Abnormal apolipoprotein B pre-mRNA splicing in patients with familial hypobetalipoproteinaemia

Background: Familial hypobetalipoproteinaemia (FHBL) is a codominant disorder characterised by fatty liver and reduced plasma levels of low-density lipoprotein (LDL) and its protein constituent apolipoprotein B ( apoB). FHBL is linked to the APOB gene in some but not all known cases. In a group of 59 patients with FHBL genotyped for APOB gene mutations, we found three novel splice-site mutations: c. 904+4AR -> G in intron 8, c. 3843-2A -> G in intron 24 and c. 4217-1G -> RT in intron 25. Objective: To assess the effects of these mutations on apoB prem-RNA splicing. Methods: ApoB mRNA was analysed in the liver of one proband and in cells expressing APOB minigenes harbouring the mutations found in the other probands. Results: In the liver of the c. 3843-2A -> RG carrier, an apoB mRNA devoid of exon 25 was identified, predicted to encode a truncated peptide of 1260 amino acids. The analysis of minigene transcripts in COS-1 cells showed that the c. 904+4A -> G mutation caused the formation of an mRNA devoid of exon 8, predicted to encode a short apoB of 247 amino acids. The minigene harbouring the c. 4217-1G -> T mutation in intron 25 generated an mRNA in which exon 25 joined to a partially deleted exon 26, resulting from the activation of an acceptor site in exon 26; this mRNA is predicted to encode a truncated protein of 1380 amino acids. All these truncated apoBs were not secreted as constituents of plasma lipoproteins. Conclusion: These findings demonstrate the pathogenic effect of rare splice- site mutations of the APOB gene found in FHBL