under 01GM1105

Partner and subprojects in IonNeurONet

 

SP 1: Coordination and management

Prof. Dr. Holger Lerche
University of Tübingen
University Department of Neurology, Department of Neurology and Epileptology and Hertie-Institute for Clinical Brain Research

Dr. Holm Graessner
University of Tübingen
Centre for rare diseases Tübingen

Summary

This subproject will develop the clinical network needed for the execution of the project and will provide an organisational structure consisting of a project manager, a project coordination board and an external advisory board. Monitoring and quality assessment procedures will be set up and implemented. Internal and external communication will be organised. Interactions with the ion channel research community and with the clinical network will be strengthened by a training and education scheme which involves a training program for clinicians and an annual meeting. The project web site will be utilised to meet communication requirements of the proposed consortium. This tool will support the communication between the members of the consortium and the scientific community and between the consortium and the public, especially patients suffering from ion channel disorders, their family members and medical practitioners.

SP 3: Pathogenesis of hypokalemic periodic paralysis (HypoPP)

PD Dr. Karin Jurkat-Rott / Prof. Dr. Dr. h.c. Frank Lehmann-Horn
University of Ulm
Division of Neurophysiology

Summary

Hypokalemic periodic paralysis (HypoPP) is a disabling muscle channelopathy affecting >1:100,000 individuals. There is no generally accepted truly effective therapy. Many patients develop a chronic progressive myopathy. HypoPP is caused by voltage sensor mutations in two channels of the skeletal muscle fibre membrane: sodium channel Nav1.4 and calcium channel Cav1.1. As recently described by our group, the weak muscles are electrically depolarized, particularly at low serum potassium, and take up sodium and water. For the most frequent HypoPP mutations we will clarify if the major pathologic currents are conducted by the central channel pore or an aberrant pore in the voltage sensor. Next we will identify agents that repolarize the muscle fibres in a newly developed cell model of HypoPP and myotubes derived from patients. To identify appropriate, available drugs among the modifiers of the central and an aberrant channel pore currents, we first investigate the depolarizing effect of the most frequent HypoPP mutations. As repolarization seems to normalize sodium and water content, it may prevent muscle fibre degeneration and facilitate regeneration. This will be tested by measuring the survival time of cultured cells. As there is considerable phenotypic variability leading to maldiagnosis, we will collect clinical data and perform Sanger and exome sequencing to unclarified patients with similar phenotypes to detect new genes involved in collaboration with the network platforms.

SP 4: Gene identification and characterization in hemiplegic migraine

Prof. Dr. Christian Kubisch
University of Ulm
Institute for Human Genetics

Summary

Hemiplegic migraine is a rare and severe form of an episodic brain disease characterized by attacks of reversible hemiplegia and severe headaches. Mainly autosomal dominant subtypes of the disease have been found to be caused by mutations in one of two voltage-gated ion channels or a sodium-potassium ATPase thereby defining this paroxysmal disease as a channelopathy of the central nervous system. However, a significant part of patients with a positive family history and the large majority of sporadic patients do not have a mutation in one of the three known genes showing an extensive further heterogeneity of the disease. The primary aim of this project is the identification of novel hemiplegic migraine gene(s) by applying high-throughput sequencing approaches in families and patients excluded for the known disease genes. Novel hemiplegic migraine genes will be further analysed by appropriate functional in vitro tests including detailed electrophysiological analysis in cooperation with other network partners. Due to the clinical overlap between hemiplegic migraine and monogenic forms of epilepsy and episodic ataxias we will furthermore study novel hemiplegic migraine genes in patients with other episodic brain diseases of this network and vice versa, which will further improve our understanding about the molecular pathophysiology of these rare and severely disabling paroxysmal diseases.

SP 5: Pathophysiology of neuronal KV7.2 channelopathies in benign familial neonatal seizures (BFNS) and epileptic encephalopathies

Dr. Snezana Maljevic
University of Tübingen
University Department of Neurology, Department of Neurology and Epileptology and Hertie-Institute for Clinical Brain Research

Summary

Mutations in the neuronal voltage-gated potassium channels KV7.2 and KV7.3, have been associated with Benign Familial Neonatal Seizures (BFNS), a well-defined, rare seizure disorder manifesting transiently in the first days and weeks of life. These mutations cause a loss of channel function, with haploinsufficiency as the major pathomechanism in BFNS. A few reports hint to more severe outcomes, such as drug-resistant seizures or psychomotor developmental delay, associated with KV7.2 mutations. To expand our understanding of the (patho)physiological role of KV7.2 and other ion channels in paroxysmal disorders in general, we will (i) provide detailed functional analysis of a cohort of KV7.2 mutations associated with epileptic encephalopathies and search for genetic modifiers associated with benign or severe outcome by exome sequencing, (ii) establish an electrophysiological platform including automated two-voltage clamp of oocytes and automated patch clamp of mammalian cells for functional characterization of other ion channel mutations detected within the consortium, and (iii) generate induced pluripotent stem (iPS) cells from fibroblasts of patients with either BFNS or epileptic encephalopathy carrying different KV7.2 mutations and differentiate them into neurons, which will be characterized by functional neurophysiological studies, immunostainings and RT-PCR. We expect to find determinants of benign or severe phenotypes associated with KV7 disorders and verify novel genetic defects in channelopathies in cooperation with other partners of the consortium.

SP 6: Mechanisms of impaired surface expression and development of bioassays to rescue trafficking defective mutants in retinal channelopathies

Prof. Dr. Bernd Wissinger
University of Tübingen
Research Institute for Ophthalmology

Summary

Defects in membrane-targeting and reduced surface expression is a common pathomechanism in channelopathies. In this project we will study the cellular mechanisms that cause this membrane-targeting defect in in vitro and in vivo systems using the mutants of the α-subunit of the cone photoreceptor cyclic nucleotide gated cation channel (cone CNG Channel) as a model system. Moreover we will develop and apply a bioassay that enables the screening of substances that improve surface expression of membrane-targeting deficient channel mutants.

Mutations in CNGA3, the gene encoding the α-subunit of the cone CNG Channel cause Achromatopsia, an autosomal recessive inherited human disease with severe visual impairment. In prior studies we could show that a considerable fraction of these mutations results in impaired membrane-targeting and reduced surface expression of the mutant channels. We propose that these mutant channels are misfolded, recognized by the cellular quality control system and subsequently targeted onto protein degradation pathways or retained in the endoplasmatic reticulum (ER) and the formation of protein aggregates. In order to test this hypothesis we will apply biochemical techniques to analyse in a cell culture system which step(s) of the process of protein maturation are impaired in CNGA3 mutants and which mechanisms of the cellular surveillance system are invoked in that process. In comparison we will study the fate and effects of membrane-targeting deficient CNG channels in true cone photoreceptor by viral gene delivery in the murine retina.

In parallel we will further improve and eventually apply a cell-based bioassay that records calcium influx through CNG channels and thus allows to assess surface expression of mutant channels in a quantitative manner. This bioassay will be used to screen for compounds that improve surface expression of mutant CNG channels and thus may have therapeutic potential.

SP 7: Genetic diagnostic and exome sequencing in ion channel disorders

Dr. Saskia Biskup
CeGaT GmbH
Center for Genomics and Transciptomics

Summary

Central to the project is the establishment and the application of novel, very fast and cost-efficient screening tools to diagnose ion channel disorders. We utilize target enrichment of either a list of selected genes or all coding regions of the genome (namely the “exome”) and massive parallel sequencing to identify the underlying genetic cause of the disease. Our aim is to implement these revolutionary novel genetic screening methods into a diagnostic setting with direct impact for the clinician. As a large number of ion channel subunits are either known disease genes or candidate genes, our method holds great promise to detect pathogenic variants in patients with neuromuscular disorders, episodic ataxias, hemiplegic migraine, seizure disorders and other ion channel diseases included within this consortium. The idea is to avoid long lasting and invasive diagnostic procedures to find the underlying cause of the disease. If the causing gene is identified, predictions about disease progression, examination of relatives and personalized treatment options according to the underlying genetic basis become possible. If no treatment is available, a gene identified by exome sequencing as cause of a disease is the first clue towards understanding the mechanism of the disease. Knowledge on a mutated gene that up to now has not been associated with ion channel disorders enables completely novel directions in research especially for partners within this consortium.