Community Research and Development Information Service - CORDIS


Executive Summary:
The PROPANE STUDY aimed identifying new variants in genes encoding for the sodium channel subunits expressed in the peripheral nociceptive pathway in order to provide diagnostic evidence in patients affected by idiopathic painful small fibre neuropathy and explain the occurrence of neuropathic pain in patients affected by diabetic neuropathy. Indeed, neuropathic pain, which can present with a large number of symptoms and signs (e.g. burning, deep pain, paroxysmal pain, itch, allodynia, hyperalgia), occurs in a percentage of patients with peripheral nerve damage, suggesting the existence of individual’s profile of susceptibility to pain that, if explained, could pave the way toward a concrete personalised medicine approach in clinical practise.
The project stemmed from our first discovery of pathogenic variants in SCN9A and SCN10A, encoding for Nav1.7 and Nav1.8, respectively, two sodium channel subunits playing a key role in the generation and propagation of pain-related signalling. The PROPANE STUDY has been designed to test the hypothesis that other sodium channel genes as well as other genes could be involved in painful neuropathy. To demonstrate it, we have built a research Consortium including neurologist, molecular biologists, system biologists and bioinformatics, basic scientists and cell neurophysiologists with the aim to determine a pipeline that, from well-characterised patients could find gene variants, define its pathogenicity, investigate their effect on in vitro and in vivo models, and verify the response to analgesic compounds.
Results have been successful. We have collected 1,238 patients, thus achieving 82% of the expected target. Among them, we have banked 647 patients with SFN and 591 patients with diabetic neuropathy (the latter group including 291 painful and 300 painful neuropathy) for the targeted genetic analysis on sodium channel genes. Moreover, we have collected and sequenced 68 familial cases and 36 sporadic early onset cases.
The first result achieved has been the discovery of 8 pathogenic variants in SCN11A, encoding for the Nav1.9 sodium channel subunit, in 12 patients with painful peripheral neuropathy identified after sequencing 345 patients without mutations in SCN9A and SCN10A. This discovery, widening the spectrum of sodium channel-related painful neuropathies and definitively confirming the new syndrome, has guided the development of recommendation for the clinical practice that the PROPANE Consortium has published the same year.
With the aim to facilitate the characterisation of sodium channel gene variants and identify the most promising candidates for cell electrophysiology assay, which can provide the biological evidence of pathogenicity but is expensive and time-consuming, we have performed a computational analysis of all published SCN9A variants and discovered that a parameter known as betweenness centrality can correctly differentiate pathogenic NaV1.7 mutations from variants not causing biophysical abnormalities (nABN) and homologous SNPs (hSNPs) with 76% sensitivity and 83% specificity. It has been a relevant scientific achievement in the field of sodium channelopathies, providing a tool that could be replicated for other sodium channel genes associated to painful neuropathies or other diseases, like epilepsy.
The solid clinical characterisation of patients allowed recognising new phenotypes besides the classical presentation of painful peripheral neuropathy. We first identified a family showing a peculiar picture characterised by autosomal dominant paroxysmal itch followed by burning pain and found a variant in SCN9A already described by our Consortium in painful and autonomic neuropathy. However, because itch had been never associated with sodium channel gene mutations, we explored the hypothesis that other causative genes could be involved. After identifying other 2 families with a similar picture of autosomal dominant itch phenotype, we performed an unbiased genetic analysis by whole exome sequencing and discovered two variants in COL6A5 cosegregating in 8 affected members of the 3 families. One of these variants has been found also in one sporadic patient with painless diabetic neuropathy. How this new gene may modulate itch will be investigated in the next future. However, this has been the first evidence that itch susceptibility can be related to a gene, opening a new scenario for potential therapies.
Following the research hypothesis to cluster neuropathy patients as painless and painful based on different genetic background, we sequenced the five sodium channel genes and identified 12 (potential) pathogenic gene variants associated with the painful phenotype and 10 (potential) pathogenic gene variants associated with the painless phenotype. In the next months, clinicians, molecular biologists and bioinformatics will define the weight of these variants in determining the phenotype in order to provide a new approach to the diagnosis and treatment of painful idiopathic and diabetic neuropathies.
The issue regarding the link between functional changes in sodium channels driven by gene variants and the degeneration of small nerve fibre, which is the diagnostic hallmark in clinical practice and cause of patients’ pain and disability, has been approached by in vitro studies. We confirmed the early reduction of neurite length of the Nav1.7 Asp623Asn mutant channels and discovered a longer and time-dependent reduction in the length of neurites of the Nav1.7 Gly856Asp mutant, suggesting that subtype-specific blockade of Nav1.7 or inhibition of reverse NCX might slow or prevent axon degeneration in SFN.

Project Context and Objectives:
Neuropathic pain is a frequent feature of peripheral neuropathy that adversely affects patients’ quality of life and highly increases health care costs. Not all individuals with neuropathy develop pain and it is not possible to predict who is more or less susceptible among those with similar risk exposure. Current inability to identify high-risk individuals hinders development and application of therapies to counteract neuropathic pain and to address targeted prevention strategies.
Current treatments for painful neuropathies are largely inadequate, being available treatments able to provide at the best 50% of pain relief in less than 50% of patients, while at least 25% of patients withdraw drugs because of unpredictable side effects. This disappointing scenario in painful neuropathies is due to the lack both of drugs acting on target sites for which there are strong evidence of pathogenicity and the inability to identify responder patients. Most of the available analgesics act at different levels (e.g. sodium channels, noradrenergic system, opioidergic system) and are prescribed to patients suffering from pain without any selection and are prescribed without any selection in terms of pathogenesis and aetiology. In the last decades, none of the new drugs that have entered the market have proven to be more effective than amitriptyline (EFNS guidelines on the pharmacological treatment of neuropathic pain. Eur J Neurol 2010), an old antidepressant with non-specific sodium channel blocking activity, the use of which in clinical practice is limited by systemic anticholinergic side effects. This unequivocally indicates that new research strategies are urgently needed to achieve a better control of neuropathic pain. So far, candidate molecules have been chosen following pharmacologic hypotheses experimented in animal models. The inability to identify high-risk individuals and the lack of drugs acting on target sites can explain the disappointing effect of current treatments.

State of the art at the time of the PROPANE Study submission
At the time in which the PROPANE Study was submitted, some preliminary findings suggested that sodium channel mutations could change the physiological properties of nociceptors and impair also the integrity of their neurites, eventually leading to the degeneration of small nerve fibres that is the clinical hallmark of most painful neuropathies and the cause of pain in patients. The identification of mechanisms underlying these changes in key neuronal structures would have allowed bridging functional abnormalities and structural changes in nociceptors, thus opening a window upon new circuitries and target druggable sites.
Previous studies had focused on Nav1.7, encoded by SCN9A, a 113.5-kb gene comprising 26 exons (OMIM 603415) that maps to chromosome 2q24, because mutations had explained conditions at the edges of the spectrum of pain disorders, from congenital insensitivity to pain (CIP) to inherited erythermalgia (IE) and paroxysmal extreme pain disorder (PEPD). Immunohistochemical studies demonstrated that Nav1.7 is expressed also in the intraepidermal nerve fibres (IENF), which are the endings of small-size DRG neurons acting as the most distal nociceptors. Nav1.7 current is tetrototoxin-sensitive (TTX-S) subunit and is characterized by slow transition of the channel into an inactive state when it is depolarized, even to a minor degree, a property that allows it remaining available for activation with small or slowly developing depolarizations, usually mimicked by electrophysiologists as ramp-like stimuli. Thus, Nav1.7 amplifies small, subtle depolarizations such as generator potentials, thereby bringing neurons to voltages that stimulate Nav1.8, which has a more depolarized activation threshold producing most of the transmembrane current responsible for the depolarizing phase of action potentials. Recent evidence also suggests that Nav1.7 may act within dorsal horn to facilitate synaptic transmission of pain signals to second-order pain-signalling neurons (Minett et al. Nature Comm 2012).
Three years prior to the submission of the PROPANE Study, our Consortium started investigating a well characterized subgroup of patients affected by chronic excruciating pain in the legs and feet caused by small fibre neuropathy (SFN) of unknown origin, a condition quite common in the general population and in patients affected by systemic diseases (Lauria et al. Curr Op Neurol 2012). Intriguingly, we identified the first 8/28 patients with idiopathic SFN harbouring novel gain-of-function mutations in Nav1.7. All these single amino acid substitutions caused a distinct profile of DRG nociceptor hyperexcitability and firing at voltage and current-clamp analysis that distinguished them from IE and PEPD.
Our findings allowed us to define the new channelopathy-related painful neuropathy syndrome (Faber et al. Ann Neurol 2012; Han et al. Neurology 2012; Hoeijmakers et al. Brain 2012) that widened the spectrum of sodium channel-associated pain disorders. These findings provided a solid background for further investigations aimed to deepen our knowledge on the relationship between pain-related sodium channel mutations and axonal degeneration leading to the development of new therapeutic strategies. At the time of the project started, targeted sequencing of Nav1.7 has allowed our Consortium to identify and profile novel gain-of-function mutations in 11/150 patients with painful predominantly small fibre neuropathy (SFN), including 2 patients with diabetic painful neuropathy.
The role of Nav1.8 in human painful neuropathy has been demonstrated for the first time by our Consortium (Faber, Lauria, Merkies et al. PNAS 2012). We described gain-of-function mutations in 3 patients with painful neuropathy, including a father–son pair. Cell electrophysiological assay provided evidence that the mutations altered the gating properties of the channel in a proexcitatory manner and increased the excitability of DRG nociceptors. In particular, after expression of the mutant channels within DRG neurons, where Nav1.8 is normally expressed (Shields et al. Pain 2012), we found by voltage clamp that it enhanced the response to depolarization thus inducing a proexcitatory effect to the channel, and by current clamp that it decreased current threshold and increased firing frequency in response to suprathreshold stimuli (L554P, A1304T), depolarized resting potential (A1304T), and induced spontaneous firing of small DRG neurons (L554P), which include nociceptors. The first three changes would be expected to lower threshold for, or increase intensity of, evoked pain, whereas the latter change would be expected to contribute to spontaneous pain. Indeed, these changes in the electrophysiological properties of DRG nociceptors reflected the features of neuropathic pain complained by these patients. Intriguingly, the patients harbouring the A1303T mutation complained of intense neuropathic pain and were diagnosed with small fibre neuropathy based on clinical picture and abnormal thermal and pain thresholds at quantitative sensory testing. Indeed, skin biopsy yielded normal IENF density, suggesting that some but not all sodium channel mutations can lead to an overt degeneration of terminal nociceptors, at least over a relatively short time from onset. At the time of the project submission, our Consortium has found novel Nav1.8 gain-of-function mutations in 9 patients, including 2 with diabetic painful neuropathy, among those negative for Nav1.7 mutations. Therefore, our functional observations strongly suggested that these Nav1.8 mutations contribute to the pathophysiology of neuropathic pain in those patients and that Nav1.8 should be considered a further important target in painful neuropathies.
Despite the substantial evidence of the involvement of the above mentioned sodium channels in neuropathic pain, our knowledge of their pathogenic role in patients remained limited, even for the most studied Nav1.7 and no specific drug was currently commercially available. Moreover, not all the patients are expected to have mutations in sodium channel genes. Therefore, the identification of genetic causes of yet unknown pathogenicity to resolve the complete genetic architecture of painful neuropathy also needed a complementary unbiased approach besides the targeted assay of sodium channel genes.
Whole exome sequencing (WES) was planned to be used in selected cohorts of patients with probable genetic neuropathy (e.g. familial cases, onset <40 years) after targeted sequencing of sodium channel genes, in order to identify novel pain-related genes.
One further unbiased approach is the analysis of the transcriptome in skin of SFN patients. This innovative strategy was planned to support the identification of new pain-related targets from the direct assay of human nociceptors in the skin. The recent evidence that transcripts can be obtained in peripheral axons (Gumy et al. RNA 2011) and that skin biopsies allow detection of transcript biomarkers in genetic neuropathies (Fledrich et al. Brain 2012) supported our strategy.

Project objectives
The PROPANE Study, starting from the hypothesis of a common origin of neuropathic pain in a cohort of patients with SFN, aimed to develop this original idea in a larger and well characterized study population, to provide evidence for the reliable stratification of patients at high risk and potential new treatments tailored on patients’ clinical features, in order to improve their quality of life. It focused on diabetic and idiopathic painful neuropathy that are among the most frequent and challenging conditions in clinical practice.
The PROPANE Study project has been an observational, non-interventional, patient-oriented research (not a clinical trial by definition aimed at understanding the molecular mechanisms underlying pain, identifying druggable targets and new molecules tailored to potentially drug-responder patients and determining their effects in pre-clinical settings.

Project Results:
Patient recruitment and assessment DNA banking, targeted and unbiased genetic analyses
(WP1, WP2)
The PROPANE Study developed through intertwined workpackages (WP) and well defined milestones (MS) that allowed achieving the expected results. The project started soon after the approval of the clinical and experimental protocols by the Ethic Committees of the participating centres by the end of month 3. In order to achieve a stratification of patients at high risk for neuropathic pain among those with diabetic and idiopathic neuropathy the consortium recruited and assessed 1261 patients. Recruitment of diabetic neuropathy patients started in October 2014 and ended on July 2016 (WP1). The development of a web-based electronic database for painful neuropathies has been completed by month 12. Genomic DNA has been extracted and stored in a DNA databank for the whole PROPANE study population.
The clinical assessment allowed the identification of new clinical phenotypes besides the classical length-dependent SFN. We therefore deepened our knowledge including within the spectrum of presentation also patients suffering from diffuse pain or specific symptoms like itch. These findings will allow applying the approach used in the PROPANE Study also in other painful disorders.
With the aim to further strengthened the role of skin biopsy in the diagnosis of painful neuropathies and its use as an outcome measure in RTCs, we investigated the consistency of the quantification of IENF density (IENFD) comparing right and left leg in SP patients and healthy controls, and comparing the data to follow-up biopsies performed after 3 week, the time of skin epidermal renewal. Our original results, showing overlapping values and excellent correlation coefficients for IENFD quantification both in healthy subjects and patients with SFN, confirmed that skin biopsy can be performed unilaterally to assess the diagnosis of SFN in individual patients and that IENFD can be reliably used as a biomarker of small nerve fibre degeneration within a 3-week period.
All patients have undergone targeted exon sequencing of sodium channel genes encoding for Nav1.7, Nav1.8, Nav1.9, Nav1.6 and Nav1.3 to reveal the frequency of mutations and variants after screening of public databases and, if needed, geographically matched healthy subjects (WP2). Genetic analyses by targeted sequencing performed in the project have identified new variants in known genes (Nav1.7 and Nav1.8) and variants in new genes (Nav1.9, Nav1.6, Nav1.3). These results together with clinical assessment and skin biopsy analyses have allowed strengthening the definition of the new syndrome in the spectrum of pain disorders: the sodium channel-related painful SFN.
Genetic analyses started by targeted sequencing of exons and exon-flanking intron sequences of the sodium channel genes SCN3A (encodes Nav1.3), SCN8A (encodes Nav1.6), SCN9A (encodes Nav1.7), SCN10A (encodes Nav1.8) and SCN11A (encodes Nav1.9) using the MiSeq sequencer and the TruSeq Custom Amplicon kit (Illumina).
In order to increase the cost-effectiveness of the genetic assay, considering the large number of patients, we have developed, standardised and validated a panel 107 candidate pain-genes to be assayed by Molecular Inversion Probes-targeted Next Generation Sequencing (MIP-NGS), a relatively cheap, flexible and reliable technique based on short oligonucleotides (Molecular Inversion Probes, MIPs) that capture genomic regions for massively parallel DNA sequencing. The validated was performed by through a 3-step pilot study (Pilot I, SCN9A; Pilot II, SCN3A, SCN7A-11A and SCN1B-SCN4B; Pilot III, 107 pain-related genes including the 10 SCN genes) and assured a performance and capture efficiency of 92.3%. Variants with possible pathogenic effect are identified using a standardized bioinformatics pipeline that the Consortium has already developed using Alamut Mutation-Interpretation Software (Interactive-Biosoftware, Rouen, France). Presence and frequency of variants are determined in public databases (HGMD, Mooney Laboratory, SM2PH-db 2.0, dbSNP, Exome Variant Server and 1000 Genomes Project). In the case of new genes or new mutations not reported in public databases, available DNA stored from 700 healthy donors geographically matched is used.
Using both the approaches, 1087 patients have been sequenced so far, and 766 have been analysed for SCN3A, SCN7A-11A, and SCN1B-4B. Variants detected were annotated according to the guidelines of the Human Genome Variation Society ( Variants with a possible pathogenic effect were identified using Alamut Mutation-Interpretation Software (Interactive-Biosoftware, Rouen, France). Classification of variants was based on the practice guidelines of the Association for Clinical Genetic Science (ACGS) and recommendations published by our Consortium (Waxman et al., Lancet Neurol 2014). Overall, sequence data of SCN3A, SCN7A-SCN11A, SCN1B-4B revealed 116 different (potential) pathogenic variants in 181 patients with idiopathic SFN and 31 different (potential) pathogenic variants were detected in 37 patients. For the painless diabetic neuropathy patients (n=198 patients), sequence data of SCN3A, SCN7A-SCN11A, SCN1B-4B revealed 27 different (potential) pathogenic variants in 33 patients, of which 10 variants are phenotype specific. In the painful idiopathic neuropathy patients (n=486 patients), 81 different (potential) pathogenic variants were detected in 109 patients. Seventy variants were phenotype specific. Of the 116 different (potential) pathogenic variants, 16 variants have been published in literature as disease causing mutation, possible pathogenic variant, variant with uncertain significance or variant unlikely to be pathogenic. Twenty-five variants were found in more than one patient. Hundred variants were novel and classified according to the practice guidelines of the ACGS as a class 3 (uncertain clinical significance) or 4 (likely to be pathogenic) variant.
The demonstration of the biological effect of sodium channel variants on DRG nociceptors is obtained by cell electrophysiology assays, which is time-consuming and needs high manpower with specific expertise. In order to fasten this process and select the most promising candidate variants, we developed a novel approach to predict the pathogenicity of Nav1.7 variants characterising the effect of the mutation on the proteins at the atomic level by a computational modelling approach. By means of in-silico mutagenesis, we solved and analysed the tertiary structure of NaV1.7 variants previously assessed to be pathogenic for IEM, PEPD and SFN, with the aim to provide a further tool able to reliably predict the potential pathogenicity of the variant and address to the cell electrophysiology study, that is time-consuming, only the stronger candidates. Through this protein and mathematical modelling, we have calculate the changes of different topological parameters, and found that one of them, known as betweenness centrality (Bct), correctly differentiated pathogenic NaV1.7 mutations from variants not causing biophysical abnormalities (nABN) and homologous SNPs (hSNPs) with 76% sensitivity and 83% specificity, using the cut-off value ±0.26 calculated by receiver operating curve analysis.
Considering that not all the cases of painful neuropathy could be explained by mutations in sodium channels, those patients negative for sodium channel mutations or with a possible genetic form (e.g. early onset of neuropathy, affected relatives) have been assayed by unbiased WES. Co-segregation of variants in new genes has been investigated in familial cases (at least 2 first-degree relatives affected with the disease), in trios of patients with peculiar phenotypes and in selected cohorts of clinically homogeneous patients screened negative for candidate pathogenic variants through MIP-NGS. WES has be performed using HiSeq2000 and NextSeq 500 sequencers (Illumina) and as enrichment protocol the SureSelect V4 exome enrichment (Agilent Technologies Inc., Santa Clara, CA). Candidate genes have be selected based on the presence of potential pathogenic mutations in different patients. The ~15,000 potential variants per exome are filtered on coverage, evolutionary conservation, mutation frequency (<1%) and in silico predicted effect (Alamut Mutation-pathogenicity prediction software suite; SIFT and PolyPhen2 scores among others) using an established bioinformatics pipeline. Genes containing 200-400 potentially causative variants are further filtered based on segregation across families, multiple occurrence among unrelated patients, tissue-expression patterns in human or in animal models, functional characteristics and using additional bioinformatics tools. WES was performed in 11 families with painful idiopathic neuropathy or other and led to the identification of five potential disease causing variants pain-related genes and variants. Among them, we examined 3 families whose 8 affected members suffered from chronic itch. WES allowed identifying two variants in COL6A5 and discovering the first gene associated with neurogenic itch, thus offering a new candidate target for therapies.
A workflow from skin biopsy collection to RNA extraction has been defined and optimised after a validation study that allowed identifying the better condition to obtain high quality RNA.
We set up an in vivo screening model of pain-related candidate mutations through the optimization of a panel of read-out parameters reflecting neuropathic pain in zebrafish (WP4). These results were validated using genetic loss of function model (scn8aa).
We completed the characterization of new Nav1.9 pain-related gene mutations by voltage and current-clamp analyses performed in transfected HEK293 and small size DRG neurons (WP5). By characterizing the pharmacology of the wildtype (WT) hNav1.7 TTX-R channel, we found that the inhibition of Nav1.7 by sodium channel blockers is voltage-dependent, with greater potency at more depolarised membrane potentials.
Results prompted the screening for the identification of candidate sodium channel blockers among existing molecules using high throughput and conventional electrophysiology (WP6), which final objective was to discover putative selective pharmacotherapies tailored on pain-related patients’ genotype. We have performed the experiments using conventional manual patch-clamp (MP) and the higher throughput QPatch (QP) platform to investigate the robustness of different assays and found that the rank order of potency obtained using the QP corresponded to that obtained in MP experiments (WP6).
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) and their efficacy comparing pain-related behaviour between mutated and wild-type animals (WP7) have been 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 used 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). By month 18, we have set up the transfection/infection protocols for DRG neurons, the cell culture system for analysis of neurite outgrowth and the protocols for axonal degeneration analyses. Using these in vitro approaches, we have assessed 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 has run for the whole project duration: project management (WP9), devoted for administration of all scientific, social and financial issues, and dissemination and exploitation (WP10), devoted to disseminate and exploit the project concept and results.
The above mentioned objectives were achieved through the following:
1) the recruitment of 591 patients with diabetic neuropathy, 300 painless and 291 painful, and 647 patients with idiopathic SFN, for a total of 1,238 patients (82% of the expected number), based on strict clinical, neurophysiological, and skin biopsy criteria, and their assessment (MS3) recorded using an electronic database (MS2) in order to profile their phenotype, after approval of the project by the Ethic Committees of the clinical centres participating in the PROPANE STUDY (MS1);
2) the identification of novel mutations in the genes encoding for Nav1.7, Nav1.8, Nav1.9, Nav1.6, and Nav1.3 sodium channels (MS4 and6) using targeted sequencing in all 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 (MS6) 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 <40 years) after negative targeted sequencing of sodium channels;
4) to generate a further list of new candidate genes (MS7) using an established bioinformatic pipeline in addition to the unbiased WES and transcriptome assay approaches (MS4 and MS6);
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 (MS8);
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 (MS9 and MS10), 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 (MS11);
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 (MS12 and MS13), 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 (MS14);
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 (MS15), 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 (MS16).

Bioinformatic and mathematical analyses
Based on the system-level co-expression with the set of genes known to be involved in small fiber neuropathies we predicted gene candidates for screening in patients. The genes were additionally inspected for the transcript presence in DRG and TG cells, leading to the final set of 20 genes potentially functionally linked with voltage-gated sodium channels.
Bioinformatics pipeline
1. Pipeline for whole-exome sequencing data of familial and early onset cases.
2. Pipeline for RNA-sequencing of transcriptomic data.
Quantitative assessment of 3D structures
We have computed the several quantitative measurements from the closed form of the mutated and wild type NaV1.7 3D structures.
Modelling of hydropathic topology of an ion channel pore
1. Analysis of hydropathic topology of the Nav1.7 channel
2. In-silico mutagenesis of the Nav1.7 channel, and comparison of different optimization techniques
3. Numerical quantification of the perturbing effect of a mutation on the stable state of the Nav1.7 molecule in terms of hydropathicity
NaV1.7 interatomic structure graph design
A total of 70 NaV1.7 tertiary structures, after in-silico mutagenesis, have been solved. These structures included 30 mutations identified in IEM, SFN and PEPD patients and confirmed by cell electrophysiology, 4 variants not causing biophysical abnormalities in the channel (N1245S, L1267V ,V1428I, T920N) and 40 SNPs identified between human and homologous mammalian NaV1.7 genes with >90% sequence identity. NaV1.7 structure was transformed into undirected graphs by the identification of hydrophobic, hydrophilic, cation-π and π-π stacking interactions among the amino acids. In the resulting graph, amino acids are the nodes and their interactions are the edges.
Gain-of-function NaV1.7 mutation cause Bct variations
We found that Bct variation (ΔBct) is a prevalent feature that significantly differentiates gain-of-function mutations in IEM, SFN and PEPD (∆Bct > 0.4; p<0.01 versus Wt) compared with nABN and hSNPs (∆Bct <0.4; p<0.05 versus Wt). Indeed, 20 out of the 30 gain-of-function NaV1.7 mutations (63%) examined showed high ΔBct values ranging from positive (>0.4) to negative (< -0.4); 6 mutations (20%) showed a mild ΔBct variation (0.4<ΔBct<0.1) whereas 4 mutations (10%) had small ΔBct values <0.1. Conversely, most of nABN and hSNPs showed ΔBct values <0.1. The values of the other topological parameters (degree, CCct, Cct and Ect) for different amino acid substitution were not significant and less informative than ΔBct.
ΔBct in distinguishing gain-of-function from nABN and hSNPs
We examined whether ΔBct might prove sufficient sensitive and specific to serve as a marker for gain-of-function mutations. Using the cut-off value (ΔBct±0.27) that maximizes sensitivity and specificity ΔBct correctly classified 37 out of 40 controls variants and 22 out of 30 gain-of-function mutations yielding 91% sensitivity and 73% specificity respectively. The area under the ROC curve analysis for the ΔBct scores was 0.83.

Zebrafish (Dario Rerio) model to screen candidate pain-related mutations
We have set up a read-out panel reflecting important clinical hallmarks of small fibre neuropathy, the panel was optimized and validated using known/published conditions and tested with known pathogenic mutations (I228M/G856D). The panel exists of behavioural assays including; touch response, temperature sensitivity and responses to irritating compounds. Additionally, one morphological read-out has been established; quantification of sensory fibres in the skin of the zebrafish. The most significant results for each parameter will be discussed in the sections below.
Touch-evoked response
Validation of this parameter has been performed using a published Scn8aa morpholino. These published results showed an aberrant touch response at 2, 3 and 4 days after knockdown of scn8aa. This knockdown phenotype could be confirmed in our hands with a touch response assay. Furthermore, we confirmed the mutant phenotype with the genetic scn8aa loss-of-function (LOF) model (genetic loss-of-function line, available from EZRC zebrafish stock centre). This scn8aa LOF model most probably represents a state of insensitivity to pain. Unfortunately, we do not observe any significant differences in touch when overexpressing pathogenic mutations.
Changes in locomotion behavior in response to temperature changes.
To test alterations in temperature sensitivity, we have conceived, tested and validated a behavioral response assay upon temperature change. To this end, we customized a commercial zebrafish behavioral monitoring device and added the possibilities to do fast temperature changes and measure the behavioral response. The results of our studies in wild-type zebrafish demonstrated that we are able to quantify zebrafish behavioral changes in response to temperature change. The behavioral response to temperature increase (35°C) of wildtype (WT) zebrafish and zebrafish in which scna8aa was knocked down using a scn8aa morpholino was assessed at 4 and 5 days post fertilization (dpf). This temperature is considered noxious but not damaging for zebrafish embryo’s. WT zebrafish respond to increasing temperature with an increased activity, while both the transient knockdown model as the genetic LOF scn8aa model both have a reduced response to a temperature change (fig. 1). This altered response relates to the altered thermal sensitivity of SFN patients.
Overexpression of the I228M pathogenic mutation affects temperature response of the mutant zebrafish. The initial response is the same for wildtype and mutant zebrafish (fig. 2A). However, at high temperatures (36°C) the activity of the mutant zebrafish is diminished indicating that thermal sensitivity is altered (fig. 2B). This could be due to reduced thermal sensitivity or by enhanced habituation to higher temperatures. We are currently testing the effects of the second pathogenic mutation (G856D).

Density of sensory neurons
The sensory nerve fibers can be observed as a network of branching fibers throughout the body of the larvae (fig. 3) in the transgenic line Sensory:GFP, which marks all sensory neurons. As the tip of the tail of a zebrafish larva exists of not more than two layers of skin and some connective tissue, the tip of the tail is a suitable area to study (image) the density of sensory neurons. This assay is very similar to the diagnostic test for SFN that measures the density of sensory fibers in the skin of patients. This nerve-density is reduced in SFN patients. The density of sensory neurons in the outer part of the tail was quantified with the particle analyzer of ImageJ as described by Campbell et al. (Campbell, P. D. et al. J. Neurosci. 34, 14717–14732 (2014) (fig. 3). We have validated this quantification method by comparing a control condition with a condition in which the larvae were heat-shocked and confirmed a significant reduction of sensory fibers in the skin as described by Malafogia et al. (Malafoglia et al. J. Cell. Physiol. 2014;229:300–8).
When we overexpress the I228M mutation we observe a significant differences in the density/complexity of networks of sensory neurons (fig. 4). This is probably due to hyperactivity and subsequent cell death of the sensory neurons.
Our future experiments will include the screening of more putatively pathogenic variants, identified in WP2, as well as validating potential roles of other, novel SFN-causing genes from WP2.
So far, the vast majority of the experiments were performed using transient expression models (morpholino knock-down and mRNA expression). The next step is generating stable genetic models. To this end, we are using the CrispR/Cas9 method to generate zebrafish that carry pathogenic mutations (I228M and G856D) in endogenous zebrafish sodium channels. Currently, we are screening the first generation of CrispR/Cas9-injected zebrafish to identify potential carriers. Until now, we have only identified a mutation that causes a loss-of-function. Demonstrating that the CrispR/Cas9 works because the Cas9 induces a double stranded break which is repaired by either non-homologous end joining repair resulting in a loss-of-function mutation or homology-directed repair which results in a knock-in mutation. The efficiency of homology-directed repair is lower; we expect to identify a carrier by screening a larger pool of putative transgenes.

Cell electrophysiology
The objective of WP5 was the investigation of the effects of variants in voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.7, Nav1.8 and Nav1.9, and other associated proteins and related targets found in subjects with peripheral painful neuropathy, and selected for functional analysis, if possible, by zebrafish modeling (WP4) and/or computational modeling (WP3). In the absence of supporting information from computer simulations or zebrafish model, in silico analysis as well as comparative analysis of similar mutations in other sodium channels were used to prioritize mutations for functional profiling. For voltage-gated sodium channels, functional analysis of the mutations on the gating properties of the channels was investigated in HEK293 or ND7/23 mammalian cell lines, or in primary sensory or sympathetic neurons from rodents (sympathetic ganglion neurons, SCG, from adult rat; dorsal root ganglion neurons, DRG, from Nav1.8-null or Nav1.9-null mouse). The effect of the expression of mutant channels and other related targets on firing behavior of neurons was conducted in small diameter DRG neurons from adult rats which carry the full complement of ion conductances. The aim is to assess potential pathogenicity of these mutations, in order to explain occurrence and features of neuropathic pain in each individual patient and to expand our knowledge on molecular mechanisms, circuitries and druggable target sites.
The effect of the amino acid substitutions on the properties of sodium channels are assessed using voltage-clamp recording of sodium currents in different cell expression systems depending on the channel isoform. Voltage-clamp recordings were obtained at room temperature (22 ± 1°C), 40–48 h after transfection, using protocols that have been optimized by the partners. The effect of the amino acid substitution on the properties of Nav1.6 was assessed in the DRG-derived cell line ND7/23, which has been previously used to study this channel. The effects of mutations on Nav1.7 were assessed in HEK293 cells which support the expression of this channel. By contrast, Nav1.8 and Nav1.9 channels do not produce reproducible data in these cell types, and primary DRG neurons from Nav1.8-null (to study Nav1.8 mutations), Nav1.9-null mouse or rat superior cervical ganglion (SCG) neurons (to study Nav1.9 mutations) were used. SCG were used in one set of experiments because it lacks endogenous TTX-R currents (Nav1.8 or Nav1.9) that makes analysis of the Nav1.9 TTX-R currents much easier. The effect of the amino acid substitution on the firing properties of adult rat DRG neurons, which expresses the full complement of ion conductance comprising the electroginosome, was assessed using current-clamp recording of action potentials. Transfection of DRG neurons by WT or mutant channels does not permit precise control of the level of expression of the transfected channels, and, by definition, produces supra-physiological levels of the channel. We therefore used dynamic clamp recordings, for some mutant channels, to introduce physiologically relevant, calibrated levels of WT and mutant currents into small DRG neuron. A kinetic computer model of the Nav1.9 conductance was built from values of gating parameters of this channel that were determined empirically. Another innovation is the use of multielectrode recordings to assess the effect of some mutations on the firing behaviour of intact DRG neurons which allows the assessment of the same neurons over time, and is a higher throughput assay compared to manual current-clamp recordings.
Using electrophysiological recordings, we functionally assessed a total of 19 mutations in Navs and one mutation in one of the auxiliary subunits (Naβ2), and showed that some of these mutations confer gain-of-function attributes on the channel and increase the excitability of DRG neurons that express the mutant protein (Table 1). The significant findings from this work are that genetic and functional observations have identified missense mutations in voltage-gated sodium channels as contributing to a painful peripheral neuropathy in human subjects, but that a substantial number of variants were found not to change channel properties or neuronal firing or conferred loss-of-function attributes on the channels, and are thus of unknown clinical significance. One mutation in Nav1.8 (Arg1582His) produced paradoxical findings with current-clamp recordings showing increased excitability while the voltage-clamp recording in Nav1.8-null DRG neurons showing loss-of-function attributes.
Our mixed findings highlight the need for functional assessment of variants in sodium channels before clinical significance may be assigned to them.
Using a high throughput screening electrophysiology platform, Convergence has developed a suite of assays to investigate the pharmacological profile of sodium channel blockers against different sodium channel variants. We have tested sodium channel blockers used in the clinic, as well as Convergence proprietary compounds, and compare their efficacy in blocking the variant and the wild type channels. We have profile a single Nav1.7 variant: I228M. The experimental data obtained on this variant show complex and subtle differences with the WT channel, which are assay and compound-dependent. The translational value of this data in terms of extrapolation to clinical efficacy is unclear. A more systematic study including several variants is needed to evaluate the potential utility of this approach to help identify potential treatments for small fiber neuropathy patients carrying pathogenic sodium channel variants.

Identification of candidate sodium channel blockers and Pre-clinical tests of new sodium channel blockers
(WP6 and WP7)
As planned in the research projects, we have profiled the dose-response pharmacology of Nav1.7-WT and Nav1.7-I228M, using a panel of clinically-available sodium channel blockers used to treat neuropathic pain (D6.1), and some of Convergence’s proprietary tool compounds (D6.2). We found that, in general the affinity of the antagonists are not altered by the presence of the mutation, since except for lamotrigine at -90mV, the amounts of inhibition are in similar ranges between the WT and I228M mutation at a specific voltage. However subtle changes were found between compounds, as some voltage-dependent inhibitions were highlighted between the closed (-90mV) and the more inactivated state (-70mV) for some compounds with the NaV-I228M mutation (CNV3000016, CNV3000098 and Lamotrigine). In some cases (CNV3000065 and Lacosamide), the voltage-dependent inhibition were observed in the WT variant, and lost for the mutant. Some compounds such as Amitriptyline, Carbamazepine and CNV1427400, did not show clear voltage-dependent activity between the two voltages used in this study. Our results represent a broad overview of the potencies of eight sodium channel blockers, but more experiments would be needed in the future to fully characterize all the compounds, with the aim to select a drug candidate for Nav1.7-I228M patients.
Our results also show that the mechanism of action of the Nav1.7 channel blockers are complex and cannot be elucidated by assessing the potency at a single voltage alone. One of the aspects of the inhibition, the voltage-dependence has been addressed in this study, but there are also other parameters to consider. For example, sodium channels have been shown to express frequency-dependent inhibition in the presence of some compounds, (Kourtney, J Pharmacol Exp Ther. 1975; Rogawski and Löscher, Nat Med. 2004; Wang et al, Plos One 2015). Inhibition thereby is more prominent when the cellular activity is increased, such as in the case of pain-related high frequency firing. Gating mechanisms of the hNav1.7 channel have been also described in the past as crucial to determine cellular excitability. For instance, modifications of properties such as the activation, steady state fast or slow inactivation, as well as the recovery from inactivation and the amount of resurgent current, have been extensively documented as associated to mutations inducing pain conditions (Dib-Hajj et al., Nat Neurosci 2013; Lampert et al, Handb Exp Pharm 2014). The process to select the best candidate to treat patients carrying the NaV1.7-I228M mutation would therefore need further experiments. We have recently developed the assays to measure most of these biophysical properties on a high throughput platform (Derjean et al, EFIC pain congress, Vienna, 2015).
In conclusion, we have developed using a high throughput screening electrophysiology platform, a method to compare the pharmacological profile of sodium channel blockers against different sodium channel variants. The experimental data obtained on the I228M variant show complex and subtle differences with the WT channel. The meaning of this data in terms extrapolating to clinical efficacy is unclear. One needs to look at several variants to evaluate the potential utility of this approach to help identify potential treatments for patients carrying pathogenic mutations responsible of small fiber neuropathy.
The aim of WP7 was to test the efficacy and toxicity of conventional sodium channel blockers and additional compounds that are used for the current treatment of SFN. Therefore, we have tested the effects of different compounds on temperature sensitivity of the zebrafish. After consulting with the other partners in this WP: P3 Yale and P8 Convergence, we compiled a panel of compounds that were tested. Our panel of compounds includes carbamazepine, lamotrigine, clonidine and amitriptyline.
For each compound the optimal concentration needed to be determined since at a high dose these compounds become toxic and/or will affect normal locomotor behavior of the zebrafish. The most optimal concentrations are listed in table 1 and were determined by performing titrations of the compound or by using data available from literature. At higher concentrations the baseline activity of the zebrafish is affected, resulting in a lower activity at the maximal temperature. This is probably due to a sedative and/or toxic effect. No differences in the slope of the curves upon temperature increase were observed (fig. 5). This demonstrates that these compounds at high concentration only affect locomotor behavior (sedative effect) rather than temperature sensitivity. Eventually, the effect on temperature sensitivity was determined for each of these compounds using the concentrations mentioned in table 2.
All four the compounds have no effect on the initial activity of the zebrafish in response to temperature change (fig. 6A) at 5dpf. Both exposed and control fish respond to the same extend to the temperature change. However, a clear difference in activity between control and zebrafish exposed to carbamazepine is observed at a noxious temperature of 36.5°C (fig. 6B), meaning that exposure to carbamazepine made the zebrafish larvae less sensitive at the highest temperature (36.5°C) or makes them habituate faster to this elevated temperature. At the moment we are testing the effects of carbamazepine on temperature sensitivity in zebrafish that express pathogenic mutations. Furthermore, we are expanding our panel with promising compounds like lacosamide.

In vitro studies of axonal degeneration
The mechanisms that underlie nerve fiber injury in SFN are incompletely understood, although roles for energetic stress have been suggested. Furthermore, small fiber neuropathy is characterized by an age-dependent manifestation, with symptoms appearing in adulthood. Our previous published studies reporting effects of the expression of mutant Nav1.7 on neurite length and integrity utilized DRG neurons in culture for only 3 days. We tested two additional mutations, Asp623Asn and Ile739Val, using this standard assay. undertook optimizing and validating new culture conditions that extended analyses for up to 30 days in culture, and used these new conditions to investigate the effect of the Gly856Asp mutation in Nav1.7 on time-dependent degeneration of neurites, levels of intracellular calcium levels [Ca2+]I, reactive oxygen species (ROS) and ATP levels. We also examined whether the reversal mode of sodium-channel exchanger contributes the impairment of neurite length in DRG neurons expressing mutant sodium channels, and investigated whether sodium channel blockers can counteract axonal degeneration.
Our data show that the expression of the Nav1.7 Asp623Asn mutant channels in DRG neurons resulted in a reduction of the neurite length after 3 days in culture. By contrast, expression of the Ile739Val channels produced a small change that did not reach statistical significance. We interpret these data to suggest that DRG neurons expressing the Ile739Val channels may require longer time to manifest their effect on neurite length, or that an additional metabolic insult is required for the full manifestation of the mutant phenotype.
Our studies on longer-term DRG cultures (18 days) show that cell bodies and neurites of DRG neurons transfected with Gly856Asp display increased levels of [Na+]i and [Ca2+]i following stimulation with high [K+] compared to WT Nav1.7-expressing neurons. Blockade of reverse mode of the sodium-calcium exchanger (NCX) or of sodium channels attenuates [Ca2+] transients evoked by high [K+] in Gly856Asp-expressing DRG cell bodies and neurites. We also show that treatment of WT or Gly856Asp-expressing neurites with high [K+] or 2-deoxyglucose (2-DG) does not elicit degeneration of these neurites, but that high [K+] and 2-DG in combination evokes degeneration of Gly856Asp neurites but not WT neurites. This finding has a theoretical basis in the requirement for energetic stores to maintain membrane potential and transmembrane ionic gradients in excitable cells such as neurons. Our results also demonstrate that zero Ca2+ or blockade of reverse mode of NCX protects Gly856Asp-expressing neurites from degeneration when exposed to high [K+] and 2-DG.
Using longer-term cultures (30 days), our data show there was a time-dependent reduction in the length of Gly856Asp -expressing neurites compared to WT neurites, with the mean neurite lengths of Gly856Asp-expressing neurons displaying a nearly 30% decrease compared to WT neurites. At 30 days in culture, smaller diameter Gly856Asp-expressing neurites displayed significantly increased [Ca2+]i compared to WT-expressing neurites, consistent with mutant channels placing an accumulating burden on DRG neurite integrity. Enhanced Na+ influx through mutant channels is likely to exert two major effects: 1) an increased demand for Na+/K+-ATPase activity to maintain ionic gradients in neurites and 2) a reversal of NCX with import of Ca2+ leading to sustained elevated levels of [Ca2+]i. In both cases, demand for energy supplies would be anticipated to increase. Particularly in smaller diameter neurites, with longer time, rising [Ca2+]i levels might trigger mitochondrial dysfunction, further compounding a time-dependent increase in intracellular calcium levels that could enhance activity in degradative pathways and culminate in neurite degeneration with a predilection for small-diameter nerve fibers. Paralleling the reduction in neurite lengths of Gly856Asp-expressing neurons, we observed a significantly increased percentage of neurites displaying signs of degeneration at 30 days. These observations in tissue culture suggest that the presence of mutant G856D Nav1.7 channels contributes in a time-dependent manner to dysfunction of neurite integrity.
These results demonstrate utility of our optimized long-term cultures for the assessment of molecular basis of axonal deficits in DRG neurons expressing mutant Nav1.7 channels. Our data point to [Na+] overload in DRG neurons expressing mutant Gly856Asp Nav1.7 that triggers reverse mode of NCX and contributes to Ca2+ toxicity, and suggest subtype-specific blockade of Nav1.7 or inhibition of reverse NCX as strategies that might slow or prevent axon degeneration in SFN. The quantitative analysis of mitochondrial morphology in rat DRG neurons co-transfected with pDsRed2-Mito and GFP plasmid, Nav1.7 wt or mutant D623N and I228M, showed altered mitochondrial shape (fig. 7).

Potential Impact:
The PROPANE Study had a strong translational value that can be identified both in the diagnostic and treatment approaches to neuropathic pain. Our proposal had a circular design, which started from the patients, undergoes functional and validation studies using in vitro and in vivo methodologies, and returned to the patients as results tailored on their genetic and clinical features. This design allowed the identification of new clinical presentations, the implementation of diagnostic tools, the identification of new genetic variants associated with the risk of neuropathic pain in individual and subgroup of patients, the discovery of new genes and of new druggable targets potentially tailored to specific patients.
The exploitable results, the exploitation possibilities, as well as the impact that could be derived by the uptake / exploitation of such results are described here below in details:
• Identification of new clinical presentations
The approach to neuropathic pain as a whole condition influenced by a genetic background, irrespective of the clinical feature, led to include subgroups of patients with peculiar phenotypes, like itch or diffuse pain, in whom the diagnosis has been defined and a new gene has been identified thus far.
• Implementation of diagnostic tools
Skin biopsy is the gold standard for diagnosing small fibre neuropathy. Our study showing the internal consistency of intraepidermal nerve fibre density quantification when compared between the right and left side, as well as after 3 weeks, which is the renewal time of the epidermis, strengthened the reliability of this tool for the clinical practice and research in RCTs.
• Identification of new genetic variants associated with the risk of neuropathic pain in individual and subgroup of patients
The identification of signatures for neuropathic pain in diabetic neuropathy, compared with painless diabetic neuropathy, can determine the level of risk in individual neuropathy patients and guide new therapeutic strategies, possibly also to prevent pain. Our findings provided a solid platform to develop a more effective the diagnostic approach in clinical practice offering patients a large screening panel of painful and painless diabetic neuropathy related gene variants. This result could be of extreme importance also in the design of RCTs on neuropathic pain. Moreover, we identified new gene included among those already thought to be involved in neuropathic pain and a new one discovered by unbiased whole exome sequencing.
• Identification of new prediction parameter for Nav1.7 pathogenic variants
Using computation modelling, we first discovered the one parameter of the topology network analysis could predict the pathogenicity of Nav1.7 in painful syndromes with high specificity and sensitivity. This finding is of extreme relevance for the prioritisation of the candidate variants for cell electrophysiology assays, which is currently used to identify the biological effect of sodium channel mutations. Our discovery will increase the cost-effectiveness of the pipeline, reducing the risk to test false positive variants.
• Zebrafish model for testing the effect of drugs on mutant sodium channels
Preclinical testing using in vivo zebrafish models have followed high throughput and conventional electrophysiology, in order to meet the need of valid in vivo models in which treatment targeted to gene variants found in patients. The readouts have been completed and represent the platform to perform preclinical test on the gene variants that will be selected as strong candidates by the genetic analysis still on course, and potentially transferable to other gene associated with painful neuropathy and diseases related to sodium channel gene mutations.
• RCTs with new sodium channel blockers
The definition of the new syndrome of sodium channel-related painful neuropathy, which has been driven by our Consortium, and the evidence from the PROPANE Study have created new potential market applications that might have an innovative impact on patients’ care. The pipeline of clinical characterisation and genetic assessment defined by the PROPANE Study led to the design on a new randomised controlled trial on painful small fibre neuropathy sponsored by a big pharmacompany, in which several partners of the PROPANE study will be involved. The trial will start in 2017 and test the efficacy of a proprietary sodium channel blocker in patients with painful idiopathic and diabetic small fibre neuropathy.
• Clinical database
A database has been developed with characteristics useful for new further studies by the research partners, or by external parties in the field of pain, upon open access disclosure. This will allow to leverage research investments in pain, by providing opportunities for scientists to address different questions with the project outcomes (dataset / cells...) to maximise access to and re-use of research data generated by projects.
In order to favour the future uptake and exploitation of the above mentioned results, the PROPANE Study concept and results have been disseminated to the scientific and clinical community through 17 publications in peer reviewed journals and within more than 50 national and international conferences and workshops, through oral presentations and posters.
We have kept updated advisory board members by organising focused meetings and presenting the results from the PROPANE STUDY at the meeeting of the scientific societies which they represent.
In the last part of the project, contacts have been also established with representatives of other funded consortia in the field of pain, in order to perform joint concertation activities.
A project website has been also created and updated.

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