Title-molecular diagnostics of dystrophinopathies in Sri Lanka towards phenotype predictions: an insight from a South Asian resource limited setting.

BACKGROUND: The phenotype of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients is determined by the type of DMD gene variation, its location, effect on reading frame, and its size. The primary objective of this investigation was to determine the frequency and distribution of DMD gene variants (deletions/duplications) in Sri Lanka through the utilization of a combined approach involving multiplex polymerase chain reaction (mPCR) followed by Multiplex Ligation Dependent Probe Amplification (MLPA) and compare to the international literature. The current consensus is that MLPA is a labor efficient yet expensive technique for identifying deletions and duplications in the DMD gene. METHODOLOGY: Genetic analysis was performed in a cohort of 236 clinically suspected pediatric and adult myopathy patients in Sri Lanka, using mPCR and MLPA. A comparative analysis was conducted between our findings and literature data. RESULTS: In the entire patient cohort (n = 236), mPCR solely was able to identify deletions in the DMD gene in 131/236 patients (DMD-120, BMD-11). In the same cohort, MLPA confirmed deletions in 149/236 patients [DMD-138, BMD -11]. These findings suggest that mPCR has a detection rate of 95% (131/138) among all patients who received a diagnosis. The distal and proximal deletion hotspots for DMD were exons 45-55 and 6-15. Exon 45-60 identified as a novel in-frame variation hotspot. Exon 45-59 was a hotspot for BMD deletions. Comparisons with the international literature show significant variations observed in deletion and duplication frequencies in DMD gene across different populations. CONCLUSION: DMD gene deletions and duplications are concentrated in exons 45-55 and 2-20 respectively, which match global variation hotspots. Disparities in deletion and duplication frequencies were observed when comparing our data to other Asian and Western populations. Identified a 95% deletion detection rate for mPCR, making it a viable initial molecular diagnostic approach for low-resource countries where MLPA could be used to evaluate negative mPCR cases and cases with ambiguous mutation borders. Our findings may have important implications in the early identification of DMD with limited resources in Sri Lanka and to develop tailored molecular diagnostic algorithms that are regional and population specific and easily implemented in resource limited settings.

Toxoplasma membrane inositol phospholipid binding protein TgREMIND is essential for secretory organelle function and host infection.

Apicomplexan parasites possess specialized secretory organelles called rhoptries, micronemes, and dense granules that play a vital role in host infection. In this study, we demonstrate that TgREMIND, a protein found in Toxoplasma gondii, is necessary for the biogenesis of rhoptries and dense granules. TgREMIND contains a Fes-CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, which binds to membrane phospholipids, as well as a novel uncharacterized domain that we have named REMIND (regulator of membrane-interacting domain). Both the F-BAR domain and the REMIND are crucial for TgREMIND functions. When TgREMIND is depleted, there is a significant decrease in the abundance of dense granules and abnormal transparency of rhoptries, leading to a reduction in protein secretion from these organelles. The absence of TgREMIND inhibits host invasion and parasite dissemination, demonstrating that TgREMIND is essential for the proper function of critical secretory organelles required for successful infection by Toxoplasma.

In silico and experimental analysis of the repeated domains in BvaP, a protein important for GBS vaginal colonization.

Streptococcus agalactiae (group B strep, GBS) infections in neonates are often fatal and strongly associated with maternal GBS vaginal colonization. Previously, we highlighted the importance of a formerly uncharacterized protein, BvaP, in GBS vaginal colonization. BvaP is highly conserved across GBS and is made up of repeated domains, with a variable number of repeats between strains. Here, we evaluate the prevalence of BvaP repeated domains and their relevance in phenotypes previously associated with vaginal colonization. Using in silico analysis, we found that the number of repeats in the BvaP protein does not generally appear to be associated with serotype, isolation site, or host. Using BvaP truncations in GBS strain A909, we determined that a smaller number of repeats was correlated with decreased bacterial chain length, but adherence to vaginal epithelial cells was complemented using BvaP containing one, two, three, or five repeats. Future research will be geared toward understanding the host immune response to BvaP in vivo and whether vaginal carriage or host response is dependent on the BvaP repeated domains.

Duchenne Muscular Dystrophy from Brain to Muscle: The Role of Brain Dystrophin Isoforms in Motor Functions.

Brain function and its effect on motor performance in Duchenne muscular dystrophy (DMD) is an emerging concept. The present study explored how cumulative dystrophin isoform loss, age, and a corticosteroid treatment affect DMD motor outcomes. A total of 133 genetically confirmed DMD patients from Sri Lanka were divided into two groups based on whether their shorter dystrophin isoforms (Dp140, Dp116, and Dp71) were affected: Group 1, containing patients with Dp140, Dp116, and Dp71 affected (n = 98), and Group 2, containing unaffected patients (n = 35). A subset of 52 patients (Group 1, n = 38; Group 2, n = 14) was followed for up to three follow-ups performed in an average of 28-month intervals. The effect of the cumulative loss of shorter dystrophin isoforms on the natural history of DMD was analyzed. A total of 74/133 (56%) patients encountered developmental delays, with 66/74 (89%) being in Group 1 and 8/74 (11%) being in Group 2 (p < 0.001). Motor developmental delays were predominant. The hip and knee muscular strength, according to the Medical Research Council (MRC) scale and the North Star Ambulatory Assessment (NSAA) activities, "standing on one leg R", "standing on one leg L", and "walk", declined rapidly in Group 1 (p < 0.001 In the follow-up analysis, Group 1 patients became wheelchair-bound at a younger age than those of Group 2 (p = 0.004). DMD motor dysfunction is linked to DMD mutations that affect shorter dystrophin isoforms. When stratifying individuals for clinical trials, considering the DMD mutation site and its impact on a shorter dystrophin isoform is crucial.

Integrated genomic, proteomic and cognitive assessment in Duchenne Muscular Dystrophy suggest astrocyte centric pathology.

Introduction: Documented Duchenne Muscular Dystrophy (DMD) biomarkers are confined to Caucasians and are poor indicators of cognitive difficulties and neuropsychological alterations. Materials and methods: This study correlates serum protein signatures with cognitive performance in DMD patients of South Asian origin. Study included 25 DMD patients aged 6-16 years. Cognitive profiles were assessed by Wechsler Intelligence Scale for Children. Serum proteome profiling of 1317 proteins was performed in eight DMD patients and eight age-matched healthy volunteers. Results: Among the several novel observations we report, better cognitive performance in DMD was associated with increased serum levels of MMP9 and FN1 but decreased Siglec-3, C4b, and C3b. Worse cognitive performance was associated with increased serum levels of LDH-H1 and PDGF-BB but reduced GDF-11, MMP12, TPSB2, and G1B. Secondly, better cognitive performance in Processing Speed (PSI) and Perceptual Reasoning (PRI) domains was associated with intact Dp116, Dp140, and Dp71 dystrophin isoforms while better performance in Verbal Comprehension (VCI) and Working Memory (WMI) domains was associated with intact Dp116 and Dp140 isoforms. Finally, functional pathways shared with Alzheimer's Disease (AD) point towards an astrocyte-centric model for DMD. Conclusion: Astrocytic dysfunction leading to synaptic dysfunction reported previously in AD may be a common pathogenic mechanism underlying both AD and DMD, linking protein alterations to cognitive impairment. This new insight may pave the path towards novel therapeutic approaches targeting reactive astrocytes.

Gene therapy for selected neuromuscular and trinucleotide repeat disorders - An insight to subsume South Asia for multicenter clinical trials.

Background: In this article, the authors discuss how they utilized the genetic mutation data in Sri Lankan Duchenne muscular dystrophy (DMD), Spinal muscular atrophy (SMA), Spinocerebellar ataxia (SCA) and Huntington's disease (HD) patients and compare the available literature from South Asian countries to identifying potential candidates for available gene therapy for DMD, SMA, SCA and HD patients. Methods: Rare disease patients (n = 623) with the characteristic clinical findings suspected of HD, SCA, SMA and Muscular Dystrophy were genetically confirmed using Multiplex Ligation Dependent Probe Amplification (MLPA), and single plex PCR. A survey was conducted in the "Wiley database on Gene Therapy Trials Worldwide" to identify DMD, SMA, SCA, and HD gene therapy clinical trials performed worldwide up to April 2021. In order to identify candidates for gene therapy in other neighboring countries we compared our findings with available literature from India and Pakistan which has utilized the same molecular diagnostic protocol to our study. Results: From the overall cohort of 623 rare disease patients with the characteristic clinical findings suspected of HD, SCA, SMA and Muscular Dystrophy, n = 343 (55%) [Muscular Dystrophy- 65%; (DMD-139, Becker Muscular Dystrophy -BMD-11), SCA type 1-3-53% (SCA1-61,SCA2- 23, SCA3- 39), HD- 52% (45) and SMA- 34% (22)] patients were positive for molecular diagnostics by MLPA and single plex PCR. A total of 147 patients in Sri Lanka amenable to available gene therapy; [DMD-83, SMA-15 and HD-49] were identified. A comparison of Sri Lankan finding with available literature from India and Pakistan identified a total of 1257 patients [DMD-1076, SMA- 57, and HD-124] from these three South Asian Countries as amenable for existing gene therapy trials. DMD, SMA, and HD gene therapy clinical trials (113 studies) performed worldwide up to April 2021 were concentrated mostly (99%) in High Income Countries (HIC) and Upper Middle-Income Countries (UMIC). However, studies on the potential use of anti-sense oligonucleotides (ASO) for treatment of SCAs have yet to reach clinical trials. Conclusion: Most genetic therapies for neurodegenerative and neuromuscular disorders have been evaluated for efficacy primarily in Western populations. No multicenter gene therapy clinical trial sites for DMD, SMA and HD in the South Asian region, leading to lack of knowledge on the safety and efficacy of such personalized therapies in other populations, including South Asians. By fostering collaboration between researchers, clinicians, patient advocacy groups, government and industry in gene therapy initiatives for the inherited-diseases community in the developing world would link the Global North and Global South and breathe life into the motto "Together we can make a difference".

Urine proteomics by mass spectrometry identifies proteins involved in key pathogenic pathways in patients with juvenile dermatomyositis.

OBJECTIVES: To identify and validate biomarkers in JDM patients using a multiplexing tandem mass tag urine proteome profiling approach. METHODS: First morning void urine samples were collected from JDM patients (n = 20) and healthy control subjects (n = 21) and processed for analysis using a standardized liquid chromatography-tandem mass spectrometry approach. Biomarkers with significantly altered levels were correlated with clinical measures of myositis disease activity and damage. A subset of candidate biomarkers was validated using commercially available ELISA kits. RESULTS: In total, 2348 proteins were detected in the samples, with 275 proteins quantified across all samples. Among the differentially altered proteins, cathepsin D and galectin-3 binding protein were significantly increased in the urine of JDM patients (adjusted P < 0.05), supporting previous findings in myositis patients. These two candidate biomarkers were confirmed with ELISAs. Cathepsin D positively correlated with Myositis Damage Index (r = 0.57, P < 0.05) and negatively correlated with the Childhood Myositis Assessment Scale (r = -0.54, P < 0.05). We also identified novel JDM candidate biomarkers involved with key features of myositis, including extracellular matrix remodelling proteins. CONCLUSION: This study confirmed the presence of several proteins in the urine of JDM patients that were previously found to be elevated in the blood of myositis patients and identified novel candidate biomarkers that require validation. These results support the use of urine as a source for biomarker development in JDM.

Evaluation of an ester-linked immunosuppressive payload: A case study in understanding the stability and cleavability of ester-containing ADC linkers.

In spite of their value in prodrug applications, the use of esters in antibody-drug-conjugate (ADC) payloads and linkers has generally been avoided due to the ubiquitous and promiscuous nature of human esterases. ADCs generally have a long circulating half life (3-7 days) that makes them susceptible to esterase-mediated metabolism. Moreover, it is largely unclear whether lysosomal and cytosolic esterases cleave ester-containing linkers upon ADC internalization. Due to our interest in the targeted delivery of immune-modulators, our team has recently prepared a series of ester-linked dexamethasone ADCs. Herein, we report our studies of the functional activity of these ADCs, with a particular focus on their catabolism in various biological milieu. We found that esters are selectively but inefficiently cleaved upon cellular uptake, likely by cytosolic esterases. Lysosomal catabolism studies indicate that, in spite of the strong proteolytic activity, very little cleavage of ester-containing linkers occurs in the lysosome. However, ADCs bearing the ester-linked payloads are active in various immune-suppressive assays, suggesting that cytosolic cleavage is taking place. This was confirmed through LCMS quantitation of the payload following cell lysis. Finally, the stability of the ester linkage was evaluated in mouse and human plasma. We found, similar to other reports, there is a significant site-dependence on the cleavage. Esters attached at highly exposed sites, such as 443C, were rapidly cleaved in plasma while esters at more hindered sites, such at 334C, were not. Together, these results help to unravel the complexities of ester-incorporation into ADC linkers and pave a path forward for their utility in ADC applications.

A single tick screening for infectious pathogens using targeted mass spectrometry.

The black-legged tick, Ixodes scapularis, is a well-known vector for the Lyme disease-causing pathogen (Borrelia burgdorferi) but can also carry other disease-causing pathogens such as Rickettsia, Anaplasma, Bartonella, Ehrlichia, and Theileria. Hence, tick screening using highly specific protein signatures for specific pathogens will help assess the prevalence of infected ticks and understand the pathogen-tick interactions in a specific geographic area. In this study, we used data-dependent acquisition to key pathogen protein signatures in black-legged ticks collected from the Southern Tier New York. Bottom-up proteomic analysis of extract from five combined ticks identified 2,052 tick proteins and 41 pathogen proteins with high confidence (≥ 99% C.I.). Results show high peptide spectral match counts for Rickettsia species and Borrelia species and lower counts for other rarer pathogens such as Anaplasma phagocytophilum. Parallel reaction monitoring performed on protein extracts from individual ticks (n = 10) revealed that 8 out of the 10 screened ticks carried Rickettsia species, 5 carried Borrelia species, 3 carried both pathogens, and only 1 tick carried no detectable bacteria. Mass spectrometry-based proteomics is a highly specific way to define the expression of different types of pathogen proteins in infected ticks. This might bring insights into the tick-pathogen interactions at the molecular level and especially expression pathogen surface proteins in ticks.

Proteomic and Bioinformatic Analysis of Decellularized Pancreatic Extracellular Matrices.

Tissue microenvironments are rich in signaling molecules. However, factors in the tissue matrix that can serve as tissue-specific cues for engineering pancreatic tissues have not been thoroughly identified. In this study, we performed a comprehensive proteomic analysis of porcine decellularized pancreatic extracellular matrix (dpECM). By profiling dpECM collected from subjects of different ages and genders, we showed that the detergent-free decellularization method developed in this study permits the preservation of approximately 62.4% more proteins than a detergent-based method. In addition, we demonstrated that dpECM prepared from young pigs contained approximately 68.5% more extracellular matrix proteins than those prepared from adult pigs. Furthermore, we categorized dpECM proteins by biological process, molecular function, and cellular component through gene ontology analysis. Our study results also suggested that the protein composition of dpECM is significantly different between male and female animals while a KEGG enrichment pathway analysis revealed that dpECM protein profiling varies significantly depending on age. This study provides the proteome of pancreatic decellularized ECM in different animal ages and genders, which will help identify the bioactive molecules that are pivotal in creating tissue-specific cues for engineering tissues in vitro.

Temporal Proteomic Profiling During Differentiation of Normal and Dystrophin-Deficient Human Muscle Cells.

BACKGROUND: Myogenesis is a dynamic process involving temporal changes in the expression of many genes. Lack of dystrophin protein such as in Duchenne muscular dystrophy might alter the natural course of gene expression dynamics during myogenesis. OBJECTIVE: To gain insight into the dynamic temporal changes in protein expression during differentiation of normal and dystrophin deficient myoblasts to myotubes. METHOD: A super SILAC spike-in strategy in combination and LC-MS/MS was used for temporal proteome profiling of normal and dystrophin deficient myoblasts during differentiation. The acquired data was analyzed using Proteome Discoverer 2.2. and data clustering using R to define significant temporal changes in protein expression. RESULTS: sFour major temporal protein clusters that showed sequential dynamic expression profiles during myogenesis of normal myoblasts were identified. Clusters 1 and 2, consisting mainly of proteins involved mRNA splicing and processing expression, were elevated at days 0 and 0.5 of differentiation then gradually decreased by day 7 of differentiation, then remained lower thereafter. Cluster 3 consisted of proteins involved contractile muscle and actomyosin organization. They increased in their expression reaching maximum at day 7 of differentiation then stabilized thereafter. Cluster 4 consisting of proteins involved in skeletal muscle development glucogenesis and extracellular remodeling had a lower expression during myoblast stage then gradually increased in their expression to reach a maximum at days 11-15 of differentiation. Lack of dystrophin expression in DMD muscle myoblast caused major alteration in temporal expression of proteins involved in cell adhesion, cytoskeleton, and organelle organization as well as the ubiquitination machinery. CONCLUSION: Time series proteome profiling using super SILAC strategy is a powerful method to assess temporal changes in protein expression during myogenesis and to define the downstream consequences of lack of dystrophin on these temporal protein expressions. Key alterations were identified in dystrophin deficient myoblast differentiation compared to normal myoblasts. These alterations could be an attractive therapeutic target.

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy.

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies -Eteplirsen, Golodirsen, Viltolarsen, and Casimersen -for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

Effects of Chronic, Maximal Phosphorodiamidate Morpholino Oligomer (PMO) Dosing on Muscle Function and Dystrophin Restoration in a Mouse Model of Duchenne Muscular Dystrophy.

BACKGROUND: Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is currently used in clinical development to treat Duchenne muscular dystrophy (DMD), with four exon-skipping drugs achieving regulatory approval. Exon skipping elicits a truncated, but semi-functional dystrophin protein, similar to the truncated dystrophin expressed in patients with Becker Muscular dystrophy (BMD) where the disease phenotype is less severe than DMD. Despite promising results in both dystrophic animal models and DMD boys, restoration of dystrophin by exon skipping is highly variable, leading to contradictory functional outcomes in clinical trials. OBJECTIVE: To develop optimal PMO dosing protocols that result in increased dystrophin and improved outcome measures in preclinical models of DMD. METHODS: Tested effectiveness of multiple chronic, high dose PMO regimens using biochemical, histological, molecular, and imaging techniques in mdx mice. RESULTS: A chronic, monthly regimen of high dose PMO increased dystrophin rescue in mdx mice and improved specific force in the extensor digitorum longus (EDL) muscle. However, monthly high dose PMO administration still results in variable dystrophin expression localized throughout various muscles. CONCLUSIONS: High dose monthly PMO administration restores dystrophin expression and increases muscle force; however, the variability of dystrophin expression at both the inter-and intramuscular level remains. Additional strategies to optimize PMO uptake including increased dosing frequencies or combination treatments with other yet-to-be-defined therapies may be necessary to achieve uniform dystrophin restoration and increases in muscle function.

Validation of Chemokine Biomarkers in Duchenne Muscular Dystrophy.

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.

Biomarker-focused multi-drug combination therapy and repurposing trial in mdx mice.

Duchenne muscular dystrophy is initiated by dystrophin deficiency, but downstream pathophysiological pathways such as membrane instability, NFĸB activation, mitochondrial dysfunction, and induction of TGFß fibrosis pathways are thought to drive the disability. Dystrophin replacement strategies are hopeful for addressing upstream dystrophin deficiency; however, all methods to date use semi-functional dystrophin proteins that are likely to trigger downstream pathways. Thus, combination therapies that can target multiple downstream pathways are important in treating DMD, even for dystrophin-replacement strategies. We sought to define blood pharmacodynamic biomarkers of drug response in the mdx mouse model of Duchenne muscular dystrophy using a series of repurposed drugs. Four-week-old mdx mice were treated for four weeks with four different drugs singly and in combination: vehicle, prednisolone, vamorolone, rituximab, ß-aminoisobutyric acid (BAIBA) (11 treatment groups; n = 6/group). Blood was collected via cardiac puncture at study termination, and proteomic profiling was carried out using SOMAscan aptamer panels (1,310 proteins assayed). Prednisolone was tested alone and in combination with other drugs. It was found to have a good concordance of prednisolone-responsive biomarkers (56 increased by prednisolone, 39 decreased) focused on NFκB and TGFß cascades. Vamorolone shared 45 (80%) of increased biomarkers and 13 (33%) of decreased biomarkers with prednisolone. Comparison of published human corticosteroid-responsive biomarkers to our mdx data showed 14% (3/22) concordance between mouse and human. Rituximab showed fewer drug-associated biomarkers, with the most significant being human IgG. On the other hand, BAIBA treatment (high and low dose) showed a drug-associated increase in 40 serum proteins and decreased 5 serum proteins. Our results suggest that a biomarker approach could be employed for assessing drug combinations in both mouse and human studies.

Serum protein biomarkers for juvenile dermatomyositis: a pilot study.

BACKGROUND: Blood accessible biomarkers to assess disease activity and their response to therapies in Juvenile Dermatomyositis (JDM) are urgently needed. This pilot study aims to identify serum protein biomarkers associated with clinical disease activity in untreated JDM and their response to medical therapy. METHODS: SomaScan® technology screened JDM patients for 1305 proteins at three points: 1) before start of treatment, 2) while on therapy, and 3) after treatment tapering when patients were clinically inactive. To define disease associated biomarkers, SomaScan® data from untreated JDM patients (n = 8) were compared to SomaScan® data from an independent age-matched healthy control group (n = 12). Longitudinal analysis defined treatment responsive proteins at three time points: untreated (7 samples), treated (7 samples), and clinically inactive (6 samples). To confirm the SomaScan® data, a subset of nine candidate proteins (CXCL11, IL-17B, IL-17D, IL-22, CXCL10, MCP-1, ANGPT2, MIF, IL-23) were tested by ELISA after adding 2 JDM (one untreated, one clinically inactive) serum samples to the same group of JDM girls (8 untreated, 7 treated; 7 clinically inactive) as well as with 17 age, gender, matched healthy controls. RESULTS: Comparison of untreated JDM versus healthy controls identified 202 elevated and 49 decreased serum proteins in JDM patients with an adjusted p-value < 0.001. Only 82 out of 251 identified biomarker candidates responded to treatment while 12 out of these 82 proteins returned to their original untreated disease levels upon therapy tapering. The ELISA testing of the untreated samples for nine candidate proteins confirmed previously known biomarkers (CXCL10 or IP-10, CXCL11 or I-TAC and MCP-1) and identified novel biomarkers including IL-22, Angiopoetin-2, and IL-17B in a cross-sectional analysis comparing 8 untreated JDM and 17 age/gender matched controls. The subsequent longitudinal data by ELISA were not concordant for some biomarkers (IL-22 and IL-17B), but the other biomarkers either normalized or rebounded concordantly. CONCLUSIONS: Blood accessible protein biomarkers reflecting JDM pathophysiology were identified; some of them rebounded after therapy was tapered. Further studies bridging these biomarkers to specific clinical features of JDM are required to confirm the clinical utility of these serum protein biomarkers.

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

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.

Comparison of Serum Pharmacodynamic Biomarkers in Prednisone-Versus Deflazacort-Treated Duchenne Muscular Dystrophy Boys.

Prednisone (Pred) and Deflazacort (Dfz) are commonly used glucocorticoids (GCs) for Duchenne muscular dystrophy (DMD) treatment and management. While GCs are known to delay the loss of ambulation and motor abilities, chronic use can result in onerous side effects, e.g., weight gain, growth stunting, loss of bone density, etc. Here, we use the CINRG Duchenne natural history study to gain insight into comparative safety of Pred versus Dfz treatment through GC-responsive pharmacodynamic (PD) biomarkers. Longitudinal trajectories of SOMAscan® protein data obtained on serum of DMD boys aged 4 to 10 (Pred: n = 7; Dfz: n = 8) were analyzed after accounting for age and time on treatment. Out of the pre-specified biomarkers, seventeen candidate proteins were differentially altered between the two drugs (p < 0.05). These include IGFBP-2 and AGER associated with diabetes complications, and MMP-3 associated with extracellular remodeling. As a follow-up, IGFBP-2, MMP-3, and IGF-I were quantified with an ELISA using a larger sample size of DMD biosamples (Dfz: n = 17, Pred: n = 12; up to 76 sera samples) over a longer treatment duration. MMP-3 and IGFBP-2 validated the SOMAscan® signal, however, IGF-I did not. This study identified GC-responsive biomarkers, some associated with safety, that highlight differential PD response between Dfz and Pred.

Serum biomarkers associated with baseline clinical severity in young steroid-naïve Duchenne muscular dystrophy boys.

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin in muscle, and while all patients share the primary gene and biochemical defect, there is considerable patient-patient variability in clinical symptoms. We sought to develop multivariate models of serum protein biomarkers that explained observed variation, using functional outcome measures as proxies for severity. Serum samples from 39 steroid-naïve DMD boys 4 to <7 years enrolled into a clinical trial of vamorolone were studied (NCT02760264). Four assessments of gross motor function were carried out for each participant over a 6-week interval, and their mean was used as response for biomarker models. Weighted correlation network analysis was used for unsupervised clustering of 1305 proteins quantified using SOMAscan® aptamer profiling to define highly representative and connected proteins. Multivariate models of biomarkers were obtained for time to stand performance (strength phenotype; 17 proteins) and 6 min walk performance (endurance phenotype; 17 proteins) including some shared proteins. Identified proteins were tested with associations of mRNA expression with histological severity of muscle from dystrophinopathy patients (n = 28) and normal controls (n = 6). Strong associations predictive of both clinical and histological severity were found for ERBB4 (reductions in both blood and muscle with increasing severity), SOD1 (reductions in muscle and increases in blood with increasing severity) and CNTF (decreased levels in blood and muscle with increasing severity). We show that performance of DMD boys was effectively modeled with serum proteins, proximal strength associated with growth and remodeling pathways and muscle endurance centered on TGFß and fibrosis pathways in muscle.