RESUMO
Duchenne muscular dystrophy (DMD) is a progressive muscle disease involving complex skeletal muscle pathogenesis. The pathogenesis is triggered by sarcolemma instability due to the lack of dystrophin protein expression, leading to Ca2+ influx, muscle fiber apoptosis, inflammation, muscle necrosis, and fibrosis. Our lab recently used two high-throughput multiplexing techniques (e.g., SomaScan® aptamer assay and tandem mass tag-(TMT) approach) and identified a series of serum protein biomarkers tied to different pathobiochemical pathways. In this study, we focused on validating the circulating levels of three proinflammatory chemokines (CCL2, CXCL10, and CCL18) that are believed to be involved in an early stage of muscle pathogenesis. We used highly specific and reproducible MSD ELISA assays and examined the association of these chemokines with DMD pathogenesis, age, disease severity, and response to glucocorticoid treatment. As expected, we confirmed that these three chemokines were significantly elevated in serum and muscle samples of DMD patients relative to age-matched healthy controls (p-value < 0.05, CCL18 was not significantly altered in muscle samples). These three chemokines were not significantly elevated in Becker muscular dystrophy (BMD) patients, a milder form of dystrophinopathy, when compared in a one-way ANOVA to a control group but remained significantly elevated in the age-matched DMD group (p < 0.05). CCL2 and CCL18 but not CXCL10 declined with age in DMD patients, whereas all three chemokines remained unchanged with age in BMD and controls. Only CCL2 showed significant association with time to climb four steps in the DMD group (r = 0.48, p = 0.038) and neared significant association with patients' reported outcome in the BMD group (r = 0.39, p = 0.058). Furthermore, CCL2 was found to be elevated in a serum of the mdx mouse model of DMD, relative to wild-type mouse model. This study suggests that CCL2 might be a suitable candidate biomarker for follow-up studies to demonstrate its physiological significance and clinical utility in DMD.
RESUMO
Blood-accessible molecular biomarkers are becoming highly attractive tools to assess disease progression and response to therapies in Duchenne muscular dystrophy (DMD) especially in very young patients for whom other outcome measures remain subjective and challenging. In this study, we have standardized a highly specific and reproducible multiplexing mass spectrometry method using the tandem mass tag (TMT) strategy in combination with depletion of abundant proteins from serum and high-pH reversed-phase peptide fractionation. Differential proteome profiling of 4 year-old DMD boys (n = 9) and age-matched healthy controls (n = 9) identified 38 elevated and 50 decreased serum proteins (adjusted P < 0.05, FDR <0.05) in the DMD group relative to the healthy control group. As expected, we confirmed previously reported biomarkers but also identified novel biomarkers. These included novel muscle injury-associated biomarkers such as telethonin, smoothelin-like protein 1, cofilin-1, and plectin, additional muscle-specific enzymes such as UTP-glucose-1-phosphate uridylyltransferase, aspartate aminotransferase, pyruvate kinase PKM, lactotransferrin, tissue alpha-l-fucosidase, pantetheinase, and ficolin-1, and some pro-inflammatory and cell adhesion-associated biomarkers such as leukosialin, macrophage receptor MARCO, vitronectin, galectin-3-binding protein, and ProSAAS. The workflow including serum depletion, sample processing, and mass spectrometry analysis was found to be reproducible and stable over time with CV < 20%. Furthermore, the method was found to be superior in terms of specificity compared to other multiplexing affinity-based methods. These findings demonstrate the specificity and reliability of TMT-based mass spectrometry methods in detection and identification of serum biomarkers in presymptomatic young DMD patients.
