Mikko Muona

A recurrent de novo mutation in KCNC1 causes progressive myoclonus epilepsy

Progressive myoclonus epilepsies (PMEs) are a group of rare, inherited disorders manifesting with action myoclonus, tonic-clonic seizures and ataxia. We sequenced the exomes of 84 unrelated individuals with PME of unknown cause and molecularly solved 26 cases (31%). Remarkably, a recurrent de novo mutation, c.959G>A (p.Arg320His), in KCNC1 was identified as a new major cause for PME. Eleven unrelated exome-sequenced (13%) and two affected individuals in a secondary cohort (7%) had this mutation. KCNC1 encodes KV3.1, a subunit of the KV3 voltage-gated potassium ion channels, which are major determinants of high-frequency neuronal firing. Functional analysis of the Arg320His mutant channel showed a dominant-negative loss-of-function effect. Ten cases had pathogenic mutations in known PME-associated genes (NEU1, NHLRC1, AFG3L2, EPM2A, CLN6 and SERPINI1). Identification of mutations in PRNP, SACS and TBC1D24 expand their phenotypic spectra to PME. These findings provide insights into the molecular genetic basis of PME and show the role of de novo mutations in this disease entity.

Segmental isotopic labeling of multi-domain and fusion proteins by protein trans -splicing in vivo and in vitro

Segmental isotopic labeling is a powerful labeling technique for reducing nuclear magnetic resonance (NMR) signal overlap, which is associated with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental isotopic labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR experiments. We described in vivo (i.e., in Escherichia coli) and in vitro protocols for segmental isotopic labeling of multi-domain and fusion proteins via protein trans-splicing (PTS) using split DnaE intein without any refolding steps or α-thioester modification. The advantage of PTS approach is that it can be carried out in vivo by time-delayed dual-expression system with two controllable promoters. A segmentally isotope-labeled protein can be expressed in Escherichia coli within 1 d once required vectors are constructed. The total preparation time of a segmentally labeled sample can be as short as 7–13 d depending on the protocol used.

Segmental Isotopic Labelling of a Multidomain Protein by Protein Ligation by Protein Trans-Splicing

Segmental isotopic labelling is a powerful method for the incorporation of stable isotopes into particular regions within proteins for NMR detection, thereby reducing the complexity of NMR spectra and offering the potential to perform sequential assignments. Here we have demonstrated segmental isotopic labelling of a domain in a multidomain protein both in vivo and in vitro through protein ligation by protein trans-splicing. This robust protein trans-splicing approach could open possibilities for studying particular domains in intact proteins without dissection into smaller globular domains. Recent advances in optimization of transverse-relaxation in NMR spectroscopy have opened avenues for study of larger molecules (close to 1 MDa). However, sequential resonance assignments in large proteins remain time-consuming and challenging because of the increased number of signals and signal overlapping. Segmental isotopic labelling is one promising approach among numerous isotope-labelling techniques, because, unlike in the case of selective amino acid labelling, segmentally isotope-labelled samples can be directly analysed by tripleresonance NMR techniques developed for sequential resonance assignments. Segmentally isotope-labelled proteins have been prepared either by expressed protein ligation (EPL), which makes use of native chemical ligation (NCL), 6] or by protein trans-splicing (PTS), through the use of artificially split protein splicing domains (inteins). 7] EPL requires the preparation of an a-thioester group from a thiol reagent and an N-terminal cysteine residue by proteolysis in vitro (Scheme 1A), which demands considerable preparation efforts, 8] although an easier approach has recently been proposed. In protein splicing, an intein catalyses protein ligation of two polypeptide fragments fused to the Nand C-terminal ends of an intein. Protein splicing could take place in trans, when an intein is split into two fragments (Scheme 1B). 10] Segmental isotopic labelling through protein trans-splicing with artificially split inteins requires no additional thiol reagent nor cofactor, but denaturation and renaturation steps are necessary before protein-splicing activity can be restored. Unlike artificially split inteins, naturally split inteins do not require any denaturation and renaturation steps for protein splicing. Therefore, these have been suggested as potentially useful for segmental isotopic labelling of multidomain proteins. Protein trans-splicing with naturally split inteins has advantages over EPL because protein ligation can be performed not only in vitro but also in vivo, making it possible to achieve segmental isotopic labelling in vivo. Despite its many potential applications, it has never been used for segmental isotopic labelling of multidomain proteins except for a fusion tag for enhancing protein solubility. This is because the protein-splicing activity of the split inteins could be negatively affected even when naturally split inteins were fused with the Scheme 1. Protein ligation by A) expressed protein ligation and B) protein trans-splicing. C) Outline of the in vivo procedure for segmental isotopic labelling used in this article.

Dravet syndrome: New potential genetic modifiers, imaging abnormalities, and ictal findings

Dravet syndrome is an autosomal dominant epileptic encephalopathy of childhood, which is caused mainly by SCN1A and PCHD19 mutations. Although Dravet syndrome is well recognized, the causes of acute encephalopathy are still elusive, and reported data on ictal electroencephalography (EEG) and structural brain abnormalities are scarce.

Mutation of the nuclear lamin gene LMNB2 in progressive myoclonus epilepsy with early ataxia

We studied a consanguineous Palestinian Arab family segregating an autosomal recessive progressive myoclonus epilepsy (PME) with early ataxia. PME is a rare, often fatal syndrome, initially responsive to antiepileptic drugs which over time becomes refractory and can be associated with cognitive decline. Linkage analysis was performed and the disease locus narrowed to chromosome 19p13.3. Fourteen candidate genes were screened by conventional Sanger sequencing and in one, LMNB2, a novel homozygous missense mutation was identified that segregated with the PME in the family. Whole exome sequencing excluded other likely pathogenic coding variants in the linked interval. The p.His157Tyr mutation is located in an evolutionarily highly conserved region of the alpha-helical rod of the lamin B2 protein. In vitro assembly analysis of mutant lamin B2 protein revealed a distinct defect in the assembly of the highly ordered fibrous arrays typically formed by wild-type lamin B2. Our data suggests that disruption of the organisation of the nuclear lamina in neurons, perhaps through abnormal neuronal migration, causes the epilepsy and early ataxia syndrome and extends the aetiology of PMEs to include dysfunction in nuclear lamin proteins.

Dysfunctional ADAM22 implicated in progressive encephalopathy with cortical atrophy and epilepsy

Objective: To identify the molecular genetic basis of a syndrome characterized by rapidly progressing cerebral atrophy, intractable seizures, and intellectual disability. Methods: We performed exome sequencing in the proband and whole-genome single nucleotide polymorphism genotyping (copy number variant analysis) in the proband-parent trio. We used heterologous expression systems to study the functional consequences of identified mutations. Results: The search for potentially deleterious recessive or de novo variants yielded compound heterozygous missense (c.1202G>A, p.Cys401Tyr) and frameshift deletion (c.2396delG, p.Ser799IlefsTer96) mutations in ADAM22, which encodes a postsynaptic receptor for LGI1. The deleterious effect of the mutations was observed in cell surface binding and immunoprecipitation assays, which revealed that both mutant proteins failed to bind to LGI1. Furthermore, immunoprecipitation assays showed that the frameshift mutant ADAM22 also did not bind to the postsynaptic scaffolding protein PSD-95. Conclusions: The mutations identified abolish the LGI1-ADAM22 ligand-receptor complex and are thus a likely primary cause of the probands epilepsy syndrome, which is characterized by unusually rapidly progressing cortical atrophy starting at 3–4 months of age. These findings are in line with the implicated role of the LGI1-ADAM22 complex as a key player in nervous system development, specifically in functional maturation of postnatal synapses. Because the frameshift mutation affects an alternatively spliced exon with highest expression in postnatal brain, the combined effect of the mutations is likely to be hypomorphic rather than complete loss of function. This is compatible with the longer survival of the patient compared to Lgi1−/− and Adam22−/− mice, which develop lethal seizures during the first postnatal weeks.

Myoclonus epilepsy and ataxia due to KCNC1 mutation: Analysis of 20 cases and K+ channel properties

To comprehensively describe the new syndrome of myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK), including cellular electrophysiological characterization of observed clinical improvement with fever.

Long-term follow-up of two siblings with adult-onset neuronal ceroid lipofuscinosis, Kufs type A

Reports on the clinical presentation of adult-onset neuronal ceroid lipofuscinoses (NCL) are scarce compared to infantile- and childhood-onset forms. Here, we aimed to present detailed temporal evolution of clinical and electrophysiological features of two siblings with adult-onset NCL and homozygous mutation in the CLN6 gene. We retrospectively analysed medical records and electrophysiological data in order to delineate evolution of clinical and electrophysiological findings. Electrophysiological studies included routine EEG and video-EEG, as well as polymyographic analysis of myoclonus and brainstem reflex studies. Both patients had seizures and cerebellar signs. Despite the slow progression of ataxia, they developed no mental deterioration, but had severe obsessive compulsive disorder and depression. EEG revealed frequent generalized spikes, polyspikes, and waves, prominent on awakening and during photic stimulation without significant change throughout the clinical course. Abnormalities concerning the blink reflex, auditory startle response, and startle response to somatosensory inputs manifested within four years. The patients underwent transient and mild improvement with valproate, whereas ataxia and seizures were dramatically ameliorated following high-dose piracetam. Patients with adult-onset NCL may present with slowly progressive ataxia, persistent photosensitivity, and seizures without dementia or extrapyramidal findings. Brainstem abnormalities become more evident with time, in line with ataxia. Piracetam is effective for both seizures and ataxia.