Treatabolome for finely targeting muscle pathology in LGMD

LGMD's current progress and classification.
In 2025, the large majority of Limb Girdle Muscular Dystrophy(LGMD) patients achieve a genetic diagnosis, due to the recent development of advanced genome sequencing techniques, and the research is open to the future development of specific therapies. Patients' management is so far limited to physical rehabilitation and clinical follow-up of cardiologic and respiratory complications, but several therapies have been tested (including corticosteroids 1 and myostatin inhibitors) in trials with variable success. In this letter, a survey of treatable progress and hurdles occurring in different types of LGMD are covered.

LGMD’s current progress and classification

In 2025, the large majority of Limb Girdle Muscular Dystrophy (LGMD) patients might achieve a genetic diagnosis, due to the recent development of advanced genome sequencing techniques, and the research is open to the future development of specific therapies. Patients’ management is limited to physical rehabilitation and clinical follow-up of cardiologic and respiratory complications. Still, several therapies have been tested (including corticosteroids and myostatin inhibitors) in trials with variable success. In this letter, a survey of treatable progress and hurdles occurring in different types of LGMD are covered.

Classification of LGMDs

Following the genetic identification in loci of several LGMDs in the late 20th century, in 1995 a consensus on the classification of LGMD subtypes based on molecular and genetic criteria; the autosomal dominant loci were designated as LGMD type 1, and the autosomal recessive loci were designated as LGMD type, avoiding imprecise and often lengthy nomenclature in use (e.g. Leyden Mobius muscular dystrophy, SCARMD).

During the following 10 years, the number of LGMD loci/genes identified increased rapidly, and occupied all the alphabetic letters (from LGMD2A to LGMD2Z); furthermore, the classification system applied by the Online Mendelian Inheritance in Man (OMIM) catalog was no longer used by the clinicians because it included also inherited myopathies with normal CK levels which could be more properly classified between categories other than LGMD (e.g. congenital myopathies, scapulo-peroneal myopathies, metabolic myopathies, etc.).

Because of these factors, it was time to update and modify the LGMD nomenclature.

An ENMC workshop, which was held in March 2017 in Naarden, in the Netherlands, on the classification and nomenclature of the LGMDs, was aimed at reaching a consensus reaching an updated definition of LGMD and reviewing and evaluating suggestions of potential new classifications of LGMD subtypes. At this meeting, the classification was revised naming the autosomal dominant LGMDs as D and numbering them from 1 to 5, and the recessive forms as R with numbers from 1 to 23. The classification of LGMD included dystrophies with a proximal or disto-proximal presentation with evidence at a biopsy of fiber degeneration and splitting, high CK, muscle MRI imaging consistent with degenerative change, and fibro-fatty infiltration.

Today, more than 30 different genetic subtypes of LGMD have been identified. Like other inherited myopathies, several clinical phenotypes might be present with the same variant(s) for epigenetic influence.

New Criteria for LGMD

The ENMC consensus on LGMD classification in 2017 ^1-2 was done according to the following criteria:

• Genetic cause of the disease
• Progressive predominantly proximal muscle weakness.
• Condition which permanently affects skeletal muscle.
• Achievement of independent walking at some point.
• Weakness is caused by the loss of muscle fibers.
• Elevated serum creatine kinase (CK) detected in the blood.
• Degenerative changes in muscle imaging as the cause of the disease.
• Change in muscle tissue (biopsy) that are in accordance.
• with dystrophic changes ultimately leading to end-stage pathology in affected muscles.
• For a new disease to be classified as LGMD the the disease must have been identified in at least two different families.

LGMD therapy for precisely targeting muscle pathology

Recessive forms have been seldom treated but several animal models are investigated for drug trials.

Calpainopathy (LGMD-R1)

This is the most frequent autosomal recessive type of LGMD in South Europe while a dominant type was identified in Denmark and other North European countries.in LGMD-R1 the myopathology is due to a proteolytic enzyme defect, i.e. muscle specific calpain-3.

Three autosomal recessive calpainopathy phenotypes have been identified based on the distribution of muscle weakness and age at onset:

Pelvifemoral limb-girdle muscular dystrophy (LGMD) (Leyden-Möbius LGMD) phenotype, the most frequently observed calpainopathy phenotype, in which muscle weakness is first evident in the pelvic girdle and later in the shoulder girdle, with onset that may occur as early as before age 12 years or as late as after age 30 years ².

Scapulohumeral LGMD (Erb LGMD phenotype), usually a milder phenotype with infrequent early onset, in which muscle weakness is first evident in the shoulder girdle and later in the pelvic girdle.

HyperCKemia is usually observed in children or young individuals, in which individuals are asymptomatic and have high serum creatine kinase (CK) concentrations.

The autosomal dominant form of calpainopathy (LGMD D4) is clinically variable, ranging from an almost asymptomatic phenotype to wheelchair dependence after age 60 years in a few individuals; the phenotype is generally milder than the recessive form.

The major sleep respiratory distressing signs are snoring, apnoeic episodes, and daytime somnolence, which indicate the possibility of obstructive sleep apnoea. If patients under-ventilate at night, the resultant hypercapnia may cause early-morning headaches, reduced concentration, or clouded consciousness. The blurring of vision from papilledema has been described but is rare and only seen in severe hypercapnia. A history of recurrent chest infections may indicate an ineffective cough. Coughing requires activation of the inspiratory muscles, closure of the glottis, and then contraction of the expiratory muscles, particularly those of the abdominal wall; finally, the expulsive phase is initiated by opening the glottis. A poor cough can result from weakness or uncoordinated contraction of the inspiratory, glottic, or expiratory muscle groups.

LGMD R1 is a frequent LGMD, but clinical trials have been delayed because there have been cardiac complications in gene transfer therapy in animal models (Tab. I).

Dysferlinopathy(LGMD-R2)

This is the second most frequent AR LGMD with two distinctive clinical phenotypes of LGMD2B/R2 and Miyoshi Myopathy (MM)which are clinically different: the first appears in the second/third decade with high CK and proximal limb-girdle involvement, the second with distal weakness, both phenotypes can be detected among patients belonging to the same family. The clinical heterogeneity might be attributed to additional epigenetic factors.

Dysferlin protein immunolocalizes the sarcolemma and has a central role in membrane fusion and repair of the sarcolemma.

Skeletal muscle lesions are generated by eccentric muscle contraction, as demonstrated by the presence of many crowded vesicles just beneath the sarcolemma ³. Several studies have reported a prominent inflammatory response in dysferlinopathy biopsy and increased ubiquitin-proteasomal and autophagic degradation secondary due to high levels of regeneration and inflammation are seen in biopsy. The detection of dysferlin deficiency by western blotting in muscle offers an important diagnostic tool.

In several patients the use of the steroids prednisone and in a clinical trial in Germany deflazacort has been unsuccessful, systemic delivery of Dysferlin overlap vectors provided long-term gene expression and functional Improvement in Dysferlinopathy animal models ⁴, however dysferlinopathy gene therapy remains complex for the relatively large size of the protein that needs a double AAV approach to produce a viable dysferlin protein in skeletal muscle.

Sarcoglycanopathies

To this group pertain several LGMDs that are due to defects in alpha, beta, and gamma delta sarcoglycans relatively named LGMDR3, R4, R5 R6.

The sarcoglycans concur to form the sarcoglycan complex that stabilizes the sarcolemma and is composed of small molecules amenable to be carried by AAV9.

However, the current available treatments are focused on heart, and coronary artery smooth muscle to prevent myocardial and skeletal muscle ischemia. Patients with alpha-sarcoglycan gene mutations appear clinically heterogeneous and show either a rapid progressive or a late-onset slow course. In the slowly evolving group, a residual alpha-sarcoglycan protein is present, and its level correlated with a milder disease course and significant later inability to stand up from the floor Most patients with beta- and gamma-sarcoglycan gene mutations presented a severe clinical course, a peculiar feature is the presence of cardiomyopathy (44% of cases), and initial cardiomyopathy (19% of cases) are frequent, as well as arrhythmias and dilated cardiomyopathy 39,40. Signs of hypoxic myocardial damage may occur in LGMD2E/R4, LGMD2F/R6, and LGMD2C/R5. Abnormal coronary smooth muscle function has been suggested to be involved in the development of cardiomyopathy in LGMD2E/R4 and LGMD2F/R6 since β-SG and δ-SG are also expressed in the coronary arteries.

Impaired vasoregulation occurs via a marked reduction in membrane-associated neuronal nitric oxide synthase (nNOS) in both cardiac and skeletal muscle. Without dystrophin, nNOS mislocalizes to the cytosol; this greater distance between nNOS and the sarcolemma may impair NO diffusion through the myocyte membrane to the microvasculature. As a consequence, insufficient NO release follows muscle contraction resulting in muscle ischemia. Unopposed vasoconstriction may explain the necrosis observed in skeletal and cardiac muscle of dystrophinopathy patients. Microvasculature abnormality might result primarily from the absence of dystrophin or sarcoglycan components in cardiomyocytes. The sarcolemmal nNOS expression correlated with the clinical severity 44 and muscle fatigue: absence or severe reduction of sarcolemmal nNOS expression was associated with a severe and childhood-onset form of muscular dystrophy and in most cases also with dilated cardiomyocytes.

Phase 1 studies of AAV-delivered gene therapy for LGMD R4 and R5 (beta and gamma sarcoglycans) have demonstrated proof-of-principle for delivery in an isolated muscle and showed sarcoglycan staining in muscle biopsies post-therapy ^5-6. It is likely that the clinical trial for Beta -sarcoglycan deficiency might show sustained beta-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type R4.

Preclinical efforts to develop gene therapies for FKRP mutations (LGMDR9) and sarcoglycan Beta mutations (LGMDR4) are underway.

FKRP related LGMD

LGMD R9 is not typically classified as a metabolic disorder, due to perturbed metabolic changes the use of ribitol supplementation is on the way in the UK in LGMD R9 and has a large number of patient requests and consensus ^7-10.

There is evidence to suggest that muscular dystrophy-dystroglycanopathies are characterized by perturbed metabolic networks. Some of the metabolic pathways have been identified previously in similar physiological disorders, including anomalies in amino acid, glucose, lipid, and mitochondrial metabolism. Experiments derived from skeletal muscle tissue extracts from FKRP-deficient mice treated with AAV9-hFKRP and the respective positive (C57BL/6) and negative (FKRPP448L) controls, have searched the current metabolic footprint of musculoskeletal disorders and identified new potential candidate biomarkers in skeletal muscle associated with FKRP deficiency and age-related muscle pathology. A list of the top 20 metabolites, which is dominated by lipids but is also highlighted by contributors of amino acid or nucleic acid metabolism has been provided ¹¹. An important treatable study has been carried out both in animal models and in man inLGMD R9/2I Limb-girdle muscular dystrophy R9 (LGMDR9) which is caused by a deficiency of FKRP. FKRP allows the addition of ribitol-5-phosphate, a molecule made from ribitol, to the sugar chain already formed by the action of other enzymes. Oral intake of BBP-418 (ribitol) is used to increase glycosylation of α-dystroglycan by saturating FKRP with the substrate. A Phase I (completed) and Phase II (ongoing) trial of ribitol have delivered initial results.

The Phase 1 trial was conducted in 85 healthy volunteers. This randomized controlled trial of a single dose and then multiple escalating doses versus placebo studied the pharmacokinetics of the molecule and found it to be very well tolerated with no signs of toxicity even at doses above the intended therapeutic doses.

Given this positive trial a Phase 2 open-label trial of BBP-418 in 14 people with LGMDR9, aged 12 to 55 years, has been designed to determine the safety and tolerability of escalating doses of BBP-418 over 5 years.

Preliminary results in the first 12 participants, aged 12 to 53 years, 9 of whom had retained walking, were presented at the Muscular Dystrophy Association 2022 conference. They show:

• a 43% increase in glycosylated alpha-dystroglycan; a mean decrease in CPK at 90 days of 70%;
• an increase in the walking speed over 10 meters at 3 and 6 months, whereas it had decreased in the 6 months of natural history study preceding the intake of ribitol.

Also B4GALNT2 (GALGT2) Gene Therapy Reduces Skeletal Muscle Pathology in the FKRP P448L Mouse Model of Limb Girdle Muscular Dystrophy R9/2I ¹⁰.

Caution has to be exerted since this type of dystrophy produces a Becker-like phenotype with lower limb involvement, calf hypertrophy, and cardiomyopathy, and in some cases, there was brain CNS involvement with visual-spatial difficulty in designing or recalling Rey’s figure.

Current trials are based on the results with Ribitol The trial with Ribitol was prematurely closed before any Italian participants could be included.

Dominant LGMD

For the dominant LGMDs which are not amenable to either drug or gene replacement therapy, there is evidence that small molecules can affect the pathology associated with DNAJB6 mutations (LGMDD1). With multiple gene replacement therapies currently in pre-clinical/phase 1 testing, there is a demand from patients to start a clinical trial.

Dominant LGMD aims to collect natural history and quality of Life (QOL) data to guide trials ¹².

In the studies in which QoL has been assessed with other instruments in patients with recessive LGMD, the survey determined the frequency and impact of symptoms on QoL in patients diagnosed with three types of LGMD.

In a survey participants with a diagnosis of LGMD due to Calpain-3 (LGMD R1), Dysferlin (LGMD R2), and FKRP (LGMD R9) completed a questionnaire to report the frequency and relative impact of themes and symptoms of LGMD. Frequency, mean impact, and population impact scores were calculated, and responses were categorized by age, symptom duration, gender, employment status, use of assistive devices, and 134 LGMD participants completed the survey. The most prevalent themes included a 100% inability to do activities, limitation with mobility, and lower extremity weakness. Themes with the greatest impact were: limitations with mobility, lower extremity weakness, and an inability to do activities. Symptom duration and the use of assistive devices were associated with the presence of multiple items.

Employment was associated with the impact of several themes with no differences in frequency. In the LMGD population, the most prevalent andwalking speed over 10 meters at 3 and 6 months, whereas it had decreased in the 6 months of natural history study preceding the intake of ribitol were also considered in a clinical setting since they might at large correlate with socioeconomic problems and the impact on ADLs. In addition, fatigue and its impact on independence and social relationships are also reported in the LGMD-D2 sample ¹².

Conclusions

Several molecular-based therapies, such as steroids and other non-specific muscular dystrophy therapies are currently in clinical trials. Such approaches may prove beneficial for LGMD. These include targeting the myostatin pathway, muscle inflammation, increasing myofiber regeneration, or reducing fibrosis. Recently completed early-phase studies of non-specific therapies in LGMD include an anti-myostatin drug to improve muscle growth (NCT02841267), a steroid to target inflammation (NCT00527228), and a tRNA synthetase to target muscle inflammation and regeneration (NCYT02836418).

Recent developments in our genetic understanding of LGMD and molecular approaches to therapy have led to proposed gene replacement therapies for at least three LGMD types: in clinical trials are LGMD R4-R5 and ribitol LGMD R9 in the recessive LGMD types ^1-3.

Calpainopathy LGMD R1, although relatively frequent trials has been delayed in gene transfer therapy since in animal models there has been a cardiac failure and dysferlinopathy awaits positive results from animal models.

LGMD R9 might obtain a favorable metabolic and clinical analysis of skeletal muscles in FKRP-deficient patients who underwent the trial with Ribitol Therapy opening a treatabolomic era for LGMD, since it determined walking speed over 10 meters at 6 months, whereas it had decreased in the 6 months of natural history study preceding the intake of ribitol.

Quality of life is improved by participating in a trial, the most impacting themes are mobility and fatigue.

History

Received: February 7, 2025

Accepted: March 10, 2025

Figures and tables

Table I. Ongoing therapy and trials in LGMD-R.

LGMD type	Protein detect	Gene trials	Drug for LGMD-FKT	Outcome
CalpainopathyLGMD R1	R1Calpain-3	Cardiac complications in gene transfer therapy in animal models	Prevent respiratoryfailure	Stable
DysferlinopathyLGMD R2	R2dysferlin	Needs a double AAV approach to produce a viable dysferlin protein	Prednisone/deflazacort	No result onsymptoms
SarcoglycanopathyLGMD R3-R6	R3 (alpha-SG)R4 (beta-SG)R5 (gamma-SG)R6 (delta SG)	Phase 1 studies of AAV-delivered gene therapy for LGMD R4 and R5 (beta and gamma sarcoglycans) demonstrated delivery in an isolated muscle and showed sarcoglycan staining in muscle biopsies post-gene therapy.	Prednisone/deflazacortHeart transplant.	Stable/Improved
Fukutin related LGMD LGMD R9	R9positiveFKRP	B4GALNT2 (GALGT2) Gene Therapy Reduces Skeletal Muscle Pathology in the FKRP P448L Mouse Model	RIBITOL/BBP-418 is used to increase glycosylation of α-dystroglycan by saturating FKRP with the substrate.	Improved in mouse model,Ongoing in patients