Accurate Detection of Hot-Spot MTOR Somatic Mutations in Archival Surgical Specimens of Focal Cortical Dysplasia by Mole
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SHORT COMMUNICATION
Accurate Detection of Hot‑Spot MTOR Somatic Mutations in Archival Surgical Specimens of Focal Cortical Dysplasia by Molecular Inversion Probes Paola Dimartino1 · Valeria Mariani2 · Caterina Marconi1 · Raffaella Minardi3 · Manuela Bramerio4 · Laura Licchetta3,5 · Veronica Menghi3,5 · Luca Morandi3,6 · Pamela Magini7 · Patrizia Mongelli7 · Francesco Cardinale2 · Marco Seri1,7 · Paolo Tinuper3,5 · Laura Tassi2 · Tommaso Pippucci7 · Francesca Bisulli3,5
© Springer Nature Switzerland AG 2020
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. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s40291-020-00488-1) contains supplementary material, which is available to authorized users. * Tommaso Pippucci [email protected] 1
Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy
2
“Claudio Munari” Epilepsy Surgery Center, Niguarda Hospital, Milan, Italy
3
IRCCS Istituto delle Scienze Neurologiche di Bologna, Epilepsy Center (Reference Center for Rare and Complex Epilepsies - EpiCARE), Bologna, Italy
4
Department of Pathology, Niguarda Hospital, Milan, Italy
5
Department of Biomedical and NeuroMotor Sciences (DIBINEM), University of Bologna, Bologna, Italy
6
Functional MR Unit, Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
7
Unità Operativa di Genetica Medica, Azienda Ospedaliero-Universitaria di Bologna, Via Albertoni 15, 40138 Bologna, Italy
Key Points Single-molecule molecular inversion probes can be an experimentally simple, cost-effective and accurate approach for the detection of clinically
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