Large-scale serum protein biomarker discovery in Duchenne muscular dystrophy Yetrib Hathouta, Edward Brodyb, Paula R. Clemensc,d, Linda Cripee, Robert Kirk DeLisleb, Pat Furlongf, Heather Gordish- Dressmana, Lauren Hachea, Erik Henricsong, Eric P. Hoffmana, Yvonne Monique Kobayashih, Angela Lortsi, Jean K. Mahj, Craig McDonaldg, Bob Mehlerb, Sally Nelsonk, Malti Nikradb, Britta Singerb, Fintan Steeleb, David Sterlingb, H. Lee Sweeneyl, Steve Williamsb, and Larry Goldb,1 aResearch Center for Genetic Medicine, Children’s National Medical Center, Washington, DC 20012; bSomaLogic, Inc., Boulder, CO 80301; cNeurology Service, Department of Veteran Affairs Medical Center, Pittsburgh, PA 15240; dUniversity of Pittsburgh, Pittsburgh, PA 15213; eThe Heart Center, Nationwide Children’s Hospital, The Ohio State University, Columbus, OH 15213; fParent Project Muscular Dystrophy, Hackensack, NJ 07601; gDepartment of Physical Medicine and Rehabilitation, University of California Davis School of Medicine, Davis, CA 95618; hDepartment of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202; iThe Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; jDepartment of Pediatrics, University of Calgary, Alberta Children’s Hospital, Calgary, AB, Canada T3B 6A8; kDivision of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Aurora, CO 80045; and lDepartment of Pharmacology & Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610 Contributed by Larry Gold, April 29, 2015 (sent for review March 6, 2015; reviewed by Allan Jacobson) Serum biomarkers in Duchenne muscular dystrophy (DMD) may minidystrophins, up-regulation of utrophin to compensate for provide deeper insights into disease pathogenesis, suggest new the missing dystrophin, and many others (17). Pharmacological therapeutic approaches, serve as acute read-outs of drug effects, strategies in development include dissociative steroid drugs, and be useful as surrogate outcome measures to predict later clin- which offer the potential of greater efficacy and lesser side ef- ical benefit. In this study a large-scale biomarker discovery was fects (18), other anti-inflammatory therapies, and effectors of performed on serum samples from patients with DMD and age- signaling pathways (19). The current primary clinical endpoint matched healthy volunteers using a modified aptamer-based pro- used for determining efficacy in the majority of these therapeutic teomics technology. Levels of 1,125 proteins were quantified in approaches for ambulatory boys with DMD is the “six-minute serum samples from two independent DMD cohorts: cohort 1 (The ” – ’ walk test (20, 21), although it is not ideal (22). Parent Project Muscular Dystrophy Cincinnati Children s Hospital Blood provides a circulating protein representation of all body Medical Center), 42 patients with DMD and 28 age-matched normal tissue in both normal and pathological conditions, and serum volunteers; and cohort 2 (The Cooperative International Neuromus- proteins are emerging as useful biomarkers for diagnosis and cular Research Group, Duchenne Natural History Study), 51 patients prognosis of a growing number of diseases (23, 24). Mass spec- with DMD and 17 age-matched normal volunteers. Forty-four pro- teins showed significant differences that were consistent in both trometry (MS)-based proteomic screens recently have proved cohorts when comparing DMD patients and healthy volunteers at successful at de novo biomarker identification in DMD (25). a 1% false-discovery rate, a large number of significant protein However, verification and validation of MS-discovered serum changes for such a small study. These biomarkers can be classified biomarkers remain challenging (24). Other approaches, such as by known cellular processes and by age-dependent changes in pro- multiplexed antibody or aptamer-based assays, are being con- tein concentration. Our findings demonstrate both the utility of this sidered for proteome screens because of their potential for unbiased biomarker discovery approach and suggest potential new higher throughput and better sensitivity, which may help overcome diagnostic and therapeutic avenues for ameliorating the burden of the validation challenges of identified biomarkers. For example, a DMD and, we hope, other rare and devastating diseases. Significance proteomics | muscular dystrophy | biomarkers | SOMAscan | SOMAmer Duchenne muscular dystrophy (DMD) is a rare and devastating here is an urgent need for a reliable surrogate biomarker or muscle disease caused by mutations in the X-linked DMD gene Tset of biomarkers for Duchenne muscular dystrophy (DMD), (which encodes the dystrophin protein). Serum biomarkers hold ideally based on readily accessible and measurable molecules (1). significant potential as objective phenotypic measures of DMD DMD is a severe form of myopathy with an incidence of about 1 disease state, as well as potential measures of pharmacological in 3,600–9,337 boys worldwide (2, 3), and is a result of different effects of and response to therapeutic interventions. Here we types of mutations in the X-linked DMD gene that abolish the describe a proteomics approach to determine serum levels of expression and biological activity of dystrophin, an essential pro- 1,125 proteins in 93 DMD patients and 45 controls. The study tein for muscle-fiber plasma membrane integrity and myofiber identified 44 biomarkers that differed significantly between function (4, 5). Clinically, the disease is characterized by pro- patients and controls. These data are being made available to gressive muscle wasting, leading to loss of ambulation by 8–15 y DMD researchers and clinicians to accelerate the search for new of age and early death from complications from respiratory, diagnostic, prognostic, and therapeutic approaches. orthopedic, and cardiac problems (2, 6). Several current drug-development programs are focused on Author contributions: Y.H., P.R.C., L.C., P.F., L.H., E.H., E.P.H., C.M., S.N., H.L.S., and L.G. slowing or preventing the progressive muscle loss in DMD either designed research; L.C., H.G.-D., E.H., A.L., and J.K.M. performed research; Y.H., E.B., R.K.D., Y.M.K., B.M., S.N., M.N., B.S., F.S., D.S., H.L.S., S.W., and L.G. analyzed data; and Y.H., E.B., in conjunction with the standard of care treatment or as stand- R.K.D., Y.M.K., S.N., F.S., D.S., H.L.S., and L.G. wrote the paper. alone therapies. Standard of care is currently chronic high-dose Reviewers included: A.J., University of Massachusetts Medical School. – glucocorticoids, which are able to prolong ambulation by 3 4y Conflict of interest statement: L.G. is the founder and a stakeholder in SomaLogic, Inc; (7, 8) and slow disease progression, but are associated with a E.B., R.K.D., B.M., M.N., B.S., F.S., D.S., and S.W. are employees and stakeholders in Soma- significant array of side effects (2, 6, 9, 10). Promising thera- Logic, Inc.; Y.M.K. has been affiliated with the Eli Lilly and Co. since October 2012. peutic approaches for DMD include restoring expression of the Freely available online through the PNAS open access option. – 1 dystrophin gene via exon-skipping strategies (11 13), viral-based To whom correspondence should be addressed. Email: [email protected]. SCIENCES gene therapies (14, 15), and nonsense suppression/read-through This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. strategies (16). Other genetic approaches include delivering 1073/pnas.1507719112/-/DCSupplemental. APPLIED BIOLOGICAL www.pnas.org/cgi/doi/10.1073/pnas.1507719112 PNAS | June 9, 2015 | vol. 112 | no. 23 | 7153–7158 Downloaded by guest on September 29, 2021 recent study using an antibody-based array against 384 target results. Our standard quality-control protocols detected no sig- proteins identified 11 protein biomarkers of disease across dif- nificant difference in the samples from the two cohorts. ferent muscular dystrophies from patient samples gathered Serum samples were tested using the SOMAscan protein from four different clinical sites (26). In addition, a modified biomarker discovery assay (SomaLogic), which detects 1,125 aptamer-based technology (the SOMAscan assay) is emerging as proteins simultaneously using 65 μL of serum. At a 1% false- another highly sensitive and multiplexed assay for biomarker discovery rate (FDR) (Materials and Methods), based on SOMAscan discovery and validation (27–29). Based on novel reagents (Slow assay data from a total of 93 DMD patients and 45 age-matched Off-rate Modified Aptamers, or SOMAmer reagents) that rec- controls from the two cohorts, we identified 44 proteins that ognize specific conformational epitopes of native 3D proteins consistently differed in the serum in both cohorts when comparing with high specificity and high sensitivity (30–32), the SOMAscan DMD patients vs. controls. The UniProt names and a measure assay measures levels of 1,125 analytes in only 65 μL of serum of differential expression [the signed Kolmogorov–Smirnov (KS) over a wide dynamic range (>8 logs of concentration). Because distance] for these 44 proteins in each cohort are shown in Table 1, the SOMAscan assay relies on the availability of the protein along with an indicator of each protein’s known enrichment in epitopes (i.e., the epitopes are not blocked by other protein muscle tissue. The entire 1,125 protein SOMAscan assay results binding, posttranslational modifications, and so forth),