|Year : 2007 | Volume
| Issue : 4 | Page : 214-224
Limb girdle muscular dystrophies: The clinicopathological viewpoint
J Andoni Urtizberea1, France Leturcq2
1 Assistance Publique - Hopitaux de Paris, Hopital Marin, BP40139, 64700 Hendaye, France
2 Assistance-Publique - Hopitaux de Paris, Hospital Cochin-Maternités, Boulevard de Port Royal, 75014 Paris, France
J Andoni Urtizberea
Assistance Publique - Hopitaux de Paris, Hopital Marin, BP40139, 64700 Hendaye
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Limb girdle muscular dystrophies (LGMD) are characterized by involvement of the pelvic and shoulder girdles, classically with an onset in the second or third decade and a slow progression as opposed to Duchenne muscular dystrophy. In fact, there are many clinical variants that are related to this broad definition. For the past 13 years and since the discovery of calpain-3 as the underlying defect in LGMD 2A in 1995, a number of different genes have been found to cause LGMD; some of whose encoding proteins are located either in the sarcolemma, nucleus, cytosol or in the extra-cellular matrix. Very little is known regarding a possible common pathogenesis between all these entities. The current nomenclature of LGMDs, although a bit confusing, is still necessary to continue the establishment of homogeneous cohorts of patients and to look for unknown genes. The diagnosis of LGMD is nowadays based on a complementary clinical, immunocytochemical and genetic approach that is best achieved in specialized myology centers. In this context, India can make a significant contribution to improve the routine diagnosis in LGMD patients and to find new LGMD genes in genetic isolates. Therapeutic prospects in LGMD, although quite exciting, remain at a preliminary stage, especially those with
Keywords: Calpainopathies, diagnosis, dysferlinopathies, genetics, limb-girdle muscular dystrophy, muscle pathology, sarcoglycanopathies
|How to cite this article:|
Urtizberea J A, Leturcq F. Limb girdle muscular dystrophies: The clinicopathological viewpoint. Ann Indian Acad Neurol 2007;10:214-24
| Introduction|| |
With respect to any other group of muscle diseases, perhaps, the historical perspective is more relevant to the understanding of the current problems and challenges in limb-girdle muscular dystrophies. The term itself, "limb-girdle muscular dystrophy" or best known as "LGMD", was coined by several authors in the early 1950s before being widely advertised in a renowned paper by Walton and Nattrass in 1954. The two authors put forward a new, more genetic-oriented classification of neuromuscular diseases.  At this instant, the definition of LGMD was rather restrictive and based on the following criteria: late onset in the first decade, symptoms of muscular weakness in shoulder and/or pelvic girdle, relatively slow progression, nevertheless leading to severe disablement and often premature death and autosomal recessive mode of inheritance. The original goal of Walton and Nattrass was to better delineate the boundaries between the X-linked forms of muscular dystrophy (Duchenne and Becker muscular dystrophies), the dominantly inherited one (facioscapulohumeral muscular dystrophy or FSHD) and the form of muscular dystrophy reported by Erb in 1884  (as a juvenile form of muscular dystrophy with recessive inheritance). Shortly after this publication, the concept of LGMD started to be heavily criticized not because of its intrinsic relevance but because of the wide spectrum of neuromuscular disorders that could be misdiagnosed with it (the most common errors were pseudomyopathic spinal muscular atrophy, acid maltase deficiency and other metabolic disorders). Michael Brooke, a prominent myologist, even questioned the accuracy of this term in his handbook that was devoted to neuromuscular disorders in 1977.  Michel Fardeau, a neurologist who pioneered myology in France, stated that LGMD was actually a "diagnostic fourre-tout" (a diagnosis meant to pool all patients with undiagnosed or misdiagnosed muscle diseases). The situation substantially moved in the early eighties with the advent of molecular testing that was applied to muscle diseases. The discovery of dystrophin as the defective protein in Duchenne/Becker muscular dystrophy in 1986 paved the way to the elucidation of other muscular dystrophies. It showed that positional cloning applied to this group of diseases could be extremely powerful, particularly to identify the candidate proteins. The recipe to identify new causative genes was, at least in principle, very straightforward: select homogeneous groups of LGMD patients (best when they belonged to consanguineous and/or genetic isolates), extract DNA and subject these batches of samples to powerful linkage analysis software programs and then to positional cloning. Hopefully for LGMD, a number of genetic niches with high preponderance of patients presenting with autosomal recessively inherited muscular dystrophy had been reported earlier in the literature in various countries and several inbred communities, notably in the Amish, Mennonites, Tunisians, Reunion Islanders and other areas in Brazil or North Africa. It was also found that in some pedigrees, the genetic transmission was not recessive but autosomal dominant, thereby suggesting the existence of dominant forms of LGMD. In parallel, and over the same decade, biochemists had dissected the molecular complex formed, at the muscle membrane level, by the dystrophin and other hitherto unknown proteins: sarcoglycans, dystroglycans, dystrobrevins, etc. In this respect, the invaluable results accomplished by the group led by Kevin Campbell in Iowa City played a pivotal role in elucidating the various subtypes of LGMD.  The combination of both approaches (linkage studies and positional cloning on one hand and biochemical dissection of the complex on the other) resulted in the discovery of an impressive series of new genes within a decade. Genetic heterogeneity was found to be considerably greater than that expected. In 1995, an important meeting was held in Naarden, the Netherlands, under the auspices of the European Neuromuscular center.  A new nomenclature of genetic loci was agreed upon on the basis of two modes of inheritance (LGMD1 for autosomal dominant forms and LGMD2 for recessive ones). In each category, the suffix numbering with letters (A, B, C, etc.) followed the chronological order of their discovery. Even at that stage, the proposed nomenclature, actually meant to facilitate the identification of new LGMD loci and genes, was heavily criticized for its rather loose clinical definition and for its tendency to lump too many distinct entities. From that instant, one started to lump clinical entities and genes that were increasingly distinct from the original clinical and histological description made by Erb  one century back and rediscovered by Michel Fardeau in the Reunion Island.  Even now, there is still a long way to go before a consensus is reached on this subject. More recently, a classification based on the location and putative functions of each LGMD gene has been proposed; however, this does not solve the question of the extremely wide clinical spectrum of all these diseases together.  Meanwhile, novel or known genes responsible for LGMD phenotypes kept appearing in the literature, thereby creating more confusion as the list seems endless [Figure - 1],[Figure - 2].
Current classifications of limb-girdle muscular dystrophies
To date, 19 LGMD genes have been mapped, 15 of which being cloned. 13 are recessive and 6 are dominant. There are different ways to categorize these genes or entities. The chronological classification is based on the series of genetic discoveries made since 1994 at a time when CAPN-3 (calpain deficiency or LGMD2A) was the very first gene ever cloned in LGMD.  The following years saw the subsequent genetic elucidation of many LGMD subtypes [Table - 1]. This process is still in progress, although it is following a decreased tempo. Thus, the latest genes to be identified were POMT1 and Fukutin (LGMD2K and LGMD2L, respectively), , and according to many experts, this inventory is unlikely to be close to completion.
Interestingly, LGMD and other muscular dystrophies, notably the ever-expanding group of congenital muscular dystrophies (MDC), clearly overlap at the genetic level. The same gene, for instance FKRP, is capable of causing either a severe form of congenital muscular dystrophy (MDC1C) or LGMD type 2I.  Similarly, POMT1 and fukutin mutations were originally described in patients with congenital muscular dystrophy before getting involved in the LGMD arena.
As LGMD acronyms are not very user friendly, one can use another terminology based on a suffix added to the name of the defective protein: calpainopathies, dysferlinopathies, sarcoglycanopathies, myotilinopathies, caveolinopathies and so on. This is not operating with LGMD 2H nor with 2I, 2J and 2K. The point is that protein names often vary. Telethonin, for instance, is now called titin-cap, a less fancy name. Moreover, a given entity is sometimes re-classified in another group of muscular dystrophies: FKRP deficiency, best known as LGMD 2I, is now a part of the emerging group of alphadystroglycanopathies. This approach is nevertheless helpful to sort all these genes out and to keep them in mind. In contrast, it is not relevant for clinicians when it comes to differentiating between them at the clinical level.
The latest attempt to categorize all the LGMDs is based on the location and/or functions of each protein involved  [Table - 2]. The first group encompasses the proteins involved, directly or indirectly, in the muscle plasma membrane (sarcolemma) machinery and/or the dystrophin-associated-complex: sarcoglycans (alpha, beta, gamma and delta) and to a lesser extent, dysferlin, caveolin-3 and presumably others to come. The disruption of the dystrophin-associated complex is still believed to be the major mechanism by which muscle fiber degeneration occurs. However, it is now established that if this pathology accounts for a great number of LGMD cases, it is not the only explanation. Very little is known about the various interactions between these proteins themselves to account for the clinical events and progression.
At present, the second group composed of cytosolic proteins is emerging: Calpain-3 and to a lesser extent, TRIM32 are either enzymes or proteins located in the cytoplasm. , Their substrates are not entirely known and the way they interact is still unclear. They nevertheless lead to the same dystrophic process as the other LGMDs.
Proteins of the third group of LGMD belong to the sarcomere , the elementary contractile unit of the muscle cell. Two gigantic proteins, titin and nebulin, act as molecular rulers around which other proteins such as myosin, actin, telethonin (titin-cap) and myotilin are interacting during muscle contraction. Usually, such defects in structural proteins give rise to congenital myopathy and not to muscular dystrophy. It was therefore surprising to find out that three of them (telethonin, myotilin, titin) could indeed be mutated in LGMD (LGMD2G, LGMD1A and LGMD2J respectively). ,, The connection with muscle membrane pathology is not yet established and further investigations are in progress.
Mutations in the LMNA gene constitutes the fourth group.  LMNA encodes a nuclear envelope protein, lamin A/C, which is also found involved in a myriad of other genetic disorders of neuromuscular ( Emery-Dreifuss muscular dystrophy More Details, axonal CMT) or non-neuromuscular nature (Dunnigan-type familial partial lipodystrophy, premature ageing syndromes, mandibuloacrodysplasia, etc.). The so-called laminopathies are extensively investigated at present, and their understanding may shed more light on the complex molecular pathways leading to cell degeneration. Interestingly, another protein of the nuclear envelope, emerin, has just been involved in two Japanese LGMD patients very recently. 
Some authors also include components of the so-called extracellular matrix to account for some LGMD cases (the fifth group).  This matrix is composed of two layers: basal lamina and collagen networks. There is an increasing evidence that defective glycosylation in the basal lamina and notably at the level of dystroglycan, its major component, can give rise to LGMD phenotype. Several enzymes or proteins can lead to this pathological phenomenon: fukutin-related protein or FKRP (LGMD2I), POMT1 (LGMD2K) and more recently fukutin (LGMD2L). Similarly, collagen-VI deficiencies occasionally mimic the LGMD phenotype in adult patients; however, this entity (called Bethlem myopathy) has not been incorporated as yet in the LGMD classification.
Autosomal recessive LGMD
LGMD 2A, the first LGMD to be described, is calpain deficiency. The clinical picture is identical to that of Erb's early description in the late last century.  The onset of symptoms occurs in the second decade, progression is slow and muscle selectivity is marked with prominent involvement of scapular fixators, harmstrings and hip adductors. Contractures are rather limited and most often confined to the Achilles tendon. Classically, cardiac and respiratory functions are never compromised. Muscle pseudohypertrophy is never a salient feature. LGMD2A is an autosomal recessive disorder by definition, and many genetic clusters have been reported in inbred communities. In the 1980s, Fardeau et al .  found a great deal of LGMD patients in the Reunion Island, a small French territory lost in the Indian Ocean, between Malagasy and Mauritius. Sixty patients sharing more or less the same ancestors developed the disease and presented, for the most part, with a typical LGMD phenotype. This cluster was used to map LGMD2A to chromosome 15q by Beckmann.  Early in the genetic studies, six different CAPN-3 mutations were identified in the Reunion gene pool  ). A recent reappraisal led us think there is no longer a "Reunion Paradox," as hypothesized earlier. Out of the six mutations present in the Island, one (c.946A) is actually very prevalent and found in 80% of LGMD 2A patients, either heterozygously or homozygously. It is likely that various past immigration waves introduced the other five mutations inside the Reunion Island.
Similar genetic isolates with private mutations have been identified in other regions of the world: the Amish communities who escaped from religious persecutions and settled in the United States three centuries ago now harbor a number of LGMD2A cases amongst them, particularly in Northern Indiana. One specific mutation has only been reported in the Amish so far and is therefore regarded as a private mutation. The Basque LGMD2A cluster is the third LGM2A isolate to ever be recorded. Close to 70 patients linked one way or another to the Northern Spanish province of Guipuzcoa share the same mutation. 
The CAPN-3 gene encodes calpain-3, a cytosolic enzyme whose likely function is to remodel pieces of the sarcomere. Over 280 different CAPN3 mutations have been reported and a couple of them correspond to private/founder mutations. Establishing the diagnosis of LGMD2A on firm grounds is difficult at times. Clinically, the involvement of the posterior compartment of the thigh is the best clue, but it may not be present at the later stages. The sole interest of routine immunocytochemistry is to demonstrate other proteins are present (dystrophin, dysferlin and sarcoglycans, notably). The only way to visualize calpain deficiency objectively is to use immunoblotting on muscle specimen. The absence or marked reduction of calpain bands on the Western blot is the best signature for LGMD2A. There are nevertheless some misleading or tricky situations; following are some of them. First, it is because of the secondary reductions of the calpain signal that have been reported in other neuromuscular diseases, including LGMD2B, LGMD2I and LGMD2J. Second, since the normal calpain immunoblot in muscle does not rule out LGMD2A, as shown by Angellini and others in 20-30% of patients with molecularly proven CAPN-3 defects.  The mutation detection in the CAPN-3 itself is rather robust and performed routinely in various laboratories. Finally, it should be mentioned that at this point in time, a major clinical study to document the natural course of LGMD2A in 100 patients is in progress in both Paris, the Rιunion Island and the Basque Country.
LGMD 2B Dysferlin is a huge protein playing a major role in membrane repair. The term dysferlinopathies refers to its deficiency and various phenotypes have been linked to it: Miyoshi-type distal myopathy, hyperCKemia, DACM (distal anterior compartment myopathy) and LGMD.  Interestingly, two (rarely three) of these phenotypes can coexist in the same kindred, thereby suggesting the possible influence of modifying genetic factors. , The LGMD2B phenotype is often "pure" (that is proximal); but sometimes one may note a certain degree of concomitant distal leg involvement, a feature best assessed by muscle imaging. This "distal touch" is important to consider and constitutes an interesting clue. Another feature is the presence of variable inflammatory infiltrates in LGMD2B muscle. Therefore, many of these patients are misdiagnosed with polymyositis and unnecessarily treated with corticoids and immunosuppressive agents. Dysferlin deficiency can readily be tested in monocytes by using a commercial kit. The ultimate clue though is the absence of dysferlin staining on muscle sections and on immunoblotting. Looking for mutations in DYSF - a 55-exon gene - remains time-consuming, labor-intensive and only done on a research basis in very few laboratories. In addition, it is common to find only one mutation out of the expected two, polymorphisms being rather common in this large gene. That is why geneticists prefer to work on RNA (extracted from muscle specimens) rather than on blood genomic DNA. In our own experience, the disease is most prevalent in the Middle-East, North Africa and in the Indian subcontinent.
LGMD 2C-2F or sarcoglycanopathies (SG)
These LGMDs are caused by mutations in one of the following four sarcoglycan genes: alpha, beta, delta and gamma. ,,,, Sarcoglycans are transmembrane proteins playing a role in signal transduction and assembly of the dystrophin-associated complex. The prevalence of each subtype remains elusive due to the paucity of epidemiological data. In the Western world, alpha and gamma-SG are the most common followed by beta and occasionally delta.  Sarcoglycanopathies are characterized by a wide clinical spectrum irrespective of their molecular subtype, from DMD-like features, with muscle pseudohypertrophy, to adult-onset LGMD with minimal symptoms.  Cardiac complications are common as the patients grow old, and this is must be considered for providing early appropriate intervention.  The diagnosis is made by immunostaining for the four forms of sarcoglycans on muscle sections as well as on immunoblots.  The interpretation of the results are sometimes very difficult as the primary deficiency of the given sarcoglycan often results in the variable reduction of the other three as well. The four sarcoglycan genes are relatively small and easy to sequence. In a few ethnic or geographical contexts, some common mutations can be detected in a straightforward manner. 
LGMD2G is quite anecdotal and caused by mutations in telethonin (titin-cap), a protein interacting with the other sarcomeric proteins. Only a couple of individuals with LGMD had this gene mutated. 
LGMD2H remains a rare entity and is caused by a single homozygous mutation in TRIM32, a cytosolic enzyme  It was originally thought to be confined to Canadian Hutterites living in Manitoba; however, very recently, V. Nigro found other mutated patients in Europe while randomly processing a large batch of LGMD samples in Naples, Italy (personal communication).
LGMD 2I, 2K and 2L are caused by mutated proteins (FKRP, POMT1 and fukutin, respectively) known to play a role in glycosylation of transmembrane muscle proteins such as dystroglycan. Despite a common denominator (reduction of the alpha-dystroglycan labeling) and a well-identified disease mechanism, they do not exactly share the same phenotype since LGMD2K and LGMD2L present with additional mental retardation unlike LGMD2I.
Autosomal dominant LGMD
Autosomal dominant LGMD are less common than their recessive counterparts (around ten times less). Amongst them, LGMD1B (lamin A/C deficiency) appears as a common diagnostic working hypothesis when the patient presents with cardiac disease and LGMD phenotype.  Cardiac disease is characterized by conduction blocks and a high risk of sudden death. Mutated lamin A/C also causes autosomal dominant Emery-Dreifuss muscular dystrophy but the contractile features are more prominent in the latter than in LGMD1B. In the absence of reliable immunostains for lamin A/C, the best way to diagnose LGMD1B is to entirely sequence the LMNA gene. There are no known hot spots in this gene.
The other dominant LGMD forms (1A, 1C, 1D-G) are more anecdotal and uncommon. Although caveolinopathy (LGMD1C) may prove to be more frequent than the others, it is due to a defective CAV-3 gene that normally encodes caveolin, a constituent of muscle membrane caveolae (little caves).  Interestingly the same gene is responsible for other phenotypes such as muscle rippling disease and isolated hyperCKemia.
Can we distinguish clinically between all these varieties of LGMD?
To be honest, that remains extremely difficult even in the most expert hands ,, Of course, clinical clues such as muscle pseudohypertrophy (of calves and/or of the tongue) plead more in favor of sarcoglycanopathies (LGMD 2C to 2F) and LGMD2I; however, many exceptions have been reported. Along the same line, a concomitant muscle wasting of the posterior compartment of the leg is highly suggestive of dysferlin deficiency. Similarly, the presence of a muscle rippling phenomenon would be indicative of caveolinopathy. The association of muscular dystrophy and severe primary cardiomyopathy with conduction defects is suggestive of laminopathy (LGMD1B); however, when a pure congestive dilated cardiomyopathy is detected, LGMD 2I, sarcoglycanopathies and LGMD 1D are more likely to be involved. The early wasting of the posterior part of the thigh is often in favor of calpain deficiency and is best assessed on muscle imaging. On the contrary, distinguishing clinically between the four different types of sarcoglycan deficiencies is almost impossible. In contrast, determining the precise ethnic or geographical background of the LGMD patient can be useful and instrumental to direct the diagnosis. For instance, autosomal recessive muscular dystrophy in Maghreb (Northern Africa) prompts to think of sarcoglycan deficiency as the prior choice, particularly the gamma-subtype (LGMD 2C). LGMD in the Canadian Hutterites are invariably due to LGMD 2I or LGMD 2H. The Amish LGMD patients, in Indiana and Pennsylvania, are preferentially at risk for LGMD2A and LGMD2E, respectively. Patients belonging to the Roma population based in Europe (Gypsies) are more often diagnosed with LGMD 2C than with any other LGMD subtype.
Which are the most relevant diagnostic tools?
Since the clinical assessment is often noncontributory, further investigations are required at the benchside in an attempt to direct mutation detection, the ultimate gold standard for LGMD diagnosis. To start with, muscle imaging may provide interesting clues: CT-scan or MRI of the lower limbs is accessible worldwide and most often affordable by patients. It does not require any use of contrast enhancing media and is not time-consuming. In addition, the acquired images can guide the pathologist to select the best site for muscle sampling. Thigh and calf muscles are the most important areas to be examined for any ongoing degenerating process. The topography of early lesions is of great interest, particularly for LGMD with high selectivity of muscle impairment (LGMD2A, LGMD2B). Other baseline investigations are less relevant: serum CK levels are classically elevated with a wide range, varying from one LGMD variety to another and from one disease stage to another, nor are they useful to monitor the course of the disease. If clinical clues point to dysferlin deficiency, a noninvasive commercial blood test is available to measure the dysferlin content within monocytes.
At this stage, there are several strategies that depend on the clinical data generated by the initial workup, the geographic/ethnic origin of the patients and the availability and quality of muscle sampling. As we know, muscle biopsy remains invasive and subject to many technical constraints. It requires great expertise in handling and interpretation. In Europe, North America and Japan, such biopsies are generally performed in specialized referral centers, where the latest technologies are available (antibodies, immunoblot). In other places, it is still an almost impossible task. That is why it might be preferable, under rare circumstances, to bypass muscle biopsy and proceed directly to DNA studies. When there is substantial evidence to point to one specific LGMD, a direct screening of the suspected defective gene can be envisaged. This is the case, for instance, when the LGMD patient belongs to a specific ethnic group in which a private mutation has already been reported (C283Y in Gypsies, del525T in Maghrebian or peri-Mediterranean communities, c.946A in the Reunion Island, etc.). At times, one specific mutation is so common in the general population at the heterozygous state that it is worthwhile screening it in the whole LGMD population prior to any muscle biopsy. That is precisely what our German colleagues did as a first-line assessment of their pool of LGMD patients to detect the L276I mutation in the FKRP gene (LGMD2I).
In general, the above mentioned situations are rather uncommon or restricted to certain geographical areas or ethnic isolates. In most cases, when one is left with a clinical picture consistent with LGMD without any specific clue or family history, the best way to proceed from there is to perform a muscle biopsy. If the muscle biopsy cannot be processed in a specialized center with the appropriate, comprehensive immunolabelling techniques, we do think it is not ethical or scientifically sound to go further. Best is to wait for better times (when the technique is locally available) or to travel to an expert myology center overseas. The biopsy site is important to consider: the muscle to be sampled must be easily accessible and reasonably affected. At late-end stages of muscular dystrophy, muscle is indeed replaced by fat, thereby precluding any accurate interpretation. In the context of LGMD, the most sampled muscles are the deltoid, quadriceps and biceps brachialis muscles. Best results are obtained when these specimens are carefully taken out by the pathologist or a dedicated neurologist and processed at the earliest in a nearby laboratory. With conventional stains (hematein-eosin, modified Gomori trichrome, oxidative enzymes), the muscle looks dystrophic, with more or less marked necrosis and degeneration of muscle fibers and increased interstitial connective tissue. From these basic findings, there is no way to predict the LGMD subtype. Therefore, immunocytochemistry will be the next essential step. Using either immunofluorescence or immunoperoxidase technique, the presence of proteins with monoclonal antibodies can be established (except for the financial consideration that immunofluorescence microscopes are costly). A panel of antibodies directed to most LGMD gene products can be studied on muscle sections and a complementary immunoblot will help in quantifying the importance of the specific protein deficiency. When the proteins are located near or inside the muscle membrane, there are easily recognizable with a positive circular membrane stain at the sarcolemma level. It is important to include controls simultaneously. The controls include applying the same antibody on muscle cryosections from individuals without this disease and omitting the primary antibody and carrying out the staining procedure. That is the best quality control so far. A few LGMD proteins are not recognized with such a first-line approach: calpain, TRIM32, FKRP, etc. That is why immunoblotting, a complementary technique that is applied to a small chunk of muscle, is crucial to detect the presence of some of these proteins when applicable. This is particularly true in calpain deficiency, quite a common cause of LGMD (calpain is mostly located in the cytosol). In the group of LGMDs related to glycosylation defects (LGMD 2I, 2K and 2L), the reduction of an indirect marker (alpha-dystroglycan) may be indicative, although this is sometimes subtle to assess. Overall, most diagnostic situations are sorted out after this two-step histopathological analysis (immunocytochemistry and immunoblotting). DNA studies can then get started to detect mutations in one targeted gene out of the 19 existing LGMD genes or loci. In fact, for logistic reasons, only a few of these genes are routinely accessible to mutation screening  Clinicians should bear in mind the fact that these DNA studies can last extremely long despite all recent advances made in sequencing techniques. In other instances, the targeting of the LGMD gene is quite uneasy, particularly when the second step of the initial workup (muscle immunoblotting) is missing. Elsewhere, the interpretation of immunostains is more difficult than predicted. For instance, the secondary reductions of other immunostains are sometimes misleading: dystrophin can be slightly decreased in primary sarcoglycanopathies, calpain stain has been shown to be occasionally reduced in dysferlin deficiency and so on. In the most difficult diagnostic situations and provided the family history is sufficiently informative at the genetic level, linkage studies based on a statistical approach can be useful to direct the mutation search. In fact, very few laboratories are keen to provide this approach unless a new genetic locus is strongly suspected.
Limb-girdle muscular dystrophies in the Indian context
Limb-girdle muscular dystrophy (LGMD) is a ubiquitous worldwide disease that suffers no exception. Whether or not LGMD is more prevalent in India than in the Western world remains to be clarified, but it is highly plausible. For the past 10 years or so, with the assistance of various clinical teams from Mumbai, Delhi, Bangalore, Chennai and now Hyderabad, we have tried to investigate the overall prevalence of this disease as well as its distribution into the various known subgroups. This constitutes a quite challenging, ongoing, lengthy task; at this stage, the only thing we can do is to make same general comments.
For many outsiders including myself, India represents a formidable opportunity to elucidate new LGMD genes or to establish large cohorts of patients with already known gene defects. From a genetic viewpoint, the clinical material in this country is highly valuable: The overall population is large, size of the families remains extended and baseline consanguinity is high, either for religious, sociological or geographical reasons. As a result, we would expect an elevated rate of autosomal recessive cases of LGMD that is likely to be true. Dominant LGMD cases probably exist in the country, although it has not been reported in literature to the best of our knowledge. In some places, although inbreeding is so high that even recessively transmitted LGMD can mimic an autosomal dominant pattern (pseudodominance). This is particularly true in South India where uncle-niece intermarriages are frequent.
An encouraging positive trend is the increasing interest expressed by a growing number of Indian clinicians involved in neuromuscular diseases in general, particularly by those involved in LGMD. ,, They know it is no longer acceptable to give the generic, vague diagnosis of "unspecific muscular dystrophy" to their patients. This is important not only for genetic counseling but also for therapeutic prospects. The patients themselves, particularly the educated ones and those who readily access the Internet, know that by targeting the right disease-causing gene, there might be a hope toward a cure in a more or less distant future. That is the reason why the diagnosis of LGMD has to be considerably accurate and documented than ever. Muscle immunocytochemistry has just been introduced in a few major centers in India. All these techniques are improving hopefully; however, immunoblotting, a pivotal semi-quantitative technique in LGMD, is not yet available in India. In fact, for the time being, there is no way to systematically or randomly screen any of the 19 existing LGMD genes/loci.
If dystrophinopathies and spinal muscular atrophies are reasonably well studied in a couple of Indian genetic centers, it is not the case in LGMD for which not a single gene is currently screened despite a substantial number of patients with this diagnosis in India. By comparison, molecular screening of most LGMDs has been structured in Europe over the years within a network of various genetic centers that specialized in some of the LGMD subtypes. This approach might certainly be a good option for India.
Despite the logistic limitations and administrative hurdles, interesting data (some of which have been published) have accumulated over the past five years. For instance, it appears, that dysferlin deficiency (LGMD 2B) could account for a great deal, if not the majority, of LGMD patients in the area. These patients have been diagnosed on clinical grounds but also on the basis of a negative immunostain with antibodies directed against dysferlin. Unfortunately, only a tiny part of this cohort of patients underwent a thorough mutation analysis for DYSF gene. Thus far, no founder effect has ever been observed in this small Indian population.
The second largest group of LGMD patients in India is represented by sarcoglycanopathies, notably the gamma-subtype (LGMD 2C). In that respect, it is noteworthy that the so-called Mediterranean mutation in the gamma-sarcoglycan gene (del525T) was recently detected in a couple of patients examined in Mumbai (Dr. Khadilkar, personal communication). We hypothesize that this mutation could have followed the Arab migration waves centuries ago. Another common pathologic change in the gamma-sarcoglycan gene, namely the C283Y missense mutation, has not been found in India as expected. In fact, this mutation was identified first in European Gypsy patients 10 years ago. At that time, it had been hypothesized that given the genetic proximity of these patients with Indian early nomadic tribes, one ought to encounter the C283Y mutation in the Indian subcontinent. Finally, delta-sarcoglycan deficiencies appear to be present in India more often than in Caucasian populations. In the absence of calpain immunoblotting, it is still impossible to assess the prevalence of LGMD2A in India, whereas at times it represents 40-50% of LGMD patients in Europe.
Two complementary strategies are necessary to take care of the LGMD patients. Daily management based on medical or surgical interventions is largely supportive and somewhat efficient. However, innovative therapies, although promising and exciting by nature, are still at a preliminary stage and have not generated any significant clinical change to date. 
A customized therapeutic approach is always preferable for each patient. This should take into account the genetic basis of the disease, its natural course, notably when various non-skeletal muscles are potentially at risk. That is the reason why documenting the type of LGMD as early as possible is critical. For instance, patients with LGMD 2I (FKRP deficiency) should be carefully monitored for cardiac disease and dilated cardiomyopathy in particular. Young patients with early onset of sarcoglycan deficiency may develop orthopedic deformities (equinovarus, kyphoscoliosis, etc.) and should be treated accordingly with orthosis and/or surgery. On average, each LGMD adult patient should be followed-up yearly in a dedicated neuromuscular clinic. The cardiorespiratory status must be checked regularly and prophylactic measures may be helpful (flu shots and bi-PAP home ventilation in case of respiratory insufficiency, angiotensin-conversion-enzyme inhibitors for early left cardiac dysfunction, etc.). Genetic counseling is also a part of medical management. Patients and relatives should be taught on the potential risk of transmitting the disease. Prenatal testing is also theoretically available when the genetic defect is known with accuracy. In fact, even in Western countries, few couples demand it. In most instances, LGMD progresses slowly indeed and is seldom life-threatening. Therefore, from an ethical viewpoint, prenatal diagnosis (PND) is questionable in most LGMD families. Although in inbred communities with a private mutation in a given LGMD gene, the detection of heterozygotes may be relevant and would be more acceptable socially and religiously. Premarital counseling for instance is something accepted in the most open-minded Amish groups living in the United States, where LGMD2A and LGMD2E are prevalent.
The other therapeutic approach in LGMD is based on innovative therapies: conventional pharmacology, gene therapies and cell therapies. In front of the huge diversity and variability of phenotypes and genotypes in LGMD, these prospects look particularly challenging. Thus far, with the exception of a possible common denominator between a few LGMDs and Duchenne muscular dystrophy (via a defective assembly of the dystrophin-associated complex), very little is known about a possible generic pathogenesis that leads to all these diseases.
A universal approach or panacea-like treatment looks unlikely or quite in the distance. More generic drugs such as corticosteroids and new anti-fibrotic agents could be useful; however, this awaits further validation. A few patients with sarcoglycanopathy improved at least transiently with corticosteroids. ACE inhibitors are certainly helpful to treat and presumably to prevent, cardiomyopathy in some specific LGMDs (LGMD2I, LGMD2C and maybe others). Anti-myostatin agents, meant to boost muscle mass, have been recently used in a couple of LGMD patients and they may also prove to be useful.
In any case, no dramatic impact is expected from these drugs and the need for a more powerful curative approach persists. In that respect, LGMD treatment will certainly benefit from advances made in other areas of myology, notably in Duchenne muscular dystrophy. Exon skipping or gene repair techniques seem to move fast in dystrophinopathies, and the data from preclinical experiments look very exciting. Some LGMD genes might be eligible for exon skipping: dysferlin and perhaps others; however, this will require a long time to materialize. Stop mutations in various LGMD genes may also benefit from forced read-through techniques with agents developed by PTC, a US biopharmaceutical company.
As far as clinical trials are concerned, another challenge in LGMD is the relative paucity of patients worldwide. In sarcoglycanopathies, which represent up to 10-20% of all recessive progressive muscular dystrophies in Western countries, the number of patients available for clinical trials is rather limited. Recruiting volunteers is not a big issue in phase I-II trials; however, it becomes critical for further phases when large cohorts of patients will be required. Nevertheless, several clinicians undertook various trials based on gene therapy or on more conventional strategies. The most advanced project in this respect relates to gamma-sarcoglycanopathy, a disorder mostly prevalent in Northern Africa (and possibly in the Indian sub-continent). Scientists of Genethon (in Evry, France) designed an adeno-associated virus (AAV) that is capable of embarking and delivering a transgene (containing the gamma-sarcoglycan gene) to the muscle fibers. This transgene is injected locally (in the radialis muscle), but systemic delivery is regarded as a plausible option in the near future. LGMD 2D (alpha-SG deficiency) is also high on the agenda as far as gene therapy developments are concerned, particularly in the US where a similar trial is in the pipeline.
These trials are nevertheless very preliminary and encounter serious obstacles at the clinical level; gene therapy is not very popular nowadays, and it is more difficult to convince regulatory agencies regarding its relevance and inocuity. These early phases are quite costly and require a constant follow-up by various outsiders and regulators. It should be noted that even clinicians find it difficult to convince their patients to get enrolled. For instance, it took 1 year to select and enroll three French patients with LGMD 2C for the first gene therapy trial at the Institut de Myologie, Paris.
| Acknowledgments|| |
We are deeply indebted to Prof. Jean-Claude Kaplan (Cochin Hospital, Paris, France) and Prof. Michel Fardeau (Institut de Myologie, Paris, France) for their contributions and guidance in the LGMD field.
| References|| |
|1.||Walton JN, Nattrass FJ. On the classification, natural history and treatment of the myopathies. Brain 1954;77:169-231. |
|2.||Erb W. Ueber die 'Juvenile Form' des progressiven Muskelatrophie ihre Beziehungen zur sogennten Psuedohypetrophie der Muskeln. Dtsch Arch Klin Med 1884;34:467-519. |
|3.||Brooke MH. A clinician's point of view of neuromuscular diseases. Williams and Wilkins: Baltimore; 1977. p. 178-81. |
|4.||Lim L, Campbell K. The sarcoglycan complex in limb-girdle muscular dystrophy. Curr Opin Neurol 1998;11:443-52. |
|5.||Bushby K, Beckmann J. Report of the 30 th and 31 st ENMC International workshop - the limb-girdle muscular dystrophies and proposal for a new nomenclature. Neuromuscul Disord 1995;5:337-43. |
|6.||Beckmann JS, Brown RH, Muntoni F, Urtizberea A, Bonnemann C, Bushby KM. Workshop report: The 66 th /67 th ENMC sponsored workshop - the limb-girdle muscular dystrophies. Neuromuscul Disord 1999;9:436-45. |
|7.||Laval SH, Bushby KM. Limb-girdle muscular dystrophies - from genetics to molecular pathology. Neuropathol Appl Neurobiol 2004;30:91-105. |
|8.||Richard I, Broux O, Allamand V, Fougerousse F, Chiannikulchai N, Bourg N, et al . Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell 1995;81:27-40. |
|9.||Balci B, Uyanik G, Dincer P, Gross C, Willer T, Talim B, et al. An autosomal recessive limb girdle muscular dystrophy (LGMD2) with mild mental retardation is allelic to Walker-Warburg syndrome (WWS) caused by a mutation in the POMT1 gene. Neuromuscul Disord 2005;15:271-5. |
|10.||Godfrey C, Escolar D, Brockington M, Clement EM, Mein R, Jimenez-Mallebrera C, et al. . Fukutin gene mutations in steroid-responsive limb girdle muscular dystrophy. Ann Neurol 2006;60:603-10. |
|11.||Brockington M, Yuva Y, Prandini P, Brown SC, Torelli S, Benson MA, et al. Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C. Hum Mol Genet 2001;10:2851-9. |
|12.||Frosk P, Weiler T, Nylen E, Sudha T, Greenberg CR, Morgan K, et al. Limb-girdle muscular dystrophy type 2H associated with mutation in TRIM32, a putative E3-ubiquitin-ligase gene. Am J Hum Genet 2002;70:663-72. |
|13.||Moreira ES, Wiltshire TJ, Faulkner G, Nilforoushan A, Vainzof M, Suzuki OT, et al. Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein telethonin. Nat Genet 2000;24:163-6. |
|14.||Hauser MA, Horrigan SK, Salmikangas P, Torian UM, Viles KD, Dancel R, et al. Myotilin is mutated in limb girdle muscular dystrophy 1A. Hum Mol Genet 2000;9:2141-7. |
|15.||Hackman P, Vihola A, Haravuori H, Marchand S, Sarparanta J, De Seze J, et al. Tibial muscular dystrophy is a titinopathy caused by mutations in TTN, the gene encoding the giant skeletal-muscle protein titin. Am J Hum Genet 2002;71:492-500. |
|16.||Muchir A, Bonne G, van der Kooi AJ, van Meegen M, Baas F, Bolhuis PA, et al. Identification of mutations in the gene encoding lamins A/C in autosomal dominant limb girdle muscular dystrophy with atrioventricular conduction disturbances (LGMD1B). Hum Mol Genet 2000;9:1453-9. |
|17.||Ura S, Hayashi YK, Goto K, Astejada MN, Murakami T, Nagato M, et al. Limb-girdle muscular dystrophy due to emerin gene mutations. Arch Neurol 2007;64:1038-41. |
|18.||Fardeau M, Hillaire D, Mignard C, Feingold N, Feingold J, Mignard D, et al. Juvenile limb-girdle muscular dystrophy: Clinical, histopathological and genetic data from a small community living in the Reunion island. Brain 1996;119:295-308. |
|19.||Beckmann JS, Richard I, Hillaire D, Broux O, Antignac C, Bois E, et al. A gene for limb-girdle muscular dystrophy maps to chromosome 15 by linkage. CR Acad Sci III 1991;312:141-8. |
|20.||Urtasun M, Saenz A, Roudaut C, Poza J, Urtizberea J, Cobo A, et al. Limb-girdle muscular dystrophy in Guipuzcoa (Basque country, Spain). Brain 1998;121:1735-47. |
|21.||Fanin M, Nardetto L, Nascimbeni AC, Tasca E, Spinazzi M, Padoan R, et al. Correlations between clinical severity, genotype and muscle pathology in LGMD2A. J Med Genet 2007;44:609-14. |
|22.||Liu J, Aoki M, Illa I, Wu C, Fardeau M, Angelini C, et al. Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy. Nat Genet 1998;20:31-6. |
|23.||Illarioshkin SN, Ivanova-Smolenskaya IA, Tanaka H, Vereshchagin NV, Markova ED, Poleshchuk VV, et al. Clinical and molecular analysis of a large family with three distinct phenotypes of progressive muscular dystrophy. Brain 1996;119:1895-909. |
|24.||Weiler T, Bashir R, Anderson L, Davison K, Moss J, Britton S, et al. Identical mutation in patients with limb girdle muscular dystrophy type 2B or Miyoshi myopathy suggests a role for modifier gene(s). Hum Mol Genet 1999;8:871-7. |
|25.||Roberds SL, Leturcq F, Allamand V, Piccolo F, Jeanpierre M, Anderson RD, et al. Missense mutations in the adhalin gene linked to autosomal recessive muscular dystrophy. Cell 1994;78:625-33. |
|26.||Noguchi S, McNally E, Ben Othmane K, Hagiwara Y, Mizuno Y, Yoshida M, et al. Mutations in the dystrophin-associated protein gamma-sarcoglycan in chromosome 13 muscular dystrophy. Science 1995;270:819-22. |
|27.||B φnnemann CG, Modi R, Noguchi S, Mizuno Y, Yoshida M, Gussoni E, et al. Beta-sarcoglycan (A3b) mutations cause autosomal recessive muscular dystrophy with loss of the sarcoglycan complex. Nat Genet 1995;11:266-73. |
|28.||Lim LE, Duclos F, Broux O, Bourg N, Sunada Y, Allamand V, et al. Beta-sarcoglycan: Characterization and role in limb-girdle muscular dystrophy linked to 4q12. Nat Genet 1995;11:257-85. |
|29.||Nigro V, de Sa Moreira E, Piluso G, Vainzof M, Belsito A, Politano L, et al. Autosomal recessive limb-girdle muscular dystrophy, LGMD2F, is caused by a mutation in the delta-sarcoglycan gene. Nat Genet 1996;14:195-8. |
|30.||Eymard B, Romero NB, Leturcq F, Piccolo F, Carriι A, Jeanpierre M, et al. Primary adhalinopathy (alpha-sarcoglycanopathy): Clinical, pathological and genetic correlation in twenty patients with autosomal recessive muscular dystrophy. Neurology 1997;48:1227-34. |
|31.||Melacini P, Fanin M, Duggan D, Freda M, Berardinelli A, Danieli G, et al. Heart involvement in muscular dystrophies due to sarcoglycan gene mutations. Muscle Nerve 1999;22:473-9. |
|32.||Anderson LV, Davison K. Multiplex Western blotting system for the analysis of muscular dystrophy proteins. Am J Pathol 1999;154:1017-22. |
|33.||Merlini L, Kaplan JC, Navarro C, Barois A, Bonneau D, Brasa J, et al. Homogeneous phenotype of the gypsy limb-girdle muscular dystrophy with the gamma-sarcoglycan C283Y mutation. Neurology 2000;54:1075-9. |
|34.||Bonne GB, Di Barletta MR, Varnous S, Bιcane HM, Hammouda EH, Merlini L, et al. Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy. Nat Genet 1999;21:285-8. |
|35.||Minetti C, Sotgia F, Bruno C, Scartezzini P, Broda P, Bado M, et al. Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy. Nat Genet 1998;18:365-8. |
|36.||Dinηer P, Leturcq F, Richard I, Piccolo F, Yalnizoglu D, de Toma C, et al. A biochemical, genetic and clinical survey of autosomal recessive limb girdle muscular dystrophies in Turkey. Ann Neurol 1997;42:222-9. |
|37.||Passos-Bueno M, Vainzof M, Moreira E, Zatz M. Seven autosomal recessive limb-girdle muscular dystrophies in the Brazilian population: From LGMD2A to LGMD2G. Am J Med Genet 1999;82:392-8. |
|38.||Moore SA, Shilling CJ, Westra S, Wall C, Wicklund MP, Stolle C, et al. Limb-girdle muscular dystrophy in the United States. J Neuropathol Exp Neurol 2006;65:995-1003. |
|39.||Dua T, Kalra V, Sharma MC, Kabra M. Adhalin deficiency: An unusual cause of muscular dystrophy. Indian J Pediatr 2001;68:1083-5. |
|40.||Khadilkar SV, Singh RK, Katrak SM. Sarcoglycanopathies: A report of 25 cases. Neurol India 2002;50:27-32. |
|41.||Sharma MC, Mannan R, Singh NG, Gulati S, Kalra V, Sarkar C. Sarcoglycanopathies: A clinicopathological study of 13 cases. Neurol India 2004;52:446-9. |
|42.||Daniele N, Richard I, Bartoli M. Ins and outs of therapy in limb girdle muscular dystrophies. Int J Biochem Cell Biol 2007;39:1608-24. |
[Figure - 1], [Figure - 2]
[Table - 1], [Table - 2]
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