Analysis of common PI3K-AKT-MTOR mutations in pediatric surgical epilepsy by droplet digital PCR reveals novel clinical and molecular insights
Analysis of common PI3K-AKT-MTOR mutations in pediatric surgical epilepsy by droplet digital PCR reveals novel clinical and molecular insights
Abstract Focal malformations of cortical development (FMCD) including focal cortical dysplasia (FCD), hemimegalencephaly (HMEG) and megalencephaly (MEG), constitute a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism, and intellectual disability. Importantly, FCD is the most common cause of intractable pediatric focal epilepsy. Gain and loss of function mutations in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we aimed to assess droplet digital Polymerase Chain Reaction (ddPCR) as a first-tier molecular diagnostic method, as well as define genotype-phenotype relationships among the most common PI3K-AKT-MTOR pathway mutations in FMCD. A total of 144 specimens, including 113 brain samples, were collected from 58 individuals with intractable focal epilepsy phenotypes including FCD, MEG, HMEG and other types of developmental cortical lesions. We designed an ultra-deep and highly sensitive molecular diagnostic panel using ddPCR for six of the most common mutations in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key phenotype, neuroimaging and neuropathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of %. Variant allele fractions (VAF) ranged from 0.1% to 22.67% across all positive samples. Our data shows that MTOR mutations are mostly associated with FCD, whereas PIK3CA mutations are more frequent in the HMEG-DMEG spectrum. The presence of one of these common PI3K-AKT-MTOR-mutations correlated with earlier onset of seizures. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or neuropathological examination. Interestingly, we could not identify the six most common pathogenic variants in other types of cortical lesions (e.g., polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the FCD-HMEG-MEG spectrum. Finally, our data suggest that ultra-deep targeted molecular analysis for the most common PI3K-AKT-MTOR mutations via ddPCR is an effective molecular diagnostic approach for FMCD phenotypes with a good diagnostic yield when paired with neuroimaging and neuropathology evaluations. The high sensitivity and low DNA input requirements suggests that ddPCR is an effective molecular diagnostic tool for disorders caused by somatic mutations with a narrow mutational spectrum, including specific subtypes of pediatric epilepsy surgical phenotypes such as FCD and HMEG.
Ojemann Jeffrey、Kapur Raj P.、Pirozzi Filomena、Pao Emily、Guerrini Renzo、Novotny Edward、Friedman Seth、Doherty Emily S.、Sulc Josef、Forzano Francesca、Dobyns William B.、Saneto Russell P.、Gonzalez Lorenzo、Berkseth Matthew、Hauptman Jason、Oyama Nora、Conti Valerio、Wright Jason N.N.、Timms Andrew E.、Shear Rylee、Mirzaa Ghayda M.、Saitta Sulagna C.
Department of Neurological Surgery, University of WashingtonDepartment of Laboratories, Seattle Children?ˉs HospitalCenter for Integrative Brain Research, Seattle Children?ˉs Research InstituteCenter for Integrative Brain Research, Seattle Children?ˉs Research InstitutePediatric Neurology, Neurogenetics and Neurobiology Unit and Laboratories, Children?ˉs Hospital A. Meyer-University of FlorenceCenter for Integrative Brain Research, Seattle Children?ˉs Research Institute||Division of Pediatric Neurology, Department of Neurology, Seattle Children?ˉs Hospital||Department of Neurology, University of WashingtonCenter for Clinical and Translational Research, Seattle Children?ˉs HospitalSection of Clinical Genetics, Carilion Clinic Children?ˉs HospitalCenter for Integrative Brain Research, Seattle Children?ˉs Research InstituteDepartment of Clinical Genetics, Guy?ˉs and St Thomas NHS Foundation Trust and King?ˉs College LondonDivision of Genetics and Metabolism, Department of Pediatrics, University of MinnesotaCenter for Integrative Brain Research, Seattle Children?ˉs Research Institute||Division of Pediatric Neurology, Department of Neurology, Seattle Children?ˉs HospitalJohns Hopkins University School of MedicineCenter for Integrative Brain Research, Seattle Children?ˉs Research InstituteDepartment of Neurological Surgery, University of WashingtonCenter for Integrative Brain Research, Seattle Children?ˉs Research InstitutePediatric Neurology, Neurogenetics and Neurobiology Unit and Laboratories, Children?ˉs Hospital A. Meyer-University of FlorenceDepartment of Radiology, Seattle Children?ˉs HospitalCenter for Integrative Brain Research, Seattle Children?ˉs Research InstituteCenter for Integrative Brain Research, Seattle Children?ˉs Research InstituteCenter for Integrative Brain Research, Seattle Children?ˉs Research Institute||Division of Genetic Medicine, Department of Pediatrics, University of Washington||Brotman-Baty Institute for Precision Medicine||Institute for Stem Cell and Regenerative Medicine, University of WashingtonDivision of Medical Genets, Department of Obstetrics and Gynecology, David Geffen School of Medicine at the University of California Los Angeles
神经病学、精神病学基础医学分子生物学
Focal Cortical DysplasiaHemimegalencephalygenotype-phenotypeddPCRseizures
Ojemann Jeffrey,Kapur Raj P.,Pirozzi Filomena,Pao Emily,Guerrini Renzo,Novotny Edward,Friedman Seth,Doherty Emily S.,Sulc Josef,Forzano Francesca,Dobyns William B.,Saneto Russell P.,Gonzalez Lorenzo,Berkseth Matthew,Hauptman Jason,Oyama Nora,Conti Valerio,Wright Jason N.N.,Timms Andrew E.,Shear Rylee,Mirzaa Ghayda M.,Saitta Sulagna C..Analysis of common PI3K-AKT-MTOR mutations in pediatric surgical epilepsy by droplet digital PCR reveals novel clinical and molecular insights[EB/OL].(2025-03-28)[2025-07-16].https://www.medrxiv.org/content/10.1101/2021.06.09.21257462.点此复制
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