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

Laurens F. Reeskamp, Manon Balvers, Jorge Peter, Laura van de Kerkhof, Lisette N. Klaaijsen, Mahdi M. Motazacker, Aldo Grefhorst, Natal A.W. van Riel, G. Kees Hovingh, Joep C. Defesche, Linda Zuurbier (Corresponding author)

Research output: Contribution to journalArticleAcademicpeer-review

7 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)14-20
Number of pages7
Publication statusPublished - Mar 2021


  • Familial hypercholesterolemia
  • Intron
  • LDL cholesterol
  • LDL receptor
  • Next-generation sequencing
  • RNA splicing


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