In vitro functional characterization of missense mutations in the LDLR gene

BackgroundMutations in the LDL receptor gene are the major cause of familial hypercholesterolaemia (FH) but it has been previously shown that the simple finding of a variation in the coding sequence of the LDLR does not confirm that it is the actual cause of FH. The pathogenicity of five missense alterations in the LDLR gene coding sequence found in a previous epidemiologic study was investigated.MethodsThe effects of the different sequence variants on LDLR expression and activity were analysed in vitro stably transfected CHO-ldlA7 cells by immunobloting of cell extracts, by uptake and degradation rates of 125I-labelled LDL and immunofluorescence microscopy of whole cells. Analysis in silico was also performed.ResultsLDLR functional assays showed that variants p.V429L, p.W490R and p.S648P of the LDLR coding sequence severely impaired receptor function, while variant p.P685S had a milder effect and cells carrying p.V859M variant had LDL clearance rates comparable to cells expressing normal LDLR. In silico analysis failed to predict correctly the effect of 4/5 alterations.ConclusionAssessing the pathogenicity of the different variants found in patients with clinical diagnosis of FH is of great importance to distinguish pathogenic mutations from rare silent variants and has clinical implications for determining the associated cardiovascular risk.

► We performed the functional characterizations of 5 LDLR missense mutations. ► We used 3 methods including 125I-labelled LDL uptake and degradation studies. ► Phenotype and genotype co relation was performed. ► One mutation is a rare polymorphism and in silico analysis failed to predict. ► For clinical purposes it's importance to distinguish mutations from silent variants.