High-throughput sequencing of the entire genomic regions of CCM1/KRIT1, CCM2 and CCM3/PDCD10 to search for pathogenic deep-intronic splice mutations in cerebral cavernous malformations

Eur J Med Genet. 2017 Sep;60(9):479-484. doi: 10.1016/j.ejmg.2017.06.007. Epub 2017 Jun 20.


Cerebral cavernous malformations (CCM) are vascular lesions of the central nervous system that can cause headaches, seizures and hemorrhagic stroke. Disease-associated mutations have been identified in three genes: CCM1/KRIT1, CCM2 and CCM3/PDCD10. The precise proportion of deep-intronic variants in these genes and their clinical relevance is yet unknown. Here, a long-range PCR (LR-PCR) approach for target enrichment of the entire genomic regions of the three genes was combined with next generation sequencing (NGS) to screen for coding and non-coding variants. NGS detected all six CCM1/KRIT1, two CCM2 and four CCM3/PDCD10 mutations that had previously been identified by Sanger sequencing. Two of the pathogenic variants presented here are novel. Additionally, 20 stringently selected CCM index cases that had remained mutation-negative after conventional sequencing and exclusion of copy number variations were screened for deep-intronic mutations. The combination of bioinformatics filtering and transcript analyses did not reveal any deep-intronic splice mutations in these cases. Our results demonstrate that target enrichment by LR-PCR combined with NGS can be used for a comprehensive analysis of the entire genomic regions of the CCM genes in a research context. However, its clinical utility is limited as deep-intronic splice mutations in CCM1/KRIT1, CCM2 and CCM3/PDCD10 seem to be rather rare.

Keywords: CCM1; CCM2; CCM3; Cerebral cavernous malformation; Deep-intronic variants; NGS.

MeSH terms

  • Adolescent
  • Adult
  • Apoptosis Regulatory Proteins / genetics*
  • Carrier Proteins / genetics*
  • Child
  • DNA Copy Number Variations
  • Female
  • Genetic Testing / methods*
  • Hemangioma, Cavernous, Central Nervous System / diagnosis
  • Hemangioma, Cavernous, Central Nervous System / genetics*
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Introns
  • KRIT1 Protein / genetics*
  • Male
  • Membrane Proteins / genetics*
  • Middle Aged
  • Mutation*
  • Proto-Oncogene Proteins / genetics*
  • RNA Splicing*
  • Sequence Analysis, DNA / methods


  • Apoptosis Regulatory Proteins
  • CCM2 protein, human
  • Carrier Proteins
  • KRIT1 Protein
  • KRIT1 protein, human
  • Membrane Proteins
  • PDCD10 protein, human
  • Proto-Oncogene Proteins