Familial hypercholesterolemia (FH) is an inherited lipid disorder characterized by increased levels of LDL cholesterol. About 5-10% of FH cases occur due to variants in the APOB gene, but these alterations can be a more common cause of FH than expected since most of APOB variants identified is still unknown their effect on the metabolism. The majority of the variants are missense but there are a few nonsense variants and small indels in exon 29 identified in individuals with hypercholesterolemia phenotype that can cause FH. The aim of this project was to functional characterize APOB variants from exon 29 identified in individuals referred to the Portuguese FH Study to assess if these are the genetic cause of disease.
LDL from index cases and relatives was isolated through sequential ultracentrifugation. ED-LDLR was purified from HEK293 cells transfected with the pcDNA3.1-EC-LDLR-His plasmid by affinity chromatography. Purified ED-LDLR fragments were coated onto 96-well plates and incubated with the different APOB variants. Antibodies were used for ligand detection, and absorbance was determined at 405 nm. CHO-ldlA7 cells were transfected with wt LDLR plasmid and incubated with FITC-labeled LDL to determine LDL binding and uptake by flow cytometry.
p.(Gln4316*) and p.(Glu4387Asnfs*7) alterations from exon 29 showed reduced affinity for the LDL receptor. Uptake and binding assays results were similar, so these variants may affect the binding of apoB to the LDL receptor. The alterations studied were not present in a normolipidemic panel.
APOB variants studied in this work produce truncated forms of apoB, but they are unlikely to lead to nonsense-mediated decay processes due to their location near the end of the gene. Functional studies can provide important evidence for variant pathogenicity assessment being these essential to provide an accurate diagnosis. These assays can confirm the clinical diagnosis by highlighting the cause of disease, and contribute to a personalized treatment and stratify patient associated cardiovascular risk.N/
