A Journey with LGMD: From Protein Abnormalities to Patient Impact.

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.

[Sarcoglycanopathies: state of the art and therapeutic perspectives]. / Les sarcoglycanopathies - État des lieux et perspectives thérapeutiques.

Sarcoglycanopathies are the third most common cause of autosomal recessive limb girdle muscular dystrophies (LGMD). They are the result of a deficiency in one of the sarcoglycans a, b, g, or d. The usual clinical presentation is that of a symmetrical involvement of the muscles of the pelvic and scapular girdles as well as of the trunk, associated with more or less severe cardio-respiratory impairment and a marked increase of serum CK levels. The first symptoms appear during the first decade, the loss of ambulation occurring often during the second decade. Lesions observed on the muscle biopsy are dystrophic. This is associated with a decrease or an absence of immunostaining of the sarcoglycan corresponding to the mutated gene and, to a lesser degree, of the other three sarcoglycans. Many mutations have been reported in the four incriminated genes and some of them are prevalent in certain populations. To date, there is no curative treatment, which does not prevent the development of many clinical trials, especially in gene therapy.

TITLE: Les sarcoglycanopathies - État des lieux et perspectives thérapeutiques. ABSTRACT: Les sarcoglycanopathies font partie des dystrophies musculaires des ceintures (LGMD) autosomiques récessives et représentent la troisième cause la plus fréquente d'entre elles. Elles sont consécutives à un déficit d'un des sarcoglycanes α, ß, γ, ou δ. La présentation clinique habituelle est celle d'une atteinte symétrique des muscles des ceintures pelvienne et scapulaire ainsi que du tronc, associée à une atteinte cardiorespiratoire plus ou moins sévère et une élévation franche des créatine-phospho-kinases (CPK). Les premiers symptômes apparaissent au cours de la première décennie, la perte de la marche survenant souvent au cours de la deuxième décennie. Les lésions sont de type dystrophique sur la biopsie musculaire. Il s'y associe une diminution ou une absence d'immunomarquage du sarcoglycane correspondant au gène muté, et dans une moindre mesure des trois autres sarcoglycanes associés. De nombreuses mutations ont été rapportées dans les quatre gènes impliqués et quelques-unes d'entre elles sont prépondérantes dans certaines populations. à ce jour, il n'existe pas de traitement curatif ce qui n'empêche pas de voir se développer de nombreux essais cliniques, notamment en thérapie génique.

LGMD. Identification, description and classification.

The term 'limb girdle muscular dystrophy' (LGMD) was first used in the seminal paper by Walton and Nattrass in 1954, were they identified LGMD as a separate clinical entity In LGMD description it is pointed out that the category of LGMD most likely comprises a heterogeneous group of disorders. After that the clinical entity was discussed but the LMGD nosography reached a permanent classification during two ENMC workshops held in 1995 and 2017, in the last one an operating definition of LGMD was agreed. This last classification included dystrophies with proximal or distal-proximal presentation with evidence at biopsy of fibre degeneration and splitting, high CK, MRI imaging consistent with degenerative changes, fibro-fatty infiltration present in individuals that reached independent walking ability. To be considered in this group at least two unrelated families should be identified. A review is done of the first genetic characterisation of a number of LGMDs during the late twentieth century and a historical summary is given regarding how these conditions were clinically described and identified, the progresses done from identification of genetic loci, to protein and gene discoveries are reported. The LGMD described on which such historical progresses were done are the recessive calpainopathy (LGMD 2A/R1), dysferlinopathy (LGMD 2B/R2), sarcoglycanopathy (LGMD 2C-2F/R3-R6) types and the dominant type due to TPNO3 variants named transportinopathy (LGMD 1F/D2). Because of new diagnostic techniques such as exome and genome sequencing, it is likely that many other subtypes of LGMD might be identified in the future, however the lesson from the past discoveries can be useful for scientists and clinicians.

[A male patient with adult-onset sporadic calpainopathy presenting with hypertrophy of the upper extremities].

A 33-year-old man presented with slowly progressive weakness in the lower extremities over 8 years. At the age of 16 years, the elevation of serum creatine kinase level was detected. Physical examination revealed scapular winging, exaggerated lumbar lordosis and tendoachilles contracture. Gowers sign was positive and proximal dominant limb weakness was noted. Hypertrophy was observed in the upper limbs such as the biceps brachii and forearm flexor muscles. Muscle biopsy showed distinct differences in size of muscle fibers and regenerating and necrotic muscle fibers. A histological study revealed decreased calpain3 expression. Gene analysis of CAPN3 revealed two known gene mutations, leading to a diagnosis of calpainopathy (limb girdle muscular dystrophy 2A; LGMD2A). We here report our patient to discuss findings of upper limb hypertrophy, which are frequently missed compared to the lower limb, but important clinical findings.

An update on diagnostic options and considerations in limb-girdle dystrophies.

INTRODUCTION: Limb-girdle muscular dystrophies (LGMDs) encompass a clinically heterogeneous group of rare, genetic progressive muscle disorders presenting with weakness and atrophy of predominant pelvic and shoulder muscles. The spectrum of disease severity ranges from severe childhood-onset muscular dystrophy to adult-onset dystrophy. Areas covered: The review presents an update of the clinical phenotypes and diagnostic options for LGMD including both dominant and recessive LGMD and consider their differential clinical and histopathological features. An overview of most common phenotypes and of possible complications is given. The management of the main clinical respiratory, cardiac, and central nervous system complications are covered. The instrumental, muscle imaging, and laboratory exams to assess and reach diagnosis are described. The use of recent genetic techniques such as next generation sequencing (NGS), whole-exome sequencing compared to other techniques (e.g. DNA sequencing, protein analysis) is covered. Currently available drugs or gene therapy and rehabilitation management are focused on. Expert commentary: Many LGMD cases, which for a long time previously remained without a molecular diagnosis, can now be investigated by NGS. Gene mutation analysis is always required to obtain a certain molecular diagnosis, fundamental to select homogeneous group of patients for future pharmaceutical and gene trials.

Untangling the complexity of limb-girdle muscular dystrophies.

The limb-girdle muscular dystrophies (LGMDs) are a group of genetically heterogeneous, autosomal inherited muscular dystrophies with a childhood to adult onset, manifesting with hip- and shoulder-girdle muscle weakness. When the term LGMD was first conceptualized in 1954, it was thought to be a single entity. Currently, there are 8 autosomal dominant (LGMD1A-1H) and 26 autosomal recessive (LGMD2A-2Z) variants according to the Online Mendelian Inheritance in Man database. In addition, there are other genetically identified muscular dystrophies with an LGMD phenotype not yet classified as LGMD. This highlights the entanglement of LGMDs, which represents an area in continuous expansion. Herein we aim to simplify the complexity of LGMDs by subgrouping them on the basis of the underlying defective protein and impaired function. Muscle Nerve 58: 167-177, 2018.

Limb girdle muscular dystrophies: classification, clinical spectrum and emerging therapies.

PURPOSE OF REVIEW: The aim of the study was to describe the clinical spectrum of limb girdle muscular dystrophies (LGMDs), the pitfalls of the current classification system for LGMDs, and emerging therapies for these conditions. RECENT FINDINGS: Close to half of all LGMD subtypes have been discovered within the last 6 years of the 21-year-period in which the current classification system for LGMD has existed. The number of letters for annotation of new recessive LGMD conditions is exhausted, and multiple already classified LGMDs do not strictly fulfill diagnostic criteria for LGMD or are registered in other classification systems for muscle disease. On the contrary, diseases that fulfill classical criteria for LGMD have found no place in the LGMD classification system. These shortcomings call for revision/creation of a new classification system for LGMD. The rapidly expanding gene sequencing capabilities have helped to speed up new LGMD discoveries, and unveiled pheno-/genotype relations. Parallel to this progress in identifying new LGMD subtypes, emerging therapies for LGMDs are under way, but no disease-specific treatment is yet available for nonexperimental use. SUMMARY: The field of LGMD is rapidly developing from a diagnostic and therapeutic viewpoint, but a uniform and universally agreed classification system for LGMDs is needed.

Limb-girdle muscular dystrophies - international collaborations for translational research.

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.

Muscle fatigue, nNOS and muscle fiber atrophy in limb girdle muscular dystrophy.

Muscle fatigability and atrophy are frequent clinical signs in limb girdle muscular dystrophy (LGMD), but their pathogenetic mechanisms are still poorly understood. We review a series of different factors that may be connected in causing fatigue and atrophy, particularly considering the role of neuronal nitric oxide synthase (nNOS) and additional factors such as gender in different forms of LGMD (both recessive and dominant) underlying different pathogenetic mechanisms. In sarcoglycanopathies, the sarcolemmal nNOS reactivity varied from absent to reduced, depending on the residual level of sarcoglycan complex: in cases with complete sarcoglycan complex deficiency (mostly in beta-sarcoglycanopathy), the sarcolemmal nNOS reaction was absent and it was always associated with early severe clinical phenotype and cardiomyopathy. Calpainopathy, dysferlinopathy, and caveolinopathy present gradual onset of fatigability and had normal sarcolemmal nNOS reactivity. Notably, as compared with caveolinopathy and sarcoglycanopathies, calpainopathy and dysferlinopathy showed a higher degree of muscle fiber atrophy. Males with calpainopathy and dysferlinopathy showed significantly higher fiber atrophy than control males, whereas female patients have similar values than female controls, suggesting a gender difference in muscle fiber atrophy with a relative protection in females. In female patients, the smaller initial muscle fiber size associated to endocrine factors and less physical effort might attenuate gender-specific muscle loss and atrophy.

RNAi-based gene therapy for dominant Limb Girdle Muscular Dystrophies.

Limb Girdle Muscular Dystrophy (LGMD) refers to a group of 25 genetic diseases linked by common clinical features, including wasting of muscles supporting the pelvic and shoulder girdles. Cardiac involvement may also occur. Like other muscular dystrophies, LGMDs are currently incurable, but prospective gene replacement therapies targeting recessive forms have shown promise in pre-clinical and clinical studies. In contrast, little attention has been paid to developing gene therapy approaches for dominant forms of LGMD, which would likely benefit from disease gene silencing. Despite the lack of focus to date on developing gene therapies for dominant LGMDs, the field is not starting at square one, since translational studies on recessive LGMDs provided a framework that can be applied to treating dominant forms of the disease. In this manuscript, we discuss the prospects of treating dominantly inherited forms of LGMD with gene silencing approaches.

Childhood onset of limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophies comprise a rare heterogeneous group of genetic muscular dystrophies, involving 15 autosomal recessive subtypes and seven autosomal dominant subtypes. Autosomal recessive dystrophy is far more common than autosomal dominant dystrophy. Typical clinical features include progressive limb muscle weakness and atrophy (proximal greater than distal), varying from very mild to severe. Significant overlap of clinical phenotypes, with genetic and clinical heterogeneity, constitutes the rule for this group of diseases. Muscle biopsies are useful for histopathologic and immunolabeling studies, and DNA analysis is the gold standard to establish the specific form of muscular dystrophy. A definitive diagnosis among various subtypes is challenging, and the data presented here provide neuromuscular clinicians with additional information to help attain that goal.

Limb girdle muscular dystrophies: update on genetic diagnosis and therapeutic approaches.

PURPOSE OF REVIEW: This review is an up-to-date analysis of the genetic diagnosis and therapeutic strategies for limb girdle muscular dystrophies (LGMDs). RECENT FINDINGS: LGMDs are an example of both clinical and genetic heterogeneity. Clinically, by the description of non-LGMD phenotypes associated with LGMD genes and of LGMD phenotypes associated with originally non-LGMD disease genes; and genetically, by the description of new LGMD genes that further increase the diagnostic complexity. Moreover, new powerful approaches for DNA analysis, such as exome sequencing, promise to revolutionize the field of heterogeneous genetic diseases, also providing information about the true penetrance of LGMD mutations. The recent inputs on novel pathogenic mechanisms and pathways in LGMD will suggest novel therapeutic approaches and future clinical trials. In addition, therapeutic approaches of gene and cell delivery into animal models show promising results that will be translated into clinical trials. SUMMARY: The genetic diagnosis of LGMD from the present home-made algorithms will move toward high-throughput diagnostic strategies based on next-generation sequencing (NGS) technologies. As therapy, new powerful drug approaches based on recent pathogenetic findings will be pushed to clinical trials. In addition, novel more efficient and safer viral vectors for gene delivery will be proposed.

Limb-girdle muscular dystrophy-associated protein diseases.

UNLABELLED: The limb-girdle muscular dystrophies are a genetically and clinically heterogeneous group of diseases. Most of these proteinopathies show wide inter- and intrafamilial phenotypic heterogeneity, so that limb-girdle involvement may be often considered as one of the possible clinical expressions of a determined protein defect. REVIEW SUMMARY: This review reports an updated and comprehensive classification of these proteinopathies according to protein defect and transmission modality and focuses on the main associated clinical pictures. CONCLUSIONS: An accurate diagnosis is often difficult because of the clinical and genetic variability characterizing this group of muscle diseases. Appropriate diagnostic approaches are essential to achieve the correct diagnosis.

[Autosomal recessive limb-girdle muscular dystrophy]. / Distrofias musculares de cinturas autosomicas recesivas.

Muscular dystrophies are a heterogeneous group of hereditary diseases characterized by loss of muscle and weakness of non neurogenic origin. They are caused by mutations in one or more genes involved in the formation of muscle cells. The discovery of several proteins in the muscle began with the discovery of dystrophin, 130 years after the clinical description of muscular dystrophy. Currently, due to a better understanding of the biology of normal and diseased muscle, has achieved a classification at the molecular level of different types of muscular dystrophies, according to the protein that is affected. This has been particularly important for limb girdle muscular dystrophies, which present clinical features that can lead to confusion with Duchenne muscular dystrophy. Moreover, in recent years has encouraged the development of therapies in the near future could provide a solution for restoring the function of the muscle fiber.

Cardiac pathology exceeds skeletal muscle pathology in two cases of limb-girdle muscular dystrophy type 2I.

Limb-girdle muscular dystrophy type 2I (LGMD-2I) is caused by mutations in the fukutin-related protein gene (FKRP) that lead to abnormal glycosylation of alpha-dystroglycan in skeletal muscle. Heart involvement in LGMD-2I is common, but little is known about a underlying cardiac pathology. Herein we describe two patients with LGMD-2I (homozygous FKRP mutation c.826C>A, p.Leu276Ile) who developed severe congestive heart failure that required cardiac transplantation. The dystrophic pathology and impairment of alpha-dystroglycan glycosylation were severe in the heart but mild in skeletal muscle, underscoring the lack of correlation between cardiac and skeletal muscle involvement in some LGMD-2I patients.

Use of SNP array analysis to identify a novel TRIM32 mutation in limb-girdle muscular dystrophy type 2H.

Molecular diagnosis of monogenic diseases with high genetic heterogeneity is usually challenging. In the case of limb-girdle muscular dystrophy, multiplex Western blot analysis is a very useful initial step, but that often fails to identify the primarily affected protein. We report how homozygosity analysis using a genome-wide SNP array allowed us to solve the diagnostic enigma in a patient with a moderate form of LGMD, born from consanguineous parents. The genome-wide scan performed on the patient's DNA revealed several regions of homozygosity, that were compared to the location of known LGMD genes. One such region indeed contained the TRIM32 gene. This gene was previously found mutated in families with limb-girdle muscular dystrophy type 2H (LGMD2H), a mild autosomal recessive myopathy described in Hutterite populations and in 4 patients with a diagnosis of sarcotubular myopathy. A single missense mutation was found in all these patients, located in a conserved domain of the C-terminal part of the protein. Another missense mutation affecting the N-terminal part of TRIM32, observed in a single consanguineous Bedouin family, was reported to cause the phenotypically unrelated and genetically heterogeneous Bardet-Biedl syndrome, defining the BBS11 locus. Sequencing of TRIM32 in our patient revealed a distal frameshift mutation, c.1753_1766dup14 (p.Ile590Leu fsX38). Together with two recently reported mutations, this novel mutation confirms that integrity of the C-terminal domain of TRIM32 is necessary for muscle maintenance.

Limb-girdle muscular dystrophy type 2A can result from accelerated autoproteolytic inactivation of calpain 3.

Loss-of-function mutations in calpain 3 have been shown to cause limb-girdle muscular dystrophy type 2A (LGMD2A), an autosomal recessive disorder that results in gradual wasting of the muscles of the hip and shoulder areas. Due to the inherent instability of calpain 3, recombinant expression of the full-length enzyme has not been possible, making in vitro analysis of specific LGMD2A-causing mutations difficult. However, because calpain 3 is highly similar in amino acid sequence to calpain 2, the recently solved crystal structure of full-length, Ca2+-bound, calpastatin-inhibited rat calpain 2 has allowed us to model calpain 3 as a Ca2+-bound homodimer. The model revealed three distinct areas of the enzyme that undergo a large conformational change upon Ca2+ binding. Located in these areas are several residues that undergo mutation to cause LGMD2A. We investigated the in vitro effects of six of these mutations by making the corresponding mutations in rat calpain 2. All six mutations examined in this study resulted in a decrease in enzyme activity. All but one of the mutations caused an increased rate of autoproteolytic degradation of the enzyme as witnessed by SDS-PAGE, indicating the decrease in enzyme activity is caused, at least in part, by an increase in the rate of autoproteolytic degradation. The putative in vivo effects of these mutations on calpain 3 activity are discussed with respect to their ability to cause LGMD2A.

How to tackle the diagnosis of limb-girdle muscular dystrophy 2A.

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.