Internatinal Symposium: Nerve biology and inherited peripheral neuropathy. From biology to therapy
Madrid, 11 y 12 de diciembre de 2014
Madrid, December 11-12, 2014
I
Charcot-Marie-Toothdisease & related neuropathies: an overview, Mary Reilly
Clinical evaluation, scales & diagnostic, Davide Pareyson
Diagnóstico Genético de la enfermedad de Charcot-Marie-Tooth, Carmen Espinós
New genes on CMT, Stephan Züchner
Pathogenic mechanisms of CMT, Vincent Timmerman
Modelos en organismos invertebrados: Drosophila,Ibo Galindo
Drug screening and Discovery, John Svaren
Biomarkers in CMT1A, Michael Sereda
Rehabilitation, Joshua Burns
Charcot-Marie-Tooth disease & related neuropathies: an overview, Mary Reilly
Charcot Marie Tooth disease (CMT) and the related neuropathies are a clinically and genetically heterogeneous group of disorders which most commonly present with a length dependent motor and sensory neuropathy often accompanied by foot deformity. Over the last two decades and especially in the last 5 years with the development of affordable next generation sequencing techniques, there have been rapid advances in the identification of the causative genes with over 80 genes now being described. Many of the causative genes for the axonal form of CMT (CMT2) remain to be identified. One interesting discovery with the identification of the latest causative genes is that genes that were traditionally thought to cause other disorders such as Hereditary Spastic Paraparesis (HSP) e.g. Atlastin and REEP1, are now occasionally described to cause an inherited neuropathy such as Hereditary Sensory Neuropathy (HSN) or distal Hereditary Motor Neuropathy (HMN) respectively.
A further consequence of the introduction of next generation sequencing to diagnostic practise is the difficulty in validating whether a mutation is pathogenic or not. With multiple genes being screened together, there are often many potential mutations in many genes identified and with the current lack of easily available functional analysis of genes it can be challenging to decide which mutation is the causative one. The same problem arises in identifying the causative gene using whole exome sequencing when looking for new genes.
Finally we still have no effective therapies for CMT or the related disorders. Two of the major challenges to developing therapies are the very slow and usually not life threatening progression of the disease with a need therefore for very safe therapies and the difficulty in identifying sensitive and responsive outcome measures for clinical trials.
This overview will cover these areas especially the consequences of next generation sequencing for the field and the progress in therapy development.
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Clinical evaluation, scales & diagnostic, Davide Pareyson
The diagnostic approach to the diagnosis of CMT is becoming more and more complicated as the associated gene number increases and the clinical characteristics of the different CMT types greatly overlap. The availability of next generation sequencing (NGS) techniques has improved the diagnostic yield and at the same time makes it fundamental to well characterize the CMT patients’ phenotype to interpret NGS results. Careful clinical evaluation of patients is therefore still fundamental, and need to be completed by the assessment of inheritance pattern and of nerve conduction studies (NCS). It is important to look for peculiar clinical features that may still be specific for certain CMT subtypes, such as optic atrophy, glaucoma, vocal cord palsy, pyramidal tract signs/spastic paraplegia, foot and hand ulcers, autonomic dysfunction, learning difficulties, predominant upper limb involvement, differences in hand musculature involvement. Nerve biopsy, now limited to selected cases, may reveal specific myelin (i.e., myelin outfoldings) or axonal (e.g., giant axons) changes. Cellular and animal model studies are demonstrating the therapeutic potential of a series of compounds making it important clinical trial readiness. However, the experience with ascorbic acid trials revealed the difficulties in translation from animals to patients and in detecting intervention efficacy, owing to slow disease progression. We therefore need to develop responsive outcome measures. The ascorbic acid trial in Italy and UK showed the limits of the CMTNS and the potentiality of foot dorsiflexion myometry (the most sensitive-to-change measure of the trial). Clinical research led to develop an updated version of the CMTNS, a novel paediatric scale (CMTPedS) based on clinimetric methods, and to test outcome measures used in other disorders (activity monitors, 6 Minutes Walking Test). Exploration of surrogate paraclinical outcome measures include studies on computerised gait analysis, quantitative MRI, and biomarkers. We measured mRNA PMP22 levels in skin biopsies from 46 patients recruited in the ascorbic acid trial at study entry and end of study: we found no change over two years and no correlation with disease severity. The skin biopsy approach allows testing other potential biomarkers.
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Diagnóstico Genético de la enfermedad de Charcot-Marie-Tooth, Carmen Espinós
La enfermedad de Charcot-Marie-Tooth (CMT) o neuropatía hereditaria sensitivo motora (HMSN, Hereditary Motor Sensory Neuropathy) está caracterizada por una amplia heterogeneidad genética: se han descrito más de 50 genes y esta cifra no cesa de crecer. La enfermedad de CMT suele dividirse en dos grandes grupos atendiendo a criterios electrofisiológicios e histológicos, CMT desmielinizante y CMT axonal. La causa más común de CMT es la duplicación CMT1A que afecta al gen PMP22 y representa aproximadamente el 80% de los casos con CMT desmielinizante. Otros genes también frecuentes son MPZ, GJB1, PMP22 y SH3TC2 relacionados con CMT desmielinizante, y MFN2, GDAP1 y GJB1 implicados en CMT axonal. El resto de genes asociados con CMT representan porcentajes muy bajos. Por todo ello, el diagnóstico genético es complicado y se suele abordar a diferentes niveles: (1) Análisis de genes más frecuentes o genes candidatos, lo que implica el análisis gen-a-gen mediante secuenciación de Sanger. Es una aproximación sencilla, pero costosa en tiempo. (2) Panel de genes que consiste en el análisis de todos los genes conocidos implicados en este grupo de trastornos mediante secuenciación masiva. Se trata de un método eficaz por el número de genes que se analizan, si bien su análisis informático es difícil por la cantidad de información que proporciona y la dificultad para establecer el posible carácter patológico de los cambios identificados. (3) Secuenciación de exoma (WES, Whole Exome Sequencing) o completa del genoma (WGS, Whole Genome Sequencing) basadas en secuenciación masiva. La complejidad de estas dos aproximaciones hace que de momento sólo se puedan abordar en el marco de proyectos de investigación y principalmente en casos de singular interés con el fin de identificar nuevos genes implicados en este grupo de neuropatías.
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New genes on CMT, Stephan Züchner
Genome-wide sequencing techniques have transformed and greatly enhanced our approach to novel disease gene identification and diagnostics. In the CMT field alone a novel gene is now identified every single month. The consequences are exciting for molecular studies and development of therapeutic targets, yet the diagnostic conundrum of genetic variants of unknown significance is at least temporarily increasing. This presentation will summarize these recent developments, opportunities and challenges based on our experiences in CMT genetic research studies, namely the Inherited Neuropathy Consortium. I will discuss ways forward to overcome current limitations and towards an increasingly powerful genetic diagnostic toolbox.
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Pathogenic mechanisms of CMT, Vincent Timmerman
Charcot-Marie-Tooth (CMT) neuropathies comprise a group of monogenic disorders affecting the peripheral nervous system. CMT is characterized by a clinically and genetically heterogeneous group of neuropathies, involving all types of Mendelian inheritance patterns. Over 1,000 different mutations have been discovered in 80 disease-associated genes. Genetic research of CMT has pioneered the discovery of genomic disorders and aided in understanding the effects of copy number variation and the mechanisms of genomic rearrangements. Clinical, molecular genetic and functional studies suggest for common pathomechanisms and gene networks for peripheral nerve degeneration. The most remarkable group of genes are those coding for three small heat shock proteins (HSPBs). Although regulated by stress, they are constitutively expressed and responsible for quality control and protein folding. The HSPBs are not only molecular chaperones but also involved in many essential cellular processes such as apoptosis, autophagy, splicing, cytoskeleton dynamics and neuronal survival. We reported that tubulin differentially interacts with mutant HSPB1. This anomalous binding leads to the stabilization of the microtubule network. We also found that mutations in HSPB1 disrupt the neurofilament network and cause their aggregation. Altogether, specific mutations in the small heat shock proteins affect the axonal transport via induced hyperphosphorylation of neurofilaments and stabilization of the microtubule network. Both pathomechanisms can be targeted by drugs in experimental models and may open possibilities for future treatment strategies.
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Modelos en organismos invertebrados: Drosophila, Ibo Galindo
Las enfermedades raras ya se cuentan por miles y el número de genes responsables conocidos sigue creciendo cada día. Sin embargo, los recursos humanos y económicos necesarios para su estudio no crecen al mismo ritmo, lo cual nos obliga a buscar estrategias con las que optimizar estos medios. Una de las estrategias que nos puede ser muy útil es identificar modelos animales no vertebrados en los cuales los procesos biológicos implicados estén conservados. La mosca de la fruta, Drosophila melanogaster, utilizada en investigación genética desde principios del siglo XX, actualmente se utiliza para estudiar numerosas patologías humanas ya que compartimos muchos mecanismos moleculares y celulares en fisiología celular, neurobiología y genética.
En el caso de la investigación en CMT, ya hay modelos en Drosophila para comprender las mutaciones de genes relacionados con la enfermedad: Gars, Yars, Rab7, MFN2 y GDAP1, este último realizado por nuestro laboratorio. Para entender la neuropatía de CMT es importante disponer de modelos animales con los que complementar los modelos celulares, ya que hay implicados procesos como la comunicación sináptica con el músculo y la degeneración neuromuscular ligada al envejecimiento, que no se pueden reproducir adecuadamente en células aisladas. A partir de estos estudios podemos elaborar modelos que reproduzcan las lesiones moleculares presentes en los pacientes para identificar nuevos biomarcadores y desarrollar nuevos tratamientos.
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Drug screening and Discovery, John Svaren
The most common cause of Charcot-Marie-Tooth (CMT) disease is a gene duplication resulting in three copies of the gene encoding the major myelin protein, Peripheral Myelin Protein 22 (PMP22). The associated neuropathy has been classified as CMT1A, and work by several groups have established that rodent models with PMP22 overexpression recapitulate many aspects of the human disease. Moreover, reduction of PMP22 levels has been shown to resolve many aspects of the peripheral neuropathy. In previous work, development of therapeutic approaches for CMT had not employed chemical genomic approaches to identify potential therapeutic compounds using unbiased screens of chemical libraries. Therefore, we have developed a series of drug screening assays to identify compounds that selectively reduce PMP22 expression to develop effective treatments for CMT1A. Our latest generation of assays has used recently developed techniques of genome editing to insert reporter genes into the endogenous PMP22 locus in a Schwann cell line that expresses high levels of PMP22. In collaboration with the group of Dr. Inglese at the National Center for Advancing Translational Sciences/NIH, these assays have been used to screen chemical libraries using a sensitive and powerful drug screening protocol that employs dose-dependent screening. In addition, follow-up assays and rodent models are being used to evaluate the effectiveness of new compounds. Overall, recent genomic studies have uncovered a number of disease states, like CMT1A, that are caused by copy number variants. Therefore, the use of genome edited assays to identify modulators of gene expression levels should be applicable to many other gene dosage disorders, as well as other types of CMT.
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Biomarkers in CMT1A, Michael Sereda
Despite its monogenetic cause, patients with CMT1A display a marked interindividual variability of disease severity. The underlying reason for this variability is largely unknown and epigenetic factors have been discussed. At present, the assessment of the individual disease severity in patients with CMT1A is performed solely by clinical and electrophysiological examinations. In light of the slow disease progression, insensitive outcome measures may increase the risk of false negative results in clinical trials and biomarkers could add powerful tools to monitor therapeutic effects. Biomarkers may not only serve as a sensitive surrogate marker of clinical severity, but may also identify responders to a putative therapy. CMT rats carrying additional copies of the Pmp22 gene recapitulate the striking disease variability observed in patients with CMT1A. In a proof of principle study, we have demonstrated that the expression levels of selected genes in sciatic nerve and skin tissue can be utilized to measure and predict the disease severity in CMT rats. Importantly, we validated these disease severity markers in skin biopsies of 46 patients with CMT1A. At the moment, these and new markers are examined with regard to disease progression within a large pan-European consortium in 277 CMT1A patients (83 from Germany, 56 from the Czech Republic, 50 from Spain, 27 from Belgium, 20 from the UK, 17 from Italy, 15 from the USA und 9 from France). In the near future we hope to provide the community with applicable biomarkers which in turn may accelerate the development of a therapy for CMT1A.
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Rehabilitation, Joshua Burns
This presentation provides an overview of evidence-based rehabilitation strategies for children and adults with Charcot-Marie-Tooth disease. Rehabilitation primarily focuses on pain and disability associated with the cavus foot deformity, foot drop, ankle contracture, chronic ankle instability, muscle cramps, sensory loss, impaired balance as well as hand and upper limb dysfunction. Strategies include stretching and strengthening exercises, orthotic and footwear therapies, rehabilitation after orthopaedic surgery and surveillance for hip dysplasia to increase each patient’s ability to perform activities of daily living and improve their quality of life.
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