In vitro functional characterization of missense mutations in the LDLR gene

Abstract Background Mutations 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 m...

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Bibliographic Details
Published in:Atherosclerosis 2012-11, Vol.225 (1), p.128-134
Main Authors: Silva, S, Alves, A.C, Patel, D, Malhó, R, Soutar, A.K, Bourbon, M
Format: Article
Language:English
Subjects:
Adolescent
Animals
Atherosclerosis (general aspects, experimental research)
Biological and medical sciences
Blood and lymphatic vessels
Blood coagulation
Cardiology. Vascular system
Cardiovascular
Cardiovascular Diseases - genetics
Child
CHO Cells
Computational Biology
Cricetinae
Familial hypercholesterolaemia
Female
Functional studies
Humans
Hyperlipoproteinemia Type II - genetics
Impaired receptor function
Investigative techniques, diagnostic techniques (general aspects)
Male
Medical sciences
Middle Aged
Mutation, Missense
Predicted analysis
Receptors, LDL - genetics
Risk Factors
Stably transfection of CHO-ldlA7 cells
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Summary:Abstract Background Mutations 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. Methods The effects of the different sequence variants on LDLR expression and activity were analysed in vitro stably transfected CHO- ldl A7 cells by immunobloting of cell extracts, by uptake and degradation rates of125 I-labelled LDL and immunofluorescence microscopy of whole cells. Analysis in silico was also performed. Results LDLR 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. Conclusion Assessing 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.
ISSN:0021-9150
1879-1484
DOI:10.1016/j.atherosclerosis.2012.08.017