Intronic variant screening with targeted next-generation sequencing reveals first pseudoexon in LDLR in familial hypercholesterolemia.

BACKGROUND AND AIMS: Familial hypercholesterolemia (FH) is caused by pathogenic variants in LDLR, APOB, or PCSK9 genes (designated FH+). However, a significant number of clinical FH patients do not carry these variants (designated FH-). Here, we investigated whether variants in intronic regions of LDLR attribute to FH by affecting pre-mRNA splicing. METHODS: LDLR introns are partly covered in routine sequencing of clinical FH patients using next-generation sequencing. Deep intronic variants, >20 bp from intron-exon boundary, were considered of interest once (a) present in FH- patients (n = 909) with LDL-C >7 mmol/L (severe FH-) or after in silico analysis in patients with LDL-C >5 mmol/L (moderate FH-) and b) absent in FH + patients (control group). cDNA analysis and co-segregation analysis were performed to assess pathogenicity of the identified variants. RESULTS: Three unique variants were present in the severe FH- group. One of these was the previously described likely pathogenic variant c.2140+103G>T. Three additional variants were selected based on in silico analyses in the moderate FH- group. One of these variants, c.2141-218G>A, was found to result in a pseudo-exon inclusion, producing a premature stop codon. This variant co-segregated with the hypercholesterolemic phenotype. CONCLUSIONS: Through a screening approach, we identified a deep intronic variant causal for FH. This finding indicates that filtering intronic variants in FH- patients for the absence in FH + patients might enrich for true FH-causing variants and suggests that intronic regions of LDLR need to be considered for sequencing in FH- patients.

A DNA microarray for the detection of point mutations and copy number variation causing familial hypercholesterolemia in Europe.

To facilitate genetic cascade screening for familial hypercholesterolemia (FH) in Europe, two versions (7 and 9) of a DNA microarray were developed to detect the most frequent point mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin 9 (PCSK9) genes. The design of these microarrays is based on LIPOchip, version 4, which detects 191 LDLR and APOB mutations identified in Spanish patients with FH. A major improvement of LIPOchip, versions 7 and 9, is the ability to detect copy number variation (deletions or duplications of entire exons) in LDLR, thus abolishing the need to perform multiplex ligase-dependent probe amplification in patients with FH. The aim of this study was to validate a tool capable of detecting point mutations and copy number variations simultaneously and to evaluate its use and the newly developed software for analysis in clinical practice by reanalysis of several patients with known mutations causing FH. With the help of these validations, several aspects were analyzed, improved, and implemented in a newer version, which was evaluated through an internal validation.