title: Functional analysis of four LDLR 5'UTR and promoter variants in patients with familial hypercholesterolaemia.
abstract: Familial hypercholesterolaemia (FH) is an autosomal dominant inherited disease characterised by increased low-density lipoprotein cholesterol (LDL-C) levels. The functionality of four novel variants within the LDLR 5'UTR and promoter located at c.-13A>G, c.-101T>C, c.-121T>C and c.-215A>G was investigated using in silico and in vitro assays, and a systemic bioinformatics analysis of all 36 reported promoter variants are presented. Bioinformatic tools predicted that all four variants occurred in sites likely to bind transcription factors and that binding was altered by the variant allele. Luciferase assay was performed for all the variants. Compared with wild type, the c.-101T>C and c.-121T>C variants showed significantly lower mean (±SD) luciferase activity (64 ± 8 and 72 ± 8%, all P<0.001), suggesting that these variants are causal of the FH phenotype. No significant effect on gene expression was seen for the c.-13A>G or c.-215A>G variants (96 ± 15 and 100 ± 12%), suggesting these variants are not FH causing. Similar results were seen for the c.-101T>C and c.-121T>C variants in lipid-depleted serum. However, a significant reduction in luciferase activity was seen in the c.-215A>G variant in lipid-depleted serum. Electrophoretic-mobility shift assays identified allele-specific binding of liver (hepatoma) nuclear proteins to c.-121T>C and suggestive differential binding to c.-101T>C but no binding to c.-215A>G. These data highlight the importance of in vitro testing of reported LDLR promoter variants to establish their role in FH. The functional assays performed suggest that the c.-101T>C and c.-121T>C variants are pathogenic, whereas c.-13A>G variant is benign, and the status of c.-215A>G remains unclear.
authors.affiliation: Centre for Cardiovascular Genetics, British Heart Foundation Laboratories, Institute Cardiovascular Science, University College London Medicine School, London, UK.
authors.forename: Amna
authors.initials: A
authors.lastname: Khamis
authors.affiliation: Centre for Cardiovascular Genetics, British Heart Foundation Laboratories, Institute Cardiovascular Science, University College London Medicine School, London, UK.
authors.forename: Jutta
authors.initials: J
authors.lastname: Palmen
authors.affiliation: NE Thames Regional Genetics Service, Great Ormond Street Hospital, London, UK.
authors.forename: Nick
authors.initials: N
authors.lastname: Lench
authors.affiliation: NE Thames Regional Genetics Service, Great Ormond Street Hospital, London, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Taylor
authors.affiliation: Centre for Cardiovascular Genetics, British Heart Foundation Laboratories, Institute Cardiovascular Science, University College London Medicine School, London, UK.
authors.forename: Ebele
authors.initials: E
authors.lastname: Badmus
authors.affiliation: Centre for Cardiovascular Genetics, British Heart Foundation Laboratories, Institute Cardiovascular Science, University College London Medicine School, London, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Leigh
authors.affiliation: Centre for Cardiovascular Genetics, British Heart Foundation Laboratories, Institute Cardiovascular Science, University College London Medicine School, London, UK.
authors.forename: Steve E
authors.initials: SE
authors.lastname: Humphries
mesh_terms: D020121:5' Untranslated Regions; D001483:Base Sequence; D045744:Cell Line, Tumor; D006801:Humans; D006937:Hypercholesterolemia / Q000235:genetics*; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D011973:Receptors, LDL / Q000235:genetics* / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; C573412:LDLR protein, human; D011973:Receptors, LDL