1. Congenital Muscular Dystrophy Biomarker Discovery
James Collins MD, PhD
Assistant Professor
Division of Neurology
Cincinnati Children’s Hospital Medical Center

2. Disclosures Research Foundation Grant:
Cure Congenital Muscular Dystrophies partnered with S.A.M. (

3. Biomarker
“A characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.”
Biomarkers Definition Working Group, NIH Clin Pharmacol Ther 2001;69:89-95

5. Biomarker Clinical practice identify risk for or diagnose a disease
assess disease severity or progression
predict prognosis
or guide treatment

6. Biomarker

7. CMD Biomarker Discovery
Liotta, et al. Nature, 2003.
Taniguchi, M. et al., Biochem Biophys Res Commun, (2): p

8. Biomarker Drug development How does a drug work in the body
Is the drug safe or effective
What dose of the drug is effective
Response to a treatment
Treatment trial - FDA regulatory approval process

9. Beneficial or Harmful Effects Not Measured by a Biomarker
Drug development
Measured to/ Substitute for
Therapeutic
Intervention
affects
Clinical
Endpoint
Biomarker
Beneficial or Harmful Effects Not Measured by a Biomarker
Effects of therapeutic interventions on biomarkers and clinical endpoints in clinical trials.
Biomarkers Definition Working Group, NIH. Clin Pharmacol Ther 2001;69:89-95

10. Biomarker development
Discovery phase
Proximal fluids, cell lines, animal models, tissue of interest
candidates
Qualification phase
Human plasma
Confirm candidate molecules
Verification phase
Population-based (specificity)
Validation and clinical assay development
Sensitivity and specificity
Rifai, N., M.A. Gillette, and S.A. Carr, Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol, (8): p

11. Biomarker discovery Genomics Proteomics Metabolomics Imaging
Relevant disease genes, expression profiles, signaling pathways
Proteomics
Protein expression and post-translational modifications
Metabolomics
small molecule metabolites specific to disease
Imaging
Imaging changes reflect disease state

12. RNA Expression Profiling of Blood from Humans
Apply RNA to MICROARRAYS
Whole Blood
GeneSpring, Partek and NCI public software to analyze data
DAVID, KEGG and others for pathways
STORE -700C
Isolate RNA

13. Cluster analysis of genes [with ≥ 2.5-fold change]
blood of DMD compared to healthy age matched males)
Healthy controls: note general gene expression in Lower Left generally higher than DMD subjects.
DMD cluster seperates out to maybe a couple different types maybe more N=34 (14= steroids and 20=not on steroids). Those on steroids maybe on DFZ or Pred
Wong, B., et al., Gene expression in blood of subjects with Duchenne muscular dystrophy. Neurogenetics, (2): p

14. DMD Gene expression profile Steroid treatment effect
Lit L. et al., Pharmacogenomics J, (6): p

15. Proteomics approach

16. Merosin-deficient mice models (dy/dy) muscle
Gene expression profiling CMD
Merosin-deficient mice models (dy/dy) muscle
contractile proteins
shift towards embryonic and perinatal myosin forms
genes involved in cellular adhesion
procollagen genes
genes related to immune response and complement activation
van Lunteren, E. et al., Physiol Genomics, (1): p

17. Gene expression profiling CMD
Fukuyama-type CMD and Merosin - deficient patients expression profile in muscle
up-regulation
extracellular matrix and
basement membrane component genes
unique expression pattern
dystrophin-deficient muscle
Unique profile of FCMD compared to MDC1A
Taniguchi, M., et al.Biochem Biophys Res Commun, (2): p

18. Proteomics - CMD no published proteomic studies
14th international congress WMS society 2009, Geneva, Switzerland
Abstract
Col6a1-/- mice vs WT using 2D-DGE showed 37 proteins differentially expressed in diaphragm
Bovelenta et al. NMD 2009 EM.P.5.01; doi: /j.nmd

19. Going Forward: “Bench to Bedside”
merosin deficient CMD (proteomic and gene expression profiling)
CMD subtypes
Verification: animal models / biobank tissues
Qualification: therapeutic interventions
Validation
Teaming up with multiple academic centers, private and governmental agencies
Biomarker correlation with natural history outcome measures, imaging, and functional mobility scales
Goal: biomarker profile assay use as surrogate end point

20. Challenges Access to patients and samples Heterogeneity
Progressive disorders
defining functional endpoints
Funding / incentives

21. Acknowledgements CMD families Carsten Bonnemann Kate Bushby
Ton DeGrauw
Prasad Devarajan
Andrew Hershey
Anne Rutkwoski
Brenda Wong
CMD families

22. References
Biomarkers Definition Working Group, NIH Clin Pharmacol Ther 2001;69:89-95
Rodland K. Systems biology and biomarker discovery. Disease Markers 2010;28:195-7
Sorani et al. Clinical and biological data integration for biomarker discovery Drug Discovery Today 2010, doi: /j.drudis
Fuller, H.R., et al., Valproate and Bone Loss: iTRAQ Proteomics Show that Valproate Reduces Collagens and Osteonectin in SMA Cells. J Proteome Res, 2010.
Hampel, H., et al., Biomarkers for Alzheimer's disease: academic, industry and regulatory perspectives. Nat Rev Drug Discov, (7): p
Rifai, N., M.A. Gillette, and S.A. Carr, Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol, (8): p
Wong, B., et al., Gene expression in blood of subjects with Duchenne muscular dystrophy. Neurogenetics, (2): p
Lit, L., et al., Corticosteroid effects on blood gene expression in Duchenne muscular dystrophy. Pharmacogenomics J, (6): p
van Lunteren, E., M. Moyer, and P. Leahy, Gene expression profiling of diaphragm muscle in alpha2-laminin (merosin)-deficient dy/dy dystrophic mice. Physiol Genomics, (1): p
Taniguchi, M., et al., Expression profiling of muscles from Fukuyama-type congenital muscular dystrophy and laminin-alpha 2 deficient congenital muscular dystrophy; is congenital muscular dystrophy a primary fibrotic disease? Biochem Biophys Res Commun, (2): p
Bovelenta et al. Gene expression and proteome profiles in Col6a1-/- mice, a model of Ullrich congenital muscular dystrophy. NMD 2009 EM.P.5.01; doi: /j.nmd
Liotta, L.A., M. Ferrari, and E. Petricoin, Clinical proteomics: written in blood. Nature, (6961): p. 905.