165th ENMC International Workshop: Distal myopathies 6–8th February 2009 Naarden, The Netherlands

Article Outline

• 1. Introduction
• 2. Session I
  • 2.1. Updates on clinical and molecular genetic findings in distal myopathies
  • 2.2. Titinopathy – tibial muscular dystrophy (TMD – Udd myopathy)
    • 2.2.1. TMD/LGMD2J complexity with the second mutation in France (French-B)
    • 2.2.2. TMD in Spain
    • 2.2.3. Severe TMD phenotype in a French TMD family with the first identified mutation in the second-last exon of titin (French-C)
  • 2.3. Myosinopathy – MYH7-mutated Laing distal myopathy
    • 2.3.1. A large cohort of patients and families in Valencia
    • 2.3.2. Later onset in an Austrian family with Glu1856Lys mutation
    • 2.3.3. New families and new mutations in different populations
  • 2.4. Review of other Distal Myopathies
    • 2.4.1. Vocal cord and Pharyngeal Distal Myopathy – caused by MATR3 mutation
    • 2.4.2. Distal Nebulin Myopathy
    • 2.4.3. The Italian 19p13-linked families
    • 2.4.4. The autosomal dominant Victoria Australia family
    • 2.4.5. Distal VCP-mutated myopathy
    • 2.4.6. Myofibrillar myopathy patients: the majority have a distal presentation
  • 2.5. Autophagy – a basic element of protein turnover
• 3. Session II: understanding the sarcomeric diseases – molecular pathomechanisms and animal models
  • 3.1. Titinopathies: the effects of C-terminal mutations
    • 3.1.1. Other new C-terminal titin interactions
    • 3.1.2. Complex processing of c-terminal titin
    • 3.1.3. The phenotype of the FINmaj knock-in mouse model
  • 3.2. MYH7 myosinopathies: the molecular pathology
  • 3.3. The distal arthrogryposes – caused by other myosin and thin filament defects: congenital distal myopathies
• 4. Session III: understanding the GNE associated myopathies (HIBM/DMRV): pathomechanisms, animal models and therapy
  • 4.1. Overview and basic facts
    • 4.1.1. GNE-associated HIBM: pathomechanisms and current enigmas
    • 4.1.2. GNE associated DMRV: spectrum of mutations and the hyposialylation defect
    • 4.1.3. Sialic acid biophysiology in the muscle
  • 4.2. Mouse models for GNE-associated myopathy
    • 4.2.1. The Japanese DMRV mouse model
    • 4.2.2. The NIH-group HIBM mouse model with a M712T KI-mutation
    • 4.2.3. The Israeli HIBM mouse model
  • 4.3. Results of ManNAc treatment in the GNE mouse models
    • 4.3.1. ManNAc and NeuAc administration to the DMRV mouse model
    • 4.3.2. ManNAc administration to the NIH HIBM mouse model with a M712T KI-mutation
    • 4.3.3. ManNAc in human trials?
  • 4.4. GNE-associated myopathy: other abnormalities than hyposialylation as parts of the pathomechanism
    • 4.4.1. GNE interactions, proteomics and gene expression profiling
• 5. Session IV: guidelines: how to reach final diagnosis
  • 5.1. Diagnostic evaluation
    • 5.1.1. Muscle imaging guiding the differential diagnosis of the myofibrillar myopathies
    • 5.1.2. Muscle imaging guiding the differential diagnosis of the other distal myopathies
    • 5.1.3. Clues guiding the differential diagnosis of the distal myopathies
  • 5.2. Diagnostic algorithms
  • 5.3. Available DNA-testing labs
• 6. Conclusions
  • 6.1. Diagnostic accuracy can be improved by muscle imaging techniques
  • 6.2. Improved DNA-diagnostics needed
  • 6.3. Research and molecular pathogenesis
  • 6.4. Therapy strategies and trials
• Participants
• Copyright

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1. Introduction 

The ENMC consortium on distal myopathies held its 3rd workshop in Naarden, The Netherlands, February 6–8th, 2009. The workshop was attended by 23 active participants from Finland, Japan, Israel, France, Germany, Australia, Sweden, Switzerland, Italy, Spain, Austria, the UK and the USA. During the last 15years, since the 1st ENMC workshop on the Distal Myopathies, the field of known and classified different distal myopathies has expanded from a handful clinical entities to more than 16 genetically defined separate diseases. For the majority of these diseases the underlying gene defect has been established and the disease mechanisms on the subcellular level are currently being explored. Due to this expansion, not only in the number of entities, but also in understanding the molecular biology of these disorders, all distal myopathies could no longer be reviewed and covered in one workshop. This 3rd workshop concentrated mainly on the diseases caused by defects in genes encoding sarcomeric proteins and/or showing the morphological feature of rimmed vacuolated muscle fibers.

The participants reported on a number of new mutations and detailed new clinical features that expand the phenotype of the known distal myopathies. Other reports included recently discovered new genes underlying distinct forms of distal myopathy, some of them yet unpublished. Considerable progress has been made in the clarification of the defects on the protein level and in the molecular pathomechanisms taking place downstream of the primary mutation. Based on these findings some treatment options have already been tested in disease models for a few forms of distal myopathy, such as the GNE-mutated HIBM/DMRV disease. The results of these cellular and animal studies indicate a therapeutic opportunity that may be applicable to the human disease and soon open the way for clinical trials in patients. The workshop agreed on guidelines for diagnostic procedures for clinicians, emphasizing the role of muscle imaging to complement and direct the investigation towards a correct molecular genetic diagnosis. Finally all the further steps to enable patient clinical trials were emphasized.

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2. Session I 

2.1. Updates on clinical and molecular genetic findings in distal myopathies 

Bjarne Udd (Finland) gave an overview of the advancing scientific progress in the field since the 1st ENMC workshop on the Distal Myopathies in 1994. That workshop delineated four distinct categories of distal myopathy based on clinical and pathology findings, which later proved to be five genetic disorders: Welander distal myopathy, titinopathy (Tibial muscular dystrophy – Udd myopathy), ZASPopathy (Markesbery-Griggs late onset distal myopathy), GNE-mutated Distal myopathy with rimmed vacuoles (Nonaka myopathy – the same disease as hereditary inclusion body myopathy HIBM) and dysferlinopathy (Miyoshi myopathy). Shortly after the 1st workshop the first distal myopathy was genetically defined by linkage to chromosome 14q, the Laing distal myopathy, which later proved to be a myosinopathy caused by mutations in the slow myosin gene MYH7. During the last 10years more than 10 additional distal myopathies have been genetically separated and defined. These can be grouped and categorized in different ways, starting by clinical data and age of onset, by morphological pathology data or by the pattern of involvement of muscles as judged by muscle imaging. Using all these criteria combined provides reasonable diagnostic algorithms to guide the diagnostic process towards molecular genetic definition and diagnosis (Fig. 1, Fig. 2, Fig. 3 and Table 1, Table 2, Table 3).

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• Fig. 1.

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• Fig. 2. 

  Diagnostic algorithm starting with clinical criteria.

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• Fig. 3. 

  Diagnostic algorithm starting with pathology criteria.

Table 1. Main parameters to be considered.

Table 2. Strategy for the most common causes.

If recessive and Onset 15–30years:

1. Dysferlinopathy: high CK level, predominant involvement of posterior compartment of legs; biopsy: dystrophic with necrosis/inflamation, without vacuoles, dysferlin deficiency (immunostaining and Western blot); Confirmation: dysferlin mutations

2. GNE-associated HIBM/DMRV: higher frequency in Japan and Middle East population; Moderate CK level; Leg anterior compartment, Quadriceps sparing; Biopsy: rimmed vacuoles without myofibrillar features. Confirmation: GNE mutations

If dominant and early childhood onset (later onset possible): Laing MYH7 myosinopathyCharacteristic features: hanging big toe, ankle dorsiflexion, finger extensor and neck flexor weakness; +/− ankle contractures; Biopsy: Type I hypotrophy as with congenital fiber type disproportion (pathognomonic: if all slow type1 fibers are hybrids expressing also fast myosin). Confirmation: mutations in MYH7.

If dominant and onset from adolescence and before age 40, cardiomyopathy and myofibrillar pathology: DesminopathyMuscle imaging: Peroneus>Tibialis anterior, Semitendinosus>Biceps femoris; EM: granulofilamentous deposits. Confirmation: Desmin mutation

If Desmin mutation negative, same imaging and EM features and +/- cataracts: alphaB-cristallinopathy

If dominant, late onset after age 40, no cardiomyopathy:By imaging: posterior>anterior lower legs, eventually hands, myofibrillar pathology: Zaspopathy and Myotilinopathy

Imaging: Soleus, medial Gastrocnemius, Biceps femoris and semimembranosus, Sartorius>Semitendinosus, Adductor magnus>Gracilis

EM: characteristic bundles

In central Europe: first ZASP then myotilin; in Spain and the UK: myotilin first

If selective anterior tibialis involvement and rimmed vacuoles and no MFM: Titinopathy (considered at first if from Finland). Confirmation: Titin gene mutation

Table 3. Strategy if no evidence for a common distal myopathy.

Miyoshi-like myopathy with normal dysferlin (unknown gene)Onset: 20–40years, sporadic or recessive, posterior calf involvement and +/− biceps brachii, very high CK level, dystrophic pathology but normal dyferlin expression

Late onset, dominant, anterior tibial involvement +/− hands, rimmed vacuolar pathology, +/− Paget and late onset frontotemporal dementia: VCP distal myopathy. Confirmation: VCP gene mutation

Recessive, sporadic, massive lipidosis: mutisystemic lipid storage myopathyConfirmation: Jordan bodies on blood smear. Adipose triglyceride lipase (PNPLA2) gene

Autosomal dominant with vocal cord and pharyngeal symptoms, after exclusion of myotilin: MATR3 gene

Early childhood to early adulthood, recessive or sporadic, anterior compartment of lower legs and +/- finger extensor weakness, and MYH7 excluded:No rods on light microscopy but rare nemaline rods on electron microscopy: Distal nebulin myopathy: missense mutations in NEB

Autosomal dominant, pes cavus, rimmed vacuolar pathology and bulbar involvement. In Italy: test 19p13 linkage

Early onset intrinsic hand muscles and +/− calf hypertrophy: CAV3 possibleTest caveolin 3 by immunohistochemistry

Welander distal myopathy was not specifically detailed in the sessions as no final data on the responsible gene was yet available. Sequencing all the genes in the linked region has failed to reveal the mutation and currently a large-scale whole genomic sequencing of the linked region is underway.

2.2. Titinopathy – tibial muscular dystrophy (TMD – Udd myopathy) 

Udd reported on the family and research performed in collaboration with Dr. Isabelle Pénisson–Besnier in Angers. The French-B titin mutation is located in the same last exon 363 as the first identified French missense mutation (French-A: L>P) and the Finnish FINmaj mutation. This new mutation is a nonsense mutation changing a glutamine to a stop (French-B: Q>X), just six residues C-terminal of the French-A mutation (Hackman et al. 2008). The clinical findings in the proband were slightly more severe than usually seen with onset after age 30, steppage gait on the left foot at age 34 and the need of the stick for walking at age 52. The other heterozygotes in the family, however, had a definitely milder presentation with no symptoms yet in the 50-year-old younger sister. The complexity in this family was shown by the deceased father who had a different, severe generalized muscle weakness and atrophy apparent already at age 25 also in the upper limbs, and progressing to loss of walking and loss of arm elevation at the age of 56years. The grandparents were known to be first cousins and the father was indeed homozygous for the French-B mutation. Since only medical records were available, no exact classification of the clinical phenotype was possible in the father, but clearly it was a different severe generalized phenotype compared to the distal phenotype in the daughter, in line with the completely different phenotypes in heterozygous TMD patients and homozygous LGMD2J patients with the Finnish FINmaj mutation.

Isabel Illa (Spain) reported on three different families in Spain with TMD titinopathy disease living in Barcelona (originating in western Andalucia), Albacete and Murcia. All patients in these families carry the same Spanish mutation in the last exon 363 in the titin gene, a single nucleotide deletion (Spanish mutation: K>NfsX9) causing frameshift and truncation of the last Ig-domain m10 of the protein seven residues C-terminal of the French-B mutation. The phenotype of the patients was usually within the range of the published common TMD phenotype (Udd et al. 2005), but very often with more variation of progression such as involvement of all lower leg muscles and marked involvement of proximal lower limb muscles at later stages after age 70 in the Albacete family. In the most recently identified Murcia family the proband had onset already at age 24, when her parents were still unaffected, indicating a digenic or other mechanism as the explanation for the unusual early onset in a heterozygote patient.

Bruno Eymard (France) described the interesting clinical outcome in a French family with a more severe TMD phenotype caused by a single nucleotide deletion and frameshift mutation, S33315QfsX10, truncating the protein at the end of the second last is7 domain (Hackman et al. 2008). The mutation causes an earlier onset after age 20 with ankle dorsiflexion weakness and a marked progression to proximal leg muscles already in the fourth decade. The more severe outcome might be due to the fact that the amount of the mutant transcript is, for some reason, higher than the normal allele causing a more than 50% reduction of the normal titin protein. This family also clarifies another previously unsettled question regarding the impact of the different M-line titin isoforms for TMD disease. The second-last exon 362, encoding is7 domain, is variably spliced, and different amounts of is7+ or is7− isoforms are expressed in different muscles. The more severe outcome of the mutation in this second-last exon determines that the disease is mediated through the is7+ isoform in skeletal muscle.

2.3. Myosinopathy – MYH7-mutated Laing distal myopathy 

Nuria Muelas Gómez (Spain) together with Juan Vílchez has studied four large dominant early onset distal myopathy families with a total of 32 affected patients originating from La Safor region in Valencia. Onset of lower leg anterior compartment muscle weakness ranged from congenital to the early 50’s. Most of the patients had neck flexor, finger extensor and mild facial weakness. Disability ranged from subjectively asymptomatic to wheelchair-confined patients. Neuropathy was not observed and cardiomyopathy was only found in one patient who had additional cardiovascular risk factors. All affected individuals in all families segregated a K1729del mutation in the MYH7 gene on a common haplotype. The most consistent pathological finding was the hypotrophy–atrophy of type 1 fibers among other frequent non-specific features such as irregularity in oxidative enzyme stainings. The same K1729del mutation was previously reported in a family originating from Italy (Hedera et al. 2004). Genotyping showed that a common founder origin remains possible with some sharing of alleles for a core haplotype.

Michaela Auer-Grumbach (Austria) reported on molecular genetic investigations in a family with onset of finger extension weakness between 20 and 40years of age and with weakness of ankle and big toe dorsiflexion on later examination. The pattern of inheritance for the distal phenotype was dominant and some patients had a history of cardiomyopathy. In one young child of on affected mother, the cardiomyopathy was present without signs of distal myopathy. Sequencing of the MYH7 gene revealed a C-terminal Glu1856Lys mutation which is in the region of the protein previously known to create myosin storage (hyaline body) myopathy.

Bjarne Udd (Finland) in collaboration with Odile Dubourg and Bruno Eymard has identified the first Laing myopathy family in France. The disease by a Glu1508del mutation showed a phenotype well in line with previous reports. There was no cardiomyopathy involvement in the family. Interestingly, exactly the same mutation was also found in a sporadic Finnish patient with a comparable phenotype. According to genetic studies in the parents the mutation occurred as a de novo mutation in the Finnish patient.

Bruno Eymard (France) reported on a Moldavian family with a c. 5401 G>A (p.Glu1801Lys) mutation causing, besides the usual early onset distal myopathy phenotype, also a later onset severe dilative cardiomyopathy. Tibialis anterior muscle pathology was considered dystrophic without vacuoles, and CK level was just in the upper range 247.

The first Norwegian Laing myopathy family has been identified in Udd’s lab in collaboration with Dr. Dahl. The disease in two brothers with healthy parents was caused by a Ala1603Pro mutation, which is thus yet another de novo mutation.

2.4. Review of other Distal Myopathies 

Jan Senderek (Germany/Switzerland) described their molecular genetic work on the original VCPDM family linked to 5q31 (Feit et al. 1998) and an additional Bulgarian family with similar clinical features. Fine mapping of the critical region and sequencing of positional candidate genes lead to the identification of the gene defect in the MATR3 gene. This gene encodes matrin 3, one of the major proteins of the nuclear matrix. An identical missense mutation, S85C, was identified in both families. Different disease related intragenic haplotype signatures in the two families provided evidence that two independent mutational events had occurred at the same position in the MATR3 gene.

Bjarne Udd (Finland) and Dr Carina Wallgren-Pettersson have, in collaboration, identified homozygous missense mutations in the C-terminus of nebulin to be the cause of a recessively inherited early onset distal myopathy in several Finnish families (Wallgren-Pettersson et al. 2008). Recessive mutations in nebulin are previously known to cause nemaline myopathy, but in this case nemaline rods were not discernable on light microscopic level, even on retrospective re-evalutation. The explanation was that in all families with severe nemaline myopathy the mutation was a truncating or otherwise disruptive mutation at least on one chromosome, whereas two missense mutations cause this mild distal myopathy with favorable disease course and without light microscopic rods.

Marina Mora (Italy) reported on the further studies in their chr 19p13-linked family with distal myopathy and rimmed vacuolated pathology (di Blasi et al. 2004). The first 19p13-linked Italian family was reported in 1999 (Servidei et al.) as a neuromyopathy with pes cavus and absence of reflexes (see the ENMC report 2002). The linked region in both families is overlapping but preliminary results suggest the haplotype may be different. There are also differences in clinical features and the pathology, as in the family studied in Milan pes cavus is not present and the vacuoles on muscle pathology are membrane-bound with sarcolemmal proteins and signs of exocytosis. All candidate genes in the linked region have been sequenced without mutations. Whether the two families have one or two diseases cannot be decided before the gene(s) are identified.

Nigel Laing (Australia) and Bjarne Udd (Finland) gave an update on the family described by Williams et al. (2005). The disease most often starts in the hands with weakness of the thenar muscles leading to reduced grip strength followed by weakness of calf muscles, easily shown by muscle imaging as a rather selective fatty degeneration of posterior compartment muscles in the lower leg. The pathology does not contain rimmed vacuoles or other specific features. Nigel Laing reported on the current linkage results in the family following reclassification of the affection status of family members by Udd, and gave a summary of the current state of candidate gene analysis. Further analysis of the family will be required to determine the precise disease gene. Identification of other families with the same phenotype and addition of these families to the gene discovery project would significantly help the project.

Bjarne Udd (Finland) described yet unpublished results on the investigations in an autosomal dominant Finnish family with onset of ankle dorsiflexion weakness after age 30 and with small muscle atrophy in the hands in some of the patients, leading clinically to the diagnosis of Welander distal myopathy. However, the family lacked the Scandinavian founder haplotype for Welander disease and the TMD-causing FINmaj titin mutation. When the oldest of three bothers died at the age of 59years in a rapidly progressive dementia, the clue for checking the VCP (valosin containing protein) gene was obvious. In this family there was no Paget disease component, and no scapular involvement, as has been reported previously with VCP-mutated IBMPFD disease. One patient was followed for 12years without development of additional features beyond lower leg distal myopathy. The phenotype was clinically perfectly well complying with distal titinopathy (TMD), even with selective anterior compartment lower leg lesions on muscle imaging, or Welander disease when atrophies in hand muscle were present.

Montse Olivé (Spain) detailed their experience with the myofibrillar myopathies (MFM). Of the genetically proven patients, myotilin mutations were the most frequent followed by desmin and ZASP. In a series of 48 patients from 37 families presenting with distal myopathy, 28 had myofibrillar myopathy. Among these, 10 patients had mutations in the desmin gene, 15 patients in myotilin and the remaining three in ZASP. Patients with desminopathy presented at a mean age of 25.2years. Anterior distal weakness in the lower extremities was the initial manifestation in six, whereas cardiac involvement was the presenting symptom in the remaining four. These later cases also developed distal lower-limb weakness eventually spreading to proximal muscles of all limbs. Respiratory insufficiency was a frequent complication. Patients with myotilin mutations presented at a mean age of 56.8years. Foot drop was the initial symptom in all of them. Five additional patients carrying myotilin mutations presented with mixed distal and proximal weakness, or proximal weakness alone. Interestingly, muscle imaging studies in these later cases revealed marked fatty degeneration of posterior calf muscles, exceeding the involvement of proximal muscles thus indicating that distal onset occurs in all myotilinopathy patients. Finally, three members of a family with a ZASP mutation presented at a mean age of 59years with distal muscle weakness in lower extremities, later involving proximal muscles of lower limbs and distal muscles of upper extremities.

2.5. Autophagy – a basic element of protein turnover 

Dieter Fürst (Germany) contributed an overview of the evolving diversification of the concept of autophagy. Defined on the basis of the presence of rimmed and non-rimmed vacuoles with lysosomal enzyme activity, autophagy has been observed in many neuromuscular diseases. In the basic science community, autophagy is defined as a lysosomal/vacuolar degradative pathway that mediates the turnover of proteins and organelles. It turned out that this process is highly dynamic and the disease-associated apparent increase in autophagic vacuoles may involve defects in autophagosome maturation, turnover or flux. A state-of-the-art overview of appropriate assays was recently published (Klionsky et al. 2008). Microtubule-associated protein light chain 3 (LC3), has been used as a marker for monitoring autophagy. LC3 is detected as two bands following SDS–PAGE and immunoblotting: the unconjugated (LC3-I) which is cytosolic, and the conjugated (LC3-II). The amounts of LC3-II correlates with the number of autophagosomes and at present antibodies against LC3-II seem to be the gold standard to monitor autophagy, since this protein is the only component known to be associated with autophagosomes during all steps of their development.

Autophagy in vacuolar compartments on one hand and the ubiquitin-proteasome system (UPS) on the other hand are two complementary pathways used by the cell to dispose of aggregated or misfolded protein. The inverse relation of both pathways is dramatically illustrated in protein aggregate myopathies as well as in numerous neurodegenerative diseases: if the UPS is overstrained, cells begin to exhibit signs of increased autophagy, as defined by the presence of rimmed and non-rimmed vacuoles. However, trials to improve the pathology by suppressing autophagy have failed. Quite contrary to expectations, a recent publication demonstrated that blocking autophagic processes in a transgenic mouse model expressing an αB-crystallin mutant leads to a dramatic increase in the severity of the cardiac pathology (Tannous et al. 2008). These findings will have to be taken into consideration in future therapeutic approaches.

In the ensuing discussion the need for more exact characterisation of the ongoing processes in rimmed-vacuolar pathologies was identified. Most rimmed vacuoles are not lined with a membrane but contain membrane-bound smaller vesicles apparently representing compartments of autophagosomal–lysosomal structures at different stages of maturation.

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3. Session II: understanding the sarcomeric diseases – molecular pathomechanisms and animal models 

3.1. Titinopathies: the effects of C-terminal mutations 

Mathias Gautel (UK) gave an extended update on the molecular events involving the C-terminus of the giant titin protein in the sarcomeric M-band. The many interacting proteins in complex networks including signaling molecules provide the basics for a concept where M-band titin is a sensor of sarcomeric mechanics capable of transferring signals for regulating transcription and protein turnover. New ligands of the last domain of titin, which is mutated in TMD/LGMD2J, were recently identified: obscurin and obscurin-like protein. These, in turn, also interact in a ternary complex with myomesin forming a structure capable of sensing mechanical shear stress in the sarcomeric M-band (Fukuzawa et al. 2008). The structure of the titin M10 domain in interaction with N-terminal obscurin has been resolved by crystallography and shows that the mutations in M10 underlying TMD/LGMD2J disease disrupt the interface and prevent this interaction. However, the location of myomesin is not dependent on correct titin M10-obscurin interaction, which to some degree may explain the much milder phenotype of the obscurin knock-out mouse compared with the myomesin KO-mouse model. The molecular construction of the M-band is one component of the always centered myosin thick filaments, resisting even asymmetric contractional forces in the sarcomere. Another link of the known titin interactions over small Ankyrin1 suggests a role in the organization of the sarcoplasmic reticulum.

Jaakko Sarparanta (Finland) reported on yet unpublished new interactions of C-terminal titin derived from a yeast-two-hybrid screen and followed by specific biochemical assays. So far, myospryn (CMYA5) has been confirmed as a ligand both for the last domain M10 of titin and for calpain 3. Since calpain 3 is a known ligand of C-terminal titin, a ternary complex including myospryn, titin and calpain3 cannot be ruled out, even though fully conclusive experimental data have not been obtained. However, in normal conditions the M-band localization of myospryn is very faint compared to the predominant para-Z-disc localization. Definite immunohistochemical abnormalities of myospryn amounts or subcellular localization in the homozygote titinopathy LGMD2J could not be discerned, which may partly due to inadequate resolution in the available LGMD2J muscle samples.

Jaakko Sarparanta (Finland) described some first unpublished details on normally occurring protein fragments of the titin C-terminus, detected with C-terminal antibodies. Using some antibodies these fragments disappear in the homozygous LGMD2J muscle, and with others some new different fragments may appear. Even if calpain 3 has been shown to cleave titin it may not be the main protease responsible for the processing of C-terminal titin under normal conditions, since the fragments are also present in the heart muscle where calpain 3 is not expressed. Preliminary experimental data suggest that ubiquitous calpains may be involved. What the biological role of these fragments might be, is not known. Further studies can be carried out using new M10 domain-specific monoclonal antibodies developed by the group.

Isabelle Richard (France) reported the first unpublished results of the effort to create suitable mouse models for TMD/LGMD2J disease with a knock-in strategy of the Finnish FINmaj mutation. There is a definite skeletal muscle phenotype showing light microscopic changes in the soleus muscle already by 1month of age in the surviving homozygous mice. In the heterozygotes changes in soleus are present at 6 mo and even later in tibial anterior, quadriceps and biceps femoris muscles. In contrast to the human pathology, the homozygous mouse develops a dilated cardiomyopathy before 1year of age, with extensive fibrosis histopathologically. The FINmaj-KI mouse thus provides a relevant TMD/LGMD2J model for deciphering the pathological mechanisms and testing therapeutic strategies.

3.2. MYH7 myosinopathies: the molecular pathology 

Nigel Laing (Australia) gave an update on the molecular pathology of MYH7 distal myosinopathy: preferred location of mutations and residues. Unpublished novel mutations associated with MYH7 distal myopathy, including those presented at the Workshop are blurring the previous distinction between MYH7 mutations causing distal myopathy, myosin storage myopathy (MSM) and cardiomyopathy. Previously, mutations associated with MYH7 distal myopathy were located between exons 32 and 36 whilst MSM mutations were located between exons 37 and 39, Now mutations causing MYH7 distal myopathy have been identified in exon 37 overlapping the location of MSM mutations. While previous MYH7 mutations causing distal myopathy had been missense mutations to proline or insertion or deletion of an amino-acid, the novel mutations include other missense mutations. MYH7 mutations causing both distal myopathy and cardiomyopathy have been identified and the biopsy phenotypes associated with MYH7 mutations expanded. Preliminary data on circular dichroism, thermal melt and tissue culture analysis of expressed MYH7mutants was presented. The distal myopathy mutations, because they involved missense mutation to proline or heptad repeat-altering amino-acid deletions or insertions, were predicted to affect the ability of the myosin tail to form its normal coiled-coil structure. The circular dichroism analysis indicated that the mutations have little, perhaps only a very localized effect on structure. Tissue culture expression of mutants indicated effects on the tertiary structures formed by MYH7. Despite this preliminary progress, the pathomechanisms governing whether an MYH7 mutation will cause distal myopathy, MSM, cardiomyopathy or which mixture of the three phenotypes, remain elusive.

3.3. The distal arthrogryposes – caused by other myosin and thin filament defects: congenital distal myopathies 

Anders Oldfors (Sweden) reported on their experience with mutated developmental isoforms of myosin genes and specific isoforms of troponin and tropomyosin underlying the peculiar congenital joint deformities called distal arthrogryposes (DA). Despite the term, the known genes involved are encoding proteins of the sarcomeric machinery, and notably two of them, MYH3 and MYH8, isoforms only expressed prenatally. Of the clinically different types, DA1 has been associated with mutations in TPM2 and TNNI2, whereas DA2A (Freeman-Sheldon) and DA 2B (Sheldon-Hall) may be the outcome of mutations in TPM2, MYH3, TNNI2 and TNNT3. DA7 (trismus-camptodactyly) was implicated with mutations in MYH8. The pathology in these disorders may not provide direct clues as many muscles are not affected at all. In one TPM2 mutated family with pes equinovarus at birth a muscle biopsy in adulthood in tibial anterior muscle showed normal type1 fibers only, despite the fact that TPM2 is known to be expressed in slow muscle fibers. Mutations in MYH3 may cause a pathology with small type 1 fibers. Some of the joint deformities and muscle weakness may improve in later development, as with the congenital contractions associated with mutation in MYH2. Rarely there is a mild progressive course of the myopathy in later adulthood.

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4. Session III: understanding the GNE associated myopathies (HIBM/DMRV): pathomechanisms, animal models and therapy 

4.1. Overview and basic facts 

Zohar Argov (Israel) updated on the clinical and genetic understanding of the Middle East cohort of HIBM patients most of whom carry the Persian-Jewish founder mutation M712T irrespective of current ethnicity of the patients. The sparing of the quadriceps muscle up to age 70–80 despite otherwise generalized muscle atrophy is still a hallmark of the disease. Also facial and respiratory muscles are spared. One exceptional patient is known having the homozygous founder mutation but no muscle weakness in late adulthood. The GNE gene product is known as a bifunctional enzyme with an epimerase and a kinase domain and being the rate-limiting enzyme in the sialic acid biosynthetic pathway. This function would imply a primarily cytosolic localization, but also nuclear localized GNE has been suggested (Krause et al. 05).

Ichizo Nishino (Japan) described the current data on Japanese and other South-East Asian populations. Of 91 Japanese patients 54.4% have the most common V572L founder mutation and 22% have the second known founder mutation D176V. The estimated total number of Japanese patients is 200–400 which indicates a prevalence of 2–3/10⁶. The spectrum of disease onset and progression is widening with also late onset patients. Loss of ambulance with the founder mutation can vary from 3 to 22years. In parallel with the founder mutation in Middle East also one Japanese patient homozygous for the D176V mutation is healthy. NCAM with reduced glycosylation on western blotting was suggested as a screening method for diagnostic purposes but proved to be inconsistent in Japanese patients. Instead using HP-lectin immunohistochemistry for diagnostic purposes is being evaluated.

Stephan Hinderlich (Germany) updated on the basic science regarding current knowledge of sialic acid functions and turnover. There are different variants of N-acetylneuraminic acid called sialic acids. Sialic acids are end groups of the sugar chains of the glycoconjugates, among other localizations also in the extracellular glycocalyx with finger print specificity for all different cell types. ManNAc is the substrate used only for sialic acid production. The product is moved to the nucleus for CMP activation and then exported to the Golgi as a substrate for sialyltransferases for sialylation of glycoconugates. The importance of GNE and sialic acid production is shown by lethality of the GNE KO-mouse. The +/− heterozygote mice show just minor sialic acid reduction. One perplexing finding is that GNE mRNA is present in normal muscle tissue but assays show practically no enzyme activity in the mature muscle. There are different GNE splice variants of which only GNE1 is known to be present in muscle. GNE2 has low epimerase activity and GNE3 has no enzyme activity.

4.2. Mouse models for GNE-associated myopathy 

Satoru Noguchi (Japan) reported on their mouse model, a transgenic mouse harboring the human GNE cDNA with the D176V mutation on the 129Sv/Ev background, resembling a Japanese founder mutation (Malicdan et al. 2007). This D176V transgenic mouse was later crossed with the Gne+/− heterozygous mouse, obtaining the model Gne-/-hGNED176V-Tg. The number of mutant offspring is reduced indicating that some mutant mice die prenatally. The mutant mice are smaller but have no apparent clinical features before ∼20weeks of age, although some mutants die of unknown reason after ∼6weeks of age. At ∼20weeks skeletal muscles show increased variation of fiber size. At ∼32weeks amyloid deposits can be seen in muscle tissue and at ∼40weeks rimmed vacuoles are detected in type 2A fibers by histology and EM.

Marjan Huizing (NIH, USA) reported that their group, in collaboration with the HIBM research group (Dr. Darvish, Encino, CA, USA), created a knock-in mouse by homologous recombination, mimicking the Persian-Jewish M712T mutation on the murine C57BL/6 background and on the FVB background (Galeano et al. 2007). Most mutant mice unexpectedly died between birth and postnatal day 3 (P3) due to severe renal glomerular disease. At day P2, the mutant mice showed no muscle phenotype, but their kidneys were hemorrhagic and the mice had proteinuria. On EM, the podocytes had severe effacement and the glomerular basement membrane showed segmental splitting. The mucin-like glycoprotein podocalyxin, which is abundant in podocyte foot process membranes, was hyposialylated in mutant mice. The muscle tissue showed no clear pathology. Tubular aggregates develop in male inbred mice between ∼age 5 and 6months. Since these aggregates appear in wild type as well as in mutant mice, histological analysis of inbred mouse muscle should be carefully assessed.

Ilan Salama (Israel) described their unpublished project to create a knock-in mouse resembling the human Persian-Jewish M712T mutation by homologous recombination on the 129X1/SvJ ICR background. This mutation and knock-in mechanism was similar to the NIH mouse model. Breeding and analyses of this knock-in mouse are still in progress, but first results also showed neonatal death due to kidney failure. However, in this model more mutant mice survive the neonatal period. Homozygous kinase mutants may show severe or milder phenotypes but may also look healthy. The severe phenotype shows marked kidney pathology, nevertheless, the oldest mild phenotype male mice survive to 8 Mo of age. The project continues with modifications to enhance the muscle phenotype.

4.3. Results of ManNAc treatment in the GNE mouse models 

May Malicdan (Japan) described their therapeutic attempts to correct the phenotype in the mouse model with early promising results. Analysis of overall sialylation in these mice revealed a remarkable reduction in total sialic acid levels in the serum and most of the organs. Of note, the reduction in sialylation is mild to moderate in the muscle. Physiological properties of isolated muscles in these mice were also investigated (Malicdan et al. 2008). At a younger age, twitch forces seem to be predominantly reduced, while tetanic forces are more affected at the age of RV formation. This may imply that atrophy could be responsible for the reduced force generation at a younger age, while the presence of cytoplasmic inclusions and RVs during the later age could impair the sarcomere itself, rendering an additional decrease in production of force.

Efforts to increase overall sialylation in these mice appear to be beneficial, implying that replenishing sialic acid in human patients could be employed in future clinical trials.

Marjan Huizing (USA) reported on their attempt to rescue mutant mice from neonatal death by supplying ManNAc in their drinking water (1g/kg/day) to the pregnant and nursing females, resulting in improved survival to about 50% of the homozygous mutant pups beyond P3. Kidney histology in surviving mutant mice that received ManNAc dramatically improved, with partial restoration of the podocyte foot processes and improvement of podocalyxin sialylation. ManNAc supplementation in surviving mutants was stopped at weaning (P21) and mutant mice continued to live, but remained smaller than their littermates and proteinuric. Most surviving mutant mice died before ∼6months of age and sporadic survivors beyond 6months were extensively examined.

HIBM/DMRV patients’ muscle tissue, cells, and mouse models show evidence of hyposialylation as one underlying mechanism of the pathology, and ManNAc supplementation studies to increase sialylation appeared convincing in vitro on patients’ myotubes (Noguchi et al. 2004) and in mouse models (Galeano et al. 2007, Nishino et al. unpublished results). These findings led to the design of a human clinical trial to supply ManNAc to HIBM patients by the National Human Genome Research Institute (NHGRI), NIH, Bethesda, USA (Drs. Gahl, Manoli, Huizing).

Evidence for the safety of the uncharged, physiological sugar ManNAc was that single-dose studies in 5 healthy individuals (5g and 10g) revealed no toxicity (How et al. 1967). In addition, treatment of mice had no adverse effects at 1g/kg/day (Galeano et al. 2007), and in other trials, humans are given gram quantities of other sugars such as mannose for the treatment of congenital disorders of glycosylation (∼100–200mg/kg/day) (Marquardt and Denecke 2003).

The clinical protocol was written and approved by the NHGRI Scientific Review Committee and the NHGRI Institutional Review Board. An Investigational New Drug (IND) application was submitted to the Food and Drug Administration (FDA). The FDA ordered a full clinical hold on the study and requested to provide extensive ManNAc toxicology studies in two species (a rodent and non-rodent) of duration similar or greater than that proposed for humans. Finding ways for funding such studies is currently underway. Nevertheless, 5–10g daily is anecdotally already used ‘off the record’ by some patients. Anecdotally, four HIBM patients had been treated with IvIg because of its rich content in sialylic oligosaccharides but without clear effect on the disease.

4.4. GNE-associated myopathy: other abnormalities than hyposialylation as parts of the pathomechanism 

Stella Mitrani–Rosenbaum (Israel) in their studies on patient muscle tissues has shown that the GNE activity is reduced by some 30% but that the amount of membrane-bound sialic acids did not show major difference in patient muscle compared to normal. Finer analysis of myoblast cultures showed no obvious differences in sialic acid levels of N- linked glycans, but slightly lower sialic acid content was observed in glycolipids of HIBM cells. However, large variation existed between different samples.

Stella Mitrani–Rosenbaum (Israel) continued reporting on their ongoing partly unpublished proteomics studies on three patient biopsies and three controls. In 2D gels, 36 spots turned out with a different expression. In a second project using the iTRAQ/MS-MS technique four samples were run in different groups of three always with one sample identical. This method identified 407 proteins in total of which 42 were differentially expressed in HIBM disease. These proteins indicated, among others, that the oxidative energy metabolism as a pathway may be involved. Many mitochondrial genes also popped up as dysregulated in gene profiling. The results using myoblasts and the 2D technique were similar.

Immunofluorescence studies on longitudinal muscle sections indicate that GNE may have a very distinct sarcomeric localization to the para-Z-disc region and to some extent in the M-band.

New Biacore in vitro binding assay and subsequent co-immunoprecipitation assays identified alpha-actinin1 as a ligand of GNE. This was most surprising as alpha-actinin1 was not known to be expressed in skeletal muscle tissue.

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5. Session IV: guidelines: how to reach final diagnosis 

5.1. Diagnostic evaluation 

Dirk Fischer (Switzerland) presented the recently published study on the differential patterns of muscle involvement caused by five different genes causing the pathology of myofibrillar myopathy. Since almost all MFM patients present with a distal clinical phenotype the imaging strategy is of high relevance for the separation and directing specific molecular genetic analyses. Based on the retrospective data of 39 patients, an algorithm for scheduling genetic analyses was generated by Rudolf Kley (Bochum, Germany). This algorithm, however, has to be proven in a prospective study.

Bjarne Udd (Finland) presented a collection of muscle imaging in 17 different distal myopathies showing a discernible pattern of muscle involvement. One group of distal myopathies have exclusively or a highly preferred involvement of the anterior compartment of the lower legs, such as titinopathy, MYH7 myosinopathy, GNE-associated myopathy, desminopathy, VCP distal myopathy and distal nebulinopathy. Other distal myopathies have exclusive or preferred involvement of the posterior calf muscles, such as dysferlinopathy, myotilinopathy, ZASPopathy, Victoria Australia distal myopathy and dynaminopathy. A third group of distal myopathies including Welander distal myopathy, the Finnish MPD3 family, oculopharyngo-distal myopathy and filaminopathy, has both anterior and posterior involvement and very often asymmetric findings.

Bruno Eymard (France) summarized the collective experience gathered over the last years regarding features in the clinical phenotype or biopsy findings that may be of help in the diagnostic process:

5.2. Diagnostic algorithms 

Fig. 2. Diagnostic algorithm starting with clinical criteria.

Fig 3. Diagnostic algorithm starting with pathology criteria.

5.3. Available DNA-testing labs 

See Table 4.

Table 4. Laboratories performing diagnostic DNA tests for distal myopathy genes.

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6. Conclusions 

6.1. Diagnostic accuracy can be improved by muscle imaging techniques 

6.2. Improved DNA-diagnostics needed 

•Since very many of the known genes associated with distal myopathy are huge in size (ie. titin 363 exons and 100kb cDNA, nebulin 180 exons, etc.), the current sequencing strategy is not always possible to apply in single patients even if the gene would be a good candidate.

6.3. Research and molecular pathogenesis 

6.4. Therapy strategies and trials 

Case-by-case therapeutic approaches will be necessary in the distal myopathies. The mode of inheritance, the functions of the deficient protein, and the nature of the pathological consequences all need to be considered. For the dominant forms one strategy would consist of eliminating either the mRNA or the deleterious protein itself. Proof of principle has been achieved in myotonic dystrophy type 1 (Langlois et al. 2005). In recessive diseases, circumventing the deficiency by expressing a normal form of the gene in the muscle (gene therapy) or injecting cells bearing the normal gene (cell therapy) might be applicable.

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Participants