Brain Advance Access originally published online on June 4, 2003
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Brain, Vol. 126, No. 7, 1545-1551,
July 2003
© 2003 Guarantors of Brain
doi: 10.1093/brain/awg162
A locus on chromosome 15q for a dominantly inherited nemaline myopathy with core-like lesions
1 Neuromuscular Center Nijmegen, Institute of Neurology, 2 Department of Pathology, 3 Department of Human Genetics, 4 Department of Otorhinolaryngology, University Medical Center Nijmegen, Nijmegen, 5 Department of Neurology, St Elisabeth Hospital, Tilburg, 6 Department of Neurology, Antonius Hospital, Nieuwegein, The Netherlands, 7 Neurogenetic Unit, Departments of Neurology and Anatomical Pathology, Royal Perth Hospital, Perth, 8 Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Nedlands, Western Australia, Australia and 9 Max-Delbrück-Centre for Molecular Medicine, Berlin, Germany *These authors contributed equally to this work
Correspondence to: B. G. M. van Engelen MD, PhD, Neuromuscular Centre, Nijmegen Institute of Neurology, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands E-mail: b.g.m.vanengelmen{at}neuro.umcn.nl
Received November 12, 2002. Revised February 28, 2003. Accepted March 3, 2003.
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Summary |
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 Top Summary IntroductionPatients and methodsResultsDiscussionReferences |
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Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness and the presence of nemaline rods. Five genes have now been associated with nemaline myopathy:
-tropomyosin-3 (TPM3), -actin (ACTA1), nebulin (NEB), ß-tropomysin (TPM2) and troponin T (TNNT1). In addition, mutations in the ryanodine receptor gene (RYR1) have been associated with core-rod myopathy. Here we report linkage in two unrelated families, with a variant of nemaline myopathy, with associated core-like lesions. The clinical phenotype consists of muscle weakness in addition to a peculiar kind of muscle slowness. A genome-wide scan revealed a locus for nemaline myopathy with core-like lesions on chromosome 15q21–q23 for both families. Combining the two families gave a two-point LOD score of 10.65 for D15S993. The -tropomyosin-1 gene (TPM1) located within this region is the strongest candidate gene. However, no mutations were found in the protein-coding region of TPM1, although small deletions or mutations in an intron cannot be excluded. The critical region contains few other candidate genes coding for muscle proteins and several genes of unknown function, and has not yet been sequenced completely. The novel phenotype of nemaline myopathy in the two presented families corresponds to an also novel, as yet uncharacterized, genotype. Keywords: nemaline; myopathy; genotype; core; chromosome 15
Abbreviations: TLN2= gene coding for talin2; TPM1 = gene coding for -tropomyosin 1
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Introduction |
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TopSummaryIntroductionPatients and methodsResultsDiscussionReferences |
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Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness and the presence of nemaline bodies, also called rods, in the muscle fibres (Wallgren-Pettersson and Laing, 1996
). The clinical appearance of the disease is variable, ranging from almost no symptoms to fetal akinesia. The mode of inheritance of nemaline myopathy can be autosomal recessive as well as autosomal dominant, and many sporadic cases have a de novo mutation (Nowak et al., 1999). Up to now, five genes have been associated with the disease: -tropomyosin-3 (TPM3) (Laing et al., 1995; Tan et al., 1999; Wattanasirichaigoon et al., 2002) can be involved in both autosomal dominant and autosomal recessive forms, as is also the case for -actin (ACTA1) (Nowak et al., 1999). Mutations in the nebulin gene (NEB) (Pelin et al., 1999) and in the troponin T gene (TNNT1) (Johnston et al., 2000) cause autosomal recessive nemaline myopathy, and mutations in the ß-tropomyosin (TPM2) (Donner et al., 2002) gene cause an autosomal dominant form of the disease. These five genes all encode components of the thin filaments of the sarcomere. Additionally, mutations have been identified in the ryanodine receptor gene (RYR1) in families with autosomal dominant core-rod disease (Monnier et al., 2000; Scacheri et al., 2000).
We recently described a large Dutch family with autosomal dominant nemaline myopathy (Gommans et al., 2002). Patients of this family suffer from mild muscle weakness in the neck flexors and proximal muscles of the limbs. Facial, ankle dorsiflexor and respiratory muscles function normally. The most remarkable feature in this family is a type of slowness in movement, which has not been reported previously in nemaline myopathy. A second, Australian–Dutch, family displayed a similar phenotype, although no prominent complaints of muscle slowness existed (P. Lamont, unpublished data). Muscle biopsies in both families showed numerous rods, but also core-like lesions. By linkage analysis, the genes which until then were known to be involved in nemaline myopathy and core-rod disease were excluded in both families (Gommans et al., 2002; P. Lamont, unpublished data). Here we report that a genome-wide scan with microsatellite markers revealed overlapping linkage regions on chromosome 15q21–23 for both families. Mutation analysis of the -tropomyosin-1 gene (TPM1), located within the critical region, failed to identify disease-associated mutations.
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Patients and methods |
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Patients
Family P97-889 (A) of Dutch origin previously was described clinically by Gommans et al. (2002
). In brief, the patients have muscle weakness in the neck flexors and proximal muscles of the limbs without involvement of facial, respiratory, ankle dorsiflexion or cardiac muscles. The muscle weakness is very slowly progressive. The most remarkable clinical feature is muscle slowness. Patients are unable to perform fast movements, e.g. running. Walking long distances is possible. Patients are unable to correct themselves from falling over when stumbling and are even too slow to stretch out their arms to break their fall. Quadriceps muscle biopsies were performed in six patients and showed type 1 fibre predominance. Modified Gomori’s trichrome stain revealed many fibres with a granular aspect suggestive of the presence of nemaline rods (Fig. 1A). ATPase and oxidative staining showed zones devoid of activity (Fig. 1B and C). By the circumscribed loss of myosin and mitochondria, these zones mimic so-called cores, but they are less sharply punched out than classical central cores. Ultrastructural examination of longitudinally cut sections revealed the presence of nemaline rods in almost all fibres. The numbers varied from a single rod in a low power field to large aggregations of rods while, inside these regions, hardly any mitochondria were observed (Fig. 1D). These fields are likely to correspond to the so-called cores. Apart from the former core-like lesions, which we prefer to call pseudocores, because they in fact consist of many rods, there were other core-like lesions, far fewer in number and consisting of irregular electron-dense material suggestive of Z-band streaming (Fig. 1E). The latter core-like regions are often bordered by the former pseudocores.
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The diagnosis was made by physical examination except for person III:38. This man initially was presented as being affected based on history and clinical examination revealing general weakness (Gommans et al., 2002
). However, since his general weakness could also be caused by his bad general condition due to severe cardiac and pulmonary problems, a muscle biopsy was taken before he was included in the linkage study. Analysis of the biopsy according to the methods described by Gommans et al. (2002) indicated that he is unaffected. Family 1481 (B) of Australian–Dutch origin showed histological and clinical characteristics comparable with those of family A, with the exception that not all patients complained of muscle slowness and the proband alone showed weakness of ankle dorsiflexion.
Microsatellite analysis
In families A and B, genomic DNA was isolated from blood according to Miller et al. (1988). Genome scans were performed with microsatellite markers with an average spacing of 11 cM (Sander et al., 2000). PCR (polymerase chain reaction) fragments were analysed using an ABI PRISM 3700 DNA analyser (Applied Biosystems). Data were analysed by Genescan 2.1 and Genotyper 2.0 software (Applied Biosystems).
The genome screen in family B was undertaken using the Cooperative Human Linkage Centre/Weber human screening set version 9aRG obtained from Research Genetics (Huntsville, AL, USA). Markers for fine mapping were obtained from the Généthon database (ftp://ftp.genethon.fr/pub/Gmap/Nature-1995/data). PCR was performed at 55°C using 1 mM MgCl2 and 20 µM dNTPs in 10 µl reactions. Products were labelled by incorporation of 32P, and electrophoresed on 6% denaturing polyacrylamide gels, dried and exposed to X-ray film using standard methods. Alleles were hand scored.
Statistical analysis
Two-point LOD scores were calculated with the MLINK subroutine of the LINKAGE package version 5.1 (Lathrop and Lalouel, 1984; Lathrop et al., 1984, 1986) for family A. For family B, two-point LOD scores were calculated using LIPED. Complete penetrance and a disease allele frequency of 0.001 were assumed for the calculations.
Mutation analysis
The protein-coding exons of the TPM1 gene expressed in striated muscle, exons 1a, 2b, 3–5, 6b, 7, 8, 9a and 9b, and the exon–intron boundaries of these exons were analysed by sequence analysis in patient III:34 of family A and patients III.1 and III.6 of family B. PCR primers were as described by Thierfelder et al. (1994). For amplification of the exon containing the stop codon and 3'-non-coding region, primers designed were: forward 5'-TTTTTTTCTCATTGTGCCACTT-3' and reverse 5'-GACAAATGCAGAGCTCAGAG-3'. For amplification of the non-muscle exon 6a, primers designed were: forward 5'-TTTGTGAATGGCCTTGTGC-3', reverse 5'-GGTTGCAGTTAAACCTAAAAACC-3'.
Sequence analysis was performed using the ABI PRISM Big Dye Terminator cycle sequencing V2.0 ready reaction kit and ABI PRISM 3700 DNA analyser (Applied Biosystems).
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Results |
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Linkage analysis
Family A
In the genome scan, no indications for linkage were obtained on chromosomes 1–14. Subsequently, marker D15S1023 on chromosome 15q gave a two-point LOD score >3. Testing of additional markers in the region and haplotype analysis (Fig. 2) defined a critical region delimited by the markers D15S1033 and D15S1041, a region of
25 cM, if only affected individuals are included. The unaffected individual V:3 defines D15S125 as the distal flanking marker of the critical interval, thereby reducing the region to 12 cM. This individual is an 18-year-old girl who is a good athlete on history and has a normal clinical examination. Combined with the absence of any indication for reduced penetrance in both families, it is unlikely that she is a carrier of the disease-causing mutation.
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Two-point LOD scores, calculated using all affected and unaffected individuals in the family, are shown in Table 1. A maximum LOD score of 9.36 was calculated for the marker D15S993.
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Family B
No indications for linkage were obtained on chromosomes 1–14 or 16–22. Analysis of microsatellite markers derived from the linkage interval determined for family A resulted, for family B, in statistically significant two-point LOD scores, with a maximum score of 3.61 for marker D15S125. Construction of the most likely haplotypes in this family resulted in a critical region delimited by the markers D15S155 and D15S131 (Fig. 3), a distance of 19 cM. The region is defined by recombinations in affected individuals. Two-point LOD scores calculated using all affected and unaffected individuals in the family are shown in Table 2.
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Combining the data of both families results in a critical region of
19 cM located between the markers D15S155 and D15S131 when only affected family members are included. When the unaffected individual V3 in family A is included, the critical region is an 12 cM interval between D15S155 and D15S125. This is a distance of 6.9 Mbp (mega base pairs) according to the June 2002 freeze of the draft sequence of the human genome. The maximum two-point LOD score obtained combining the two families is 10.65 for D15S933.
Haplotype analysis
Since both families are of Dutch origin and because of the rareness of the phenotype, we hypothesized that the families might have a common founder, although this was not suggested by genealogical analysis. Allele sharing was tested for the markers D15S155, D15S643, D15S1036, D15S993, D15S125, TPM1, D15S1015, D15S983, D15S100 and D15S131, which are derived from the critical region and are spaced at between 0 and 5 cM intervals. Since no sharing of alleles in the disease-associated chromosomes of both families was seen, we have no indications for a common founder for the families.
Mutation analysis of the TPM1 gene
A survey of the known genes in the linkage interval revealed that the fast -tropomyosin gene TPM1 is located within the critical region determined using only affected individuals or including affected and non-affected individuals. Mutations in this gene previously have been shown to be causative only for hypertrophic cardiomyopathy (Thierfelder et al., 1994), but mutations in two other tropomyosin genes, TPM3 (Laing et al., 1995) and TPM2 (Donner et al., 2002), have been shown to be causal for nemaline myopathy. TPM1 therefore was a strong candidate gene for the nemaline/core myopathy localized to this region. Sequence analysis of the protein-coding exons of the striated muscle isoform, the non-muscle exon 6a, the exon containing the stop codon, the 3'-non-coding region and all the exon–intron boundaries of these exons did not, however, identify a disease-associated mutation in either family.
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Discussion |
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TopSummaryIntroductionPatients and methodsResultsDiscussionReferences |
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Here we describe two nemaline myopathy families clinically characterized by proximal muscle weakness without weakness of the facial, ankle dorsiflexion (except in one patient) and respiratory muscles, which discriminates this phenotype from the classical nemaline myopathy. In addition, the patients of family A complained of muscle slowness, whereas only a few patients in family B complained of this phenomenon.
Muscle histology in both families showed numerous rods and a few core-like lesions on electron microscopy. The ‘pseudocores’ seen on light microscopy seem to be large areas of rods with loss of muscle structure (Fig. 1).
After specifically excluding the known genes for nemaline myopathy and core-rod myopathy (Gommans et al., 2002), linkage was shown to chromosome 15q21–23 in both families. A critical region of 19 cM, located between markers D15S1033 and D15S131, was determined when only affected individuals were included. Inclusion of the unaffected individual V:3 (family A) reduces the critical region to 12 cM.
All genetic causes for nemaline myopathy known so far are mutations in genes encoding components of the thin filaments of the sarcomere (Wallgren-Pettersson and Laing, 2001), while mutations associated with core-rod disease have been identified in RYR1, the gene for the sarcoplasmic calcium channel (Monnier et al., 2000; Scacheri et al., 2000). Therefore, we hypothesize that the present type of nemaline myopathy may also be caused by a mutation in a gene encoding a protein of the thin filaments or a gene involved in calcium metabolism. The TPM1 gene, encoding fast -tropomyosin, is located in the critical region. We regarded the TPM1 gene as a candidate gene, since mutations in two other tropomyosin genes TPM2 and TPM3 have been associated with nemaline myopathy (Laing et al., 1995; Tan et al., 1999; Donner et al., 2002; Wattanasirichaigoon et al., 2002). Until now, mutations in the TPM1 gene have only been associated with familial hypertrophic cardiomyopathy (Thierfelder et al., 1994). However, other specific TPM1 mutations might be causative for nemaline myopathy without a heart defect, which is the phenotype in the present families. We excluded mutations in the exons of TPM1 encoding the isoform present in striated muscles and non-muscle exon 6a. The heterozygous presence of a polymorphism in exon 4 also excludes a deletion of the entire gene. Small deletions, or duplications or intronic mutations leading to aberrant splicing cannot yet be excluded.
Besides the TPM1 gene, no other known genes encoding muscle filament proteins are located in the interval between D15S1033 and D15S131. Because of the muscle slowness, genes involved in excitation–contraction coupling are candidate genes. However, no such genes, apart from TPM1, are known to be located in the defined region [Human Genome Project Working draft (http://genome.cse.ucsc.edu)]. Also, other genes coding for specific muscle proteins or genes involved in energy metabolism were not found. The defined region contains two genes encoding myosins: MYO1E and MYO9A. However both these myosins are unconventional myosins and not known to be involved in the thick filament or in excitation–contraction coupling. Another possible candidate gene is that for talin2 (TLN2). Talin is an important component of focal adhesion plaques that link integrin to the actin cytoskeleton. It is highly expressed in the myotendinous junction (Tidball et al. 1986). As tenotomy is known to evoke rods in rats (Shafiq et al., 1969) and rods and cores in cats (Engel et al., 1966), a defective myotendinous junction might also cause rods. Multiple transcripts were detected for TLN2, and expression levels varied greatly across different tissues, with highest levels in the heart (Monkley et al. 2001). Three TLN2 transcripts are known for skeletal muscle; however, although these three transcripts are also found in heart, brain and lung (Monkley et al. 2001), as with TPM1, this does not preclude TLN2 being a candidate for a skeletal muscle disease. Several genes of unknown function are reported for the critical region, and the DNA sequence of the critical region has not yet been completed. Selection of new candidate genes for analysis can be based on homology to known genes and expression profiling of predicted genes from the sequence available thus far and emerging in the future.
In conclusion, we describe two dominant nemaline myopathy families with an unusual phenotype including core-like lesions. Both families showed linkage on chromosome 15q21–23 revealing a novel locus for nemaline myopathy with core-like lesions. Although no mutations were found in the protein-coding region, TPM1 has not yet been completely excluded as being the causative gene. There are few other plausible candidate genes within the critical region. Our results indicate that the novel nemaline core-like myopathy in the two families corresponds to a novel, as yet uncharacterized, genotype.
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Acknowledgements |
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We wish to thank the families for their participation, the Prinses Beatrix Fonds: Grant 00-0209 to B.G.M.v.E. for financial support, and the Australian National Health and Medical Research Council for project grants 139039 and 110242. Part of this work was supported by a grant of the German Human Genome Project to A.R.
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