Abstract
Background and aimsFamilial 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. MethodsLDLR 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. ResultsThree 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. ConclusionsThrough 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.
Highlights
Familial hypercholesterolemia (FH) is a common autosomal genetic disorder characterized by high levels of low-density lipoprotein cholesterol (LDL-C) resulting in an increased risk for premature car diovascular disease
The average number of intronic variants in patients groups did not differ per FH+ genotype (i.e., low-density lipoprotein receptor gene (LDLR), apolipoprotein B100 gene (APOB), proprotein convertase substilisin kexin type 9 gene (PCSK9)) or FH- phenotype (Supplementary Table 4 and Supplementary Figure 2)
We used a novel approach to detect potential FH variant-negative (FH-)causing deep intronic variants in LDLR and identified a variant, c.2141-218G>A, which causes the inclusion of a pseudoexon between exon 14 and exon 15 in the LDLR mRNA, which includes a premature stop codon
Summary
Familial hypercholesterolemia (FH) is a common autosomal genetic disorder characterized by high levels of low-density lipoprotein cholesterol (LDL-C) resulting in an increased risk for premature car diovascular disease. Familial hypercholesterolemia (FH) is caused by pathogenic variants in LDLR, APOB, or PCSK9 genes (designated FH+). 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). 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. 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 FHcausing variants and suggests that intronic regions of LDLR need to be considered for sequencing in FH- patients
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