Focal malformations of cortical development including focal cortical dysplasia, hemimegalencephaly and megalencephaly, are a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism and intellectual disability. Importantly, focal cortical dysplasia is the most common cause of focal intractable paediatric epilepsy. Gain and loss of function variants in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we performed deep molecular profiling of common PI3K-AKT-MTOR pathway variants in surgically resected tissues using droplet digital polymerase chain reaction (ddPCR), combined with analysis of key phenotype data. A total of 159 samples, including 124 brain tissue samples, were collected from 58 children with focal malformations of cortical development. We designed an ultra-sensitive and highly targeted molecular diagnostic panel using ddPCR for six mutational hotspots 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 clinical, neuroimaging and histopathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions ranged from 0.14 to 22.67% across all mutation-positive samples. Our data show that pathogenic MTOR variants are mostly associated with focal cortical dysplasia, whereas pathogenic PIK3CA variants are more frequent in hemimegalencephaly. Further, the presence of one of these hotspot mutations correlated with earlier onset of epilepsy. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or histopathology. Importantly, we could not identify these mutational hotspots in other types of surgically resected epileptic lesions (e.g. polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the focal cortical dysplasia-hemimegalencephaly spectrum. Finally, our data suggest that ultra-sensitive molecular profiling of the most common PI3K-AKT-MTOR mutations by targeted sequencing droplet digital polymerase chain reaction is an effective molecular approach for these disorders with a good diagnostic yield when paired with neuroimaging and histopathology.

Background Background: Neuropsychiatric and affective symptoms are prevalent in prodromal and clinical Parkinson's disease (PD). Some evidence suggests that they may also signify risk for motor complications (motor fluctuations and dyskinesias) of dopamine replacement therapy (DRT). Objective Objective: To determine whether neuropsychiatric symptoms present in de novo PD (ie, before DRT initiation) predict the severity of eventual motor complications of DRT. Methods Methods: We used clinical, demographic, neurobehavioral, and neuroimaging data from the Parkinson's Progression Markers Initiative (PPMI), a multicenter observational PD study. Participants were unmedicated at enrollment and 361 initiated DRT during PPMI follow-up. We used Cox proportional hazard and multivariate ordinal mixed-effects regression models to evaluate the relationship between baseline neuropsychiatric symptoms and motor complications as measured by the Movement Disorders Society-revised Unified Parkinson's Disease Rating Scale (MDS-UPDRS). Results Results: The cumulative incidences of dyskinesias and motor fluctuations during follow-up (6.0 AE 1.5 years) were 34.3% and 59.9%, respectively. Both apathy and high trait-anxiety (top quartile) conveyed over twofold increases in hazard for dyskinesia onset and for adverse impact on activities of daily living caused by both dyskinesias and motor fluctuations. The longitudinal severity of motor fluctuations and dyskinesias was significantly predicted by baseline trait-anxiety and apathy, but not depression. Models were adjusted for dimensionally related symptoms (eg autonomic dysfunction) and potential confounding variables (eg DRT dose). Conclusions Conclusions: Apathy and anxiety levels in de novo PD may be neuropsychiatric biomarkers of vulnerability to earlier and more disabling motor complications of DRT.

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 29.31%. 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.