the molecular diagnosis for ALPS. This approach is particularly valuable when RNA samples are unavailable from affected patients, enabling a more comprehensive genetic evaluation of ALPS.