The Workplan

The PROPANE STUDY will develop through intertwined workpackages (WP) and well defined milestones (MS) which will allow to achieve the expected results. The project will start soon after the approval of the Ethic Committees (WP1; MS1), and development of an electronic database (WP1; MS2) with the clinical part that represents the platform for developing the second part focused on the genetic analysis using targeted and unbiased approaches. Indeed, the aim to achieve a stratification of patients at high risk for neuropathic pain among those will diabetic and idiopathic neuropathy must include the recruitment and assessment of patients based on well defined criteria over a definite period like we have planned (WP1; MS3). Thereafter, all patients will undergo targeted exon sequencing of sodium channel genes encoding for Nav1.7, Nav1.8, Nav1.9, Nav1.6 and Nav1.3 that will reveal the frequency of mutations and variants after screening of public databases and, if needed, geographically matched healthy subjects (WP2; MS4 and MS6). Considering that not all the cases of painful neuropathy could be explained by mutations in sodium channels, those patients negative for sodium channel mutations and with a possible genetic form based on the early onset of neuropathy and/or the presence of affected relatives will be assayed by unbiased WES and skin nerve transcriptome analysis, starting from an already available cohort of 100 selected patients (WP2; MS5). This complementary approach to the identification of new candidate genes will be completed by bioinformatic and mathematical analyses (WP3; MS7), which will also characterize the effect of mutations on the proteins and create mathematical models for predicting the pathogenicity and severity of painful neuropathy (WP3; MS8). Cell electrophysiology studies used to confirm the pathogenicity of sodium channel mutations are expensive and time consuming, and the number of possible pathogenic gene mutations generated in WP2 is expected to be high. The zebrafish model, for which a panel of rapid read-outs reflecting neuropathic pain and morphological features will be first optimized (WP4; MS9) will be used to screen the pathogenicity of sodium channel mutations and other candidate pain-related gene mutations found by unbiased approached in WP2 and WP3 (WP4; MS10). Those mutations most likely associated with functional changes in the nociceptive pathways will be assayed by voltage-clamp in transfected HEK293 cells or, if needed, in small-diameter DRG neurons, and by current-clamp in DRG neurons in order to profile the functional changes of the mutated channel and the firing propertied of the cell (WP5; MS11). Results will prompt the screening for the identification of candidate sodium channel blockers among existing molecules and a range of novel proprietary Convergence molecules using high throughput and conventional electrophysiology (WP6; MS12 and MS13), which final objective is to discover putative selective pharmacotherapies tailored on pain-related patients’ genotype. Using the same behavioural read-out panel as that used to select the most promising candidate gene mutations for confirmatory tests by cell electrophysiology, toxicity and metabolism of selected sodium channel blockers (WP7; MS14) and their efficacy comparing pain-related behaviour between mutated and wild-type animals (WP7; MS14) will be assessed. Since the pathogenesis of IENF loss, namely of the most terminal nociceptors of small-size DRG neurons, in patients with painful neuropathy, especially if idiopathic, is unknown, following our preliminary findings we will use in vitro approaches to investigate the relationship between sodium channels mutations which alter the functional properties of small-size DRG neurons, the ability of neurite outgrowth and the occurrence of axonal degeneration (WP8; MS15). Using these in vitro approaches, we will assess whether the impairment of the sodium-calcium exchanger contribute to the pathogenesis of the axonal damage by increasing intracellular calcium levels and if reverse action of this exchanger as well as conventional and novel sodium channel blockers and can protect and/or recover DRG neuron and axon integrity (WP8; MS16). Two horizontal work packages that will run for the whole project duration. Project Management (WP9) is devoted for administration of all scientific, social and financial. Dissemination and Exploitation (WP10) is devoted to disseminate the project concept. Moreover, WP10 includes standardization, IPR and exploitation issues.

PERT:

Graphical presentation of the components showing their interdependencies