Identification of novel, therapy-responsive protein biomarkers in a mouse model of Duchenne muscular dystrophy by aptamer-based serum proteomics.

Anna M L Coenen-Stass,Amer F Saleh,Michael J Gait,Matthew J A Wood,Graham Mcclorey,Corinne A Betts,Hanns Lochmüller,Alison Blain,Raquel Manzano,Thomas C Roberts

doi:10.1038/srep17014

Abstract

There is currently an urgent need for biomarkers that can be used to monitor the efficacy of experimental therapies for Duchenne Muscular Dystrophy (DMD) in clinical trials. Identification of novel protein biomarkers has been limited due to the massive complexity of the serum proteome and the presence of a small number of very highly abundant proteins. Here we have utilised an aptamer-based proteomics approach to profile 1,129 proteins in the serum of wild-type and mdx (dystrophin deficient) mice. The serum levels of 96 proteins were found to be significantly altered (P < 0.001, q < 0.01) in mdx mice. Additionally, systemic treatment with a peptide-antisense oligonucleotide conjugate designed to induce Dmd exon skipping and recover dystrophin protein expression caused many of the differentially abundant serum proteins to be restored towards wild-type levels. Results for five leading candidate protein biomarkers (Pgam1, Tnni3, Camk2b, Cycs and Adamts5) were validated by ELISA in the mouse samples. Furthermore, ADAMTS5 was found to be significantly elevated in human DMD patient serum. This study has identified multiple novel, therapy-responsive protein biomarkers in the serum of the mdx mouse with potential utility in DMD patients.

Highlights

While hyperCKemia is indicative of Duchenne Muscular Dystrophy (DMD)-associated muscle damage, serum creatine kinase (CK) levels may decline in older patients with more advanced disease on account of reduced muscle mass[9]
There is an urgent need for minimally-invasive biomarkers that can be used as outcome measures in pre-clinical studies and clinical trials for DMD therapeutics
We identified CYCS, TPI1 and THBS4 as being elevated in dystrophic serum, consistent with the findings of Hathout et al utilising a mass spectrometry-based approach[22] (Supplementary Table S1). All of these proteins were restored towards wild-type levels following dystrophin restoration, supporting their usefulness for monitoring the effectiveness of therapeutic interventions in DMD patients

Summary

Introduction

The six minute walk test, in which the distance walked by a patient within 6 minutes is recorded before and after treatment[6], has limitations including high inter-patient variability and an unclear relationship to disease natural history. Magnetic Resonance Imaging (MRI) is another method of monitoring pathology in DMD by revealing the extent of muscle degeneration (i.e. muscle wasting, fibrosis and deposition of adipose tissue) but does not provide a direct read-out of muscle function per se. This approach is subject to a number of limitations such as high cost, low through-put and a requirement for specialist personnel trained in interpreting the MRI data. Protein-based biomarkers are perhaps preferable on account of the ease with which they can be integrated into existing clinical biochemistry workflows (i.e. measurement by Enzyme-Linked Immunosorbent Assay (ELISA) or standard colourimetric/turbidimetric assays)

Methods

Results

Conclusion