Distal arthrogryposis (DA) is a collection of rare developmental disorders characterized by congenital joint contractures. Most arthrogryposis mutations are in muscle- and joint-related genes, and the anatomical defects originate cell-autonomously within the musculoskeletal tissues. However, gain-of-function (GOF) mutations in PIEZO2, a principal mechanosensor in somatosensation, cause DA subtype 5 via unknown mechanisms. We show that expression of a GOF PIEZO2 mutation in proprioceptive sensory neurons mainly innervating muscle spindles and tendons is sufficient to induce DA5-like phenotypes in mice. Overactive PIEZO2 causes anatomical defects via increased activity within the peripheral nervous system during postnatal development. Surprisingly, overactive PIEZO2 is likely to cause joint abnormalities via increased exocytosis from sensory neuron endings without involving motor circuitry. This reveals a role for somatosensory neurons: excessive mechanosensation within these neurons disrupts musculoskeletal development. We also present proof-of-concept that Botox injection or dietary treatment can counteract the effect of overactive PIEZO2 function to evade DA-like phenotypes in mice when applied during a developmental critical period. These approaches might have clinical applications. Beyond this, our findings call attention to the importance of considering sensory mechanotransduction when diagnosing and treating other musculoskeletal disorders.

Acknowledgements: Our work is supported by National Institutes of Health grant (R35 NS105067, R01 DE022358, R25 SC3GM127195, R25 GM07138, R01GM133845, intramural) and Howard Hughes Medical Institute.

Musculoskeletal diseases are leading causes of disability, morbidity and economic loss across the globe today. Yet for much of the world's population access to cheap, safe and effective intervention is lacking, while others choose not to accept best practice and best evidence, or significantly more expensive treatment. Great advances have been made in some diseases like rheumatoid arthritis, but the cost of many new treatments is unaffordable, and individuals, insurance and governments struggle to, or cannot fund it. Anchor bias and politics determines national policies and research funding, often favouring other illnesses while musculoskeletal disorders lack the support proportional to their frequency and impact. This is not appreciated by policy makers and governments, and the consequences of lack of care or poor-quality care. The need has never been greater for a treatment for osteoarthritis, the most common disease in the world; but the search for a cure needs funding, and if discovered, who will pay for it?

Introduction: Monoclonal antibodies (mAbs) recognizing linear sulphation motifs in keratan sulphate (KS) were first developed in the early 1980’s. Over the years, ELISAs using 5-D-4 or other related anti-KS mAbs have been used in many studies monitoring increased cartilage aggrecan degradation with the onset of degenerative joint diseases. However, whilst these studies have in general been useful for monitoring some aspects of disease progression (usually in parallel with other biomarker assays), many longitudinal studies have shown efficacy in only the transient (early, mid or late) stages of the degenerative joint disease process. During the onset of degenerative joint disease, the pathological tissue attempts to repair/regenerate the cartilage, the chondrocytes thus synthesizing cartilage aggrecan with KS substitution [and chondroitin sulphate (CS) isomer composition] that is more like that found in developing or immature cartilage. This immature cartilage aggrecan contains much less KS substitution with shorter chain size and less linear sulphation motifs. Thus, during the different stages of degenerative joint disease progression one would expect to find variable changes in different linear sulphation epitopes present in the serum or synovial fluids. The aim of this study was to investigate the use of several monoclonal antibodies that recognise different sulphation epitopes [high sulphation (5-D-4), low sulphation (1-B-4) and KS-stubs (BKS-1)] to see if patterns of their expression could be used to distinguish different stages of degenerative joint disease. We have also developed ELISAs using mAbs recognising the KS-proteoglycans, keratocan (Ker 1) and lumican (Lum 1) for their quantification as potential biomarkers of osteoarthritis.

Methods: Competitive ELISAs were developed using monoclonal antibodies (mAbs) 5-D-4, 1B4, BKS-1, Ker-1 and Lum-1. Bovine corneal KS-proteoglycans pre-treated with keratanase were used as both the coating antigen and “standard” antigen on the same ELISA plate. Blood, synovial fluid and cartilage samples (surgical waste) obtained from patients undergoing arthroplasty with different Kellgren & Lawrence grades were analysed.

Results and Discussion: 5-D-4 and BKS-1 showed similar inhibition curves and relative 50% inhibition points. However, the curve obtained with 1B4 indicated lower relative expression of 1B4 epitope. Analysis of serum and synovial fluid sample with 5-D-4 mAb showed the presence of the epitope in both samples, but there was significantly less KS in serum than in the synovial fluid. Our results show that competitive ELISA for quantification of several different KS sulphation or “stub” epitopes and two KS-proteoglycans can all be quantified and compared using the same experimental conditions. These studies are ongoing as part of an Arthritis Research Campaign (UK) funded study. In addition the data indicates that keratocan and lumican are also increased in their expression with the progression of disease. Future studies will be performed in an attempt to quantify increased keratocan and lumican expression as potential biomarkers of degenerative joint disease.