Uncommon nucleotide excision repair phenotypes revealed by targeted high-throughput sequencing

Orphanet J Rare Dis. 2016 Mar 22;11:26. doi: 10.1186/s13023-016-0408-0.

Abstract

Background: Deficient nucleotide excision repair (NER) activity causes a variety of autosomal recessive diseases including xeroderma pigmentosum (XP) a disorder which pre-disposes to skin cancer, and the severe multisystem condition known as Cockayne syndrome (CS). In view of the clinical overlap between NER-related disorders, as well as the existence of multiple phenotypes and the numerous genes involved, we developed a new diagnostic approach based on the enrichment of 16 NER-related genes by multiplex amplification coupled with next-generation sequencing (NGS).

Methods: Our test cohort consisted of 11 DNA samples, all with known mutations and/or non pathogenic SNPs in two of the tested genes. We then used the same technique to analyse samples from a prospective cohort of 40 patients. Multiplex amplification and sequencing were performed using AmpliSeq protocol on the Ion Torrent PGM (Life Technologies).

Results: We identified causative mutations in 17 out of the 40 patients (43%). Four patients showed biallelic mutations in the ERCC6(CSB) gene, five in the ERCC8(CSA) gene: most of them had classical CS features but some had very mild and incomplete phenotypes. A small cohort of 4 unrelated classic XP patients from the Basque country (Northern Spain) revealed a common splicing mutation in POLH (XP-variant), demonstrating a new founder effect in this population. Interestingly, our results also found ERCC2(XPD), ERCC3(XPB) or ERCC5(XPG) mutations in two cases of UV-sensitive syndrome and in two cases with mixed XP/CS phenotypes.

Conclusions: Our study confirms that NGS is an efficient technique for the analysis of NER-related disorders on a molecular level. It is particularly useful for phenotypes with combined features or unusually mild symptoms. Targeted NGS used in conjunction with DNA repair functional tests and precise clinical evaluation permits rapid and cost-effective diagnosis in patients with NER-defects.

Keywords: Cockayne syndrome; ERCC2; ERCC3; ERCC5; ERCC6; ERCC8; NER; NGS; POLH; xeroderma pigmentosum.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cockayne Syndrome / genetics
  • DNA Helicases / genetics
  • DNA Repair / genetics*
  • DNA Repair Enzymes / genetics
  • DNA-Binding Proteins / genetics
  • DNA-Directed DNA Polymerase / genetics
  • Endonucleases / genetics
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Mutation
  • Nuclear Proteins / genetics
  • Phenotype
  • Poly-ADP-Ribose Binding Proteins
  • Transcription Factors / genetics
  • Xeroderma Pigmentosum Group D Protein / genetics

Substances

  • DNA excision repair protein ERCC-5
  • DNA-Binding Proteins
  • ERCC8 protein, human
  • Nuclear Proteins
  • Poly-ADP-Ribose Binding Proteins
  • Transcription Factors
  • DNA-Directed DNA Polymerase
  • Rad30 protein
  • Endonucleases
  • DNA Helicases
  • ERCC6 protein, human
  • Xeroderma Pigmentosum Group D Protein
  • ERCC2 protein, human
  • DNA Repair Enzymes