Accurate Detection of Hot-Spot MTOR Somatic Mutations in Archival Surgical Specimens of Focal Cortical Dysplasia by Molecular Inversion Probes

Paola Dimartino, Valeria Mariani, Caterina Marconi, Raffaella Minardi, Manuela Bramerio, Laura Licchetta, Veronica Menghi, Luca Morandi, Pamela Magini, Patrizia Mongelli, Francesco Cardinale, Marco Seri, Paolo Tinuper, Laura Tassi, Tommaso Pippucci, Francesca Bisulli

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Formalin-fixed, paraffin-embedded brain specimens are a potentially rich resource to identify somatic variants, but their DNA is characterised by low yield and extensive degradation, and matched peripheral samples are usually unavailable for analysis. Methods: We designed single-molecule molecular inversion probes to target 18 MTOR somatic mutational hot-spots in unmatched, histologically proven focal cortical dysplasias from formalin-fixed, paraffin-embedded tissues of 50 patients. Results: We achieved adequate DNA and sequencing quality in 28 focal cortical dysplasias, mostly extracted within 2 years from fixation, showing a statistically significant effect of time from fixation as a major determinant for successful genetic analysis. We identified and validated seven encompassing hot-spot residues (found in 14% of all patients and in 25% of those sequenced and analysed). The allele fraction had a range of 2–5% and variants were absent in available neighbouring non-focal cortical dysplasia specimens. We computed an alternate allele threshold for calling true variants, based on an experiment-wise mismatch count distribution, well predicting call reliability. Conclusions: Single-molecule molecular inversion probes are experimentally simple, cost effective and scalable, accurately detecting clinically relevant somatic variants in challenging brain formalin-fixed, paraffin-embedded tissues.

Original languageEnglish
Pages (from-to)571-577
Number of pages7
JournalMolecular Diagnosis and Therapy
Volume24
Issue number5
DOIs
Publication statusPublished - Oct 1 2020

ASJC Scopus subject areas

  • Molecular Medicine
  • Genetics
  • Pharmacology

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