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Optimization of Next-Generation Sequencing Technologies for von Hippel Lindau (VHL) Mosaic Mutation Detection and

Lucie Coppin1, Pascal Plouvier2, Michel Crépin2

  • 1UMR-S 1172-Jean-Pierre Aubert Research Center, University of Lille, Lille, France; INSERM, UMR-S 1172, Team on Mucins, Epithelial Differentiation and Carcinogenesis, Lille, France; Department of Biochemistry and Molecular Biology, Centre Hospitalier Régional et Universitaire de Lille, Lille, France.

The Journal of Molecular Diagnostics : JMD
|February 8, 2019
PubMed

Insights

Next-generation sequencing effectively detects mosaic mutations in Von Hippel-Lindau (VHL) disease, a genetic disorder causing tumors. This advanced approach improves diagnosis for patients with VHL phenotypes but lacking detectable mutations via conventional methods.

Area of Science:

  • Genetics
  • Oncology
  • Molecular Biology

Background:

  • Von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations in the VHL tumor suppressor gene.
  • A subset of patients with a VHL phenotype lack detectable mutations using standard genetic testing, suggesting potential mosaicism.

Purpose of the Study:

  • To develop and validate a next-generation sequencing (NGS) strategy for detecting VHL mosaic mutations.
  • To optimize NGS for improved variant detection sensitivity in VHL mosaicism.

Main Methods:

  • Implementation of an optimized NGS strategy with targeted amplicon enrichment.
  • Utilized SNaPshot and droplet digital PCR for sensitive confirmation of low-allelic-ratio mosaic mutations.
  • Generated VHL mutant plasmids for assay validation and quality control.

Main Results:

  • The developed NGS strategy successfully identified mosaic mutations in 8.5% of patients with suspected VHL disease.
  • Confirmed mosaic mutations with allelic ratios as low as 1% using droplet digital PCR.
  • Established a robust workflow for VHL mosaic mutation detection in a clinical setting.

Conclusions:

  • NGS combined with droplet digital PCR and SNaPshot provides a powerful tool for diagnosing VHL mosaicism.
  • This approach enhances diagnostic yield in patients with VHL phenotypes but negative conventional genetic testing.
  • Enables accurate identification of VHL mosaic mutations, improving patient management and genetic counseling.

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