Hypobetalipoproteinemia (HBL) is characterized by decreased plasma concentrations of low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (ApoB) below the fifth percentile for age and sex. The most common monogenic form of primary hypobetalipoproteinemia is caused by premature truncating variants (PTV) of the APOB gene (Familial Hypobetaliproteinemia 1, FHBL1; OMIM 615558), now called FHBL-SD2 according to the new classification [1]. The intestine produces ApoB-48, essential for secretion of exogenous lipids through chylomicron production and the liver produces full length ApoB (ApoB-100), the major structural component of very-low-density lipoproteins (VLDL) and LDL. In FHBL-SD2, ApoB truncation hampers ApoB secretion. The prevalence of heterozygote (Het) FHBL-SD2 in the general population is estimated between 1/1000 and 1/3000 [2] although this disease remains largely underdiagnosed in adulthood.

Indeed, most of Het-FHBL-SD2 cases are asymptomatic. However, because ApoB is mandatory for the transport and the metabolism of cholesterol and triglycerides (TG) in triglyceride-rich lipoproteins (TGRL) in blood, its defect may result in decreased LDL-C and triglycerides in the plasma and TG accumulation in the hepatocyte. Therefore FHBL-SD2 is associated with more than 6-fold increased risk of steatotic liver disease (SLD), that may lead to metabolic dysfunction-associated steato-hepatitis (MASH), cirrhosis and hepatocellular carcinoma[[3], [4], [5], [6]]. The rare much more symptomatic homozygous (Ho) form, is detected earlier in children, due to severe liposoluble vitamins and essential fatty acids deficiencies [7].

The gold-standard diagnosis is made by molecular analysis. Then, variants are classified according to the American College of Medical Genetics (ACMG) and Association of Medical Pathologists guidelines [8]. Nowadays, approximately 200 different pathogenic APOB variations have been characterized. Most of them are nonsense, frameshift and splice site mutations resulting in PTV encoding ApoB from 2 % to 89 % of ApoB-100 size, but also <20 activation of cryptic splice sites (review in Ref. [9]).

Some variations can be pathogenic by causing the appearance of a cryptic splicing site or the abolition of a canonical splicing site. Functional studies are required to establish its deleterious effect when the variant occurs outside the canonical natural splice site. As APOB is not expressed in easily accessible tissue, an in vitro approach using a minigene splicing reporter assay is required to study the variant's effect on splicing.

In this work, we aim to explore four variants found in four HBL probands. In silico, all of these were supposed to abolish the canonical acceptor or donor site and to result in the creation of a cryptic acceptor or donor site of intermediate strength. Therefore, we decided to study their effect on splicing by minigene splicing reporter assay.