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 Pippucci8, Francesca Bisulli3,5. 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. 8. Unità Operativa di Genetica Medica, Azienda Ospedaliero-Universitaria di Bologna, Via Albertoni 15, 40138, Bologna, Italy. tommaso.pippucci@unibo.it.
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.
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.
Authors: Renzo Guerrini; Mara Cavallin; Tommaso Pippucci; Anna Rosati; Francesca Bisulli; Paola Dimartino; Carmen Barba; Rita Garbelli; Anna Maria Buccoliero; Laura Tassi; Valerio Conti Journal: Neurol Genet Date: 2020-12-08
Authors: Wei Shern Lee; Sara Baldassari; Sarah E M Stephenson; Paul J Lockhart; Stéphanie Baulac; Richard J Leventer Journal: Int J Mol Sci Date: 2022-01-25 Impact factor: 5.923