Background and Aims

Hypobetalipoproteinemia (HBL) is characterized by plasma concentrations of LDL-cholesterol and apolipoprotein B (ApoB) below the 5^th percentile for age and gender. Familial HBL (FHBL) is mostly caused by premature stop codon on APOB. Nevertheless, some families carry APOB missense variations, classified as Variations of Unknown Significance (VUS), and express HBL phenotype. We aim to develop stable human cell lines carrying these VUS to assess their pathogenicity.

Methods

Five APOB VUS were selected from FHBL patients: p.(Cys97Gly), p.(Asp2182Tyr) and p.(Leu351Arg) or literature : p.(Ala58Pro) and p.(Leu351Met) (PMID: 20032471). Nonsense variations (knock-out, KO) generated using CRISPR editing will serve as FHBL controls.

ApoB secreting HuH7 cell lines were chosen. Genome engineering was performed by CRISPR-Cas9: guides RNA and ssODN were designed and produced. Optimal transfection conditions were determined. Cell lines were selected and isolated by dilution.

APOB transcription was assessed by digital droplets PCR. Cell culture conditions were optimized to improve ApoB secretion and estimate VLDL secretion.

Results

Until now, only KO variations were induced in HuH7 cells as p.(Arg356Glufs*5) (homozygous and heterozygous). APOB expression is decreased by 50% in heterozygous KO and almost abolished in homozygous KO.

Improvements of this strategy are currently on going in order to increase homologous recombination efficiency in order to obtain stable missense variation generation in HuH7 cells.

Conclusions

This method will be useful to explore functionality of APOB VUS, found in FHBL patients. It will be subsequently enlarged to explore VUS in other dyslipidemia.