FUNCTIONAL EFFECT OF NOVEL AMINO ACID VARIANTS OF APOLIPOPROTEIN B IN FAMILIAL HYPOBETALIPOPROTEINEMIA

Introduction. Familial Hypobetalipoproteinemia (FHBL) is acodominant disorder characterized by reduced plasma levels ofLDL-C and apolipoprotein (apo) B. In 50% of cases FHBL is due tomutations in APOB gene resulting in truncated apoBs of varioussize. Some mutations in APOB gene resulting in non-conservativeamino acid substitutions were reported to cause FHBL. In vitro,these mutations induce the retention of the mutant apoB in theendoplasmic reticulum (ER) and impair the secretion of apoB-containinglipoproteins. In two FHBL subjects we identified two novelamino acid variants (Thr26_27delinsAsn and Tyr102Cys) locatedin the N-terminal 1000 amino acids of mature apoB.Methods. To investigate the functional effect of these mutationswe constructed plasmids containing human apoB-48 cDNAs harbouringthe mutations. McA-RH7777 rat hepatoma cells weretransiently and stably transfected with wild type or mutant humanapoB-48. The secretion efficiency of human apoB-48 was determinedby immunoblotting. To evaluate whether the mutant apoB-48 was able to form apoB-containing lipoproteins, the incubationmedia were ultracentrifuged to separate the lipoprotein classes.Immunocytochemistry was used to assess the intracellular localizationof the mutant proteins.Results. The mutation Thr26_27delinsAsn strongly reduces thesecretion of apoB-48 from the transfected cells. The mutant apoB-48 appears to be retained in ER as demonstrated by the confocalimages showing co-localization of the mutant apoB with the ERmarker. In stably transfected cells the defect of mutant apoB-48secretion was confirmed by the absence of apoB-48 containinglipoproteins in the medium. These observations suggest thatThr26_27delinsAsn alters the structure of the beta-barrel of N-terminaldomain of apoB (the first 267 amino acids of mature protein)preventing the secretion of apoB-containing lipoproteins. By contrastthe mutation Tyr102Cys had no effect on apoB-48 secretion.Conclusions. This finding supports the notion that Thr26_27delinsAsn is the cause of FHBL.