David Sterling

Large-scale serum protein biomarker discovery in Duchenne muscular dystrophy

Significance Duchenne muscular dystrophy (DMD) is a rare and devastating muscle disease caused by mutations in the X-linked DMD gene (which encodes the dystrophin protein). Serum biomarkers hold significant potential as objective phenotypic measures of DMD disease state, as well as potential measures of pharmacological effects of and response to therapeutic interventions. Here we describe a proteomics approach to determine serum levels of 1,125 proteins in 93 DMD patients and 45 controls. The study identified 44 biomarkers that differed significantly between patients and controls. These data are being made available to DMD researchers and clinicians to accelerate the search for new diagnostic, prognostic, and therapeutic approaches. Serum biomarkers in Duchenne muscular dystrophy (DMD) may provide deeper insights into disease pathogenesis, suggest new therapeutic approaches, serve as acute read-outs of drug effects, and be useful as surrogate outcome measures to predict later clinical benefit. In this study a large-scale biomarker discovery was performed on serum samples from patients with DMD and age-matched healthy volunteers using a modified aptamer-based proteomics technology. Levels of 1,125 proteins were quantified in serum samples from two independent DMD cohorts: cohort 1 (The Parent Project Muscular Dystrophy–Cincinnati Children’s Hospital Medical Center), 42 patients with DMD and 28 age-matched normal volunteers; and cohort 2 (The Cooperative International Neuromuscular Research Group, Duchenne Natural History Study), 51 patients with DMD and 17 age-matched normal volunteers. Forty-four proteins showed significant differences that were consistent in both cohorts when comparing DMD patients and healthy volunteers at a 1% false-discovery rate, a large number of significant protein changes for such a small study. These biomarkers can be classified by known cellular processes and by age-dependent changes in protein concentration. Our findings demonstrate both the utility of this unbiased biomarker discovery approach and suggest potential new diagnostic and therapeutic avenues for ameliorating the burden of DMD and, we hope, other rare and devastating diseases.

Association of growth differentiation factor 11/8, putative anti-ageing factor, with cardiovascular outcomes and overall mortality in humans: analysis of the Heart and Soul and HUNT3 cohorts

AIMS Growth differentiation factor 11 and/or its homologue growth differentiation factor 8 (GDF11/8) reverses age-related cardiac hypertrophy and vascular ageing in mice. We investigated whether GDF11/8 associates with cardiovascular outcomes, left ventricular hypertrophy (LVH), or age in humans. METHODS AND RESULTS We measured plasma GDF11/8 levels in 928 participants with stable ischaemic heart disease in the Heart and Soul study. We adjudicated heart failure hospitalization, stroke, myocardial infarction, death, and their composite endpoint. Left ventricular hypertrophy was evaluated by echocardiography. We used multivariable Cox proportional hazards models to compare rates of cardiovascular events and death across GDF11/8 quartiles and logistic regression models to evaluate the association between GDF11/8 and LVH. Four hundred and fifty participants (48.5%) experienced a cardiovascular event or death during 8.9 years of follow-up. The adjusted risk of the composite endpoint was lower in the highest compared with the lowest GDF11/8 quartile [hazard ratio (HR), 0.45; 95% confidence interval (CI), 0.33-0.60; P < 0.001]. We replicated this relationship of GDF11/8 to adverse events in 971 participants in the HUNT3 cohort (adjusted HR, 0.34; 95% CI, 0.23-0.51; P < 0.001). Left ventricular hypertrophy was present in 368 participants (39.7%) at baseline. Participants in the highest quartile of GDF11/8 were less likely to have LVH than those in the lowest quartile (adjusted OR, 0.55; 95% CI, 0.35-0.86; P = 0.009). GDF11/8 levels were lower in older individuals (P < 0.001). CONCLUSION In patients with stable ischaemic heart disease, higher GDF11/8 levels are associated with lower risk of cardiovascular events and death. Our findings suggest that GDF11/8 has similar cardioprotective properties in humans to those demonstrated in mice.

Highly Multiplexed Proteomic Analysis of Quantiferon Supernatants To Identify Biomarkers of Latent Tuberculosis Infection

ABSTRACT The tests for diagnosing latent tuberculosis infection (LTBI) are limited by a poor predictive value for identifying people at the highest risk for progressing to active tuberculosis (TB) and have various sensitivities and specificities in different populations. Identifying a more robust signature for LTBI is important for TB prevention and elimination. A pilot study was conducted with samples from immigrants to the United States that were screened for LTBI by the three commercially approved tests, namely, the tuberculin skin test (TST), the Quantiferon-TB Gold in-tube (QFT-GIT), and the T-SPOT.TB (T-SPOT). QFT-GIT supernatants from 13 people with concordant positive results and 26 people with concordant negative results were analyzed via the highly multiplexed SOMAscan proteomic assay. The proteins in the stimulated supernatants that distinguished LTBI from controls included interleukin-2 (IL-2), monocyte chemotactic protein 2 (MCP-2), interferon gamma inducible protein-10 (IP-10), interferon gamma (IFN-γ), tumor necrosis factor superfamily member 14 (TNFSF14, also known as LIGHT), monokine induced by gamma interferon (MIG), and granzyme B (P <0.00001). In addition, antigen stimulation increased the expression of heparin-binding EGF-like growth factor (HB-EGF) and activin AB in LTBI samples. In nil tubes, LIGHT was the most significant marker (P <0.0001) and was elevated in LTBI subjects. Other prominent markers in nonstimulated QFT-GIT supernatants were the complement-3 components C3b, iC3b, and C3d, which were upregulated in LTBI and markedly decreased upon stimulation. We found known and novel proteins that warrant further studies for developing improved tests for LTBI, for predicting progression to active disease, and for discriminating LTBI from active TB.

Discovery and Validation of a Six-Marker Serum Protein Signature for the Diagnosis of Active Pulmonary Tuberculosis

ABSTRACT New non-sputum biomarker tests for active tuberculosis (TB) diagnostics are of the highest priority for global TB control. We performed in-depth proteomic analysis using the 4,000-plex SOMAscan assay on 1,470 serum samples from seven countries where TB is endemic. All samples were from patients with symptoms and signs suggestive of active pulmonary TB that were systematically confirmed or ruled out for TB by culture and clinical follow-up. HIV coinfection was present in 34% of samples, and 25% were sputum smear negative. Serum protein biomarkers were identified by stability selection using L1-regularized logistic regression and by Kolmogorov-Smirnov (KS) statistics. A naive Bayes classifier using six host response markers (HR6 model), including SYWC, kallistatin, complement C9, gelsolin, testican-2, and aldolase C, performed well in a training set (area under the sensitivity-specificity curve [AUC] of 0.94) and in a blinded verification set (AUC of 0.92) to distinguish TB and non-TB samples. Differential expression was also highly significant (P < 10−20) for previously described TB markers, such as IP-10, LBP, FCG3B, and TSP4, and for many novel proteins not previously associated with TB. Proteins with the largest median fold changes were SAA (serum amyloid protein A), NPS-PLA2 (secreted phospholipase A2), and CA6 (carbonic anhydrase 6). Target product profiles (TPPs) for a non-sputum biomarker test to diagnose active TB for treatment initiation (TPP#1) and for a community-based triage or referral test (TPP#2) have been published by the WHO. With 90% sensitivity and 80% specificity, the HR6 model fell short of TPP#1 but reached TPP#2 performance criteria. In conclusion, we identified and validated a six-marker signature for active TB that warrants diagnostic development on a patient-near platform.

LOW LEVELS OF GROWTH DIFFERENTIATION FACTOR 11 AND HIGH LEVELS OF ITS INHIBITOR FOLLISTATIN-LIKE 3 ARE ASSOCIATED WITH ADVERSE CARDIOVASCULAR OUTCOMES IN HUMANS

In mice, age-related decline in Growth Differentiation Factor 11 (GDF 11) leads to cardiac hypertrophy and vascular dysfunction, but information is lacking about its role in humans. We thus investigated the associations of GDF11 and its inhibitor Follistatin-like 3 (FSTL3) with cardiovascular (CV)

Correction for De Groote et al., “Highly Multiplexed Proteomic Analysis of Quantiferon Supernatants To Identify Biomarkers of Latent Tuberculosis Infection”

Mary Ann De Groote,a,b Michael Higgins,c Thomas Hraha,a Kirsten Wall,a Michael L. Wilson,c David G. Sterling,a Nebojsa Janjic,a Randall Reves,d Urs A. Ochsner,a Robert Belknapd SomaLogic, Inc., Boulder, Colorado, USAa; Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USAb; Department of Pathology and Laboratory Services, Denver Health, Denver, Colorado, USAc; Denver Health and Hospital Authority and Denver Public Health, Denver, Colorado, USAd