Background: Becker muscular dystrophy (BMD) is a rare and heterogeneous form of dystrophinopathy caused by expression of altered dystrophin proteins, as a consequence of in-frame genetic mutations. The majority of the BMD biomarker studies employ targeted approaches and focus on translating findings from Duchenne Muscular Dystrophy (DMD), a more severe disease form with clinical similarities but caused by out-of-frame mutations in the dystrophin gene. Importantly, DMD therapies assume that disease progression can be slowed by promoting the expression of truncated dystrophin comparable to what occurs in BMD patients. In this study, we explore similarities and differences in protein trajectories over time between BMD and DMD serum, and explore proteins related to motor function performance.

Methods: Serum samples collected from 34 BMD patients, in a prospective longitudinal 3-year study, and 19 DMD patients, were analyzed by using Data Independent Acquisition Tandem Mass Spectrometry (DIA-MS). Subsequent normalization, linear mixed effects model was employed to identify proteins associated with physical tests and dystrophin expression in skeletal muscle. Analysis was also performed to explore the discrepancy between DMD and BMD biomarker abundance trajectories over time.

Results: Linear mixed effects models identified 20 proteins with altered longitudinal signatures between DMD and BMD, including creatine kinase M-type (CKM) pyruvate kinase (PKM), fibrinogen gamma chain (FGG), lactate dehydrogenase B (LDHB) and alpha-2-macroglobulin (A2M). Furthermore, several proteins related to innate immune response were associated with motor function in BMD patients. In particular, A2M displayed an altered time-dependent decline in relation to dystrophin expression in the tibialis anterior muscle.

Conclusions: Our study revealed differences in the serum proteome between BMD and DMD, which comprises proteins involved in the immune response, extracellular matrix organization and hemostasis but not muscle leakage proteins significantly associated with disease progression in DMD. If further evaluated and validated, these biomarker candidates may offer means to monitor disease progression in BMD patients. A2M is of particular interest due to its association with dystrophin expression in BMD muscle and higher abundance in DMD patients in comparison to BMD. If validated, A2M could be used as a pharmacodynamic biomarker in therapeutic clinical trials aiming to restore dystrophin expression.

Introduction: Identifying serum biomarkers that reflect the restoration of dystrophin in skeletal muscle is important for eval-uating the effect of dystrophin-restoring therapies in preclinical and clinical trials. Many potential blood biomarkers have beenidentified in Duchenne muscular dystrophy (DMD) patients, which change with disease progression or respond to pharmacologi-cal treatment. In this study, it was suggested that a panel of such blood biomarker candidates could be used to monitor dystrophinrescue in mdx mice treated with microdystrophin based therapies.Methods: Plasma samples from mdx mice treated with the microdystrophin therapy SGT- 001 were analysed with an antibodysuspension bead array consisting of 87 antibodies. The array targets 83 unique proteins previously identified as biomarker can-didates for DMD. Each sample was assayed at two different plasma dilutions to cover a broader concentration range. Proteinconcentrations estimated as Median fluorescent intensities (MFI) were correlated to dystrophin expression in muscle tissue, asmeasured by immunohistochemistry and Western blot. Thirteen of the targets were selected and analysed in a DMD and Beckermuscular dystrophy (BMD) longitudinal natural history cohort using a suspension bead array.Results: Ten proteins were found to be significantly elevated in untreated mdx mice compared with C57 wild- type mice andto correlate with dystrophin expression (Spearman's correlation, FDR < 0.05) upon gene transfer in mdx mice. Translatability ofthese biomarkers from animal models to patients was evaluated by exploring abundance trajectories over time in both DMD andBMD patients and association with dystrophin expression in BMD patients. Consistent with the observations in mouse, six ofthese biomarker candidates were more abundant in DMD patients compared with controls, and six were also differentially abun-dant between BMD and DMD patients. Among them, serum titin was shown to be associated with dystrophin expression in BMDpatients, having a steeper decline over time in patients with low dystrophin expression in tibialis anterior compared with patientswith high expression. Myosine light chain 3 had a steeper decline with time in DMD patients compared with BMD patients.Conclusions: The 10 biomarker candidates identified in mouse plasma are related to muscle contraction, glycolysis, microtubuleformation and protein degradation. Human titin and myosine light chain 3 were the most interesting candidates as explorativebiomarkers to monitor microdystrophin expression in gene therapies. If confirmed, these biomarkers could be used to detect restoration of dystrophin expression per se, monitor changes in dystrophin expression over time and potentially confirm diseasephenotype changes from severe to mild disease forms.

Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease caused by pathogenic variations in the DMD gene. There is a need for robust DMD biomarkers for diagnostic screening and to aid therapy monitoring. Creatine kinase, to date, is the only routinely used blood biomarker for DMD, although it lacks specificity and does not correlate with disease severity. To fill this critical gap, we present here novel data about dystrophin protein fragments detected in human plasma by a suspension bead immunoassay using two validated anti-dystrophin-specific antibodies. Using both antibodies, a reduction of the dystrophin signal is detected in a small cohort of plasma samples from DMD patients when compared to healthy controls, female carriers, and other neuromuscular diseases. We also demonstrate the detection of dystrophin protein by an antibody-independent method using targeted liquid chromatography mass spectrometry. This last assay detects three different dystrophin peptides in all healthy individuals analysed and supports our finding that dystrophin protein is detectable in plasma. The results of our proof-of-concept study encourage further studies in larger sample cohorts to investigate the value of dystrophin protein as a low invasive blood biomarker for diagnostic screening and clinical monitoring of DMD.

Background

Molecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, their translation to clinical use is difficult due to the lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy was developed and used to assess the reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage that currently lacks reliable and specific disease monitoring tools.

Methods

Two technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Parallel Reaction Monitoring Mass Spectrometry assay (PRM-MS).

Results

Five, out of ten biomarkers previously identified by affinity-based proteomics methods, were confirmed to be associated with DMD using the mass spectrometry-based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, were quantified with two independent methods, sandwich immunoassays and PRM-MS, with Pearson correlations of 0.92 and 0.946 respectively. The median concentrations of CA3 and LDHB in DMD patients was elevated in comparison to those in healthy individuals by 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas those of LDHB vary between 15.1 and 0.8 ng/ml.

Conclusions

These results demonstrate that orthogonal assays can be used to assess the analytical reliability of biomarker quantification assays, providing a means to facilitate the translation of biomarkers to clinical practice. This strategy also warrants the development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are two rare genetic disorders of the family dystrophinopathy. They are both caused by the lack of, or reduced production of, the protein dystrophin. Due to abnormal dystrophin expression, patients experience progressive loss of muscle mass and cardio-, respiratory- and sometimes cognitive complications. DMD is the more severe form of dystrophinopathy, which manifests in young children and leads to wheelchair confinement in early teens followed by bed confinement and a shortened life expectancy. Dystrophin expression is absent or at less than 3% in DMD patients, often due to frame-shift mutations which cause protein expression to stop pre-maturely. BMD patients, on the other hand, display a higher but variable expression of dystrophin, often with large internal truncations. This partial expression results in a milder phenotype than DMD with sometimes unaffected life expectancies compared to healthy individuals. In the past decade, there has been substantial research into therapies aiming at increasing dystrophin expression in DMD patients and thereby prolonging ambulation, with the first gene-therapy gaining regulatory approval from the U.S. Food and Drug Administration (FDA) in June 2023. Current regulatory approvals for treatments of DMD patients have relied on dystrophin quantification in muscle biopsies as a biomarker and surrogate endpoint to predict a possible benefit from treatment, but these tests require repeated collection of muscle biopsies. 

Biomarkers are biochemical or physiological laboratory tests that measure a biological processes or condition. There is a need for monitoring biomarkers in dystrophinopathies, as well as biomarkers which can be used to predict outcome in clinical trials. As patients are often young children, it is important to develop biomarkers from less invasive and more readily available biological samples than muscle biopsies, such as blood or urine.

In this thesis, we have used affinity proteomics and mass spectrometry to identify and validate biomarkers for monitoring disease progression in DMD and BMD patients from serum or plasma. The overall aim has been to identify biomarkers capable of distinguishing between patients with different levels of dystrophin expression and rates of disease progression in order to suggest gene-therapy pharmacodynamic biomarkers. In Paper I, we used suspension bead array (SBA) technology to identify biomarker candidates which reflect disease progression in DMD. Ten proteins were identified as related to disease progression. The ten biomarker candidates identified in Paper I were further analytically validated in Paper II using two orthogonal and absolute quantitative methods, parallel reaction monitoring mass spectrometry (PRM-MS) and sandwich immunoassays, which resulted in five analytically validated disease monitoring biomarkers for DMD (CA3, MYL3, LDHB, COL1A1 and FGG).

Dystrophin-restoring therapies build on the hypothesis that increasing expression of internally deleted dystrophin reduces disease severity. In BMD patients, partial expression of short dystrophin molecules results in a milder phenotype than in DMD patients lacking expression of dystrophin. However, there are some differences in the nature of disease progression between DMD and BMD. In Paper III, we used Data Independent Acquisition Mass Spectrometry (DIA-MS) to study proteomic similarities and differences between DMD and BMD disease progression. This study revealed some discrepancies between disease progression biomarker candidates in the two related disorders.

In Paper IV, we searched for blood biomarkers capable of reflecting changes in dystrophin expression during gene-therapy clinical trials. We identified ten proteins which correlated with dystrophin or microdystrophin expression in DMD mouse models. Out of these ten proteins, we identified that myosin light chain 3 (MYL3) declined steeper over time in dystrophinopathy patients with low or no dystrophin expression compared to patients with higher dystrophin expression. Two more biomarkers were identified in Paper IV as potentially related to dystrophin expression in muscle biopsies from both mouse models and patients. These were titin (TTN) and, interestingly, serum leakage of dystrophin. 

The possible presence of dystrophin in blood has not been well studied, and only one prior publication suggests that dystrophin may be a biomarker for DMD. Many DMD therapies currently in clinical trials aims at restoring dystrophin production and for those trials, the possibility of monitoring dystrophin leakage into blood could provide valuable information on therapeutic efficacy. In Paper V, we designed a proof-of-principle study to explore if dystrophin in blood can be a DMD biomarker. 

In conclusion, this thesis explores disease progression monitoring biomarkers and gene-therapy pharmacodynamic biomarkers for DMD and BMD. Three proteins, MYL3, TTN, and serum levels of dystrophin, are here suggested as possible gene-therapy pharmacodynamic biomarkers. 

Duchenne muskeldystrofi (DMD) och Becker muskeldystrofi (BMD) är två ovanliga, genetiska sjukdomar som går under samlingsnamnet dystrofinopatier. Dystrofinopatier orsakas av att proteinet dystrofin antingen saknas helt eller uttrycks vid längre koncentration i muskler och hjärta än normalt. På grund utav onormalt dystrofinsyntes så förlorar patienter muskelmassa över tid samt upplever påverkan på hjärta och lungor. Ibland har patienter även nedsatt kognitiv förmåga. DMD är den allvarligare formen av dystrofinopati. De första symptomen märks redan i små barn och leder till förlorad förmåga att gå i yngre tonåren samt förkortad livslängd. I DMD uttrycks dystrofin i mindre än 3% än uttrycket i friska muskler, oftast som följd av mutationer som orsakar tidigt avslut av proteinsyntes (s.k. ramförskjutningsmutationer). BMD-patienter, å andra sidan, har ett något högre men variabelt uttryck av dystrofin jämfört med DMD och ofta i kombination med stora interna deletioner. Detta reducerade uttryck av dystrofin resulterar i en mildare fenotyp än DMD. Under det senaste decenniet har forskare fokuserat på att utveckla behandlingsmetoder som kan öka uttrycket av dystrofin i DMD-patienter och därmed förlänga patienternas livslängd samt även förlänga tiden då patienten kan gå självständigt. Den första genterapin för DMD godkändes av USA:s läkemedelsmyndighet Food and Drug Administration (FDA) i juni 2023, men saknar än så länge myndighetsgodkännande i Europa. Myndighetsgodkännanden för behandlingar av DMD har i flera fall baserats på att ett ökat inducerat dystrofinuttryck observerats i muskelbiopsier efter behandling, vilket enligt FDA sannolikt tyder på en klinisk fördel från behandlingen. Dessa tester kräver dock att patienten donerar upprepade muskelbiopsier. Dystrofinuttryck i muskel används här som en s.k. biomarkör. 

Biomarkörer är biokemiska eller fysiologiska laborativa tester som mäter en biologisk process eller tillstånd. Det finns idag ett behov av nya biomarkörer för att följa sjukdomsförlopp i dystrofinopatier samt biomarkörer som kan användas till att förutsäga utfall i kliniska studier. Eftersom denna patientgrupp ofta består av unga individer så är det viktigt att utveckla nya biomarkörer från mindre invasiva prov-material än muskelbiopsier, så som blod eller urin.

I denna avhandling så har vi använt oss utav affinitetsproteomik samt masspektrometri för att identifiera och validera blodbiomarkörer för DMD och BMD. Det övergripande målet har varit att identifiera biomarkörer med förmågan att särskilja patienter med olika nivåer av både dystrofinuttruck samt progression, för att senare kunna identifiera biomarkörer för att övervaka effekt av genterapier i DMD. I Artikel I använde vi oss utav en så kallad ”suspension bead array” (SBA)-teknologi för att identifiera biomarkörskandidater för sjukdomsprogression i DMD. Vi identifierade tio proteiner relaterade till progression. Analytisk validering av dessa tio biomarkörskandidater genomfördes i Artikel II med hjälp av två ortogonala, absolutkvantitativa metoder, ”Parallell Reaction Monitoring” masspektrometri (PRM-MS) samt ”sandwich immunoassay”. Detta resulterade i analytisk validering av fem biomarkörer för att följa sjukdomsutveckling i DMD (CA3, MYL3, LDHB, COL1A1 samt FGG).

Även om både DMD och BMD orsakas av mutationer i samma gen, så finns det skillnader i både sjukdomsprogression och symptom mellan de två patientgrupperna. I Artikel III använde vi masspektrometri för att studera skillnader och likheter i blodproteomet mellan DMD och BMD över tid, vilket visade på diskrepanser i vilka biomarkörskandidater som är lämpliga för att följa sjukdomsprogression i dessa två sjukdomar.

I Artikel IV letade vi efter blodbiomarkörer med förmågan att reflektera förändrat dystrofinuttryck i kliniska studier för genterapier. Vi identifierade tio proteiner som korrelerade med dystrofin- samt mikrodystrofinuttryck i plasma från en DMD-musmodell behandlad med en mikrodystrofinterapi. Av dessa tio proteiner konstaterade vi att myosin lätt kedja 3 (MYL3) minskar snabbare över tid i serum från dystrofinopati-patienter med lågts eller inget dystrofinuttryck än i patienter med högre uttryck. Två ytterligare proteiner, titin (TTN) och serum-läckage av dystrofin, var associerade med dystrofinuttryck i muskler.  

Endast en tidigare publikation påvisar att dystrofin kan vara en blodbiomarkör för DMD. Då många terapier för DMD i kliniska studier har som syfte att öka dystrofinuttrycket så skulle möjligheten att detektera läckage av dystrofin i blod kunna bidra med värdefull information om terapeutisk effekt. I Artikel V designade vi därför en ”proof of principle”-studie för att utforska om dystrofin kan detekteras i blod och i så fall om det kan utgöra en rimlig biomarkörskandidat.

Sammanfattningsvis utforskar denna avhandling biomarkörer för att följa sjukdomsförlopp och utvärdera genterapier i DMD och BMD. Från våra resultat föreslår vi tre biomarkörskandidater: MYL3, TTN samt dystrofin i serum.

Duchenne muscular dystrophy (DMD) is a rare genetic disease due to dystrophin gene mutations which cause progressive weakness and muscle wasting. Circadian rhythm coordinates biological processes with the 24-h cycle and it plays a key role in maintaining muscle functions, both in animal models and in humans. We explored expression profiles of circadian circuit master genes both in Duchenne muscular dystrophy skeletal muscle and in its animal model, the mdx mouse. We designed a customized, mouse-specific Fluidic-Card-TaqMan-based assay (Fluid-CIRC) containing thirty-two genes related to circadian rhythm and muscle regeneration and analyzed gastrocnemius and tibialis anterior muscles from both unexercised and exercised mdx mice. Based on this first analysis, we prioritized the 7 most deregulated genes in mdx mice and tested their expression in skeletal muscle biopsies from 10 Duchenne patients. We found that CSNK1E, SIRT1, and MYOG are upregulated in DMD patient biopsies, consistent with the mdx data. We also demonstrated that their proteins are detectable and measurable in the DMD patients' plasma. We suggest that CSNK1E, SIRT1, and MYOG might represent exploratory circadian biomarkers in DMD.

Longitudinal and high-dimensional measurements have become increasingly common in biomedical research. However, methods to predict survival outcomes using covariates that are both longitudinal and high-dimensional are currently missing. In this article, we propose penalized regression calibration (PRC), a method that can be employed to predict survival in such situations. PRC comprises three modeling steps: First, the trajectories described by the longitudinal predictors are flexibly modeled through the specification of multivariate mixed effects models. Second, subject-specific summaries of the longitudinal trajectories are derived from the fitted mixed models. Third, the time to event outcome is predicted using the subject-specific summaries as covariates in a penalized Cox model. To ensure a proper internal validation of the fitted PRC models, we furthermore develop a cluster bootstrap optimism correction procedure that allows to correct for the optimistic bias of apparent measures of predictiveness. PRC and the CBOCP are implemented in the R package pencal, available from CRAN. After studying the behavior of PRC via simulations, we conclude by illustrating an application of PRC to data from an observational study that involved patients affected by Duchenne muscular dystrophy, where the goal is predict time to loss of ambulation using longitudinal blood biomarkers.

Abstract

Background

Duchenne muscular dystrophy (DMD) is a fatal disease for which no cure is available. Clinical trials have shown to be largely underpowered due to inter‐individual variability and noisy outcome measures. The availability of biomarkers able to anticipate clinical benefit is highly needed to improve clinical trial design and facilitate drug development.

Methods

In this study, we aimed to appraise the value of protein biomarkers to predict prognosis and monitor disease progression or treatment outcome in patients affected by DMD. We collected clinical data and 303 blood samples from 157 DMD patients in three clinical centres; 78 patients contributed multiple blood samples over time, with a median follow‐up time of 2 years. We employed linear mixed models to identify biomarkers that are associated with disease progression, wheelchair dependency, and treatment with corticosteroids and performed survival analysis to find biomarkers whose levels are associated with time to loss of ambulation.

Results

Our analysis led to the identification of 21 proteins whose levels significantly decrease with age and nine proteins whose levels significantly increase. Seven of these proteins are also differentially expressed in non‐ambulant patients, and three proteins are differentially expressed in patients treated with glucocorticosteroids. Treatment with corticosteroids was found to partly counteract the effect of disease progression on two biomarkers, namely, malate dehydrogenase 2 (MDH2, P = 0.0003) and ankyrin repeat domain 2 (P = 0.0005); however, patients treated with corticosteroids experienced a further reduction on collagen 1 serum levels (P = 0.0003), especially following administration of deflazacort. A time to event analysis allowed to further support the use of MDH2 as a prognostic biomarker as it was associated with an increased risk of wheelchair dependence (P = 0.0003). The obtained data support the prospective evaluation of the identified biomarkers in natural history and clinical trials as exploratory biomarkers.