98th ENMC International Workshop on Congenital Muscular Dystrophy (CMD), 7th Workshop of the International Consortium on CMD, 2nd Workshop of the MYO CLUSTER project GENRE:

Article Outline

• 1. Introduction
• 2. CMD with rigidity of the spine
  • 2.1. RSMD1
    • 2.1.1. Clinical phenotype
    • 2.1.2. Pathological features
    • 2.1.3. Muscle imaging data
    • 2.1.4. Genetic analysis
    • 2.1.5. Summary
  • 2.2. Ullrich CMD (UCMD)
    • 2.2.1. Clinical phenotype of cases secondary to collagen 6 abnormalities
    • 2.2.2. Pathological features
    • 2.2.3. Genetic analysis
    • 2.2.4. Summary
• 3. Glycosyltransferases and congenital muscular dystrophy: MDC1C and MEB
  • 3.1. MDC1C
    • 3.1.1. Clinical features
    • 3.1.2. Pathological features
    • 3.1.3. Genetic analysis
    • 3.1.4. Summary
  • 3.2. Muscle eye brain disease (MEB)
    • 3.2.1. Clinical features
    • 3.2.2. Muscle pathology
    • 3.2.3. Molecular genetics
    • 3.2.4. Summary
  • 3.3. Congenital glycosylation disorders (CGD)
• 4. Animal model of CMD
• 5. List of participants
• Acknowledgements
• References
• Copyright

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1. Introduction 

The ENMC Consortium on Congenital muscular dystrophy (CMD) held its seventh meeting in Naarden during the weekend of October 26–28, 2001. It was attended by 23 participants from seven countries, which included Denmark, France, Germany, Italy, Turkey, the United Kingdom and the United States. This workshop was sponsored by the European Community and represented the second meeting of the Myocluster project ‘GENRE’ (Genetic Resolution of Congenital Muscular Dystrophy).

This meeting focused on three main areas: (1) CMD syndromes characterized by rigidity of the spine (RSS), with or without distal laxity, including therefore CMD with rigidity of the spine mapped to chromosome 1p (RSMD1) and Ullrich CMD (UCMD); (2) the identification of the genes (two glycosyltransferases) responsible for Muscle Eye Brain Disease (MEB) and for a novel form of CMD characterized by muscle hypertrophy (MDC1C); (3) the presentation of various animal models relevant for CMD or its treatment (myd and dy/dy mice).

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2. CMD with rigidity of the spine 

2.1. RSMD1 

The main clinical features of RSMD1 were presented by various participants, including Merlini (Bologna, Italy), Straub (Essen, Germany), Muntoni (London, UK), Quijano-Roy (Garche, France) and Haliloglu (Ankara, Turkey). Mutation analysis was performed by the laboratory of Guicheney (Paris, France) in each case.

The ‘classical’ phenotype is that of a patient born without contractures who may be moderately floppy in the first few months of life and acquire independent ambulation with some delay, or otherwise walk at a normal age. The first concern for those children with normal motor milestones can be the development of cervical-dorsal spine stiffness (invariable in the first decade of life) and scoliosis. Most patients eventually develop a progressive scoliosis; only occasionally, this can be absent and a marked lordosis present instead [1], [2], [3].

Merlini presented the natural history of spinal deformity in eight patients with RSMD1. Six patients showed a childhood onset of progressive lordoscoliosis with lateral bending and forward flexion of the trunk. This pattern is typical of the severe rigid spine syndrome and reflects the early severe involvement of the axial muscles and the mechanical consequences of the spine growth hampered by the fibrotic axial muscles [4]. These patients required mechanical ventilation at the age of 10–14 years. Two patients with a milder clinical picture had a mild scoliosis without lateral bending and forward flexion of the trunk.

The weakness in these patients and the remaining ones presented is predominantly axial, and, in the limbs, the proximal muscles are more affected than the distal muscles. The muscle built is slender, without significant muscle hypertrophy or atrophy. Strength remains on the whole stable. There is usually mild facial weakness and nasal voice and a typical facial appearance with mild mid-face hypoplasia. Significant limb contractures are unusual; a few patients may develop elbow and Achilles tendon contractures, potentially leading to confusion in the differential diagnosis with Emery–Dreifuss muscular dystrophy (EDMD). As a general rule, contractures are usually milder and of later onset in RSMD1 compared to EDMD patients. Axial weakness precedes spinal stiffness and vertebral deformity, and therefore early orthopaedic intervention should be attempted by the means of a corrective thoraco-lumbar orthosis to minimize the deformity and improve later on the results of surgical fixation (arthrodesis) after the growth period is finished, if possible. Otherwise, the development of a severe scoliosis may lead to the loss of walking due to orthopaedic difficulties. Surgical approach on the other hand can be difficult in those patients with a severe and very early onset respiratory insufficiency. The restrictive respiratory syndrome leads typically to early respiratory failure, due to a combination of stiffness of the rib cage and diaphragmatic weakness. Diurnal hypercapnia and nocturnal central apnoeas are early signs that need to be surveyed regularly. Most patients need nocturnal ventilation by the early teens, but some cases as early as 4 years of age. Non-invasive (nasal) ventilation appears appropriate as a long-term solution for the great majority of RSMD1 patients. Intelligence and cardiac function are normal. Serum CK levels are normal or only mildly elevated.

A review of the muscle pathology of the cases in London (C. Sewry) revealed myopathic changes with no necrosis and little regeneration. Endomysial connective tissue was occasionally mildly increased. Unevenness of oxidative enzyme staining was common.

The muscle biopsy findings vary from minimal changes to myopathic; necrosis and regeneration are seen only rarely although dystrophic changes can be observed in the paraspinal muscles. Unevenness of staining with oxidative enzyme histochemistry is a feature in some patients while expression of the standard sarcolemmal and extracellular matrix proteins associated with various types of muscular dystrophy is invariably normal.

Muscle imaging data (MRI and CT scanning) were presented by Mercuri (London, UK) and Merlini, who reported a specific pattern of leg muscle involvement. The most affected muscles in the thighs were adductors, sartorius and biceps femoris, while vastus lateralis, intermedius and medialis, semimembranosus and semitendinosus were also affected but to a lesser extent. Rectus femoris and gracilis were always relatively spared. In the lower leg the gastrocnemius was the most involved muscle, followed by extensor digitorum and peroneal. Tibialis anterior and soleus were less affected. Merlini in addition showed that there is an early involvement of the posterior spinal muscles, particularly at the thoracic and lumbar levels, and that the axial muscles are the muscles most severely affected in each patient.

Guicheney described the spectrum of mutations identified in patients with RSMD1. The gene responsible is SEPN1, encoding for the recently described selenoprotein N1. This gene is composed of 13 exons and a total of 16 mutations have been identified in 22 families so far. These include loss of function causing mutations (non-sense, frameshift) and missense and splicing mutations. Guicheney and Topaloglu (Ankara, Turkey) also reported the first prenatal diagnosis of RSMD1.

Guicheney finally reported data on genetic heterogeneity as several cases with a phenotype apparently indistinguishable from typical RSMD1, but without mutations in the coding region of the SEPN1 gene or unlinked to 1p, were identified.

RSMD1 is a well defined form of RSS; its diagnosis can be suspected on clinical grounds in patients with RSS and a predominantly axial myopathy with mild facial weakness, nasal voice and restrictive respiratory compromise. Muscle imaging with either CT or MRI may help in confirming the selective pattern of involvement described in RSMD1. The long-term prognosis of these patients mostly relies on the recognition and early treatment of the respiratory complications and of the associated scoliosis.

Corrective thoraco-lumbar orthosis and close surveillance of the progressive scoliosis is important in RSMD1. Respiratory insufficiency typically leads to nasal nocturnal ventilation in the first decade of life or early teens. In general no significant deterioration of the weakness is observed, but some patients may lose the ambulation especially if they develop severe deformities. Spinal surgery (vertebral arthrodesis) in these advanced cases might further compromise the functional and respiratory abilities of some of these severely affected patients and the pros and cons of surgical intervention will have to be carefully evaluated in each individual case.

So far, attempts to generate diagnostic antibodies that could be used in immunocytochemistry or Western blot analysis have not been successful. The diagnosis can therefore only be confirmed by appropriate molecular genetic testing. A relatively small proportion of patients with a similar phenotype to RSMD1 have been shown not to link to this locus nor to have mutations in the SEPN1 coding region, suggesting genetic heterogeneity. Prenatal diagnosis for RSMD1 is now possible.

2.2. Ullrich CMD (UCMD) 

This session was started by Bertini (Rome, Italy); additional cases were presented by Haliloglu, Mercuri, Bushby (Newcastle, UK), Straub (Essen, Germany), Bönnemann (Philadelphia, USA), Quijano-Roy and Merlini.

The discussion focused on patients with the classical features of UCMD (proximal contractures, distal laxity, rigidity of the spine and respiratory complications) with evidence of primary involvement of collagen VI demonstrated either by mutations in one of the COL6A genes; linkage to one of these genes in informative families; and/or reduced collagen VI expression in muscle biopsies.

The main clinical features of UCMD patients were onset at birth with hypotonia and/or contractures, including congenital arthrogryposis, extended talipes, torticollis or hip dislocation [5]. The frequent occurrence at birth of transient kyphotic deformity was highlighted by Bertini. The maximal functional abilities varied significantly: while several patients acquired the ability to walk, usually late, some never acquired ambulation. Increasing contractures of the proximal joints and of the Achilles tendons were frequently observed, leading to progressive loss of functional abilities. In some patients the distal laxity did not persist in the late stages of the disease; marked finger flexion contractures appeared. In a few patients with collagen VI involvement at the severe end of the spectrum, laxity was never a prominent feature, not even at presentation. Other distinctive features were a rounded facial appearance with prominent ears, posterior calcaneus protrusion, thickening of the subcutaneous tissue on the sole of the feet and follicular hyperkeratosis. Respiratory problems were frequently observed in terms of restrictive syndrome leading to respiratory failure in the first or second decade of life. This was managed non-invasively with satisfactory results in most cases but not always, and invasive ventilation may be required in the more severe cases. Intellectual and cardiac functions were normal in all cases with collagen VI involvement. Interestingly, three unrelated families with an UCMD phenotype but in addition with mild mental retardation, short stature and a progeroid facial appearance were reported by Mercuri, Bushby and Straub. Primary collagen VI involvement was excluded in the informative families, suggesting this represent a novel clinical entity.

The range of the muscle biopsy changes observed in cases with primary collagen VI involvement were reported by Bertini, Topaloglu, Sewry (Oswestry, UK), Brown (London, UK), Bushby, Squarzoni (Bologna, Italy); Straub and Bönnemann. Regarding the degree of pathological changes, these vary from myopathic to mildly dystrophic, depending on the timing and site of the muscle biopsy. Bertini reported immunocytochemical studies with antibodies COL6–140 kDa (2C6 antibody [6]), while Brown and Sewry used two commercially available antibodies raised against total collagen type VI. Squarzoni used α3 chain, globular domain antibodies, while Bönnemann used MAB3303 Chemicon, raised against total collagen type VI.

Bertini discussed the results obtained in patients with proven COL6 mutations. The expression of collagen VI varied from mild reduction to absent, without any obvious correlation between the severity of the collagen depletion and the clinical features of the patients affected. Brown and Sewry summarized the immunocytochemical data of collagen VI in the series of cases studied at the Hammersmith Hospital; only linkage analysis was available for these cases.

Equivalent endomysial and perimysial labelling was observed with both antibodies, and the endomysial/basal lamina labelling was considered to be of most diagnostic relevance. Two genetically linked cases showed unequivocal results with absence of sarcolemmal labelling in one and a pronounced reduction in the other. Four linked cases showed equivocal results in which a possible reduction in labeling was more difficult to assess. As normal expression of collagen VI has been observed in cases with UCMD, is not yet clear if collagen VI expression will prove to be a consistently reliable marker or if this is a reflection of genetic heterogeneity. The exclusion of linkage to collagen VI in a significant number of UCMD suggests that the latter possibility accounts for at least a proportion of cases.

The use of antibodies to other extracellular matrix proteins such as collagen IV or V to control for the preservation of the basal lamina were discussed and recommended. Studies of collagen VI expression in a chorionic villus sample showed that immunocytochemistry could be a useful aid for prenatal diagnosis. The use of controls, such as collagen IV expression, and knowledge of expression in the proband were recommended in the context of prenatal diagnosis.

Further immunohistochemical and ultrastructural studies were reported by Squarzoni and Bönnemann. Squarzoni discussed the immunohistochemical and electron microscopy investigations of collagen 6 expression in a patient affected by UCMD, genetically related to a recessive mutation in the Col6 A3 gene (R465X). This patient had an unusually mild clinical phenotype. Immunohistochemical analyses were made in skeletal muscle and skin biopsies as well as in cultured skin fibroblasts. Electron microscopy (EM) study was done in fibroblast cultures by the rotary-shadowed replica method. A reduction of Col 6 was found in the endomysium between muscle fibres, with the exception of capillary basal lamina and fibrotic areas. Remarkably, Col 6 labelling was specifically decreased in the papillary dermis and around skin hair follicles, while it was preserved at normal levels in the dermal structures, which included smooth erector pili muscle, glands, vessels and peripheral nerves. Perlecan, dystrophin related proteins, laminin α2 and collagens 3 and 7 were normally expressed in the tissues examined, while laminin β1 only was slightly decreased. In cultured fibroblasts, there was a marked reduction of collagen 6 labelling in the extracellular matrix, and a total loss of the normal three-dimensional fibrillar network. The residual labelling pattern consisted of fluorescent elongated spots between cells. EM rotary-shadowing replica method analysis showed a reduction of assembled collagen 6 in the extracellular matrix and the absence of complex three-dimensional microfibrillar networks. The ultrastructural analysis of collagen 6 filaments demonstrated alterations in the globular domain pacing and the filament structure was often coarser than normal. Regular, parallel-running filaments were generally absent, often substituted with fused ones. A peculiar tendency to develop circular arrays of filaments was evident. These arrays failed to connect with other ECM components and the general impression at the EM was that the ECM in toto was scarcely organized.

Bönnemann reported ultrastructural findings on skin biopsies from seven patients with various degrees of muscle weakness in conjunction with hyperlaxity in all and contractures in some. The most severe group (three patients) could be classified as UCMD. In this group there were two patients with a mild reduction in collagen VI immunoreactivity, all the other patients had normal collagen VI immunohistology. Findings on electron microscopy of skin biopsy specimens revealed collagen fibrils with abnormal shapes and diameters in five patients (resembling ultrastructural findings seen in classical Ehlers–Danhlos syndrome), and abnormally loose bundles with increased ground substance in two. In the two patients with partial deficiency of collagen type VI, there were fibrillar abnormalities in one and loose packaging of fibrils with increased ground substance in the other. The heterogeneity within this group of patients suggests that there may be additional entities causing abnormalities of the collagen complement.

Brown reported the expression of collagen VI in the human trophoblast from 12 weeks of gestation onwards.

Linkage analysis studies were reported by Brockington to the COL6A1 and two genes in two families and to COL6A3 in one family. Linkage analysis excluded these loci in three additional UCMD families.

Mutation analysis of the collagen 6 genes was reported by Bertini and Guicheney. Bertini reported four mutations in the COL6A 2, three mutations in Italian patients and the fourth one in a Japanese patient: a homozygous C insertion in exon 13 in a patient with consanguineous parents; a heterozygous −2 A→G substitution in IVS 17 in two other unrelated patients one of which carries a second mutation, a −1 G→A substitution in IVS23 while the second mutation of the other patient is still unknown; a homozygous 26 nt DNA deletion in exon 14 was found in a Japanese Ullrich patient with consanguineous parents. All these mutations, identified in the laboratory of Dr Pepe, in Rome, cause a frameshift and a downstream premature termination codon, which results in a truncated collagen VI α2 chain, impairing assembly of tetramers that are supposed to be degraded intracellularly.

Guicheney reported three mutations in COL6A3: (i) a homozygous nonsense mutation leading to a complete deficiency, (ii) a homozygous nonsense mutation in the N-terminal domain inducing in frame exon skipping and resulting in a partial collagen type VI deficiency and a mild phenotype, and (iii) a homozygous splice mutation leading to an in-frame deletion in the distal part of the helical domain and also to only a partial deficiency and an intermediate phenotype [7].

Brown, Brockington and Bushby reported the application of linkage analysis and immunocytochemical studies in the prenatal diagnosis of a UCMD family linked to the COL6A3 gene.

UCMD is a common CMD syndrome and an involvement of collagen VI can be demonstrated in a significant portion of cases. However, a primary role of this protein or the corresponding genes was excluded in several cases, suggesting that there is genetic heterogeneity. Moreover, a novel ‘UCMD plus’ syndrome, characterized by short stature and mild mental retardation was reported for the first time.

Immunocytochemical studies in cases with COL6 gene mutations was variable: in some cases it showed a striking reduction or absence of protein but in several patients there were mild changes were not easy to evaluate. Ultrastructural studies or studies on collagen expression in cultured fibroblasts might provide additional information as they appear to show more clear abnormalities. The data available at the moment suggest that skin biopsy might be considered a useful diagnostic tool in UCMD.

Further studies on the sensitivity and specificity of the reported changes are currently under way.

Prenatal diagnosis is now feasible for UCMD, when clear biochemical and genetic data are available.

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3. Glycosyltransferases and congenital muscular dystrophy: MDC1C and MEB 

3.1. MDC1C 

Muntoni, Voit, Quijano-Roy and Romero (Paris, France) presented the clinical features of children with mutations in the fukutin-related protein (FKRP): mutations in this gene were recently shown to underlie a novel form of CMD, named MDC1C [8], [9] and the mutation analysis in all reported cases was performed in London in the laboratory of Muntoni. Characteristic clinical features of this form of CMD are presentation at birth or in the first few weeks of life with hypotonia and weakness but no contractures. Pseudohypertrophy of the leg muscles, facial weakness and severe axial and proximal weakness with marked delay of motor milestones and inability to stand or walk are invariably found. Muscle weakness and wasting involves the arms more than the legs. Progressive pseudohypertrophy of the tongue required partial glossectomy in a few cases in the second decade of life. Serum CK is markedly elevated while intelligence and brain imaging is usually normal. Affected children develop respiratory failure in the second decade of life and echocardiography evidence of left ventricular dilatation is also common. Peripheral nerve studies are normal.

Muntoni, Voit, Romero and Quijano-Roy presented also cases with later presentation (but within the first year of life) who acquired the ability of walking, but developed rapidly progressive weakness with loss of independent ambulation in the first decade of life, followed by respiratory failure. These cases appear to have an ‘intermediate’ phenotype between MDC1C and limb girdle muscular dystrophy 2I (LGMD2I), a milder allelic variant of MDC1C [10].

Topaloglu presented the features of two unrelated families with very recently identified FKRP mutations in whom, in addition to the muscle weakness, there was severe mental retardation and structural brain involvement, in the form of cerebellar cysts. One of these families had been previously described as a novel form of CMD with central nervous system involvement [11].

Muntoni and Voit briefly presented the clinical features of other CMD variants characterized by secondary merosin deficiency and α-profound α-dystroglycan depletion that are genetically separate from MDC1C: some of these families link to the MDC1B locus on chromosome 1q42 [12]. Others do not link to either the MDC1B and MDC1C loci.

The muscle pathology features of children with MDC1C were presented by Brown, Sewry, Voit, Topaloglu and Romero. These show an active dystrophic process with prominent necrosis and regeneration. Immunohistochemical studies often showed a significant reduction of laminin α2. The most striking abnormality, however, was the reduced detection of α-dystroglycan, using a commercially available antibody (V1A1-4). In a few cases labelling of muscle fibres was virtually absent. Furthermore a decrease in the molecular weight of α-dystroglycan on Western blot was documented in the cases that could be studied with this technique, suggesting that this molecule is abnormally glycosylated in MDC1C. The immunocytochemical expression of other dystrophin-associated proteins such as the sarcoglycans and β-dystroglycan is apparently normal both at the immunocytochemical and Western blot level.

Brockington reported the identification of the fukutin-related-protein gene (FKRP) in collaboration with Blake (Oxford, UK). Sequence comparison suggests that FKRP contains a hydrophobic transmembrane-spanning region followed by a ‘stem region’ and the putative catalytic domain. A similar molecular organization is found in several Golgi-resident glycosyltransferases, therefore in enzymes involved in the glycosylation of other molecules. The genomic organization of the FKRP is characterized by 3 non-coding exons and a single large exon of 3.8 kb that contains part of the 5′ untranslated region and the entire open frame and 3′ untranslated region.

A total of 14 mutations were identified in ten families. These were 11 missense and three nonsense. To date we have not observed two null alleles suggesting that a total absence of FKRP function may be embryonic lethal. The mutations identified in the two families reported by Topaloglu with cerebellar cysts [11] were missense mutations which were not located in a distinct region of the gene.

Brockington also reported the identification of allelic mutations in patients with a much milder phenotype, giving rise to a form of limb girdle muscular dystrophy that had been mapped before to an identical locus (LGMD2I). Mutations were reported in 17 LGMD2I families [10].

Brockington finally briefly updated the status of the investigations regarding another CMD locus characterized by secondary merosin and α-dystroglycan deficiency (MDC1B [12]): he reported the refinement of the MDC1B locus on 1q42 as a result of a recombinant event identified in a German family recently studied in collaboration with Voit.

MDC1C is a severe form of CMD; diagnostic clues are the significant elevation of serum CK, the leg muscle pseudohypertrophy (followed by tongue enlargement in the late stage of the disease), and the severity of weakness that includes the face and the respiratory muscles. Mutations in the same gene have been recently associated with the milder allelic variant LGMD2I, and patients with an ‘intermediate’ phenotype between MDC1C and LGMD2I were reported. These latter patients may acquire the ability to walk, but show subsequently a rapidly progressive course, loosing all motor acquisitions and becoming ventilation dependent. Cardiac involvement is possible.

Intelligence is usually preserved, although two cases with mental retardation and cerebellar cysts were identified very recently, raising the question of the spectrum of the phenotype of this condition.

Secondary merosin deficiency is a feature of MDC1C; however, this is not always a striking feature. More dramatic abnormality of α-dystroglycan expression was constantly identified in all cases with MDC1C, and also in most of the LGMD2I patients. This is in keeping with the recently reported abnormalities of α-dystroglycan expression in Fukuyama CMD [13]. The result of the biochemical studies in MDC1C and the sequence comparison of FKRP suggest that this protein is involved in the glycosylation of α-dystroglycan.

Mutation analysis is available for this form of CMD: all mutations identified in MDC1C so far lie in the open reading frame.

Other variants of CMD with secondary merosin deficiency and profound α-dystroglycan depletion not linked to the FKRP locus exist, suggesting further heterogeneity of this group of conditions. Further studies aimed at clarifying the spectrum of conditions characterized by deficiency of α-dystroglycan are under way.

3.2. Muscle eye brain disease (MEB) 

Topalglu and Voit reported the clinical features of four families with molecularly confirmed MEB.

The cases reported all had severe hypotonia, feeding difficulties, and failure to thrive in the first months. Generalized weakness and marked delayed motor development are invariable, with only a few children eventually achieving ambulation. Mental retardation is a constant feature and it is generally severe, with only few cases achieving limited speech. Epilepsy was also common. Brain MRI showed grossly abnormal gyral formation, with frontal, temporal, and parietal pachygyria and occipital polymicrogyria. Enlarged ventricles, brainstem hypoplasia and cerebellar hypoplasia were also often present. Periventricular white matter changes are often present but limited. Regarding the ocular involvement, this included severe myopia, retinal hypoplasia, glaucoma, nystagmus and cataracts. Serum CK levels were elevated in all cases, approximately ten times normal values.

The muscle biopsy shows a classical dystrophic picture, with variation in fibre size and necrotic and regenerative fibres in the infantile period and progressive increase in connective tissue and adipose tissue at a later stage. Laminin α2 chain was reduced. The expression of α-dystroglycan was virtually absent in the cases studied.

The MEB gene has recently been identified [14]. This encodes a glycosyltransferase, protein-O-mannose-β1,2-N-acetylglucosaminyltransferase (POMGnT1) which participates in the protein-O-mannosyl glycan synthesis. This gene is composed of 22 exons and the mutations identified so far include missense, splicing and frameshift mutations. Homozygosity for loss-of-function causing mutations was reported, suggesting that total absence of POMGnT1 is compatible with life. Many of the identified mutations apparently lead to a reduction in enzymatic activity. The abnormal immunolabelling of α-dystroglycan in MEB suggests that POMGnT1 might be involved in O-mannosylation of α-dystroglycan.

The association of severe muscle weakness in a child with marked elevation of serum CK, absent speech, severe myopia and cortical pachygyria, enlarged ventricles and brainstem and cerebellar hypoplasia should alert clinicians of the possibility of MEB. Immunolabelling of laminin α2 is reduced and that for α-dystroglycan is virtually absent. The POMGTn1 gene appears to be a glycosyltransferase which very likely is involved in α-dystroglycan processing. The condition is, however, genetically heterogeneous.

Muntoni, Merlini, Bertini, Wever and Voit reported the clinical features of patients with similarities to MEB but in whom the MEB locus or gene had been excluded. These included patients with the previously reported syndrome ‘Microcephaly-muscle hypertrophy-cerebellar hypoplasia’, described in families from Italian descent [15]. Two more families with this syndrome were identified in South France by Quijano-Roy and another one in Sardinia by Muntoni. These children and the ones with another ‘MEB-like’ condition described before by Voit et al. [16] were all found to share a profound depletion of α-dystroglycan. Considering that FCMD, MEB and MDC1C were excluded in these patients, further heterogeneity of this ‘MEB-like’ group of condition is clear. The profound depletion of α-dystroglycan might suggest the involvement of a yet unidentified glycosyltransferase in these cases.

3.3. Congenital glycosylation disorders (CGD) 

In view of the growing numbers of CMD due to mutations in putative glycosyltransferases, a session of the workshop was devoted to congenital disorders of glycosylation (CDG). Körner (Göttingen, Germany) provided an overview on this subject. CDG comprise a group of recently identified inherited diseases which affect genes required for the biosynthesis or the processing of the oligosaccharide moiety of newly synthesized glycoproteins in humans [17]. CDG mostly present as multisystemic disorders including severe neurologic deficiencies. The disorders are divided into two subgroups. CDG I comprises all defects which affect the biosynthesis of dolichol-linked oligosaccharides and the transfer onto newly synthesized proteins. CDG II includes all defects in the processing of protein bound oligosaccharides. So far the molecular defects of nine different CDG have been identified [18].

In several cases of CDG syndromes, muscle weakness has been described, generally secondary to neurogenic involvement. The group of Körner, however, has recently described a novel type of CDG (CGD-IID), characterized by mental retardation and Dandy–Walker malformation, in which severe muscle hypotonia appears to be the result of a primary myopathic involvement, also confirmed by the marked elevation of serum creatine kinase [19]. In contrast to normal human serum transferrin which carries two complex biantennary N-glycan chains with four terminal sialic acid residues, CGD-IID patient's transferrin showed the loss of sialic acid- and galactose residues due to the impaired activity of β-1,4-galactosyltransferase. The enzyme, which normally catalysis the transfer of galactose residues from UDP-galactose onto terminal N-acetylglucosamine residues of complex-type oligosaccharides in newly synthesized glycoproteins in the Golgi apparatus, lacks the C-terminal 50 amino acids due to a single nucleotide insertion mutation and is therefore retained in the endoplasmic reticulum [20].

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4. Animal model of CMD 

Fiszman (Paris, France) gave an overview of the current used therapeutic strategies in the dy/dy mouse. This is an animal model for merosin deficient CMD. At INSERM the team of Fiszman is working on the dy/dy and dy^2j/dy^2j mouse models using naked laminin α2 chain DNA injection together with electroporation as a tool to reintroduce the defective gene [21].

In order to monitor for the efficiency of this procedure, in addition to look at direct effect on laminin α2 chain expression in muscle, mice are also studied using a 4 Tesla magnetic resonance imaging (MRI). The mice receive an infusion of gadolinium salts that normally does not penetrate into muscle. However, it will do so after the muscle has been partially damaged following electroporation. Using tibialis anterior muscle as the recipient muscle, it was found that the MRI observation allows to precisely visualize the muscle territory affected by the electric field and also to show that 40–50% of the muscle fibres are transduced and express the newly introduced gene.

Fiszman also highlighted a protein upregulation approach recently reported by the group of Ruegg in the dy/dy mouse [22]. These authors reasoned that it might be possible to take advantage of the upregulation of the laminin α4 chain that occurs in the dy/dy mouse muscle, despite the weak binding capacity to α-dystroglycan of this protein compared to laminin α2 chain. They engineered a miniaturized agrin molecule, which retained high-affinity binding sites for the laminin α4 chain and α-dystroglycan. The muscle pathology in these transgenic mice was significantly improved, suggesting that this strategy of creating an artificial link between the basement membrane and α-dystroglycan can be used as a therapeutic tool in merosin deficiency.

Wewer (Copenhagen, Denmark) gave an overview of her work on the function of tetranectin. This protein belongs to the C-type lectin family and is important for mesenchymal cell differentiation, including osteogenesis and muscle cell development and differentiation [23]. The expression pattern of tetranectin mRNA suggests that this gene is present in all tissues at all time, although immunocytochemical studies in adult muscle show virtually absent protein levels. Wewer recently generated tetranectin null mice and analysed the resultant phenotype [24]. Interestingly, the muscle of the limbs were normally formed in these mice and there was no associated clear phenotypic abnormality in the first 6 months of life. However, from the age of 6 months these mice develop a severe kyphosis affecting mostly the thoracic spine. Detailed X-ray and histological analysis showed a combined defect in the bones and intervertebral discs with growth plate disorganization. This gene can therefore be a candidate for kyphosis in the human.

Hewitt (Nottingham, UK) gave an update on her work focused on the isolation of the gene responsible for the myd mouse [25]. This mouse was considered for a long time an animal model for FSH muscular dystrophy but was recently shown by this group to have a mutation in a gene named Large. This protein product of this gene shows homology to glycosyltransferases and is ubiquitously expressed in the mouse, with highest levels in skeletal muscle and brain. The mutation is a deletion that causes loss of the open reading frame. The human homologue consist of 16 exons and maps to chromosome 22q. Further support to the putative glycosyltransferase role of this protein came from the demonstration that α-dystroglycan was virtually absent in muscle extracts from the myd mice. Furthermore, Hewitt presented data showing loss of laminin 1 binding in myd mutant muscle tissue using an overlay blot assay. This suggests that the deficiency of the Large gene, a putative glycosyltransferase, results in abnormal glycosylation of α-dystroglycan; this in turn results in loss of linkage between this molecule and laminin α2 chain, as the interactions between these two molecules occurs in a glycosylated epitope of α-dystroglycan.

Since the Large gene is expressed also in brain, and abnormal glycosylation of α-dystroglycan was also reported in the brain of the myd mouse, this animal might also represent an interesting model for further understanding the role of α-dystroglycan in normal brain.

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5. List of participants 

F. Muntoni (London, UK)

E. Bertini (Rome, Italy)

C. Bönnemann (Philadelphia, PA, USA)

M. Brockington (London, UK)

S. Brown (London, UK)

K. Bushby (Newcastle upon Tyne, UK)

M. Fiszman (Paris, France)

C. Körner (Göttingen, Germany)

E. Mercuri (London, UK)

L. Merlini (Bologna, Italy)

J. Hewitt (Nottingham, UK)

S. Quijano-Roy (Garches, France)

N. Romero (Paris, France)

S. Squarzoni (Bologna, Italy)

C.A. Sewry (London, UK and Oswestry, UK)

V. Straub (Essen, Germany)

H. Topaloglu (Ankara, Turkey)

G. Haliloglu (Ankara, Turkey)

T. Voit (Essen, Germany)

U. Wewer (Copenhagen, Denmark)

P. Guicheney (Paris, France)

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Acknowledgements 

This workshop was made possible thanks to European Community grant (QLG1-CT-1999-00870) Myocluster-GENRE and to the logistic support of the European Neuromuscular Centre (ENMC) and its main sponsors and associated members: Association Française contre les Myopathies (France), Deutsche Gesellschaft für Muskelkranke (Germany), Telethon Foundation (Italy), Muscular Dystrophy Campaign (UK), Muskelsvindfonden (Denmark), Prinses Beatrix Fonds (Netherlands), Schweizerische Stiftung für die Erforschung der Muskelkrankheiten (Switzerland), Verein zur Erforschung von Muskelkrankheiten bei Kindern (Austria), Vereniging Spierziekten Nederland (Netherlands), and ENMC associate member: Muscular Dystrophy Association of Finland. The authors wish to thank Professors Victor Dubowitz and Andoni Urtizeberea who also attended the meeting as ENMC representatives.

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