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 were 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 to 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.

The UCL low-density lipoprotein receptor gene variant database: pathogenicity update

“…20 Both programs give predictive scores for splice acceptor and donor sequences for wild-type and variant sequences. The pathogenic impact of LDLR variants in the promoter and 5′ untranslated region of the gene have recently been published 21. Where appropriate, structural conservation scores have been given to variants (see online supplementary table S3).…”

Clinical utility of the polygenic LDL-C SNP score in familial hypercholesterolemia

“…These methods produce a large amount of sequence data, requiring statistical and bioinformatics analytical approaches, and this has increased the number of occasions whereby a Variant of Uncertain Significance (VUS) is identified [32]. This creates a diagnostic conundrum which clearly cannot be reported as FH-causing to the clinician or patient, but which requires either in vitro molecular assays to examine impact on transcription [33] or splicing [34], or by family studies to see if the variant tracks with high LDL-C levels in the family, while the relatives without the inherited variant have normal levels of LDL-C. However, a number of relatives in a single family, or many families with the same variant, are needed to use co-segregation as a functional proof of disease-causing, and such families are not always available.…”

Identification of the Functional Variant(s) that Explain the Low-Density Lipoprotein Receptor (LDLR) GWAS SNP rs6511720 Association with Lower LDL-C and Risk of CHD

“…It also acts as a modulator of alterative exon splicing, which can lead to a shift in the reading frame and an altered gene transcript [8–11]. Non-coding SNPs in LDLR have also been reported to be functional, for example, in the promoter region c.-139C>G [12], c.-101T>C, c.-121T>C [13], and -49C>T [14], and rs17248720 in the intergenic region [15] are involved in regulation of gene expression and have been reported to cause FH.…”