Functional characterization of splicing and ligand-binding domain variants in the LDL receptor

Abstract

Familial hypercholesterolemia (FH) is an autosomal dominant disorder mostly caused by mutations in the LDLR gene. Although the detection of functional mutations in the LDLR gene provides an unequivocal diagnosis of the FH condition, there are many variants whose pathogenicity is still unknown. The aims of this study were to set up a rapid method to determine the effect of LDLR mutations, thereby providing an accurate diagnosis of FH, and to functionally characterize six LDLR mutations detected at high frequency by the LIPOchip(®) platform (Progenika Biopharma, Spain) in the Spanish population. LDLR expression and activity were analyzed by one-single-step flow cytometry assay and confocal microscopy. Splicing effects were determined by sequencing reverse transcription polymerase chain reaction products. The analysis of three heterozygous variants with a single point mutation within the low-density lipoprotein binding domain allowed us to classify the c.806G>A variant as nonpathogenic, and c.862G>A and c.895G>A variants as causative of FH. The results obtained for three variants affecting donor splice sites of the LDLR mRNA, c.313+2dupT, c.1186+5G>A, and c.1845+1G>C, demonstrated that these mutations are pathogenic. These results expand our knowledge of mutations responsible for FH, providing an accurate diagnosis and leading to early treatment to reduce the risk of premature cardiovascular events.

The importance of an integrated analysis of clinical, molecular, and functional data for the genetic diagnosis of familial hypercholesterolemia

“…Because the p.Arg406Thr variant retains 60% activity, it can be considered a mild mutation, and the variation in the phenotype of carriers can be attributed to environmental factors that are known to affect the phenotype, even in patients with FH. 30 The cutoff value for determining whether an LDLR variant is considered a functional mutant by in vitro studies has not been established, but, based on several published studies, 1,27,29,[31][32][33] in vitro LDLR activity less than 70-80% (either in expression, binding, or internalization), corresponding to 85-90% total LDLR activity, could classify a variant as pathogenic.…”

“…23,[27][28][29] Biochemical characteristics of each mutation carrier are shown in Supplementary Table S1 online. Only data for adults without treatment were included (except lipoprotein(a)) because no values were registered for the majority of pediatric patients.…”

Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment

“…For many years the reference method to estimate LDLR activity has been the radioactive assay [39]. However approaches using fluorescently-labelled LDL have also been described and allow determination of LDL binding and uptake by flow cytometry [11], [14], [30]. Radioactive assay has the advantage of being very sensitive, but it also has serious drawbacks such as the risk of exposure to radioisotopes for experimenters and colleagues, or the difficulties and ethical considerations of nuclear waste elimination procedures.…”

“…Therefore, to determine the amount of internalized LDL, Trypan blue solution (Sigma-Aldrich, Steinheim, Germany) was added to a final concentration of 0.2% directly to the samples, eliminating the extracellular signal due to the non internalized LDLR-LDL complexes. Fluorescence intensities were measured by flow cytometry, in a Facscalibur Flow cytometer according to the manufacturer instructions as previously described [30]. For each sample, fluorescence of 10,000 events was acquired for data analysis.…”

Identification of the gene defect responsible for severe hypercholesterolaemia using whole-exome sequencing

“…Over 1700 LDLR mutations have been reported worldwide and are distributed among 18 exons, introns and promoter regions, including point mutations, such as substitutions, insertions, and deletions, and copy number variants. Each of these mutations influences the LDLR residual activities to a different degree26. However, many mutations that are located in introns with uncertain biological consequences have been identified as the causative mutations of FH27.…”