CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants.

Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous -VUS (p.Leu351Arg), in a FHBL family. We generated knock-out (KO) and -p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The expression was decreased by 70% in the heterozygous -KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The -p.Leu351Arg homozygous cells presented with a 40% decreased expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of -missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.