Background Childhood‐onset dystonia is often genetically determined. Recently, KMT2B variants have been recognized as an important cause of childhood‐onset dystonia. Objective To define the frequency of KMT2B mutations in a cohort of dystonic patients aged <18 years at onset, the associated clinical and radiological phenotype, and the natural history of disease. Methods Whole‐exome sequencing or customized gene panels were used to screen a cohort of 65 patients who had previously tested negative for all other known dystonia‐associated genes. Results We identified 14 patients (21.5%) carrying KMT2B variants, of which 1 was classified as a variant of unknown significance. We also identified 2 additional patients carrying pathogenic mutations in GNAO1 and ATM. Overall, we established a definitive genetic diagnosis in 23% of cases. We observed a spectrum of clinical manifestations in KMT2B variant carriers, ranging from generalized dystonia to short stature or intellectual disability alone, even within the same family. In 78.5% of cases, dystonia involved the lower limbs at onset, with later caudocranial generalization. Eight patients underwent pallidal DBS with a median decrease of Burke‐Fahn‐Marsden Dystonia Rating Scale‐Motor score of 38.5% in the long term. We also report on 4 asymptomatic carriers, suggesting that some KMT2B mutations may be associated with incomplete disease penetrance. Conclusions KMT2B mutations are frequent in childhood‐onset dystonia and cause a complex neurodevelopmental syndrome, often featuring growth retardation and intellectual disability as additional phenotypic features. A dramatic and long‐lasting response to DBS is characteristic of DYT‐KMT2B dystonia. © 2019 International Parkinson and Movement Disorder Society

Mutations in PARK2, encoding Parkin, cause an autosomal recessive form of juvenile Parkinson Disease (JPD). The aim of the present study was to investigate the impact of PARK2 mutations on mitochondrial function and morphology in human skin fibroblasts. We analyzed cells obtained from four patients clinically characterized by JPD, harboring recessive mutations in PARK2. By quantitative PCR we found a reduction (<50%) of PARK2 transcript in all patients but one; however Western Blot analysis demonstrated the virtual absence of Parkin protein in all mutant fibroblasts. Respiration assays showed an increment of oxygen consumption, which was uncoupled to ATP cellular levels. This finding was probably due to presence of altered mitochondrial membrane potential (ΔΨm), confirmed by JC-1 analysis. The mitochondrial network was comparable between mutant and control cells but, interestingly, a “chain-like” network was found only in mutant fibroblasts. Dissipation of ΔΨm usually leads to mitochondrial fragmentation in healthy cells and eventually to mitophagy; however, this behavior was not observed in patients' fibroblasts. The absence of mitochondrial fragmentation in mutant Parkin fibroblasts could results in accumulation of damaged mitochondria not targeted to mitophagy. This condition should increase the oxidative stress and lead to cellular dysfunction and death. Our results suggest that PARK2 mutations cause mitochondrial impairment, in particular reduction in ATP cellular levels and alteration of ΔΨm, even in non-neuronal cells and confirm the hypothesis that Parkin holds a pivotal role in pro-fission events.

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Multiple System Atrophy is a severe neurodegenerative disorder which is characterized by a variable clinical presentation and a broad neuropathological spectrum. The pathogenic mechanisms are almost completely unknown. In the present study, we established a cellular model of MSA by using fibroblasts' primary cultures and performed several experiments to investigate the causative mechanisms of the disease, with a particular focus on mitochondrial functioning. Fibroblasts' analyses (7 MSA-P, 7 MSA-C and 6 healthy controls) displayed several anomalies in patients: an impairment of respiratory chain activity, in particular for succinate Coenzyme Q reductase (p < 0.05), and a reduction of complex II steady-state level (p < 0.01); a reduction of Coenzyme Q10 level (p < 0.001) and an up-regulation of some CoQ10 biosynthesis enzymes, namely COQ5 and COQ7; an impairment of mitophagy, demonstrated by a decreased reduction of mitochondrial markers after mitochondrial inner membrane depolarization (p < 0.05); a reduced basal autophagic activity, shown by a decreased level of LC3 II (p < 0.05); an increased mitochondrial mass in MSA-C, demonstrated by higher TOMM20 levels (p < 0.05) and suggested by a wide analysis of mitochondrial DNA content in blood of large cohorts of patients. The present study contributes to understand the causative mechanisms of Multiple System Atrophy. In particular, the observed impairment of respiratory chain activity, mitophagy and Coenzyme Q10 biosynthesis suggests that mitochondrial dysfunction plays a crucial role in the pathogenesis of the disease. Furthermore, these findings will hopefully contribute to identify novel therapeutic targets for this still incurable disorder.

BackgroundWe present a group of patients affected by a paediatric onset genetic encephalopathy with cerebral calcification of unknown aetiology studied with Next Generation Sequencing (NGS) genetic analyses.MethodsWe collected all clinical and radiological data. DNA samples were tested by means of a customized gene panel including fifty-nine genes associated with known genetic diseases with cerebral calcification.ResultsWe collected a series of fifty patients. All patients displayed complex and heterogeneous phenotypes mostly including developmental delay and pyramidal signs and less frequently movement disorder and epilepsy. Signs of cerebellar and peripheral nervous system involvement were occasionally present. The most frequent MRI abnormality, beside calcification, was the presence of white matter alterations; calcification was localized in basal ganglia and cerebral white matter in the majority of cases. Sixteen out of fifty patients tested positive for mutations in one of the fifty-nine genes analyzed. In fourteen cases the analyses led to a definite genetic diagnosis while results were controversial in the remaining two.ConclusionsGenetic encephalopathies with cerebral calcification are usually associated to complex phenotypes. In our series, a molecular diagnosis was achieved in 32% of cases, suggesting that the molecular bases of a large number of disorders are still to be elucidated. Our results confirm that cerebral calcification is a good criterion to collect homogeneous groups of patients to be studied by exome or whole genome sequencing; only a very close collaboration between clinicians, neuroradiologists and geneticists can provide better results from these new generation molecular techniques.

Background Dystonia is a clinically and genetically heterogeneous movement disorder characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive, movements and/or postures. Heterozygous variants in lysine methyltransferase 2B (KMT2B), encoding a histone H3 methyltransferase, have been associated with a childhood-onset, progressive and complex form of dystonia (dystonia 28, DYT28). Since 2016, more than one hundred rare KMT2B variants have been reported, including frameshift, nonsense, splice site, missense and other in-frame changes, many having an uncertain clinical impact. Results We characterize the genome-wide peripheral blood DNA methylation profiles of a cohort of 18 patients with pathogenic and unclassified KMT2B variants. We resolve the “episignature” associated with KMT2B haploinsufficiency, proving that this approach is robust in diagnosing clinically unsolved cases, properly classifying them with respect to other partially overlapping dystonic phenotypes, other rare neurodevelopmental disorders and healthy controls. Notably, defective KMT2B function in DYT28 causes a non-random DNA hypermethylation across the genome, selectively involving promoters and other regulatory regions positively controlling gene expression. Conclusions We demonstrate a distinctive DNA hypermethylation pattern associated with DYT28, provide an epigenetic signature for this disorder enabling accurate diagnosis and reclassification of ambiguous genetic findings and suggest potential therapeutic approaches.

Pantothenate kinase-associated neurodegeneration (PKAN) is a rare disorder associated with brain iron accumulation. The brain MRI abnormality consists of T2 hypointensity in the globus pallidus with a small hyperintensity in its medial part, called the "eye-of-the-tiger" sign. We report on 2 patients affected by PKAN, in whom MRI examination did not demonstrate the "eye-of-the-tiger" sign in the early stages; the typical abnormalities were detected only in the following examinations. Case 1 is a 4-year-old boy first studied at age 2 years for psychomotor delay. The brain MRI was normal. In the following 2 years, the motor impairment progressed. The second brain MRI at age 4 years demonstrated the "eye-of-the-tiger" sign. Molecular analysis of the PANK2 gene revealed a missense mutation F228S in exon 2 in homozygosis. Case 2 is a 6-year-old boy first studied at age 2 years because of psychomotor delay. His brain MRI did not demonstrate abnormalities in the globus pallidus. In the following years spastic-dystonic tetraparesis became evident. A brain MRI at age 4 years demonstrated the "eye-of-the-tiger" sign. Molecular analysis of the PANK2 gene revealed a missense mutation in exon 5 (N501I). Our 2 cases demonstrate that the observation of a normal globus pallidus in the early stage of the disease does not exclude the diagnosis of classic PKAN.