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Communications Orales

IPS cells differentiated into motor neurons to better understand mitochondrial behavior in peripheral nerve cells

Pierre-Antoine FAYE (Limoges)

Induced pluripotent stem cells (iPSCs) are a highly interesting solution to create and observe the behavior of specific and unattainable cells from a patient. Our team is interested in genetic peripheral nerve diseases and particularly in Charcot-Marie-Tooth pathology (CMT), which is the most predominant hereditary peripheral neuropathy. More than 90 genes are currently known to be responsible for this disease. We focused on one of them: GDAP1, which is responsible for a CMT axonal form and encodes a mitochondrial outer membrane protein, whose function is still unclear. To better understand its pathophysiology, we aimed to develop motor neurons models from patients using the iPSCs strategy.
iPSCs have been obtained starting from patients dermal fibroblasts (DFs) and following Yamanaka protocol. We optimized the differentiation into motor neurons with our specific protocol and investigated GDAP1 functions by immunocytochemistry, electron
and confocal microscopy. Five healthy persons and two homozygous patients carrying homozygous GDAP1 nonsense mutations have been sampled.
We successfully generated iPSCs and motor neurons. We observed that patients motor neurons derived from iPS have the same mitochondrial morphology compared to patients nerve biopsies analysed by the National Reference Center of Peripheral Neuropathies in Limoges. Mitochondria are small, round without crest and the mitochondrial network is splited. This defect is not observed neither incontrol motor neurons nor in patients and controls fibroblasts.
Generation of motor neurons using axonal CMTpatients-derived iPSCs was a first crucial step to better understand the role of GDAP1 in this pathology. In contrast to other models, such as human fibroblasts or animal models, our motor neurons derived from human iPSc showed exactly the same mitochondrial morphological defects than those observed in neuronal biopsies of human CMT patients and provides then an accurate model to precise GDAP1 function.
Taken together, these results showed that IPSCs strategy is an interesting tool to investigate peripheral neuropathic diseases, explore physiopathological pathways and then perform drug screening.

Clinical and paraclinical patterns of positive anti-FGFR3 antibody sensory neuronopathies in Brazil and in Europe

Yannick THOLANCE (Saint-Etienne)

We have reported that an antibody reacting with the intracellular domain of the Fibroblast Growth Factor Receptor 3 (FGFR3) identifies a subgroup of patients with sensory neuronopathy (SNN).1 Here we report the results of a prospective and international study in which 43 positive anti-FGFR3 antibody SNN patients were identified in Brazilian and European centers. 
The study was a prospective international study conducted in 35 European centers and 1 in Brazil. As all Brazilian patients fulfilling the diagnostic criteria of SNN, we selected only the European SNN cases for comparisons. The detection of anti-FGFR3 antibodies
in serum was performed by ELISA analysis.1 Once anti-FGFR3 antibodies were detected, a detailed clinical and paraclinical form was sent to the physician for clinical pattern collection.
The incidence of the serum anti-FGFR3 antibodies was higher in Brazilian patients than in European one (15/42, 35.7% vs 27/211, 12.8%). We found that positive Brazilians were younger at the onset of the neuropathy and the presence of an autoimmune context was more frequent in Brazilians than in Europeans. The topography of the sensory symptoms as the nature ofclinical signs was
clearly different between the two groups.
This prospective study brings new information on anti-FGFR3 antibodies by identifying two different clinical patterns in two different continents. Several mechanisms may explain this data. Among them we may mention an environmental factor acquired early in life or a specific genetic background that could be confirmed by genotyping of the HLA. The Brazilian patients showed high
prevalence of polyautoimmunity supporting the idea that different autoimmune phenotype may share common susceptibility variants.
This prospective study identified a striking difference in the prevalence of anti-FGFR3 antibodies and in the clinical and paraclinal pattern for the Brazilian and European SNN patients which suggests that environmental or genetic factors may lead to an autoimmune reaction against FGFR3.

Individualised immunoglobulin treatment vs. standard dosing for chronic inflammatory demyelinating polyneuropathy: clinical and cost comparison

Yusuf RAJABALLY (Birmingham, Royaume-Uni)

The clinical and economic implications of an individualised intravenous immunoglobulin (IVIg) protocol for chronic inflammatory demyelinating polyneuropathy (CIDP) are unknown.

We retrospectively studied 47 IVIg-treated subjects with CIDP over four years with an individualised, outcomemeasured, dose-modifying protocol. We compared clinical benefits and costs with those reported with standard dosing at 1gram/kg every 3 weeks.
The IVIg-responder rate was 83% and the 4-year remission rate was 25.6%. Mean IVIg dose requirements were 22.06 grams/week (S.D.:15.29) in patients on ongoing therapy. Dose range was wide (5.83-80 grams/week). Mean infusion frequency was every 4.34 weeks (S.D.:1.70) and infusion duration of 2.79 days (S.D.:1.15). Mean modified-INCAT (Inflammatory Neuropathy Cause and Treatment) score improvement was similar (p=0.47) and mean MRC sum score improvement greater (p<0.001) in our cohort, compared to the IVIg-treated arm of the ICE Study. Mean drug costs were GBP 37,660/patient/year (€ 43,309) and mean infusion-related costs of GBP 17,115/patient/year (€ 19,682), totalling GBP 54,775/patient/year (€ 62,991). Compared to standard dosing using recorded weight, mean savings were of GBP 13,506/patient/year (€ 15,532). Compared to standard dosing using dosing weight, savings were of GBP 6,506/patient/year (€ 7,482).
This study is the first to compare clinical and economic aspects with different IVIg treatment strategies for CIDP. Clinical equivalence as well as cost savings were demonstrated with our individualised protocol in comparison to standard dosing regimens at 1 g/kg/3 weeks.
An individualised IVIg treatment protocol is clinically non-inferior and 10-25% more costeffective than standard dosing in CIDP.

Connexin32 genomic stability, involvement in CMTX1 phenotype

Michel FONTES (Marseille)

CMTX1 is caused by mutations in the GJB1 gene coding for connexin 32, a protein involved in gap junction. Physiopathology is poorly understood. No treatment could be proposed to patients.
We created 5 transgenic lines by inserting a human BAC carrying the GJB1 gene, in which two different mutations (G12S and S26L in connexin 32 (Cx32) observed in patients were introduced. Nuclear volume, carotype and centrosmes has been explored using classical cytology. Connexon activity has been evaluated using lucifer Yellow internatisation. Locomotor behavior has been evaluated using the rotarod test.
All 5 lines present mitotic instability (polyploidy, nuclear anomalies and centrosomes overduplication). This phenotype suggests thus that CX32 is implicated in the control of mitotic stability. This has been confirmed by results of the Mitocheck project. Connexon activity is partially inhibited and locomotor performances are impaired in all lines. In addition, we observed that transgenic cells exhibit CamKII over-stimulation. Moreover, treatment by CamKII inhibitors reverts mitotic instability. We also observed that connexon activity is partially restored by treatment with CamKII inhibitors. Regarding in vivo phenotype, we observed that degradation of locomotor performances is stopped by treatment with a CamKII inhibitor (KN93). Finally, the same
phenotype has been observed in cells from patients and this cellular phenotype is reverted by KN93. 
Impact of mutations in Cx32 on peripheral nerve and CNS will be discussed. We could notice that mice invalidated for CamKII present a defect in CNS myelination.
These data allowed us to propose CamKII inhibitors as drugs to treat CMTX1. EU has granted KN93 as an ODD. In addition, we are thus currently screening chemical libraries to identify molecules able to correcto connexon activity,

Polyradiculonévrite inflammatoire démyélinisante chronique (PIDC) forme motrice pure chez 17 patients : caractéristiques cliniques, étude électrophysiologique et réponse au traitement

Antoine PEGAT (Lyon)

La polyradiculonévrite inflammatoire démyélinisante chronique de forme motrice pure (mPIDC) est une forme atypique de PIDC.
Nous rapportons les caractéristiques d’une cohorte rétrospective de patients atteints de mPIDC (EFN/PNS 2010, certaine ou probable) ; avec ou sans anomalies sensitives à l’ENMG. 
17 patients ont été inclus, ils étaient majoritairement de sexe masculin (71%). L’âge médian au début des symptômes était de 48 ans. 10 patients (63%) débutaient par une faiblesse aux membres inférieurs, 5 patients (29%) avaient une évolution par poussées. L’ONLS médian était de 4 (intervalle 3-10) au pic de sévérité, et de 3 (intervalle 0-5) au dernier suivi. Il existait 3 patients (18%) avec
une PIDC paranéoplasique. Pour les traitements de la PIDC, 12/16 (75%) patients traités par IgIV et 4/5 patients (80%) traités par corticostéroïdes présentaient une amélioration clinique. En ENMG, les blocs de conduction (BC) (82% des patients) et les anomalies des ondes F (88%) étaient les anomalies les plus fréquentes. Les BC étaient le plus souvent sur le nerf ulnaire. Une latence distale allongée (18% des patients) et un ralentissement de la vitesse de conduction motrice (35% des patients) étaient rares, et dans 80% des cas sur un nerf avec un BC. L’amplitude motrice distale était réduite chez 94% des patients, 65% avaient des activités de fibrillations au repos, 35% des fasciculations dans au moins un muscle.
Il n’existait pas de spécificité clinique ou paraclinique entre les patients avec ou sans anomalie sensitive à l’ENMG en dehors du développement de signes sensitifs distaux discrets au cours du suivi chez 40% de ceux avec anomalies sensitives en ENMG. Nos patients pouvaient répondre aux corticoïdes, ils ne doivent donc pas être formellement contre indiqué. Les maladies du deuxième
motoneurone sont l’un des principaux diagnostics différentiels de ces formes de PIDC. La NMM à BC est une entité à bien individualiser de ces formes de PIDC. 
Notre étude suggère que la mPIDC est caractérisée par des éléments cliniques (prédominance masculine, âge jeune, association fréquente à des cancers), ENMG (BC et anomalies des ondes F) et thérapeutiques (sensibilité aux corticoïdes) propres.

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