Duchenne muscular dystrophy is a devastating progressive muscle disease that causes loss of ambulation by age 12 and premature death in early thirties. New gene therapy approaches are extending life span, but the physical assessments currently used make disease progression and therapy monitoring challenging, if not impossible. CLI chatted with Professor Yetrib Hathout and Ahmed Naveed to find out more about how the hunt for blood-based biomarkers is providing a new resource for improved evaluation of progression and therapy response – which is crucial for the development of new therapies.

What is Duchenne muscular dystrophy?

Pathology and symptoms

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder characterized by progressive muscle weakness and primarily affects males. Mutations in the DMD gene result in the production of little or no functional dystrophin protein. Dystrophin is a cytoskeletal protein that links the cytoskeleton of muscle fibres to the extracellular matrix, which maintains their integrity during the cycles of contraction and relaxation.

Without it, the sarcolemma becomes leaky and highly susceptible to injury during mechanical stress. This increases its permeability to calcium ions which can be toxic to mitochondria, leading to muscle cell dysfunction, and ultimately, muscle cell death. This triggers a cascade of muscle fibre necrosis, inflammation, followed by gradual replacement of muscle by fat and fibrous tissue. Overtime, these changes result in progressive muscle wasting and weakness.

The first signs often appear in early childhood. A child may be late to walk, fall frequently, have difficulty running or climbing stairs. A decline in muscle mass is seen by the age of seven and then loss of ambulation occurs by about the age of 12. As the condition progresses, it also affects breathing muscles and the heart. Treatments can slow disease progression and improve quality of life, but DMD remains a serious lifelong condition. Patients used to live into their late twenties but lifespan can now be prolonged to 30–35 years.

Treatment

Corticosteroids
Treatment involves corticosteroids to reduce the inflammation that is associated with muscle fibre death. These are typically prednisone and deflazacort, administered daily, which unfortunately have many side effects including weight gain, bone fractures, growth stunting, and so on. More recently, in 2023, vamorolone (a dissociative corticosteroid) received FDA approval. Vamorolone acts like other corticosteroids but has fewer side effects as revealed through biomarker studies (Tobin et al., 2026).

Gene therapy
As it is a genetic mutation that causes loss of dystrophin, one idea was to restore dystrophin expression. There are a few treatments available. One is called exon skipping. This is where an antisense oligonucleotide is used to allow the translation machinery to skip over the targeted exon and return the reading frame to normal, allowing improved protein expression. This requires weekly or biweekly injections, and doesn’t cure the disease but does reduce its severity, helps to preserve muscle mass and slow progression of the disease.

Additionally, AAV microdystrophin is another gene therapy approach where the adeno-associated virus (AAV) is used to deliver healthy, working genes into a patient’s cells. However, because the DMD gene has 79 exons it is too large to be encapsulated in the AAV in full, but a short form can be introduced that can still link the extracellular matrix to the intracellular cytoskeleton of the muscle fibre. Again, this therapy restores dystrophin and stabilizes muscle to a certain extent – not to that of a healthy control – and slows disease progression.

Muscle degeneration and weakness due to the alterations of the dystrophin protein
Lack of the dystrophin protein in muscle cells causes them to be fragile an easily damaged (Shutterstock)

How is diagnosis normally achieved?

Diagnosis usually starts with the clinical picture. A clinician may notice delayed motor milestones, difficulty rising from the floor, calf enlargement, an unusual walking pattern, or a family history of DMD. A blood test for creatine kinase (CK) is then commonly used. CK is an enzyme that leaks from damaged muscle, so a very high level is an important warning sign, although it is not specific to DMD. The diagnosis is confirmed by genetic testing of the DMD gene. Muscle biopsy is also used, although less often. The biopsy can show whether dystrophin is absent or greatly reduced.

What are the limitations of the current methods?

One of the main challenges in clinical trials is monitoring disease progression and treatment response in a way that is objective, sensitive, and practical for every patient. Clinical trials and routine care use tests such as the North Star Ambulatory Assessment (NSAA), the 6-minute walk test (6MWT), and timed activities such as rising from the floor or walking 10 metres. These measures are clinically meaningful, but performance may be influenced by age, motivation, fatigue, understanding of the instructions, and differences between assessors. The tests can also be tiring, and some cannot be used  in very young children or in people who have lost the ability to walk. Furthermore, young children may initially show improvement while the disease process continues. Later, decline becomes more obvious. That natural pattern makes small treatment effects difficult to detect, especially in short or relatively small clinical trials.

How can the situation be improved?

Because of the limitations of the disease/therapy monitoring methods mentioned above, we became very interested in trying to find blood biomarkers. These offer a non-invasive alternative to traditional functional assessments. However, robust serum biomarkers with prognostic value remain elusive. CK is useful in diagnosis but is not an ideal monitoring marker as its level varies and can fall as muscle mass is lost, so a lower CK result does not automatically mean that the disease has improved.

The advantage of a serum-based biomarker is that blood collection is much less burdensome than muscle biopsy and can be repeated over time. One challenge, though, is that blood is highly complex: a small number of very abundant proteins can hide lower-abundance proteins that may carry useful information about muscle injury, repair, inflammation, or treatment response.

Mass spectrometry (MS) is a promising tool that can be used in de novo biomarker discovery by quantification of proteins in serum with high specificity. In our study, we used tandem mass tag (TMT)-MS for relative quantification. We first reduced the influence of the most abundant serum proteins by using serum depletion kit, then digested the remaining proteins into peptides. Each sample received a different TMT-tag, the tagged samples were combined, separated into fractions, and analysed by LC-MS/MS. The instrument identifies the peptides and quantifies the intensity of the tags, allowing us to compare protein levels across samples in the same analysis.

We applied this method to 42 serum samples from 14 participants in the FOR-DMD study. Each participant contributed samples at baseline, 12 months, and 24 months. This longitudinal design was important because we could follow changes within the same person and compare those protein changes with changes in motor function. We then used a linear mixed-effects model to assess the protein association with functional outcome measures.

We found 26 proteins that were associated with at least two outcomes. The majority of these proteins were associated the NSAA and the 6MWT. The proteins reflected several disease-relevant categories, including muscle damage, inflammation, transport, blood coagulation, and protection against oxidative stress. Collectively, the signatures of these candidate serum proteins may provide a valuable tool for monitoring disease progression in DMD. For example, in Duchenne patients, there was a large category of biomarkers coming from muscle leakage, such as creatine kinase, but there are many others that behave like creatine kinase, in that their levels are very high in the early stage of the disease and then start declining over time if the patient is not treated or stabilized. The decline is not because the patient is getting better; it is because of the loss of muscle mass. You can estimate from these biomarkers how much muscle mass patients are losing every year. There are also metabolites such as the creatine-to-creatinine ratio. Creatine is used by muscle and forms creatinine, so if patients are losing muscle mass, they will have less creatinine.

There are also inflammation markers that can be monitored because all patients are treated with anti-inflammatory agents such as corticosteroids, known as pharmacodynamic biomarkers.

Biomarkers, therefore, have several contexts of use, including monitoring and measuring the pharmacodynamic response to a treatment.

To date, a large catalogue of circulating biomarkers has been established for DMD and other muscular dystrophies using a combination of MS-based proteomics and highly multiplexed SomaScan technology. A publicly available database [Tu et al., 2026] provides information on biomarker levels in individuals with DMD compared with healthy controls, as well as their trajectories over time, enabling clinicians and researchers to further evaluate the clinical utility of these biomarkers.

What future developments do you envisage in this field?

Advances in mass spectrometry methods, including newer data-independent acquisition (DIA) platforms, enable the quantification of a larger number of proteins while reducing missing data. These advances may further enhance biomarker discovery, development, and validation. However, greater emphasis should be placed on transitioning candidate biomarkers that demonstrate high reproducibility and sensitivity to disease progression and treatment response from the research setting to clinical practice. This will require the development of highly specific and sensitive assays that are analytically and clinically validated.

Bibliography
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The interviewees

Prof. Yetrib Hathout PhD
Graduate Program Director and Professor

School of Pharmacy and Pharmaceutical Sciences, Binghamton University, Binghamton, NY 13902, USA

Email: yhathout@binghamton.edu

Ahmed Naveed B. Tech
PhD Candidate

School of Pharmacy and Pharmaceutical Sciences, Binghamton University, Binghamton, NY 13902, USA

Email: anaveed1@binghamton.edu

For further information, see:

Naveed A, Recinos E, Chow D, Degan C, Tsonaka R et al.; FOR-DMD investigators of the Muscle Study Group; Hathout Y. Quantitative tandem mass tag-based serum proteomics for longitudinal biomarker monitoring in Duchenne muscular dystrophy. Clin Proteomics. 2026;23(1):42. PMID: 42249273 (https://doi.org/10.1186/s12014-026-09613-4).