Minigene splicing reporter assay: a high-stake tool for genetic diagnosis in familial hypobetalipoproteinemia.

BACKGROUND & AIMS: Familial hypobetalipoproteinemia 1 (FHBL-SD2) is the most common monogenic form of primary hypocholesterolaemia, related to truncating variants in the APOB gene encoding apolipoprotein B. Due to its high level of complexity, variants of uncertain significance (VUS) require further investigations. This study aims to demonstrate the value of setting minigene assays in the FHBL-SD2's genetic diagnosis.

METHODS

Four APOB VUS occurring in patients with a FHBL-SD2 phenotype were considered. In silico analysis were performed with six software programs supposed to predict the potential splicing effect. Then, functional consequences were studied in vitro using a minigene splicing reporter assay.

RESULTS

An effect on splicing was predicted in silico for the 4 variants, with the activation of a cryptic acceptor site for c.694-13A>G and c.1471-6A>G variants, and the use of a cryptic donor site for c.1123A>G and c.1470G>A variants. Minigene study showed a complete effect on splicing for 3 mutations, confirming the in silico predictions. All of these transcripts result in premature truncated variants. Therefore, these variants were reclassified as likely pathogenic and causative of FHBL-SD2. However, no effect was shown either in HeLA and HuH7 cells for the c.1470G>A variant.

CONCLUSIONS

Minigene study appears to be a promising and valuable tool to enhance the diagnostic accuracy of FHBL-SD2. It emphasizes the challenge in interpreting VUS and underscores the importance of establishing a clear strategy to assess their significance. Therefore, promoting minigene studies would be beneficial to understand precisely the impact of splicing variants.