The Project Objectives
Aims of our project are: (A) to resolve the genetic architecture of painful neuropathy in order to achieve a stratification of patients at high risk for neuropathic pain by novel biomarkers and to enhance our understanding of underlying mechanisms, circuitries and target druggable sites; (B) to identify new molecules tailored on potentially responder patients and their effects in pre-clinical settings; and (C) to translate functional changes in nociceptors caused by pain-related sodium channel and novel gene mutations to axonal degeneration in patients.
These will be achieved through:
1) The recruitment of 1,504 patients affected by painful or painless neuropathy (50% diabetic and 50% idiopathic) based on strict clinical, neurophysiological, and skin biopsy criteria, and their assessment using an electronic database in order to profile their phenotype, after approval of the project by the Ethic Committees of the clinical centres participating in the PROPANE STUDY;
2) The identification of novel mutations in the genes encoding for Nav1.7, Nav1.8, Nav1.9, Nav1.6, and Nav1.3 sodium channels using targeted sequencing in all 1,504 patients, in order to describe their frequency in the different subgroup of painful and painless neuropathies and provide a list of candidate pain-related genes;
3) The identification of pain-related genes and variants using unbiased WES and transcriptome analysis in cohorts of candidate patients with a likely genetic origin of the painful neuropathy (e.g. familial cases, onset 4) to generate a further list of new candidate genes using an established bioinformatic pipeline in addition to the unbiased WES and transcriptome assay approaches;
5) The characterization of the effects of sodium channel mutations on the proteins identified in painful and painless neuropathy patients in order to generate prediction models and to provide protein modelling and mathematical modelling enabling postdiction and prediction;
6) The validation of an in vivo screening model of pain-related candidate mutations through the optimization of a panel of rapid read-out parameters reflecting neuropathic pain in zebrafish and the assessment of the pathogenicity of sodium channel mutations found in patients, in order to provide a list of those strongest ones on which confirmatory studies using cell electrophysiology, an expensive and time consuming approach, can more reliably focus;
7) The characterization of the functional changes in the physiological properties of sodium channels and DRG nociceptors produced by mutations found in subjects with painful neuropathy and previously selected, if possible, by zebrafish and/or computational modeling using voltage and current clamp in transfected HEK293 and small-size DRG neurons;
8) The identification and assessment of specific sodium channel blockers targeted on mutations found in patients by screening existing molecules and a range of novel proprietary Convergence molecules using high throughput and conventional electrophysiology, in order to discover putative selective pharmacotherapies tailored on pain-related patient’s genotype;
9) The achievement of preclinical read-outs on toxicity and metabolism of selected sodium channel blockers using the zebrafish model and the characterization of new compounds for druggable target sites identified by WES using the same in vivo approach;
10) The identification of sodium channel mutations which alter the physiological properties of DRG nociceptors affect also the integrity of small-size DRG neurons and their axons, either in terms of neurites outgrowth impairment or induction of axonal degeneration, or both, and the assessment of the contribution of reverse sodium calcium exchange and of the effect of conventional and novel sodium channel blockers on neurite integrity.