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

Abstract

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 APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB 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 APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB 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 APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.

CRISPR/Cas9‐based functional genomics strategy to decipher the pathogenicity of genetic variants in inherited metabolic disorders

“…Moreover, most of these methods are focused on strategies specifically designed for particular diseases rather than providing generic approaches. [35][36][37][38][39][40][41] Although several studies have highlighted the usefulness of CRISPR/Cas9 for genome editing and cell model generation, the potential of this technology for functional validation of VUS remains underexploited in the field of IMDs. [42][43][44][45][46] Here we propose a generic approach to assess the functional impact of genetic variants based on the generation of homozygous knock-in cell line models using CRISPR/Cas9.…”

“…Commonly used strategies are mainly based on the expression of the mutant alleles of interest in knock‐out cell line systems or the use of non‐human cell models, such as yeast or bacteria. Moreover, most of these methods are focused on strategies specifically designed for particular diseases rather than providing generic approaches 35–41 . Although several studies have highlighted the usefulness of CRISPR/Cas9 for genome editing and cell model generation, the potential of this technology for functional validation of VUS remains underexploited in the field of IMDs 42–46 …”

Applications and Research Advances in the Delivery of CRISPR/Cas9 Systems for the Treatment of Inherited Diseases

“…While APOB expression was 70% lower in heterozygous APOB-KO cells, it was almost nonexistent in homozygous APOB-KO cells. This work highlights CRISPR/Cas9's capability to treat FHBL [98].…”