Bioinformatics approaches to identify pain mediators, novel LncRNAs and distinct modalities of neuropathic pain

This thesis presents a number of studies in the general subject of bioinformatics and functional genomics. The studies were made in collaboration with experimental scientists of the London Pain Consortium (LPC), an initiative that has promoted collaborations between experimental and computational scientists to further understanding of pain. The studies are mainly concerned with the molecular biology of pain and deal with data gathered from high throughput technologies aiming to assess the transcriptional changes involved in well induced pain states, both from animal models of pain and human patients. We have analysed next generation sequencing data (NGS data) in order to assess the transcriptional changes in rodent’s dorsal root ganglions under well induced pain states. We have also developed a customised computational pipeline to analyse RNA- sequencing data in order to identify novel Long non-coding RNAs (LncRNAs), which may function as mediators of neuropathic pain. Our analyses detected hundreds of novel LncRNAs significantly dysregulated between sham-operated animals and animal models of pain. In addition, in order to gain valuable insights into neuropathic pain, including both its molecular signature, somatosensory profiles and clusters of individuals related to pain severity, we analysed clinical data together with data obtained from quality of life pain-questionnaires. Based on this study, we were able to identify distinct pain modalities associated with the intensity of neuropathic pain. Our results will be useful for the understanding of neuropathic pain and its future treatment

Mechanisms of neurodynamic treatments (MONET): a protocol for a mechanistic, randomised, single-blind controlled trial in patients with carpal tunnel syndrome

Background: Physiotherapeutic management is the first-line intervention for patients with entrapment neuropathies such as carpal tunnel syndrome (CTS). As part of physiotherapy, neurodynamic interventions are often used to treat people with peripheral nerve involvement, but their mechanisms of action are yet to be fully understood. The MONET (mechanisms of neurodynamic treatment) study aims to investigate the mechanisms of action of neurodynamic exercise intervention on nerve structure, and function. Methods: This mechanistic, randomised, single-blind, controlled trial will include 78 people with electrodiagnostically confirmed mild or moderate CTS and 30 healthy participants (N = 108). Patients will be randomly assigned into (1) a 6-week progressive home-based neurodynamic exercise intervention (n = 26), (2) a steroid injection (= 26), or (3) advice (n = 26) group. The primary outcome measure is fractional anisotropy of the median nerve at the wrist using advanced magnetic resonance neuroimaging. Secondary outcome measures include neuroimaging markers at the wrist, quantitative sensory testing, electrodiagnostics, and patient reported outcome measures. Exploratory outcomes include neuroimaging markers at the cervical spine, inflammatory and axonal integrity markers in serial blood samples and biopsies of median nerve innervated skin. We will evaluate outcome measures at baseline and at the end of the 6-week intervention period. We will repeat questionnaires at 6-months. Two-way repeated measures ANCOVAs, followed by posthoc testing will be performed to identify differences in outcome measures among groups and over time. Discussion: This study will advance our understanding of the mechanisms of action underpinning neurodynamic exercises, which will ultimately help clinicians to better target these treatments to those patients who may benefit from them. The inclusion of a positive control group (steroid injection) and a negative control group (advice) will strengthen the interpretation of our results. Trial registration: NCT05859412, 20/4/2023

Shared genetic susceptibility between trigger finger and carpal tunnel syndrome: a genome-wide association study

Background: Trigger finger and carpal tunnel syndrome are the two most common non-traumatic connective tissue disorders of the hand. Both of these conditions frequently co-occur, often in patients with rheumatoid arthritis. However, this phenotypic association is poorly understood. Hypothesising that the co-occurrence of trigger finger and carpal tunnel syndrome might be explained by shared germline predisposition, we aimed to identify a specific genetic locus associated with both diseases. Methods: In this genome-wide association study (GWAS), we identified 2908 patients with trigger finger and 436 579 controls from the UK Biobank prospective cohort. We conducted a case-control GWAS for trigger finger, followed by co-localisation analyses with carpal tunnel syndrome summary statistics. To identify putative causal variants and establish their biological relevance, we did fine-mapping analyses and expression quantitative trait loci (eQTL) analyses, using fibroblasts from healthy donors (n=79) and tenosynovium samples from patients with carpal tunnel syndrome (n=77). We conducted a Cox regression for time to trigger finger and carpal tunnel syndrome diagnosis against plasma IGF-1 concentrations in the UK Biobank cohort. Findings: Phenome-wide analyses confirmed a marked association between carpal tunnel syndrome and trigger finger in the participants from UK Biobank (odds ratio [OR] 11·97, 95% CI 11·1–13·0; p1·04, p<0·02). Interpretation: In this GWAS, the DIRC3 locus on chromosome 2 was significantly associated with both carpal tunnel syndrome and trigger finger, possibly explaining their co-occurrence. The disease-protective allele of rs62175241 was associated with increased expression of long non-coding RNA DIRC3 and its transcriptional target, IGBP5, an antagonist of IGF-1 signalling. These findings suggest a model in which IGF-1 is a driver of both carpal tunnel syndrome and trigger finger, and in which the DIRC3-IGFBP5 axis directly antagonises fibroblastic IGF-1 signalling. Funding: Wellcome Trust, National Institute for Health Research, Medical Research Council

Comprehensive analysis of Long non-coding RNA expression in dorsal root ganglion reveals cell type specificity and dysregulation following nerve injury

Dorsal root ganglion (DRG) neurons provide connectivity between peripheral tissues and spinal cord. Transcriptional plasticity within DRG sensory neurons after peripheral nerve injury contributes to nerve repair but also leads to maladaptive plasticity, including the development of neuropathic pain. This study presents tissue and neuron specific expression profiling of both known and novel Long Non-Coding RNAs (LncRNAs) in rodent DRG following nerve injury. We have identified a large number of novel LncRNAs expressed within rodent DRG, a minority of which were syntenically conserved between mouse, rat and human and which including both- intergenic and antisense LncRNAs. We have also identified neuron type-specific LncRNAs in mouse DRG, and LncRNAs that are expressed in human IPS cell-derived sensory neurons. We show significant plasticity in LncRNA expression following nerve injury, which in mouse is strain and gender dependant. This resource is publicly available and will aid future studies of DRG neuron identity and the transcriptional landscape in both naïve and injured DRG. We present our work regarding novel antisense and intergenic LncRNAs as an online searchable database, accessible from PainNetworks (http://www.painnetworks.org/). We have also integrated all annotated gene expression data in PainNetworks so they can be examined in the context of their protein interactions

Nav1.7 is required for normal C-low threshold mechanoreceptor function in humans and mice

Patients with bi-allelic loss of function mutations in the voltage-gated sodium channel Nav1.7 present with congenital insensitivity to pain (CIP), whilst low threshold mechanosensation is reportedly normal. Using psychophysics (n = 6 CIP participants and n = 86 healthy controls) and facial EMG (n = 3 CIP participants and n = 8 healthy controls) we have found that these patients also have abnormalities in the encoding of affective touch which is mediated by the specialised afferents; C-low threshold mechanoreceptors (C-LTMRs). In the mouse we found that C-LTMRs express high levels of Nav1.7. Genetic loss or selective pharmacological inhibition of Nav1.7 in C-LTMRs resulted in a significant reduction in the total sodium current density, an increased mechanical threshold and reduced sensitivity to non-noxious cooling. The behavioural consequence of loss of Nav1.7 in C-LTMRs in mice was an elevation in the von Frey mechanical threshold and less sensitivity to cooling on a thermal gradient. Nav1.7 is therefore not only essential for normal pain perception but also for normal C-LTMR function, cool sensitivity and affective touch

An iPSC model of hereditary sensory neuropathy-1 reveals L-serine-responsive deficits in neuronal ganglioside composition and axoglial interactions.

Hereditary sensory neuropathy type 1 (HSN1) is caused by mutations in the SPTLC1 or SPTLC2 sub-units of the enzyme serine palmitoyltransferase, resulting in the production of toxic 1-deoxysphingolipid bases (DSBs). We used induced pluripotent stem cells (iPSCs) from patients with HSN1 to determine whether endogenous DSBs are neurotoxic, patho-mechanisms of toxicity and response to therapy. HSN1 iPSC-derived sensory neurons (iPSCdSNs) endogenously produce neurotoxic DSBs. Complex gangliosides, which are essential for membrane micro-domains and signaling, are reduced, and neurotrophin signaling is impaired, resulting in reduced neurite outgrowth. In HSN1 myelinating cocultures, we find a major disruption of nodal complex proteins after 8 weeks, which leads to complete myelin breakdown after 6 months. HSN1 iPSC models have, therefore, revealed that SPTLC1 mutation alters lipid metabolism, impairs the formation of complex gangliosides, and reduces axon and myelin stability. Many of these changes are prevented by l-serine supplementation, supporting its use as a rational therapy

The genetics of neuropathic pain from model organisms to clinical application

Neuropathic pain (NeuP) arises due to injury of the somatosensory nervous system and is both common and disabling, rendering an urgent need for non-addictive, effective new therapies. Given the high evolutionary conservation of pain, investigative approaches from Drosophila mutagenesis to human Mendelian genetics have aided our understanding of the maladaptive plasticity underlying NeuP. Successes include the identification of ion channel variants causing hyper-excitability and the importance of neuro-immune signaling. Recent developments encompass improved sensory phenotyping in animal models and patients, brain imaging, and electrophysiology-based pain biomarkers, the collection of large well-phenotyped population cohorts, neurons derived from patient stem cells, and high-precision CRISPR generated genetic editing. We will discuss how to harness these resources to understand the pathophysiological drivers of NeuP, define its relationship with comorbidities such as anxiety, depression, and sleep disorders, and explore how to apply these findings to the prediction, diagnosis, and treatment of NeuP in the clinic

A behavioural SIR model and its implications for physical distancing

The paper proposes a behavioural-compartmental-epidemiological model with heterogenous agents who choose whether to enact physical distancing practices. Motivated by the evidence on individual physical distancing behaviour during the COVID-19 outbreak, our model extends the standard compartmental-epidemiological models by including endogenous physical distancing behaviour, drawing on discrete choice theory. This approach can account for two important factors:(i) the limited information about the contagion dynamics available for individuals and (ii) the heterogeneity in the individual ability and preferences concerning physical distancing. Despite its simplicity, the model provides policy indications about the timing and size of mitigating policies and the level and quality of information available for the public