Protein Biomarker Discovery in Duchenne Muscular Dystrophy
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1 Protein Biomarker Discovery in Duchenne Muscular Dystrophy Sally K. Nelson, Ph.D. June 28 th, , SomaLogic, Inc.
2 WHAT WE DO 2013, SomaLogic, Inc. 2
3 Why proteins? Proteins respond to changes in health status and drug administra2on Readily accessed via the circula2on Measuring mul2ple proteins simultaneously can capture the contribu2on of different and some2mes unexpected biological processes Discovery has been limited by technology un*l now 2013, SomaLogic, Inc. 3
4 What we do Using our proprietary SOMAscan technology, we measure over a thousand proteins in a single assay Across 8 logs of concentration In as little as 65 µl of biological sample In multiple sample types (blood, CSF, synovial fluid, cell or tissue extracts, etc.) In hundreds of samples a day Accurately, reproducibly, and cost-effectively 2013, SomaLogic, Inc. 4
5 OUR TECHNOLOGY 2013, SomaLogic, Inc. 5
6 Bringing SOMAmers into the multiplex omics era A new class of modified DNA- based affinity reagents is the key: Slow Off- rate Modified Aptamers SOMAmer reagents can quan2fy 1129 (SOMAscan) proteins down to sub- pm concentra2on in volumes as low as 65 µl from biological fluids and cells The complete proteome is our goal L.Gold et.al. (2010) PLOS ONE 5,e , SomaLogic, Inc. 6
7 Structural studies reveal basis for SOMAmer reagent specificity Modified bases (violet) interacting with target protein 2013, SomaLogic, Inc. 7
8 Therapeu9c Areas Cellular Processes Current collection: 1129 proteins measured Aging Cancer Cardiovascular Metabolic/Endocrine Inflamma2on Neurological Pulmonary Renal Cri2cal Care Signal transduc2on Stress response Proteolysis Phosphoryla2on Intracellular transport Immune Process Cell differen2a2on Cell Adhesion # of SOMAmers Protease Inhibitors 5% Growth Factors 13% Proteases 17% Hormones 3% Intracellular 25 % Extracellular Domains 28 % Kinases 20% Receptors 21% Cytokines 20% Secreted 47 % Structural Proteins 1% SOMAmer reagents cover a broad array of proteins associated with cellular processes and disease pathophysiology 2013, SomaLogic, Inc. 8
9 SOMAscan assay offers empirical biomarker discovery in <5 weeks or + 100s of Biological Samples 1129 SOMAmer Reagents Automated SOMAscan Sensitivity AUC = 0.91 AUC = Specificity Training Verification Biomarker Discovery * * * * * Data Reduction Primary Data 2013, SomaLogic, Inc. 9
10 A pilot study to discover biomarkers of DMD using SOMAscan Cincinnati Children s Hospital Medical Center (CCHMC) Patient recruitment, sample collection The Parent Project Muscular Dystrophy (PPMD) Funding and spiritual guidance 2013, SomaLogic, Inc. 10
11 We addressed the following questions: 1. Are there substantial differences in blood protein concentrations (the protein signature) in patients with DMD compared with healthy controls? 2. Do we see proteins that change with patient age? 3. Do we see biology that leads us to new ideas about disease progression or intervention for DMD patients? 4. Do steroids affect proteins involved in DMD? 2013, SomaLogic, Inc. 11
12 Study population Groups Number of Subjects(n) DMD + Steroids 28 DMD no Steroids 14 Controls 28 Total 70 Cases were subjects with DMD and no other diagnosis Ages ranged from 2 to 27 years 50% between 9 and 11 Controls were siblings or healthy children from DMD community 2013, SomaLogic, Inc. 12
13 126 proteins are significantly increased in DMD This is roughly 11% of measured proteins DMD Total degree of separation (green) exceeding the false discovery rate (beige) for each of the top proteins Example of the shift in the population concentration for a typical protein (red = DMD, blue = control) 2013, SomaLogic, Inc. 13
14 101 proteins are significantly decreased in DMD This is roughly 8% of measured proteins DMD Total degree of separation (green) exceeding the false discovery rate (beige) for each of the top proteins Example of the shift in the population concentration for a typical protein (red = DMD, blue = control) 2013, SomaLogic, Inc. 14
15 Some proteins (18) change with age in DMD but do not change in the controls Almost no proteins change with age in controls but not DMD 47 Proteins change with age in DMD and in Controls 18 Proteins change with age in DMD but not Controls 2013, SomaLogic, Inc. 15
16 The 18 DMD age-specific proteins are elevated early and decrease with age Serum creatine kinase level reaches a peak between 3 and 5 years old and then reduces with increasing age in children with DMD with clinical progression and age, with a yearly average rate of decline of 8.7%. Sun SC, Peng YS, He JB. Zhongguo Dang Dai Er Ke Za Zhi Feb;10(1):35-7. PMID: , SomaLogic, Inc. 16
17 Finding biologic processes in DMD: Clusters in agerelated proteins Re-order 137 age dependent proteins in DMD and use same ordering in Controls More proteins move together (and in opposite directions) in DMD than in Controls 2013, SomaLogic, Inc. 17
18 Dissecting the steroid effect 10 Proteins affected by steroids Proteins that are also DMD markers Proteins not involved in DMD DMD proteins moving towards normal DMD proteins moving away from normal 2 Evidence of steroid benefit False positive or steroid harm Proteins of Pharmacology , SomaLogic, Inc. 18
19 Conclusions from the early work Many novel proteins were affected by DMD. They represent known proteins (muscle cell death, inflammation) and the unexpected kind (developmental proteins, metabolic, and mitochondrial proteins). Age-related analyses showed the paradoxical effect of many proteins getting more normal with age. Cluster analysis showed patterns of change that were internally consistent and relate to known and to new physiology. A clear effect of steroids was shown, which points to a beneficial effect of steroids on some DMD proteins. 2013, SomaLogic, Inc. 19
20 Acknowledgements Patricia Furlong and PPMD Linda Cripe and CCMHC SomaLogic Larry Gold-CEO David Sterling-Bioinformatics Stephen Williams-CMO 2013, SomaLogic, Inc. 20
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