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.

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

Dimartino P.;Minardi R.;Licchetta L.;Morandi L.;Magini P.;Seri M.;Tinuper P.;Pippucci T.;Bisulli F.
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.
Dimartino P.; Mariani V.; Marconi C.; Minardi R.; Bramerio M.; Licchetta L.; Menghi V.; Morandi L.; Magini P.; Mongelli P.; Cardinale F.; Seri M.; Tinuper P.; Tassi L.; Pippucci T.; Bisulli F.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/781785
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