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Tandem Mass Tag-Based Serum Proteome Profiling for Biomarker Discovery in Young Duchenne Muscular Dystrophy Boys.

ABSTRACT: 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.

SUBMITTER: Alayi TD

PROVIDER: S-EPMC7581259 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Tandem Mass Tag-Based Serum Proteome Profiling for Biomarker Discovery in Young Duchenne Muscular Dystrophy Boys.

Alayi Tchilabalo D TD Tawalbeh Shefa M SM Ogundele Michael M Smith Holly R HR Samsel Alison M AM Barbieri Marissa L ML Hathout Yetrib Y

ACS omega 20201006 41

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 reverse ...[more]

PMID: 33110978

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Project description:Background and objectivesDuchenne muscular dystrophy (DMD) is a paediatric neuromuscular disorder caused by mutations in the dystrophin gene. Geneotype-phenotype associations have been examined in glucocorticoid treated boys, but there are few data on the young glucocorticoid-naïve DMD population. A sample of young glucocorticoid-naïve DMD boys is described and genotype-phenotype associations are investigated.MethodsScreening and baseline data were collected for all the participants in the Finding the Optimum Corticosteroid Regime for Duchenne Muscular Dystrophy (FOR-DMD)study, an international, multi-centre, randomized, double-blind, clinical trial comparing three glucocorticoid regimens in glucocorticoid-naïve, genetically confirmed boys with DMD between 4 and <8 years of age.ResultsOne hundred and ninety-six boys were recruited. The meanage at randomization (+ standard deviation) was 5.8+ 1.0 years. The predominant mutation type was out of frame deletions 67.4%, (130/193) of which 68.5% (89/130) were amenable to exon skipping. The most frequent mutations were deletions amenable to exon 51 skipping 13.0% (25/193). Stop codon mutations accounted for 10.4% (20/193).The mean age at first parental concerns was 29.8 + 18.7 months, the mean age at genetic diagnosis was 53.9 + 21.9 months and the mean diagnostic delay was 25.9 + 18.2 months. The mean diagnostic delay for boys diagnosed following an incidental finding of isolated hyperCKemia (n=19) was 6.4 + 7.4 months. The mean ages at independent walking and talking in sentences were 17.1 + 4.2 and 29.0 + 10.7 months, respectively. Median height percentiles were below the 25th percentile regardless of age group. No genotype-phenotype associations were identified expect for boys with an exon 8 skippable deletions who had better performance on time to walk/run 10 meters (p=0.02)compared to boys with deletions not amenable to skipping.DiscussionThis study describes clinical and genetic characteristics of a sample of young glucocorticoid-naïve boys with DMD. A low threshold for CK testing can lead to an earlier diagnosis. Motor and speech delay were common presenting symptoms.The effects of low, pre-treatment height on growth and adults height requires further study. These findings may promote earlier recognition of DMD and inform study design for future clinical trials.

Project description:BackgroundClinical management of boys with Duchenne muscular dystrophy (DMD) relies on in-depth understanding of cardiac involvement, but right ventricular (RV) structural and functional remodeling remains understudied.PurposeTo evaluate several analysis methods and identify the most reliable one to measure RV pre- and postcontrast T1 (RV-T1) and to characterize myocardial remodeling in the RV of boys with DMD.Study typeProspective.PopulationBoys with DMD (N = 27) and age-/sex-matched healthy controls (N = 17) from two sites.Field strength/sequence3.0 T using balanced steady state free precession, motion-corrected phase sensitive inversion recovery and modified Look-Locker inversion recovery sequences.AssessmentBiventricular mass (Mi), end-diastolic volume (EDVi) and ejection fraction (EF) assessment, tricuspid annular excursion (TAE), late gadolinium enhancement (LGE), pre- and postcontrast myocardial T1 maps. The RV-T1 reliability was assessed by three observers in four different RV regions of interest (ROI) using intraclass correlation (ICC).Statistical testsThe Wilcoxon rank sum test was used to compare RV-T1 differences between DMD boys with negative LGE(-) or positive LGE(+) and healthy controls. Additionally, correlation of precontrast RV-T1 with functional measures was performed. A P-value <0.05 was considered statistically significant.ResultsA 1-pixel thick RV circumferential ROI proved most reliable (ICC > 0.91) for assessing RV-T1. Precontrast RV-T1 was significantly higher in boys with DMD compared to controls. Both LGE(-) and LGE(+) boys had significantly elevated precontrast RV-T1 compared to controls (1543 [1489-1597] msec and 1550 [1402-1699] msec vs. 1436 [1399-1473] msec, respectively). Compared to healthy controls, boys with DMD had preserved RVEF (51.8 [9.9]% vs. 54.2 [7.2]%, P = 0.31) and significantly reduced RVMi (29.8 [9.7] g vs. 48.0 [15.7] g), RVEDVi (69.8 [29.7] mL/m2 vs. 89.1 [21.9] mL/m2 ), and TAE (22.0 [3.2] cm vs. 26.0 [4.7] cm). Significant correlations were found between precontrast RV-T1 and RVEF (β = -0.48%/msec) and between LV-T1 and LVEF (β = -0.51%/msec).Data conclusionPrecontrast RV-T1 is elevated in boys with DMD compared to healthy controls and is negatively correlated with RVEF.Level of evidence1 TECHNICAL EFFICACY: Stage 2.

Project description:BackgroundDuchenne muscular dystrophy (DMD) exhibits substantial variability in rates of disease progression and response to treatment. This has hindered treatment development and complicated interpretation of drug effects in clinical trials.ObjectiveWe hypothesized that a multivariate combination of early-age clinical outcome measurements can explain differential disease progression.MethodsData on boys with DMD (ages 4-<10 years), both treated with steroidal anti-inflammatories and untreated, were obtained from CINRG Duchenne Natural History Study (n = 209) and vamorolone VBP15-002/003/LTE (n = 46) studies. Velocities from three timed function tests (TFTs; stand from supine, run/walk 10 meters, and climb 4 stairs) were simultaneously modeled in a longitudinal latent class analysis.ResultsThree classes of differentially progressing early age DMD motor trajectories were identified. Quicker decline/progression was associated with lower baseline TFT velocities, earlier loss of ability to finish a TFT, and lower predicted velocities. Earlier substantial steroid exposure was associated with greater TFT velocities while the moderate progression class was observed to have the largest difference in performance between boys treated early with steroids vs. not. Sample size calculations with the class showing the largest treatment response showed a large reduction in required sample size as compared to using summaries from all participants. Gene mutations were also investigated in post-hoc analyses, with mutations near the beginning of the DMD gene (Dp427 absent and Dp140/Dp71 present) found to be enriched in the slowest progressing class.ConclusionsThis study provides insight into the variation in DMD progression through a latent class analysis. Our findings show class-related trajectories of motor outcomes and pharmacological response to corticosteroids, and suggest that enrichment strategies and/or subgroup analyses could be considered further in design of therapeutic interventions in DMD.

Dataset Information

Tandem Mass Tag-Based Serum Proteome Profiling for Biomarker Discovery in Young Duchenne Muscular Dystrophy Boys.

Publications

Tandem Mass Tag-Based Serum Proteome Profiling for Biomarker Discovery in Young Duchenne Muscular Dystrophy Boys.

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