Report of the 70th ENMC International Workshop: Nemaline myopathy, 11–13 June 1999, Naarden, The Netherlands

Article Outline

• 1. Introduction
• 2. Actin (ACTA1)-related disorders
• 3. Ultrastructural studies
• 4. Nebulin (NEB)-related disorders
• 5. Other Z-disc proteins and their interactions
• 6. Tropomyosin 3 (TPM3)–related disorders
• 7. Presentations of new clinical series
• 8. Clinical classification of nemaline myopathy
• 9. Workshop participants
• Acknowledgements
• Appendix A. Clinical categories of nemaline myopathy
  • A.1. Severe congenital nemaline myopathy
  • A.2. Intermediate congenital nemaline myopathy
  • A.3. The typical form of congenital nemaline myopathy
  • A.4. Mild childhood or juvenile onset nemaline myopathy
  • A.5. Adult-onset forms of nemaline myopathy
  • A.6. Other forms of nemaline myopathy
• References
• Copyright

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1. Introduction 

Twenty-four clinical and basic scientists from eleven countries gathered in Naarden, The Netherlands, on 11–13th June 1999 for a workshop on nemaline myopathy. Research activities have been lively during the 3 years since the first ENMC workshop on this disease was held and the International Consortium was formed. International collaboration has brought about a clinical classification and the identification of three genes causing various forms of this muscle disorder.

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2. Actin (ACTA1)-related disorders 

Kristen Nowak, Perth, Australia and Duangrurdee Wattanasirichaigoon, Boston, MA described mutations found in the actin gene in patients with either congenital myopathy with excess of thin filaments, severe nemaline myopathy or mild nemaline myopathy. Sequencing of genomic PCR products from exons two to seven, the six coding exons of the actin gene, revealed 15 different single missense mutations in a total of 18 patients from 14 families. The mutations were distributed throughout the six coding exons, some of them involving known functional domains of actin. In seven of the families, studies of parental DNA indicated that the mutations were new dominant mutations, and in a further four, where parental DNA was not available for study, the patients were likewise singleton cases. One family showed dominant inheritance, one showed findings compatible with autosomal recessive inheritance and in one, mutations in an affected sib pair were likely to result from somatic mosaicism in the father [1].

Carina Wallgren-Pettersson, Helsinki, Finland had compiled clinical data contributed by clinicians in different countries of the families in which mutations had been found in the actin gene (Table 1). The patients either showed more severe or milder features than those with the typical form.

Table 1. Clinical findings in patients in whom mutations have been identified

Kathryn North, Sydney, Australia described the immunocytochemical and clinical features of the family with a dominantly inherited actin mutation, in which the mother and two children (one male and one female) were affected. The mother as an infant had poor suck and respiratory problems and has never been able to run although her motor milestones were not delayed, her daughter showed childhood onset of a relatively mild disorder, while the son showed congenital onset and achieved walking at the age of 2 years. A muscle biopsy from the daughter (quadriceps, at age 17 years) demonstrated nemaline bodies in all fibres. Ten percent of fibres were atrophic. The fibres were uniformly of type 1 (by ATPase and myosin immunocytochemistry). EM showed disorganised myofibrils with increase in intermyofibrillar space and ‘whorling’ of thin filaments. In congruency with the patients described by Goebel and colleagues, also shown to have actin mutations, this patient's biopsy showed areas of thin filament accumulation.

Victor Dubowitz, London, UK, presented details of their 37-year-old patient with nemaline myopathy found to have a mutation in the actin gene. She was initially seen at 17 years of age, with a history of weakness and difficulty with running and going up stairs. Muscle biopsy showed atrophy of type 1 fibres which were selectively affected with multiple rods, and some areas with loss of structure. She subsequently remained stable in her muscle function but at 36 years was admitted to hospital with life-threatening respiratory failure following pneumonia. She was admitted to intensive care and has remained well since on nocturnal positive pressure mask ventilation (BIPAP). She was recently reviewed (Professor Muntoni) and can still walk up to a mile on level ground but has had increasing difficulty climbing stairs and getting up from the floor. A repeat biopsy of the quadriceps is under review by Dr Sewry. It still shows type 1 atrophy with rods in type 1 fibres. The family history is negative. The patient's parents have both died. She has one sister and brother, both unaffected, and two unaffected children.

Hans Goebel, Mainz, Germany described the morphology of muscle biopsy specimens from three patients with congenital myopathy with excess of thin filaments. Two patients showed masses of actin filaments within muscle fibres, one of them also showing intranuclear and some sarcoplasmic nemaline bodies. The third patient showed intranuclear nemaline bodies with masses of thin filaments in several but not all muscles.

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3. Ultrastructural studies 

Lars-Eric Thornell, Umeå, Sweden, described the Z-disc and the structure of nemaline bodies.

Bharat Jasani and Geoffrey Newman, Cardiff, UK, reported preliminary results of their ultrastructural studies of cases with nebulin mutations.

Caroline Sewry, London, UK presented observations on ultrastructural features in cases of nemaline myopathy seen at Hammersmith Hospital. The possibility of relating Z-line width to the number of nebulin repeats was discussed. Preliminary studies of archival micrographs showed variability in Z-line width in myofibrils with and without rods, in the same fibre. Although this might relate to changes in fibre type, the difficulties in interpretation were highlighted. Further studies may be informative, if material is obtained specifically for this study, but it was concluded that any differences might be due to several factors. Dr Sewry also illustrated a variety of Z-line abnormalities, in addition to rods, that can be seen even within one case of nemaline myopathy. For example, core-like regions with gross disruption of myofibrils and accumulation of Z-line like material, was observed in the very mild case from Hammersmith that is now known to have a mutation in the actin gene.

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4. Nebulin (NEB)-related disorders 

Katarina Pelin, Helsinki, Finland, described the nebulin gene, its protein structure and polymorphisms (Table 2) and mutations found to date. The 3′ end of NEB, encoding the last two super-repeats (S21–S22), the simple repeats M163–M185, and the unique end domains, comprises at least 46 exons, 93–184 bp in size. Several differentially expressed exons have been identified in the region encoding (Table 3) the Z-disc part of nebulin. Evidence for differentially expressed exons encoding the central part of the molecule also exists. The exons have been screened for mutations by SSCP (single strand conformation polymorphism) analysis in 37 families with nemaline myopathy. To date 11 different mutations have been identified in 11 families of different ethnic origins. The patients are compound heterozygotes for the mutations in six of the families, and homozygous in five. The majority of the mutations are small deletions or insertions causing frameshifts, or base substitutions causing stop codons [2].

Table 2. Sequence variants identified in the nebulin cDNA by RT-PCR and direct sequencing (GenBank AC X83957)^a

Table 3. Sequence variants identified in the 3′ end of the nebulin gene by genomic PCR and SSCP (genomic sequence, GenBank AF117661-AF117675, AC009497)

Nigel Laing, Perth, Australia, presented five novel nebulin exons detected in the mRNA of one Australian patient with a severe form of nemaline myopathy. These extra exons lie between repeats 177 and 178 in the nebulin cDNA sequence (Accession number X83957) described by Labeit and Kolmerer [3] (1995, #2016). The five novel exons have been numbered 177b–d and 178a. To date, four of the five exons have been identified in genomic DNA by Katarina Pelin in Helsinki. The physiological role of these exons remains unknown.

Alan Beggs, Boston, MA, presented polymorphisms found in the nebulin gene by Dr Dungrurdee Wattanasirichaigoon and himself (Table 3).

Carina Wallgren-Pettersson presented the clinical spectrum in patients with mutations detected in the nebulin gene (Table 1). Mutations had been identified in 15 patients from 11 families. Nine of these patients had the typical form of nemaline myopathy, three had the severe form, two had the mild form and one the intermediate form (see Appendix A). Eight of the families showed autosomal recessive inheritance, while three cases were sporadic.

Caroline Sewry discussed her immunocytochemical studies of nebulin in cases with known nebulin mutations, using three polyclonal antibodies to different C-terminal domains, kindly made available by Dr Siegfried Labeit. These antibodies showed differences in labelling in controls; the antibody to the M176-M181 repeat region showed more pronounced labelling in fibres with slow myosin, while the antibodies to the SH3 region and serine rich domains showed uniform labelling of all fibres. All cases of nemaline myopathy showed the presence of nebulin, but there was variability between cases and differences in the pattern compared with controls. In particular, one case with a homozygous stop mutation in exon 185 showed a total absence of labelling with the SH3 antibody. This indicates that in some cases, studies of nebulin may have a role in directing mutational analysis. Several cases showed unevenness of immunolabelling for nebulin in some fibres. The fibre-typing patterns with nebulin were altered in two other cases but the abundance of fetal myosin and co-expression of fast and slow myosin isoforms made it difficult to type the fibres. Correlations with antibodies to alpha-actinin 2 and alpha-actinin 3, kindly provided by Dr Alan Beggs, also revealed differences in cases of nemaline myopathy. In particular, the presence of alpha-actinin 3 in fibres with slow myosin (this antibody recognises a subset of fast fibres in control muscle). These results suggest that changes of isoforms in myofibrillar proteins is occurring, but the pathological relevance still has to be established. Dr Sewry observed no enhancement of nebulin immunolabelling in rods, similarly no absence was observed. The labelling of Z-lines, however, may make the rods difficult to observe, and conventional fluorescence microscopy is probably not adequate to examine nebulin in rods.

Claudio Graziano, Florence, Italy presented a series of Italian patients with nemaline myopathy, based on a collaborative effort by Berardino Porfirio, also present at the Workshop, and other colleagues in Italy. All patients had congenital nemaline myopathy, one severe nemaline myopathy, one intermediate, and all others representing the typical form of congenital nemaline myopathy. In one family with an affected sib pair there was lack of reactivity to nebulin antibodies in some of the type 2 fibres.

Siegfried Labeit, Heidelberg, Germany together with Marie-Louise Bang of the same group presented their work on nebulin. Nebulin is a giant (about 800 kDa) large filamentous protein of vertebrate skeletal muscle. Its C-terminal end is located at the Z-lines, whereas its N-terminal end is at the pointed end of the thin filament. The group have raised nebulin-specific antibodies which are directed to the C-terminal SH3 domain and the M176-M181 repeats. Immunoelectron microscopy with these two antibodies has demonstrated that the M176-SH3 segment of nebulin resides within Z-lines. The nebulin filament inserts about 25 nm inside the Z-line but does not traverse it. The M176-SH3 segment is also the part of the nebulin filament which is differentially expressed. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies on a panel of 12 rabbit skeletal muscle tissues has shown that a larger number of different repeat-sized isoforms are expressed in the rabbit skeletal muscles. This finding appears to be in agreement with the data from Laing, Akkari, Wallgren-Pettersson, Pelin and colleagues that alternative exons locate in the M177/M178 region in the human nebulin gene.

To further increase our knowledge of nebulin's layout in Z-lines, and to obtain better tools for the immnohistochemical analysis of nemaline myopathy biopsies, Drs Jasani and Newman (Cardiff, University of Wales) together with Dr Labeit are planning a collaborative effort to develop nebulin-specific antibodies by phage display technology. Using the nebulin C-terminal cDNA fragments coding for the M160-M185- SH3 segment, Siegfried Labeit and Marie-Louise Bang have applied the yeast two-hybrid approach in search of the interactions made by the Z-line nebulin. When screening a human skeletal muscle library, they have identified two potential ligands: desmin and a novel protein, which has a molecular weight of 145 kDa. Current work focuses on fine-mapping studies to identify which respective parts of desmin, p145 and nebulin may interact with. Furthermore, using p145 as bait, they are searching for additional proteins which may interact with p145.

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5. Other Z-disc proteins and their interactions 

Olli Carpén, Helsinki, Finland, presented his work on myotilin, a novel protein expressed in skeletal and cardiac muscle, colocalizing with alpha-actinin in the sarcomeric I-bands and directly interacting with alpha-actinin. The myotilin gene is composed of 10 exons, the protein is 57 kDa and the gene is localized on chromosome 5q31 between the markers AFM350yB1 and D5S500. The locus of a dominantly inherited limb-girdle muscular dystrophy (LGMD1A) resides in an overlapping narrow segment. The muscle specificity of myotilin and its apparent role as a sarcomeric structural protein raises the possibility that defects in the myotilin gene may cause muscle disease [4].

Kati Donner, Helsinki, Finland presented data from a preliminary screen for mutations in the recently described myotilin gene. Nine patients with clinically different types of nemaline myopathy (typical, severe or other forms) were chosen for the study. The ten myotilin exons were analysed by SSCP. The exons were also sequenced in one patient with a severe form of nemaline myopathy. This patient was heterozygous for a silent polymorphism (G/A) in exon VI. No mutations were found in any of the patients.

Alan Beggs described 3c8, a novel sarcomeric alpha-actinin binding protein whose expression is restricted to skeletal muscle Z lines. This 32 kDa protein was identified in a yeast two-hybrid screen of skeletal muscle cDNAs using a portion of alpha-actinin-2 as bait and it binds to repeats 3 and 4 of the central rod regions of both alpha-actinins-2 and -3. Indirect immunofluorescence demonstrates 3c8 associated with nemaline rods in a pattern similar to that of alpha-actinin. Mutation analysis of the 3c8 gene in patients with nemaline myopathy and other neuromuscular disorders is ongoing, although no probable mutations have been found to date.

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6. Tropomyosin 3 (TPM3)–related disorders 

Nigel Laing and Alan Beggs provided an update of mutations found in the tropomyosin 3 gene. To date, still only three mutations have been identified. The first is the Met9Arg mutation found in the Australian dominant nemaline myopathy family [5], the second the homozygous missense mutation in one patient with presumed recessive nemaline myopathy [6] and the third a point mutation changing the termination codon to a serine codon in one family from the USA, where the disease also appears to be recessive and the other, as yet unidentified mutation appears to be a functional knockout of the other TPM3 gene.

Kathryn North presented detailed LM and EM studies of muscle biopsies from two members of the big Australian family in which mutations in this gene were originally described. Clinically, the patients show childhood onset, selective involvement of the distal muscles of the lower limbs, mimicking Charcot-Marie–Tooth disease (CMT1), and slow progression. Biopsies from both family members (aged 22 and 46 years) showed two populations of muscle fibres, normal or hypertrophied fibres which expressed fast myosin only or coexpressed fast and slow myosin, and atrophied fibres which expressed slow myosin only. Nemaline bodies were observed only in fibres expressing slow myosin exclusively, i.e. in fibres expected to express the mutant, slow tropomyosin. The younger patient demonstrated marked hypertrophy of type 2 fibres, which may compensate for the type 1-specific pathological process. Immunocytochemical studies of fibre-type-specific proteins suggested that the predominance of type 1 fibres was due to a combination of abnormal maturation and fibre-type conversion.

Edna Hardeman, Sydney, Australia reported on the transgenic mouse model for TPM3 expressions.

Transgenic mice that express a dominant negative mutation (Met9Arg) in alpha-TPM_slow revealed muscle pathology consistent with that observed in humans. In addition, two structural abnormalities that are not characteristic of nemaline pathology were identified: tubular aggregates of the sarcoplasmic reticulum and cytoplasmic bodies. Cytoplasmic bodies are closely associated with rods and have now been detected in a human nemaline sample. Interestingly, both structures have been found in only one of the ten mouse muscles examined, the superficial gastrocnemius (SG) of the lower hindlimb. Using a combination of isoelectric focusing gels and Western blotting, Dr. Hardeman's group has detected the mutant protein in all muscles examined. A striking feature of this model is that although the level of mutant protein is similar in the different muscles, the pathology varies. For example, the extensor digitorum longus (EDL) and SG muscles have similar fibre composition and levels of mutant protein. However, at 6 months of age, 33% of fibres in the EDL contain rods vs. 4% in the SG. Analysis of a range of muscles from the forelimb, trunk and hindlimb supports the dissociation of mutant protein level with severity of pathology. This incomplete penetrance could reflect differential tolerance of the mutated protein as a result of differential muscle usage.

Another feature of this myopathy, type I fibre predominance, was addressed in this model. Using antibodies that discriminate between the adult myosin heavy chains present in the fibres, IIB, IIX, IIA and I, the research group examined the fibre type composition of the EDL at 1, 2, 6 and 12 months of age. Their results are consistent with a failure of fibre maturation rather than conversion of mature IIB to type I fibres. They propose that this mechanism is responsible in part for the type I fibre predominance observed in humans. At present, there is no obvious clinical muscle weakness. Since it has been observed in humans that atrophy appears irreversible in patients with nemaline myopathy, the group is employing established methods to induce atrophy in specific muscles of these mice. In addition, they are increasing the load experienced by muscles thus increasing the slow fibre composition in order to determine whether this leads to the muscle weakness and perhaps fibre type conversion observed in humans.

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7. Presentations of new clinical series 

Ikuya Nonaka, Tokyo, Japan, presented clinical and pathological findings in 80 Japanese patients with nemaline myopathy. These were among the 348 patients diagnosed at the National Centre of Neurology and Psychiatry between 1978 and 1998 as having a congenital myopathy. Out of the 80 patients, 28 had the severe neonatal form, two had the intermediate form and 33 had typical nemaline myopathy. Three of the patients with the severe form have been shown by Dr Laing's group to have different mutations in the actin gene to those identified in Caucasian patients. These mutations have not been seen in Caucasian controls, but Japanese control DNA samples have yet to be screened. Among the 33 patients with the typical form, one family showed autosomal dominant inheritance, one showed autosomal recessive inheritance and all others were sporadic cases. Eight had late respiratory failure and four had deceased. Most patients either had smallness of type 1 fibres and type 1 predominance, or uniformly showed type 1 fibres.

Kathryn North summarized the clinical and light-microscopic pathological findings in 63 Australian patients, of whom clinical and pathological data as well as muscle biopsy samples have been collected. Twenty-seven cases were sporadic, there were eight families with autosomal dominant inheritance, and three families with autosomal recessive inheritance. Inheritance was unclear in a further five families (including two patients who had asymptomatic parents with abnormal biopsies). About 50% of patients presented in the first 4 weeks of life, however it was not possible to predict outcome (in terms of survival) until 9 months of age. The best predictor of survival was achievement of motor milestones less than 12 months of age. Progressive respiratory insufficiency and failure to achieve motor milestones was highly predictive of early mortality. There was greater clinical heterogeneity in patients presenting after the neonatal period. In familial cases, there was considerable variation between family members in clinical course and outcome, suggesting an influence of stochastic factors or modifying genetic loci.

Nigel Laing and Norma Romero, Paris, France provided an update of families with core-rod myopathy with dominant inheritance. Norma Romero updated results on a large French family in which the disease was previously shown to be linked to the ryanodine receptor (RYR1) region on chromosome 19.

Nigel Laing described two West Australian families, one of Belgian extraction and the other originating from The Netherlands, showing unstructured cores and rods. These two families have a total of 12 clinically affected individuals, the larger showing tentative exclusion of the RYR1 gene region [7].

Carina Wallgren-Pettersson presented clinical and genetic linkage data on three families not showing linkage to any of the three known loci for nemaline myopathy [8]. All had severe nemaline myopathy.

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8. Clinical classification of nemaline myopathy 

Carina Wallgren-Pettersson presented her suggestion for a clinical classification of nemaline myopathy based on correlations from the international database on nemaline myopathy, now containing entries on more than 170 patients from various parts of the world. Among the database entries, the typical form is most frequent, but there may be a bias in terms of very severe cases and very mild cases being underdiagnosed. The most common mode of inheritance is autosomal recessive.

The clinical categories were discussed and consensus was achieved. Nemaline myopathy was divided into (1) severe congenital nemaline myopathy, (2) the typical form of nemaline myopathy, (3) intermediate congenital nemaline myopathy, (4) mild, childhood- or juvenile-onset nemaline myopathy, (4) adult forms of nemaline myopathy and a category of (5) other forms of nemaline myopathy ( see Addendum). The categories were defined using the typical form as a starting-point and the other categories were then defined on the basis of different ages of onset or grades of severity, progression or because of unusual associated features.

Please observe that one inclusion criterion is enough for a patient's nemaline myopathy to be assigned to categories 1, 3, 4 or 5; not all patients fulfil all inclusion criteria, but all differ in at least one major respect from the typical cases.

A word of caution is warranted in using the classification: Age of onset is clearly not always the same as age at presentation, and careful history taking and scrutinising of clinical notes is necessary to determine age of onset in individual cases. Early signs not always noted are feeding difficulties, floppiness and delay in attaining motor milestones. Also, the classification is age-dependent, so that the intermediate form can only be distinguished from the typical form at the age where motor milestones would normally have been reached.

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9. Workshop participants 

Dr Marie-Louise Bang, Heidelberg, Germany

Dr Alan Beggs, Boston, MA, USA

Dr Olli Carpén, Helsinki, Finland

Dr Kati Donner, Helsinki, Finland

Professor Victor Dubowitz, London, UK

Dr Marc Fiszman, Paris, France

Professor Dr Hans Goebel, Mainz, Germany

Dr Claudio Graziano, Florence, Italy

Dr Edna Hardeman, Wentworthville, Australia

Dr Bharat Jasani, Cardiff, UK

Dr Siegfried Labeit, Heidelberg, Germany

Professor Nigel Laing, Perth, Australia

Dr Martin Lammens, Leuven, Belgium

Dr Carmen Navarro, Vigo, Spain

Dr Geoffrey Newman, Cardiff, UK

Professor Ikuya Nonaka, Tokyo, Japan

Dr Kathryn North, Sydney, Australia

Dr. Kristen Nowak, Perth, Australia

Dr Katarina Pelin, Helsinki, Finland

Dr Berardino Porfirio, Florence, Italy

Dr Norma Romero, Paris, France

Dr Caroline Sewry, London, UK

Professor Lars-Eric Thornell, Umeå, Sweden

Dr Carina Wallgren-Pettersson, Helsinki, Finland

Dr Duangrurdee Wattanasirichaigoon, Boston, MA, USA

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Acknowledgements 

This Workshop was made possible thanks to the financial support of the European Neuromuscular Centre (ENMC) and its main sponsors: Association Française contre les Myopathies (France); Deutsche Gesellschaft für Muskelkranke (Germany); Telethon Foundation (Italy); Muscular Dystrophy Group of Great Britain and Northern Ireland (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's associate member, Muscular Dystrophy Association of Finland.

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Appendix A. Clinical categories of nemaline myopathy 

A.1. Severe congenital nemaline myopathy 

Inclusion criteria:

Exclusion criteria:

A.2. Intermediate congenital nemaline myopathy 

Inclusion criteria:

A.3. The typical form of congenital nemaline myopathy 

Inclusion criteria:

Exclusion criteria:

A.4. Mild childhood or juvenile onset nemaline myopathy 

A.5. Adult-onset forms of nemaline myopathy 

Inclusion criterion:

A.6. Other forms of nemaline myopathy 

Inclusion criteria:

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References 

1. Nowak KJ, Wattanasirichaigoon D, Goebel HH, et al.  Mutations in the skeletal muscle alpha actin gene in patients with actin myopathy and nemaline myopathy. Nat Genet. 1999;23:208–212
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2. Pelin K, Hilpelä P, Sewry C, et al.  Mutations in the nebulin gene associated with autosomal recessive nemaline myopathy. Proc Natl Acad Sci USA. 1999;96:2305–2310
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3. Labeit S, Kolmerer B. The complete primary structure of human nebulin and its correlation to muscle structure. J Mol Biol. 1995;248:308–315
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5. Laing NG, Wilton SD, Akkari PA, et al.  A mutation in the alpha-tropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy NEM1. Nat Genet. 1995;9:75–79
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6. Tan P, Briner J, Boltshauser E, Davis MR, Wilton S, Wallgren-Pettersson C, et al. Homozygosity for a nonsense mutation in the alpha-tropomyosin gene TPM3 in a patient with severe congenital nemaline myopathy. Neuromusc Disord. 1999;9:573–579
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7. Laing NG, Mastaglia FL, Fabian V, et al.  Genetic studies in a family with autosomal dominant rod and core disease. Neuromusc Disord. 1998;8:279–280
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