66th/67th ENMC Sponsored International Workshop: The Limb-Girdle Muscular Dystrophies 26–28 March 1999, Naarden, The Netherlands

Article Outline

• 1. Introduction
• 2. Autosomal dominant LGMD
• 3. Session 2: autosomal recessive LGMD
  • 3.1. Calpainopathies
    • 3.1.1. Primary calpainopathies
    • 3.1.2. Secondary calpainopathies
  • 3.2. Dysferlinopathy
  • 3.3. Sarcoglycan deficiency
  • 3.4. Other linked forms of ARLGMD
• 4. Conclusions
• 5. Workshop participants
• Acknowledgements
• References
• Copyright

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1. Introduction 

The workshop, which was the third of a series of ENMC sponsored LGMD workshops, was convened by Dr. Kate Bushby (UK), Dr. Jacqui Beckmann (France) and Dr. Bob Brown (USA). It was attended by 27 participants from 12 countries (listed at the end of the report). The first workshop had concentrated on the collection of clinically heterogeneous cases prior to the localization of most of the LGMD genes [1]. The second workshop had reviewed the status of the linkage data as it stood in 1995, and suggested a new gene-and locus based classification for the group [2]. The goal for the present workshop as introduced by Professor Alan Emery and Dr. Kate Bushby was to revisit the clinical findings in patients where, through the use of new diagnostic methodologies, the underlying diagnosis could be clarified. This allows the production of detailed genotype–phenotype correlations for the various groups. The collection of such data is clearly important for the provision of accurate prognostic information and genetic counselling. Widespread dissemination of the clinical findings will allow more rational use of the diagnostic techniques available.

Bert Bakker (The Netherlands) described the Leiden MD pages which act as a repository for information on all types of muscular dystrophy. These pages are collated by Johann Den Dunnen (The Netherlands), and are accessible through www.dmd.nl. The database already collects information on mutations and polymorphisms of unknown status in all of the LGMD genes. The objective (with appropriate funding) is eventually to transform this into a more comprehensive database, allowing the establishment of genotype-phenotype relationships for all these diseases. The database will therefore serve as a reference point for up-to-date information (including unpublished data) and exchange of data, as long as adequate primary criteria for access are established.

The clinical definition of muscular dystrophy was discussed by Professor M. Fardeau (Paris, France) and Dr. Bushby. The importance of careful clinical evaluation was emphasized, with various methods discussed to allow precise clinical characterization. The rate and nature of the evolution of the disease and the pattern of muscle involvement are key determinants of the ‘gestalt’ of a particular disorder. A mathematical method to evaluate relative muscle involvement was proposed by Professor Fardeau and requires further validation.

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2. Autosomal dominant LGMD 

The first session concentrated on the autosomal dominant forms of LGMD (Table 1). Michael Hauser (Durham, NC, USA) reviewed the latest data on the mapping of LGMD1A [3], for which a candidate gene is currently being evaluated.

Table 1. Dominant disease^a

Gene Jackson (Michigan, USA) summarized the findings in another chromosome 5q-linked family where distal myopathy is associated with altered speech and pharyngeal involvement. The candidate region for this disorder overlaps with LGMD1A [4].

Anneke van der Kooi (The Netherlands) described the phenotype observed in LGMD1B, an autosomal dominant LGMD linked to chromosome 1q [5]. This disorder is characterized by weakness of proximal leg muscles, slow progression and mild or late contractures. AV conduction disturbances were found in 25 of the 45 affected members for whom cardiological information was available. Two patients had a dilated cardiomyopathy. Cardiological abnormalities worsened with progression of the disease.

The area of chromosome 1q to which the LGMD1B gene has been linked encompasses the lamin A gene recently shown to be involved in autosomal dominant Emery–Dreifuss muscular dystrophy (ADEDMD) as described by Giselle Bonne (Paris, France) [6]. This disorder is characterized by AV conduction disturbance, early contractures of Achilles tendons, elbows and spine and a slowly progressive muscle wasting and weakness of predominantly humeroperoneal distribution. In one large family several members presented with pure cardiac symptoms and no muscle involvement thereby establishing variable expression of the symptoms, though no families with pure muscular weakness without cardiac involvement were seen. The finding of lamin A mutations establishes this disorder as a disease of the nuclear envelope, like X-linked EDMD.

In at least one family with a lamin A mutation, some patients had secondary laminin-β1 deficiency on ICC, though it seems so far that not all laminin-β1-deficient patients have lamin A mutations. The phenotype of these patients was reviewed by Christine Pollitt (Newcastle upon Tyne, UK) and Francesco Muntoni (London, UK) emphasising the consistency of the disease which could include early onset and severe contractures. This phenotype shows overlap with Bethlem myopathy (reviewed by Frank Baas, The Netherlands) where muscle weakness and contractures are seen in the absence of cardiac involvement. In Bethlem myopathy onset is congenital or in early childhood (5 years) with slow progression. Mutations may be found in collagen 6A1, 6A2 or 6A3. Analysis of more patients with a phenotype characterized by prominent contractures will be necessary to elucidate the relationships of these various disorders more fully.

Elisabeth McNally (Chicago, IL, USA) reviewed the current state of knowledge in LGMD1C, where caveolin 3 mutations have been described. Both missense and null mutations are reported in this muscle-restricted member of the caveolin family. The reports of possibly heterozygous and homozygous mutations in this gene have yet to be resolved [7], [8].

Two further dominant disorders were discussed. The disorder (linked to chromosome 6q13) designated LGMD1D was described by Elisabeth McNally [9]. Here, families have proximal muscle weakness with facial sparing and CK at the upper limit of normal. Like LGMD1B, cardiac involvement is very important in this group with a high frequency of cardiomyopathy, heart block and sudden cardiac death. Males are more severely affected for both skeletal muscle and cardiac disease. A candidate gene is currently being investigated.

Michael Hauser (Durham, NC, USA) described the localization of a new form of ADLGMD to chromosome 7q (LGMD1E). These families show onset in young adulthood with no cardiac or other complications [10].

The current state of knowledge with respect to all of these dominant diseases is summarized in Table 1.

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3. Session 2: autosomal recessive LGMD 

Given the much broader accumulated experience of the participants in the autosomal recessive forms of LGMD, the three main groupings calpainopathy (LGMD1A) [11], dysferlinopathy [12], [13] (LGMD1B/Miyoshi myopathy) and sarcoglycanopathy (incorporating separately deficiencies of α-, β-, γ- and δ-sarcoglycan) [14], [15] were each discussed in two sets of parallel workshop sessions. All participants took part in two of the three workshop sessions, and a wealth of clinical data was presented. The data were then presented to the whole meeting and consensus statements produced. The salient features are summarized in Table 2 and below.

Table 2. Recessive disease^a

3.1. Calpainopathies 

This session was co-ordinated by Dr. Jacques Beckmann and Dr. Andoni Urtizberea. Over 150 unrelated cases from Spain, France, Turkey, The Netherlands, the UK and the US, including the Amish, Basque and La Reunion isolates were reported.

There was an overall consensus as to the general features of these patients (despite occasional outliers) as well as the world-wide distribution of this disorder. Calpainopathy is characterized by an onset usually during the second decade, relatively slow progression, and a marked selectivity in muscle involvement.

The age of onset varies from 3 to 25 years of age with an average in the early teens. Occasionally, delayed motor milestones can be noted whereas asymptomatic young children can be picked up by early CK screening. Life expectancy is generally within a normal range and no intellectual impairment is noted.

The progression of the disease is usually relatively slow and not always linear as periods of stability and of rapid deterioration can alternate.

Muscle impairment is present in limb girdle muscles and follows a highly selective pattern. At an early stage, the selective involvement and wasting of hip extensors and adductors is highly suggestive of calpainopathy and can be visualized by muscle imaging such as CT-scanning. The posterior compartment of the lower leg is also affected but to a lesser degree. In the upper limb, weakness of the serratus, latissimus dorsi and inferior trapezius muscles contrasts with a relative preservation of the infra, supraspinatus and deltoid muscles. Contractures are usually mild and muscle pseudohypertrophy is rare. Cardiac function is normal.

Exceptions to this clinical pattern were reported in a few patients genetically proven as calpainopathy. Two brothers were presented with early involvement of the tibialis anterior. An Emery–Dreifuss-like appearance (with early contractures of both ankle and elbow but without any cardiac abnormalities) was noticed in several patients from various countries. Marked finger contractures can occasionally be seen as well as macroglossia and calf hypertrophy. Facial weakness was present in only three patients. Respiratory insufficiency can exist in some patients as well as non-specific ECG abnormalities.

Histologically, calpainopathies are characterized by a necrotic-regenerative pattern but without morphological specificity. At times, a large number of lobulated fibres can be detected. Two calpain 3 monoclonal antibodies are now used as part of a routine diagnostic procedure, thereby facilitating the detection of calpainopathies. To date, these antibodies only work on western blotting. One recognizes both the 94- and 30-kDa bands, the second the 94- and 60-kDa bands. Some patients biopsies show no calpain 3 bands whereas others show a decrease in the intensity of the corresponding bands or have normal bands.

Over 100 distinct calpain 3 (CAPN3) mutations have been identified this far and are dispersed throughout the entire length of the coding sequence. In addition, some neutral polymorphisms have also been reported, stressing the importance of assessing the pathogenic nature of the observed calpain 3 variants before concluding a molecular diagnosis. All these variants and mutations have been deposited in the Leiden MD web pages. It was important to note that observation of patients with suggestive clinical or protein data but with no detectable calpain 3 mutations leads one to also consider the possibility of secondary calpainopathy. The collation of those many calpain 3-deficient cases was convincing that a differential clinical diagnosis is now feasible. Just to cite a few examples, it is easy clinically or especially with CT scans, to distinguish calpainopathies from BMD, sarcoglycanopathy or dysferlinopathy. So for example the quadriceps is less affected in calpainopathies than the biceps femoris (the reverse from sarcoglycanopathy and BMD). In addition the posterior and anterior thigh involvement is identical in sarcoglycanopathies, while in calpainopathy the posterior thigh is more prominantly involved.

Bjarne Udd described a large tibial muscular dystrophy (TMD) pedigree with remote consanguinity, in which eight patients in three families have an LGMD-like presentation. Since all these patients were old when first seen (the youngest one was 45 years old), there is no documentation on the early stages of the disease. Onset is reported to be between 5 and 10 years of age, for severely affected patients, who become WCB at 30. Milder affected LGMD patients have onset at 20, and are still ambulatory. Both the biceps and triceps are equally involved (unlike calpainopathies). There is no facial weakness or cardiac involvement, and the distal muscles are less affected. CT scan shows complete loss of the thigh muscles and involvement of the tibialis anterior. In the severe cases, the only spared muscle is the tibialis posterior.

The distal TMD phenotype is linked to 2q31 (possibly the titin locus). Five of the LGMD patients are homozygous for this region. Titin staining with several antibodies is not abnormal. Preliminary data suggest that homozygous patients lack the calpain 3 94 kDa band on western blots, showing only the 30 kDa. These results are compatible with this being possibly a secondary calpainopathy.

3.2. Dysferlinopathy 

The dysferlinopathy session was convened by Dr. Kate Bushby and Dr. Bob Brown (Boston, MA, USA).

Patients with dysferlinopathies typically have normal motor development and function during their early years. The mean age of onset of weakness is about 20±5 years; weakness from dysferlin mutations has not been documented before 10 years. In all cases, the initial weakness in the legs and may be asymmetrical. Its distribution is variable. In some cases it may affect the proximal hip girdle muscles. In others it is distal at onset. Less frequently, the initial distribution is both proximal and distal. When the weakness begins distally, it is usually posterior, with prominent gastrocnemius muscle weakness (‘Miyoshi myopathy’ phenotype). In such cases, plantar–flexion weakness may cause difficulty walking on the toes; women may have difficulty wearing high-heeled shoes. In rare cases, the anterior tibial muscles may be more affected. Infrequently, gastrocnemius weakness is preceded by swelling and tenderness that may last several weeks to months. Early in the illness, there may also be focal asymptomatic biceps weakness with rather striking atrophy of part of the biceps muscle.

An invariant feature of the dysferlinopathies is marked elevation of serum CK levels. At the time the weakness develops, the CK may be 4–200-fold elevated. The combination of a markedly elevated CK in an individual with gastrocnemius weakness beginning in early adulthood is almost pathognomonic for the Miyoshi form of dysferlinopathy.

Regardless of the pattern of weakness at onset most of the lower limb muscles later become involved and weakness in the upper extremities develops as the disease progresses. This prominently involves the humeral muscles, while the deltoid and scapular muscles are usually relatively spared. In general, the rate of progression is slow. Most dysferlin patients walk until the mid-thirties or beyond. However, there are some cases with typical age of onset but a more fulminant course.

Electromyographic studies in these patients reveal myopathic features; fibrillations are often evident. Muscle biopsies of affected muscles reveal dystrophic features. In addition, it is not uncommon to find mild to moderate degrees of inflammation, sometimes in a perivascular distribution. Dystrophin and other components of the dystrophin complex are normal. By contrast, levels of dysferlin are markedly reduced. Mutations in the dysferlin gene (DYS) are variable and widespread and so far no clear genotype–phenotype correlations are apparent.

Dysferlinopathy patients do not develop cardiomyopathies, respiratory or facial muscle weakness, or cognitive impairment. Contractures almost never occur.

It is now evident that some cases with the Miyoshi myopathy phenotype arise from genetic defects other than dysferlinopathies. Some families with this phenotype are linked to chromosome 10. Clinical findings do not distinguish these subsets of Miyoshi myopathy, although it appears that the chromosome 10 cases may have a somewhat older age onset (late 20’s).

3.3. Sarcoglycan deficiency 

The sarcoglycan session was convened by Professor Francesco Muntoni (London, UK) and Dr. Carsten Bonnemann (Göttingen, Germany). It was clear that while a number of characteristics can be discussed with respect to this group as a whole, data collection needs to continue with respect to the four different entities (α, β, γ and δ sarcoglycan deficiency) in this group to ensure the disorders are not inappropriately lumped together prematurely. Having said that, across the group some general conclusions could be reached.

The sarcoglycanopathies are extremely variable though overall onset tends to be earlier than in calpainopathy. This variability is especially marked in the rate of disease progression, which may even be very variable between siblings. There is generally a poor correlation between the age at onset and outcome. A few patients may show an even more rapid deterioration than DMD though this is rare.

Delayed motor milestones are less frequent than in DMD, — even if severity later is very marked. Symptoms at presentation include weakness, toe walking and muscle pain/cramps which may be more common than in DMD. The earliest reported age of wheelchair confinement was 10 years. The variability towards the milder end is very marked indeed. A few patients are still ambulant beyond 40s and occasionally asymptomatic people are detected even at a very late age. Alpha-sarcoglycan deficiency especially may be extremely mild.

Age at death is usually >30 years, with respiratory failure probably usually later than DMD. The pattern of muscle involvement was studied to try and discern differences to DMD. Shoulder girdle involvement was usually more significant as was scapular involvement. Hamstrings were more involved than quadriceps. Foot drop was seen in some before loss of mobility which is rare in dystrophinopathy.

Hypertrophy and macroglossia do not distinguish sarcoglycanopathy and DMD. Lordosis may be important and makes rehabilitation in callipers difficult. Scoliosis may be present, but seems less prevalent and less severe than DMD. There is no intellectual retardation. Cardiac involvement is not invariable as in dystrophinopathy but may be important, though this appears to vary between sarcoglycanopathies so that in alpha-sarcoglycanopathy it is usually not present (reported in one Dutch patient), while in delta, beta and gamma-sarcoglycanopthy it may be more prevalent.

From the diagnostic perspective it is important to note that a secondary reduction in dystrophin in muscle may be present. The diagnostic scheme should include a muscle biopsy using a range of antibodies and also take into account the ethnic background of the patient, as certain mutations are particularly prevalent in specific populations. Even in patients with suggestive sarcoglycan staining patterns, in a significant proportion (25–30%) the mutations cannot currently be identified.

3.4. Other linked forms of ARLGMD 

Klaus Wrogemann (Winnipeg, Canada) presented data on LGMD2H, a form of LGMD seen in the Hutterite population of Manitoba [16]. The LGMD (LGMDH) family, whose ancestry can be traced 11 generations back to 1800, contains over 60 patients. Their LGMD is a relatively mild disorder, with proximal muscle weakness, and dystrophic muscle biopsy, CK>15× normal, even in asymptomatic individuals. This locus maps in the vicinity of the Fukuyama locus, but in a distinct region D9S1855 and 1802, or possibly between D9S241 and D92154.

Mayana Zatz (Sao Paulo, Brazil) described the LGMD2G locus on chromosome 17 [17]. In one family with six patients, most patients were WCB between 31 and 40. None had calf hypertrophy. Calf hypertrophy in one patient from a second family was however noted.

Several recessive families unlinked to any of the as yet known loci remain and were presented by Mayana Zatz, Klaus Wrogemann and Haluk Topaloglu (Ankara, Turkey). Many of these families are small and appear to be clinically heterogeneous. Nonetheless, it would appear that other loci remain to be described. The importance of the use of a full range of diagnostic investigations was emphasized by Dr. Topaloglu who described an LGMD-like phenotype in patients with partial merosin deficiency.

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4. Conclusions 

The workshop ended with a session agreeing the content of the summary tables collating the clinical data which was the primary focus of this workshop (Table 1, Table 2). The meeting concluded that a characteristic clinical phenotype could be identified for each of the genetically determined subtypes of LGMD. Muscle imaging techniques such as CT scanning may be very useful in delineating patterns of muscle involvement. While there is a definable phenotype which is seen in the majority of patients with any particular type of LGMD, it is also important to recognize that exceptions to these general rules may be seen, and that in all groups intrafamilial variability may be marked. For these reasons a full diagnostic evaluation will continue to be imperative in all suspected cases of LGMD, preferably through the analysis of a muscle biopsy using a range of antibodies followed by directed mutation analysis.

Efforts to utilize the facilities of the Leiden database were discussed as a repository for information relating to genotype, protein information and phenotype. Curators for the various disorders were discussed, on the understanding that all information contributed would have to be coordinated and its validity ensured. Bruno Eymard (Paris, France) and Carsten Bonneman were nominated as curators for the sarcoglycanopathies, Jacqui Beckmann and Christine Pollitt for calpainopathy and Bob Brown and Kate Bushby for dysferlinopathy. Louise Anderson (Newcastle upon Tyne, UK) agreed to co-ordinate collection of protein data.

Future targets were set as moving towards an understanding of the pathophysiology of the different forms of LGMD. To this end, the consortium will look into the prospects of obtaining European or other funding and plan a further ENMC workshop in due course. We are very grateful to ENMC for the opportunity to exchange data in this forum.

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5. Workshop participants 

Dr. Louise Anderson, UK 

Dr. Z. Argov, Israel 

Dr. Rumaisa Bashir, UK 

Professor J.S. Beckmann, France 

Professor M. Ben Hamida, Tunisia 

Dr. G. Bonne, France 

Dr. C. Bonnemann, Germany 

Dr. R.H. Brown, USA 

Dr. K.M.D. Bushby, UK 

Professor M. de Visser, Holland 

Professor M. Fardeau, France 

Dr. M. Hauser, USA 

Dr. S. Illarioshkin, Russia 

Dr. E. Jackson, USA 

Professor J-C. Kaplan, France 

Dr. A. Lopez de Munain, Spain 

Dr. I. Mahjneh, Finland 

Dr. E. McNally, USA 

Professor F. Muntoni, UK 

Dr. Christine Pollitt, UK 

Dr. H. Somer, Finland 

Dr. V. Straub, Germany 

Dr. H. Topaloglu, Turkey 

Dr. B. Udd, Finland 

Dr. A. Urtizberea, France 

Professor K. Wrogemann, Canada 

Dr. M. Zatz, Brazil 

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Acknowledgements 

This workshop was made possible thanks to the financial support of the European Neuromuscular Centre (ENMC) and ENMC main sponsors:

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