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The limb-girdle muscular dystrophies (LGMD) are a collection of genetic diseases united in their phenotypical expression of pelvic and shoulder area weakness and wasting. More than 30 subtypes have been identified, five dominant and 26 recessive. The increase in the characterization of new genotypes in the family of LGMDs further adds to the heterogeneity of the disease. Meanwhile, better understanding of the phenotype led to the reconsideration of the disease definition, which resulted in eight old subtypes to be no longer recognized officially as LGMD and five new diseases to be added to the LGMD family. The unique variabilities of LGMD stem from genetic mutations, which then lead to protein and ultimately muscle dysfunction. Herein, we review the LGMD pathway, starting with the genetic mutations that encode proteins involved in muscle maintenance and repair, and including the genotype–phenotype relationship of the disease, the epidemiology, disease progression, burden of illness, and emerging treatments.

Key Points New sequencing technologies, including whole-exome and whole-genome sequencing, combined with increased data sharing and international research collaborations, are resulting in the identification of new limb-girdle muscular dystrophy (LGMD) subtypes and diagnosis of additional patients These technologies are also providing evidence that added complexities, including intronic and regulatory mutations, oligogenic inheritance and modifier gene effects, could have a role in the hardest-to-diagnose forms of LGMD The increased molecular and pathogenetic understanding of LGMD subtypes is now calling into question their original classification by phenotype, and new systems-based and pathway-based classification systems may be required 'Trial readiness', which includes preparation of patient cohorts, care standards, outcome measures, and biomarkers stratified according to genes and/or pathways, is a key concept for translation of basic research to the clinic Collaboration between patients, industry and academia is essential in the translational pathway towards therapy development and clinical trials The limb-girdle muscular dystrophies (LGMDs) comprise more than 30 genetically defined neuromuscular diseases that share the determining features of weakness and wasting of the pelvic and shoulder girdle muscles. Next-generation sequencing (NGS) technologies are continuing to expand the range of genes and phenotypes associated with the LGMDs. In this article, Thompson and Straub review the international collaborations that are addressing translational research issues in the LGMDs, and the lessons learned from large-scale NGS programmes. The limb-girdle muscular dystrophies (LGMDs) are a diverse group of genetic neuromuscular conditions that usually manifest in the proximal muscles of the hip and shoulder girdles. Since the identification of the first gene associated with the phenotype in 1994, an extensive body of research has identified the genetic defects responsible for over 30 LGMD subtypes, revealed an increasingly varied phenotypic spectrum, and exposed the need to move towards a systems-based understanding of the molecular pathways affected. New sequencing technologies, including whole-exome and whole-genome sequencing, are continuing to expand the range of genes and phenotypes associated with the LGMDs, and new computational approaches are helping clinicians to adapt to this new genomic medicine paradigm. However, 60 years on from the first description of LGMD, no curative therapies exist, and systematic exploration of the natural history is still lacking. To enable rapid translation of basic research to the clinic, well-phenotyped and genetically characterized patient cohorts are a necessity, and appropriate outcome measures and biomarkers must be developed through natural history studies. Here, we review the international collaborations that are addressing these translational research issues, and the lessons learned from large-scale LGMD sequencing programmes.

Limb-girdle muscular dystrophy (LGMD) has been linked to 15 chromosomal loci, 7 autosomal-dominant (LGMD1A to E) and 10 autosomal-recessive (LGMD2A to J). To determine the distribution of subtypes among patients in the United States, 6 medical centers evaluated patients with a referral diagnosis of LGMD. Muscle biopsies provided histopathology and immunodiagnostic testing, and their protein abnormalities along with clinical parameters directed mutation screening. The diagnosis in 23 patients was a disorder other than LGMD. Of the remaining 289 unrelated patients, 266 had muscle biopsies sufficient for complete microscopic evaluation; 121 also underwent Western blotting. From this combined evaluation, the distribution of immunophenotypes is 12% calpainopathy, 18% dysferlinopathy, 15% sarcoglycanopathy, 15% dystroglycanopathy, and 1.5% caveolinopathy. Genotypes distributed among 2 dominant and 7 recessive subtypes have been determined for 83 patients. This study of a large racially and ethnically diverse population of patients with LGMD indicates that establishing a putative subtype is possible more than half the time using available diagnostic testing. An efficient approach to genotypic diagnosis is muscle biopsy immunophenotyping followed by directed mutational analysis. The most common LGMDs in the United States are calpainopathies, dysferlinopathies, sarcoglycanopathies, and dystroglycanopathies.

Mutations in the fukutin-related protein (FKRP) have recently been demonstrated to cause limb girdle muscular dystrophy type 2I (LGMD2I), one of the most common forms of the autosomal recessive LGMDs in Europe. We performed a systematic clinical and muscle MRI assessment in 6 LGMD2I patients and compared these findings with those of 14 patients with genetically confirmed diagnosis of other forms of autosomal recessive LGMDs or dystrophinopathies. All LGMD2I patients had a characteristic clinical phenotype with predominant weakness of hip flexion and adduction, knee flexion and ankle dorsiflexion. These findings were also mirrored on MRI of the lower extremities which demonstrated marked signal changes in the adductor muscles, the posterior thigh and posterior calf muscles. This characteristic clinical and MRI phenotype was also seen in LGMD2A. However, in LGMD2A there was a selective involvement of the medial gastrocnemius and soleus muscle in the lower legs which was not seen in LGMD2I. The pattern in LGMD2A and LGMD2I were clearly different from the one seen in alpha-sarcoglycanopathy and dystrophinopathy type Becker which showed marked signal abnormalities in the anterior thigh muscles. Our results indicate that muscular MRI is a powerful tool for differentiating LGMD2I from other forms of autosomal recessive LGMDs and dystrophinopathies.

Limb-girdle muscular dystrophy (LGMD) 2A (calpainopathy) is the most frequent form of LGMD in many European countries. The increasing demand for a molecular diagnosis makes the identification of strategies to improve gene mutation detection crucial. We conducted both a quantitative analysis of calpain-3 protein in 519 muscles from patients with unclassified LGMD, unclassified myopathy and hyperCKemia, and a functional assay of calpain-3 autolytic activity in 108 cases with LGMD and normal protein quantity. Subsequently, screening of CAPN3 gene mutations was performed using allele-specific tests and simplified SSCP analysis. We diagnosed a total of 94 LGMD2A patients, carrying 66 different mutations (six are newly identified). The probability of diagnosing calpainopathy was very high in patients showing either a quantitative (80%) or a functional calpain-3 protein defect (88%). Our data show a high predictive value for reduced-absent calpain-3 or lost autolytic activity. These biochemical assays are powerful tools for otherwise laborious genetic screening of cases with a high probability of being primary calpainopathy. Our multistep diagnostic approach is rational and highly effective. This strategy has improved the detection rate of the disease and our extension of screening to presymptomatic phenotypes (hyperCKemia) has allowed us to obtain early diagnoses, which has important consequences for patient care and genetic counseling.

Mutations in the caveolin‐3 gene (CAV3) cause limb girdle muscular dystrophy (LGMD) type 1C (LGMD1C) and other muscle phenotypes. We screened 663 patients with various phenotypes of unknown etiology, for caveolin‐3 protein deficiency, and we identified eight unreported caveolin‐deficient patients (from seven families) in whom four CAV3 mutations had been detected (two are unreported). Following our wide screening, we estimated that caveolinopathies are 1% of both unclassified LGMD and other phenotypes, and demonstrated that caveolin‐3 protein deficiency is a highly sensitive and specific marker of primary caveolinopathy. This is the largest series of caveolinopathy families in whom the effect of gene mutations has been analyzed for protein level and phenotype. We showed that the same mutation could lead to heterogeneous clinical phenotypes and muscle histopathological changes. To study the role of the Golgi complex in the pathological pathway of misfolded caveolin‐3 oligomers, we performed a histopathological study on muscle biopsies from caveolinopathy patients. We documented normal caveolin‐3 immunolabeling at the plasmalemma in some regenerating fibers showing a proliferation of the Golgi complex. It is likely that caveolin‐3 overexpression occurring in regenerating fibers (compared with caveolin‐deficient adult fibers) may lead to an accumulation of misfolded oligomers in the Golgi and to its consequent proliferation. Hum Mutat 25:82–89, 2005. © 2004 Wiley‐Liss, Inc.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder caused by mutations in the CAPN3 gene. Its definitive diagnosis is laborious, since the clinical phenotype is often similar to other types of muscular dystrophy and since the CAPN3 gene encompasses a large genomic region with more than 300 pathogenic mutations described to date. In fact, it is estimated that nearly 25% of the cases with a phenotype suggestive of LGMD2A do not have mutations in the CAPN3 gene and that, in up to 22% of the cases, only one mutation is identified. In the present work, we have characterised CAPN3 messenger RNA (mRNA) expression in peripheral blood, and we have performed a retrospective diagnostic study with 26 LGMD2A patients, sequencing a transcript of CAPN3 present in white blood cells (WBCs). The 25% of the mutations presented in this paper (7/28) act modifying pre-mRNA splicing of the CAPN3 transcript, including the first deep-intronic mutation described to date in the CAPN3 gene. Our results determine that the sequencing of CAPN3 transcripts present in WBCs could be applied as a new approach for LGMD2A diagnosis. This method improves and simplifies diagnosis, since it combines the advantages of mRNA analysis in a more accessible and rapidly regenerated tissue. However, the lack of exon 15 in the CAPN3 isoforms present in blood, and the presence of mRNA degradation make it necessary to combine mRNA and DNA analyses in some specific cases.

LGMD refers to a class of muscular dystrophies with onset in the proximal muscles. They are genetically heterogeneous, with both autosomal recessive and dominant forms. The autosomal recessive forms are more common and in general follow a more severe course compared to the dominant forms. It is important to reach a specific genetic diagnosis beyond making a group diagnosis of LGMD to provide adequate genetic counseling, to predict risks for the patient such as the development of cardiomyopathy, and to be able to take advantage of specific treatments when they become available. Establishing a specific diagnosis requires knowledge about the individual clinical features, expert analysis of the muscule biopsy, and the guided initiation of appropriate genetic testing.

Muscular dystrophies are characterized by a progressive loss of muscle tissue and/or muscle function. While metabolic alterations have been described in patients’‐derived muscle biopsies, non‐invasive readouts able to describe these alterations are needed in order to objectively monitor muscle condition and response to treatment targeting metabolic abnormalities. We used a metabolomic approach to study metabolites concentration in serum of patients affected by multiple forms of muscular dystrophy such as Duchenne and Becker muscular dystrophies, limb‐girdle muscular dystrophies type 2A and 2B, myotonic dystrophy type 1 and facioscapulohumeral muscular dystrophy. We show that 15 metabolites involved in energy production, amino acid metabolism, testosterone metabolism and response to treatment with glucocorticoids were differentially expressed between healthy controls and Duchenne patients. Five metabolites were also able to discriminate other forms of muscular dystrophy. In particular, creatinine and the creatine/creatinine ratio were significantly associated with Duchenne patients performance as assessed by the 6‐minute walk test and north star ambulatory assessment. The obtained results provide evidence that metabolomics analysis of serum samples can provide useful information regarding muscle condition and response to treatment, such as to glucocorticoids treatment.

Matriglycan of alpha dystroglycan (α-DG) serves as a receptor for extracellular matrix proteins. Hypoglycosylation of α-DG underlies specific types of muscular dystrophy, dystroglycanopathy. Fukutin Related Protein ( FKRP ) gene encodes a glycosyltransferase that adds ribitol-5-phosphate to the core glycan of α-DG and enables the synthesis of matriglycan. Mutations in the FKRP gene are a common cause of dystroglycanopathies. Ribitol is able to restore matriglycan in diseased muscles with FKRP mutations, but this effect relies on partial function of mutant FKRPs. Different mutations affect FKRP function differently, which could affect the efficiency of ribitol treatment. Here we examined the long-term effect of ribitol in mice with FKRP C826A ( L276I) mutation, the most common genotype in patient population of LGMD2I/R9. Oral administration of ribitol significantly enhances expression of matriglycan in both cardiac and skeletal muscles up to 40% of normal muscle levels. Importantly, matriglycan is homogeneously expressed in almost all muscle fibers with similar levels especially in cardiac muscle. Consistently, muscle degeneration and regeneration are greatly attenuated with reduced central nucleation and fibrosis especially in the diaphragm. This is associated with improvements in muscle functions, although the diseased mice only show limited deficiency when compared to wild type C57 mice. The higher level of restoration in matriglycan in L276I mice than in P448L mice is consistent with the hypothesis that therapeutic potential of ribitol treatment may depend on the remaining function of mutant FKRPs. These results support clinical trials of ribitol to the majority of patients with FKRP mutations.