Distal myopathy caused by homozygous missense mutations in the nebulin gene
1Department of Medical Genetics, University of Helsinki, 2The Folkhälsan Institute of Genetics, 3Department of Pathology, University of Helsinki, 4Department of Pathology, University of Turku, Finland, 5Department of Pediatrics, Hammersmith Hospital, Imperial College Faculty of Medicine, Hammersmith Hospital, London, Centre for Inherited Neuromuscular Disorders, Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, UK, 6Department of Biological and Environmental Sciences, University of Helsinki, and 7Departments of Neurology, Vasa Central Hospital and University of Tampere, Finland
Correspondence to: Carina Wallgren-Pettersson, M.D., The Folkhälsan Department of Medical Genetics, P.O. Box 211, FIN-00251 Helsinki, Finland E-mail: carina.wallgren{at}helsinki.fi
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We describe a novel, recessively inherited distal myopathy caused by homozygous missense mutations in the nebulin gene (NEB), in which other combinations of mutations are known to cause nemaline (rod) myopathy (NM). Two different missense mutations were identified in homozygous form in seven Finnish patients from four unrelated families with childhood or adult-onset foot drop. Both mutations, when combined in compound heterozygous form with more disruptive mutations in NEB, are known to cause NM. Hitherto, no patients with NM have been found to have two missense mutations in NEB. Muscle weakness predominantly affected ankle dorsiflexors, finger extensors and neck flexors, a distribution different both from the patterns of weakness seen in NM caused by NEB mutations, and those of the known recessively inherited distal myopathies. Singleton cases need to be distinguished from the Laing type of distal myopathy. Histologically, this myopathy differs from NM in that nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even with electron microscopy. Rimmed vacuoles, commonly seen in other distal myopathies, were not a feature. We conclude that homozygous missense mutations in NEB cause a novel distal myopathy, predominantly involving lower leg extensor muscles, finger extensors and neck flexors.
Key Words: distal myopathy; nebulin gene; mutation; missense; recessive
Abbreviations: NEB, nebulin gene; NM, nemaline myopathy; TMD, Tibial muscular dystrophy
Received January 24, 2007. Revised February 22, 2007. Accepted March 26, 2007.
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Introduction |
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Mutations in the gene for the giant sarcomeric protein nebulin (NEB, OMIM #161650) are a main cause of autosomal recessive nemaline (rod) myopathy (NM, OMIM #256030) (NM), defined clinically by weakness of voluntary muscles and histopathologically by disorganization of the sarcomeric Z discs and accumulation in the muscle fibres of nemaline bodies or rods, i.e. aggregates of Z-disc and thin filament proteins (Pelin et al., 1999
; Wallgren-Pettersson and Laing 2000; Pelin et al., 2002; Wallgren-Pettersson et al., 2004a). In the typical form of this disorder, muscle weakness is initially proximal but later also distal (Wallgren-Pettersson 1989; Wallgren-Pettersson et al., 1989).
Distal myopathies are a clinically and genetically heterogeneous group of muscle disorders characterized by predominantly distal muscle weakness (Udd and Griggs 2004). Tibial muscular dystrophy (TMD) or Udd myopathy is the most common distal myopathy in Finland, where it was first described (Udd et al., 1993; Hackman et al., 2002; Udd et al., 2005). Thus, it is the first diagnosis to consider in a Finnish patient with such a myopathy. This muscle disorder, caused by heterozygous mutations in the titin gene (OMIM #600334), presents after the age of 35 years with foot drop. Progression is slow. Histological features are myopathic with normal fibre type distribution. Necrotic fibres are rare. In the primarily affected tibialis anterior muscle rimmed vacuoles occur frequently (Udd et al., 2005).
The gigantic protein nebulin (600–900 kDa) is expressed in the thin filaments of striated muscle. It is one of the biggest actin-binding proteins in the muscle sarcomere; one nebulin molecule spans the thin filament having the potential to bind 200 actin monomers (Wang et al., 1996). The differences in the lengths of expressed nebulin molecules correspond to the lengths of the thin filaments. Nebulin is required for the proper assembly of thin filaments, for the maintenance of their lengths and for their contractile function (Chen et al., 1993; Chen and Wang, 1994; McElhinny et al., 2005; Bang et al., 2006; Witt et al., 2006).
The nebulin gene (NEB), located in the chromosomal region 2q21–q22, comprises 183 exons spanning 249 kb of genomic sequence (Donner et al., 2004). The translation initiation codon is in exon 3, and the stop codon and the 3'UTR are in exon 183. The structure of nebulin is highly repetitive; it consists of simple repeat modules, each 30–35 amino acid residues long, forming simple repeats and super repeats. Each of the simple repeat modules binds actin. The C-terminus of nebulin is anchored to 
-actinin in the Z disc of the sarcomere via myopalladin (Bang et al., 2001). Alternatively spliced exons in four regions of the gene, two in the 3' end and two in the central region, give rise to a great variety of different nebulin isoforms (Donner et al., 2004; Donner et al., 2006).
In 1997, NEB was localized to the linkage region for autosomal recessive NM (Wallgren-Pettersson et al., 1995; Pelin et al., 1997), leading to the identification of the first mutations in NEB associated with this rare muscle disorder (Pelin et al., 1999). To date 64 different NEB mutations have been identified in 55 families with NM (Pelin et al., 1999; Pelin et al., 2002; Lehtokari et al., 2006). Hitherto, mutations in this enormous gene have not been associated with any other disorders. Here we describe a novel distal myopathy caused by homozygous missense mutations in NEB, distal nebulin myopathy.
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The seven patients from four different Finnish families included in this study (Figs 1–4
) had presented with foot drop, and six of them had been referred for medical examination because of a clinical suspicion of an unusually early form of TMD (Udd et al., 1993). The seventh patient (Patient 2.II.3), the deceased father of Patient 2.III.2, brother of Patient 2.II.2, had had similar clinical features. All six living patients were re-examined by two of the authors (BU and CWP). The pedigrees are shown in Fig. 5.
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The diagnostic muscle biopsies, 1–4 biopsies per patient, were re-evaluated in detail by light and electron microscopy by two of the authors (CS and HK). Light microscopic stains included haematoxylin and eosin, Gomori trichrome and PAS stains, as well as oxidative enzyme stains and myosin ATPase enzyme reactions. Immunolabelling was performed for alpha–actinin, desmin as well as fetal, neonatal slow and fast myosin isoforms (Novocastra, Newcastle, UK). Specimens for electron microscopy were fixed in glutaraldehyde, postosmicated and embedded in epon. Semithin sections were stained with toluidine blue, and on the basis of these, regions of interest were selected for thin sectioning and examination by electron microscopy.
Molecular genetic studies
Families 1 and 2 with affected sib pairs originated from the same geographic region in western Finland. Because of their geographic origin, their DNA samples were used as pathological controls in the analysis of the origin and age of a specific haplotype of the genetic region harbouring NEB. The DNA samples were analysed using single strand conformation polymorphism (SSCP) and sequencing to create an intragenic haplotype of known single nucleotide polymorphisms (SNP) in NEB.
Polymerase chain reaction
For analysis of NEB (GenBank NT_005151.12) exons 122 and 151 by SSCP and sequencing we used primers located in introns to amplify exons 121 and 122 in one and exon 151 in another fragment. The primer data is available upon request. PCR reactions were performed in 96-well plates each 35 µl reaction mix containing 60–90 ng of genomic DNA (3 µl), 10 x PCR buffer supplied with the AmpliTaq Gold containing 15 mM MgCl2, 5 nmol each of dNTP, 20 pmol forward primer, 20 pmol reverse primer and 0.8 U AmpliTaq Gold polymerase enzyme (Applied Biosystems, Roche Molecular Systems, Inc., Branchburg, USA). The reaction was carried out in a PTC-225 DNA Engine Tetrad Thermocycler (MJ Research, Waltham, USA) starting with denaturation for 10 min at 95°C followed by annealing at 59°C, and extension at 72°C. The lengths of the denaturation, annealing and extension steps were 45 s each. A final extension was performed at 72°C for 10 min. Amplification of the PCR products was confirmed by agarose gel electrophoresis before SSCP and sequence analysis.
SSCP analysis
Fragments containing exons 121 and 122 in one PCR product and exon 151 in another were screened by SSCP analysis in order to analyse known SNP markers. These PCR products were run on 0.5 – 1X MDETM-gels (FMC Bioproducts, Rockland, USA). The gels were run at room temperature for 17–19 h at 3–5 W, fixed in 10% citric acid (Riedel de Haën AG, Seelze, Germany) for 20 min, stained for 30 min in a 0.1% silver nitrate (Riedel de Haën AG, Seelze, Germany) solution supplemented with 0.15% formaldehyde (Merck, Darmstadt, Germany), and developed in a 3% sodium carbonate (Riedel de Haën AG, Seelze, Germany) solution supplemented with 0.15% formaldehyde and 0.0002% sodium thiosulphate (Merck, Darmstadt, Germany).
Sequencing
Samples showing abnormal migration on SSCP gels were sequenced. The PCR products were purified using Exonuclease I and shrimp alkaline phosphatase (USB Corporation, Cleveland, USA), and the purified products were sequenced using BigDye version 3.1 sequencing chemistry and an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, USA). Sequences were analysed by the Sequencher 4.1. software.
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Clinical and histological findings
Family 1
Patient 1.II.1: Both parents of Family 1 with an affected sib pair (Patients 1.II.1 and 1.II.6) were healthy on examination at the ages of 76 and 80 years, respectively, and came from the Southern Ostrobothnia region in western Finland (Fig. 5). Patient 1.II.1 was not known to have had any abnormality of his motor development and he reportedly still walked normally during his military service as a young adult. In his thirties he noted foot drop. His weakness has progressed slowly, he retired at the age of 44 years and at age 57 he walks good distances on even ground but only 100 m on uneven ground. He has had no swallowing difficulties.
The patient sought medical attention at the age of 40 years because of chest pain. This led to investigation of his foot drop. Electromyography at the age of 45 showed small polyphasic motor unit potentials while nerve conduction velocities were normal. Serum activities of creatine kinase were normal.
On examination at the age of 57 years he was a man of normal build with atrophy of the sternocleidomastoid muscles, shoulder region, anterior thigh muscles and dorsiflexors of the feet. He walked with a foot drop. There was moderate symmetric pseudohypertrophy of the calves. The palate was slightly high-arched and there was slight facial weakness. Sensation was normal. Muscle weakness on the MRC scale was of grade 4 in the flexors of the neck and trunk, and of grade 0 in the dorsiflexors of the feet. Strength was normal in all other muscle groups. Forced vital capacity was normal. A CT scan showed fatty replacement of the muscles of the anterior compartments of the lower legs.
A muscle biopsy from the tibialis anterior muscle at the age of 46 years showed severe myopathic changes with marked size variability and fibrosis (Fig. 6A). Hypertrophic fibres, up to 100 µm in diameter, were immunopositive for both slow (MHCs) and for fast (MHCf) myosin, often simultaneously, explaining the poor distinction at ATPase staining of type 1 and type 2 fibres (Fig. 6A). The numerous severely atrophic fibres were usually MHCf positive and they sometimes appeared in groups. Many small fibres were positive for neonatal myosin (MHCn) whereas very few tiny fibres were immunopositive for fetal myosin (MHCd). Internal nuclei were common in hypertrophic fibres and there was occasional fibre splitting, but no rimmed vacuoles were observed (Fig. 6A). The intermyofibrillar network was coarse and lobulated, and there were a few ring fibres (Fig. 6B) and some cytochrome oxidase (COX)—negative fibres. No nemaline bodies were detected with Gomori staining at the time of the diagnostic biopsy, neither were any identified with certainty on re-examination after the discovery of the NEB mutation, nor in additional thin frozen sections. However, in toluidine blue-stained semithin sections of epon embedded samples a few tiny nemaline bodies could be identified in <10% of the fibres (cf. Fig. 6D). The presence of these rods was confirmed by electron microscopy, which disclosed occasional scattered typical nemaline bodies (Fig. 6C). Z discs were out of register and there was focal loss of myofibrils. The distribution of mitochondria was markedly uneven.
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Patient 1.II.6: This patient, brother of Patient 1.II.1, had no motor difficulty before a foot drop was noted around the age of 10 years. He has had no problems with breathing or swallowing. At the age of 35 he was examined because of the myopathy diagnosed in his brother. Electromyography at that time showed small polyphasic potentials in the anterior tibial muscles. At the age of 45 he was re-examined because his legs tired easily and he had an increasing tendency to stumble and fall. Findings at electromyography remained myopathic and nerve conduction velocities were normal. A CT scan showed fatty replacement of the muscles of the anterior compartment of the lower leg (Fig. 7).
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On examination at the age of 48 years he was of heavy build with good muscle bulk except for the neck flexors and the anterior tibial muscles. He had symmetric hypertrophy of the calves. The palate was somewhat high-arched, there was mild dysarthria and weakness of the tongue, which was slightly furrowed, and facial weakness was mild. Muscle weakness was of grade 4 in the neck flexors and finger extensors, of grade 2 in the toe extensors and of grade 0 in the ankle dorsiflexors. Forced vital capacity was normal.
A muscle biopsy was taken from the vastus lateralis at the age of 45. The pathological alterations were mild (cf. Fig. 6E). There was abnormal variability of fibre size, with both MHCf and MHCs positive fibres up to 100 µm in diameter. Atrophic fibres were scarce, though a few nuclear clumps were detected. Occasional internal nuclei were present, but the intermyofibrillar network was regular and there were no COX-negative fibres. On re-examination of very thin Gomori-stained sections a few nemaline bodies were observed. No MHCd or MHCn positive fibres were present. Semithin sections (Fig. 6D) and electron microscopy showed scattered nemaline bodies, and Z discs appeared widened focally.
Family 2
Patient 2.II.2: This family came from the same geographic region as Family 1. The parents of the affected brothers 2.II.2 and 2.II.3 (Fig. 5) were healthy by history and distantly related. Patient 2.II.2 had abnormal gait from the beginning and as a child he often tripped. He has had no difficulties with breathing or swallowing. Working as a woodman he tired easily and had difficulty walking in deep snow. He did not seek medical attention for his muscle weakness, but was referred for examinations at the age of 57 years through the occupational health care system. He was found to have weakness of the facial muscles, shoulder girdle and lower leg muscles. Serum activities of creatine kinase were normal. CT scans of the muscles at the ages of 57 and 64 years showed fatty degeneration in the anterior compartment of the lower legs and in the medial parts of the gastrocnemii. Electromyography showed small polyphasic potentials.
When examined at the age of 70 years he had for some years had a valgus deformity of the knees. He was of slender build and walked with stable hips but lifting his feet high and shifting his weight from side to side. He had mild asymmetric pseudohypertrophy of the calves and atrophy of the neck flexors, shoulder muscles, thighs and the anterior compartments of the lower legs. Muscle weakness of grade 4 was found in the neck flexors, limb girdle muscles, knee and elbow flexors and finger extensors, while the weakness of the ankle dorsiflexors was of grade 0. Facial weakness was mild and he had no dysarthria or swallowing difficulties. Forced vital capacity was normal.
The first biopsy from the vastus lateralis at the age of 57 showed only mild myopathic alterations with marked hypertrophy and indistinct core-like areas with NADH-TR staining. The second biopsy from the tibialis anterior disclosed marked myopathic changes as in Patient 1.1 (cf. Fig. 6A).
Patient 2.III.2 was the niece of Patient 2.II.2 (Fig 5). Her healthy mother (2.II.4) came from the same geographic region, a genetic isolate, as her affected father (2.II.3). Her motor milestones were not observed to be delayed. When she was aged six her parents noticed that she had a foot drop and she remembered tripping and falling at least from the age of seven. She had never been a fast runner. As an adult she noted some weakness of her forearms and hands.
On examination at the age of 8 years she had isolated weakness of the ankle dorsiflexors and the toe extensors. Electromyography showed loss of motor units, motor unit potentials of variable size, and long duration of some of the potentials. Surgical interventions to correct her foot drop were unsuccessful. At the age of 29 years an MRI scan showed fatty degeneration of the tibialis anterior muscles while the other muscles of the lower limbs showed normal findings. Electromyography showed small polyphasic potentials in the tibialis anterior muscles while findings in other muscles were normal. Nerve conduction velocities were normal, as were serum activities of creatine kinase.
When examined at the age of 42 years she was of normal build with foot drop and inability to walk on her heels. She had slight facial weakness with high-arched palate and small mouth, but no swallowing difficulties. Muscle bulk was small in the anterior muscles of the lower legs, the neck flexors and the thenar and hypothenar muscles. Muscle weakness was of grade 4 in the elbow flexors and the wrist and finger extensors and in the intrinsic muscles of the hands, of grade 2 in the neck flexors, and of grade 0 in the ankle dorsiflexors. Otherwise, muscle strength was normal, as was forced vital capacity.
A muscle biopsy was taken at the age of 29 from the tibialis anterior muscle. The findings were similar to those in Patients 1.II.1 and 3.II.1 with severity intermediate between these (cf. Fig. 6A and E). The intermyofibrillar network was irregular as in Patient 1.II.1, but less severely, with core-like structures. No nemaline bodies were detected in Gomori-stained frozen sections, but in semithin epon sections a few tiny nemaline rods were suspected on re-examination, and verified by EM.
Patient 2.II.3 was the deceased father of Patient 2.III.2. He had been examined at the age of 59 years because of a myopathy being investigated in his brother and daughter. According to the hospital records he had had mild muscle weakness from childhood and steppage gait. On examination he had not shown obvious facial weakness. There was mild atrophy of the muscles of the shoulder region and the thighs, but he was able to rise from the squatting position. Low field MRI scans showed atrophy of the vastus lateralis muscle and fatty degeneration of the sartorius and calf muscles, while the tibialis anterior muscles showed complete fatty replacement. Serum concentrations of creatine kinase were normal and electromyography showed myopathic changes in both proximal and distal muscles. A non-diagnostic biopsy from the tibialis anterior muscle showed end-stage pathology.
Family 3
Patient 3.II.1 (Fig. 5) was from a region close to the one from which Families 1 and 2 originated. Her healthy parents were second cousins. A paternal aunt and uncle were said to have had foot drop, but they were unavailable for examination. The patient attained her motor milestones normally but soon after achieving walking she was noted to have foot drop. She felt that the weakness in her lower limbs had remained unchanged, whereas she had noted a slow deterioration in hand strength.
When examined at the age of 14 years she had no movement in the anterior tibial muscles. She had slight facial weakness and atrophy of the thenar and hypothenar muscles. Electromyography recorded myopathic features in the anterior tibial muscles, and a second examination of the small muscles of the hands and feet at the age of 16 was interpreted as showing neurogenic features. Creatine kinase levels were normal or just above normal. At the age of 28 years MRI imaging showed fatty degeneration of the tibialis anterior muscles.
On examination at the age of 37 years she was of slender build and walked with a foot drop. She was unable to walk on her heels. Muscle bulk was notably small in the neck flexors, hands, forearms and anterior compartment of the lower legs, while calf muscle bulk was slightly diminished. She had a 90° contraction of the Achilles tendons. Facial weakness was moderate and the facies was myopathic with small mouth, high-arched palate and small mandible, but she had no swallowing difficulties. Muscle weakness was of grade 4 in the finger flexors, of grade 1–3 in the finger extensors, the index fingers being slightly stronger than the others, and of grade 0 in the ankle dorsiflexors. Forced vital capacity was normal.
This patient underwent four muscle biopsies, at the ages of 14, 16, 27 and 29 years. The first two biopsies showed only non-specific findings. The third biopsy was taken from the extensor carpi radialis muscle and the findings were minor (Fig. 6E), similar to those described for Patient 1.II.6. Neither semithin nor electron microscopy sections revealed nemaline bodies. In the fourth biopsy from the gastrocnemius muscle there were markedly more severe alterations, now similar to those in Patient 1.II.1 (cf. Fig. 6A). In addition, MHCd and MHCn immunopositive fibres were common. In Gomori-stained frozen sections no nemaline bodies were detected, but in epon sections, wavy streaming of Z-discs was observed (Fig. 6F). With electron microscopy no definite rods were identified, but there was diffuse widening and streaming of Z discs, which were often out of register (Fig. 6G).
Family 4
Patient 4.II.2 (Fig. 5): This patient was born to non-consanguineous parents originating from the South of Finland. She had no neonatal problems and her motor development was normal. Her speech has always been slightly dysarthric. At the age of 30 years she experienced difficulty waking up after anaesthesia.
The patient came to medical attention aged 30 years after her second pregnancy because of an increasing tendency to stumble. Her arms and hands had become weaker over the years and she had had to stop playing the piano. Electromyography of the tibialis anterior muscles showed small polyphasic motor unit potentials and fibrillations. MRI revealed fatty degeneration of the anterior compartment of the lower legs, while the other muscles of the lower limbs showed normal results. Serum activities of creatine kinase were normal.
On examination at the age of 42 years the patient had myopathic facies with high-arched palate and small mouth. Her facial weakness was moderate and her speech slightly dysarthric, worsening after ingestion of cold food or in cold weather, but she did not report any swallowing difficulties. The finger and knee flexors showed weakness of grade 4, neck flexors of grade 2, and finger extensors of grade 1–3 with better preservation of index finger extension. The weakness was more pronounced in the right hand. Ankle dorsiflexion and toe extension was of grade 0. She has inversion deformity of both metatarsi and slight asymmetry of calf muscle bulk. Her forced vital capacity was 50% of the reference value.
A muscle biopsy was taken at the age of 30 years from the tibialis anterior muscle. The findings were similar to those in the fourth biopsy of Patient 1.II.6 but somewhat less severe. In additional Gomori-stained frozen sections as well as in semitihin epon sections done after the discovery of the NEB mutations, tiny nemaline bodies were observed (Fig. 6H), and verified by electron microscopy. In a couple of fibres a few vacuoles were encountered containing myeloid bodies and other cell debris corresponding to small rimmed vacuoles, and some of these vacuoles were also associated with tubular aggregates (Fig. 6I).
Summary of the histological findings
The muscle biopsies, in two patients multiple, did not indicate any specific muscle disorder. In none of the 10 biopsies from the six patients from whom representative biopsies were available were nemaline bodies detected in the routine Gomori-stained frozen sections. On closer scrutiny, after the identification of the mutations, a few tiny nemaline bodies were observed in some of these sections. In longitudinal toluidine blue-stained semithin epon sections viewed at high power small nemaline bodies were discovered and verified by electron microscopy in three of the four patients from Families 1 and 2 who had the NEB exon 151 mutation. In Families 3 and 4 with the NEB exon 122 mutation, nemaline bodies were identified in Patient 4.II.2, whereas patient 3.II.1 had only diffuse Z-disc pathology. Thus, electron microscopy showed Z-disc alterations in all, and a few nemaline bodies in 4/5 patients studied using this method. The patient in whom none were found had undergone as many as four biopsies. The nemaline bodies were not discernible with antibodies against alpha–actinin.
The general myopathic pattern was similar in all four families, although the severity of the pathology varied markedly, being more striking in biopsies from the more severely affected anterior tibialis and forearm extensor muscles. The pathological features included marked variability in fibre size. Type 1 fibre predominance was not a feature. There were large hypertrophic fibres with an increase in internal nuclei, as well as very small nuclear clump fibres expressing MHCn, suggesting chronic atrophy. MHCd-positive fibres were scarce indicating that myofibre necrosis was not a prominent feature, although connective tissue and adipose tissue, and occasional necrotic fibres were seen in more advanced cases. Clusters of cell debris corresponding to small rimmed vacuoles and also containing tubular aggregates were detected in a couple of fibres in one biopsy only, from Patient 4.II.2 Irregular intermyofibrillar network were observed in three patients carrying the NEB exon 151 mutation, one (1.II.1) with lobulated fibres and two (2.II.2 and 2.II.3) with core-like structures.
Molecular genetic findings
The founder haplotype associated with TMD in Finnish patients had been excluded in all four families, and in two patients, the corresponding mutation, FINmaj in the titin gene (Hackman et al., 2002) was excluded.
In Families 1 and 2 an abnormal SSCP band was observed in exon 151 of NEB. Sequencing confirmed the homozygous presence of a missense mutation previously identified in patients with NM. In the NM patients, this mutation had been observed in compound heterozygous form, together with another, more disruptive (other than missense) mutation. We investigated this further by sequencing samples from seven additional families included on the basis of similar clinical features (early or adult-onset foot drop in families with pedigree data compatible with recessive inheritance) to screen for the nine pathogenic NEB mutations previously identified in Finnish families. In this series, the patients in Families 3 and 4 showed homozygosity for another missense NEB mutation in exon 122.
In Families 1 and 2, a homozygous missense mutation was found at g.207181A>C in exon 151 of NEB (GenBank NT_005403 [GenBank] ), causing an amino acid change, threonine to proline (p.Thr5681Pro, protein sequence ID P20929 [GenBank] ). This mutation was homozygous in all the patients of these families (1.II.1, 1.II.6, 2.II.2, 2.II.3 and 2III.2) and heterozygous in healthy carriers (1.I.1, 1.I.2, 1.II.3, 1.II.4, 1.II.8, 1.II.9 and 2.II.4). The healthy sibling of family 1, 1.II.2 was not found to carry the mutation. From the remaining family members DNA was not available for mutation analysis (Fig. 5). The mutation in exon 151 had previously been identified in compound heterozygous form together with a more disruptive mutation (g.171895_171904delACAGACACGC in exon 122, and g.92509_90512dupTACT in exon 61) on the other allele in patients from two Finnish families with NM, in which the parents were healthy heterozygotes. Three parents and two siblings in three Finnish NM families were healthy heterozygous carriers of the missense mutation in exon 151. The alteration was not found in 300 Finnish control chromosomes.
In Families 3 and 4, a homozygous missense mutation was identified in exon 122 at the site g.171944G>T (GenBank NT_005403
[GenBank]
), causing a substitution of the amino acid serine for isoleucine (p.Ser4665Ile, protein sequence ID P20929
[GenBank]
). This mutation was identified in homozygous form in the Patients 3.II.1 and 4.II.2, and in heterozygous form in the healthy parents of Family 3 (3.I.1 and 3.I.2). DNA was not available for study from other members of these families (Fig. 5). The exon 122 missense mutation had been similarly identified in compound heterozygous form together with a more disruptive mutation, in this case g.82777G>A, a splice site mutation in intron 53, g.225116C>G, a nonsense mutation in exon 163, and g.225095_225098dupGTTT in exon 163, on the other allele in patients from three Finnish families with NM (Pelin et al., 2002; Lehtokari et al., 2006). Seven parents and three other family members in nine Finnish NM families were healthy heterozygous carriers of the missense mutation in exon 122, while in two of the NM families DNA from the parents was not available for study. This alteration was not found in 188 Finnish control chromosomes, nor in 90 additional control chromosomes from the same geographic region as the patients.
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We have described a novel hereditary muscle disease entity, distal nebulin myopathy, in seven patients from four families. The distribution of muscle weakness in these patients was rather uniform, and clearly different both from the pattern of weakness typical of NM caused by more disruptive mutations in NEB, and from that seen in TMD caused by mutations in the titin gene. The most severely affected muscles were the ankle dorsiflexors, the finger extensors and the neck flexors. None of the patients were able to walk on their heels. The two patients with the exon 122 mutation had moderate facial weakness of early onset and severe impairment of finger extension, while these features were milder in the patients with the exon 151 mutation. In some of the older patients, there was an additional component of proximal muscle weakness. Mild respiratory problems such as shortness of breath on strenuous exercise were experienced by three of six patients, but only one had abnormally small vital capacity. However, in view of the serious respiratory impairment with insidious onset in ambulant patients with NM, we recommend that patients with distal nebulin myopathy be monitored regularly for vital capacity, although at long intervals if no reduction is observed (Wallgren-Pettersson et al., 2004b
).
The selective involvement of the distal muscles in the patients described here is unexplained, but it is possible that it could be due to different usage of nebulin isoforms in different muscles (Donner et al., 2004; Bang et al., 2001), also proposed for a number of other muscle disorders including TMD (Udd et al., 2005). This pattern of muscle involvement separates the present disorder clinically from the typical form of NM caused by more disruptive mutations in NEB, where muscle involvement is selective also, but much more widespread (Wallgren-Pettersson et al., 2004a; Wallgren-Pettersson 1989; Wallgren-Pettersson et al., 1990; Jungbluth et al., 2004). Another difference is the asymmetry of muscle involvement in three of six patients of the present series. Weakness in NM caused by NEB mutations is mostly generalized and symmetric, with a predilection for the neck flexors and the facial, bulbar, axial and limb girdle muscles, and later severe impairment of ankle dorsiflexion. In distal muscles, the patients may at later ages show so-called neurogenic features at electromyography (Wallgren-Pettersson et al., 1989). Heart muscle is usually not affected in NM nor in this novel distal myopathy, because nebulin is only minimally expressed in heart muscle (Kazmierski et al., 2003).
In TMD, onset is later than in most of the patients with distal nebulin myopathy, and there is no involvement of the neck flexors, finger extensors or facial muscles (Udd et al., 1993).
Two distal myopathies with autosomal recessive inheritance have been previously described: Miyoshi myopathy caused by mutations in the dysferlin gene at chromosome 2p12–14 (Miyoshi et al., 1977; Bejaoui et al., 1995; Bashir et al., 1998; Liu et al., 1998), and Nonaka distal myopathy caused by mutations in the GNE gene at 9p1–q1 (Nonaka et al., 1985; Mitrani-Rosenbaum et al., 1996; Ikeuchi et al., 1997; Eisenberg et al., 2001). In Miyoshi myopathy, there is weakness and atrophy of the calf muscles. In Nonaka myopathy, as in the present new entity of distal nebulin myopathy, foot drop is the presenting feature. The course is progressive, with loss of ambulation 10–15 years after onset (Nonaka, 1999). The abundance of rimmed vacuoles distinguishes Nonaka distal myopathy histopathologically from distal nebulin myopathy, since no vacuoles were detected in the patients with the exon 151 mutation and only a couple of fibres with a few vacuoles were found in one of the patients with the exon 122 mutation.
A clinical differential diagnosis in sporadic cases is the Laing type of distal myopathy (MPD1, OMIM #160500) caused by dominant mutations in the myosin gene MYH at chromosome 14q11.2 (Laing et al., 1995a; Meredith et al., 2004). This myopathy presents in childhood or young adulthood with weakness of the anterior leg muscles and neck flexors (Lamont et al., 2006). Later, the finger extensors become weak, and in some there is an additional mild involvement of shoulder and hip muscles. Histological examination shows a variety of non-specific myopathic changes, but in the target muscle tibialis anterior there is group atrophy of fibres expressing both fast and slow myosins; rimmed vacuoles are infrequent and there are no sarcoplasmic inclusion bodies (Lamont et al., 2006).
A further disorder, dominantly inherited but clinically similar, is the one described in a large Australian family with NM caused by a missense mutation in the -tropomyosin gene TPM3. The ankle dorsiflexors are affected first, but then weakness spreads more rapidly to generally involve the lower limbs (Laing et al., 1992; 1995b). Thus, in singleton cases, this is a differential diagnosis.
The age of onset and early symptoms may be similar in desminopathy also (Milhorat and Wolff, 1943; Horowitz and Schmalbruch, 1994; Sjöberg et al., 1999). However, histopathological findings indicating myofibrillar myopathy, later frequent involvement of cardiac muscle and the dominant mode of inheritance are distinguishing features.
The histopathological findings in the present series differ from those of NM in that nemaline bodies were absent, or so few and tiny that they are not discernible in routine light microscopic frozen sections, especially in transversely sectioned myofibres. Nemaline bodies were inconspicuous or absent even in toluidine blue-stained semithin sections and at electron microscopy, and one of the patients had undergone no less than four biopsies, all of which were negative for nemaline bodies. The number of rods can vary in NM, even between muscles, and in some patients, an initial biopsy has shown no nemaline bodies at all. In the majority of cases they are, however, abundant. Predominance of type 1 fibres is a common feature in NM, but not in the present series. Rimmed vacuoles, common in the majority of the previously described distal myopathies, were very infrequent in this disorder, despite extensive searching.
The relatively small number of patients and the different muscles histopathologically analysed in the present series do not permit a definite determination of the full pattern of structural alterations in distal nebulin myopathy. The site of the homozygous mutation may also affect the histopathological pattern. For example, core-like changes devoid of oxidative enzyme activity and lobulated fibres with irregular distribution of mitochondria were present in Families 1 and 2 with the exon 151 mutation but not in Families 3 and 4 with the exon 122 mutation. A consistent feature was the presence of hypertrophic fibres of both fibre types as well as of internal nuclei. These were often associated with groups of atrophic fibres, sometimes weakly reminiscent of neurogenic atrophy. Other histological features were variable, and uncommon in NM. In particular, an increase in fibrous and adipose tissue, and in the number of internal nuclei are not usually seen in diagnostic biopsies of patients with NM, although such features can occur in follow-up biopsies (Wallgren-Pettersson et al., 1988). Core-like changes devoid of oxidative enzyme activity and lobulated fibres with irregular distribution of mitochondria are also not common.
Recessive mutations in NEB are a known cause of NM. Most mutations identified to date in this gene are either nonsense, frameshift or splice-site mutations generating structurally abnormal protein products, while missense mutations are rare. Patients with NM are often compound heterozygotes for two different mutations, in a few cases for one of the missense mutations described here and another, more disruptive mutation. In our large international series of samples from patients with NM, none of the 55 patients with mutations identified in NEB were homozygous or compound heterozygous for two missense mutations (Lehtokari et al., 2006). The majority of the mutations in NM patients are frameshift or nonsense mutations predicted to cause mRNA instability or premature truncation of nebulin (Pelin et al., 1999, 2002; Donner et al., 2004; Lehtokari et al., 2006). Point mutations or small deletions affecting conserved splice signals are predicted in the majority of cases to cause in-frame exon skipping. Patients with more severe clinical pictures tend to have mutations predicted to be more disruptive than patients with milder forms. Immunohistochemical labelling shows that the nebulin protein is present in the muscles of NM patients, despite their truncating mutations. Given the enormous size of the gene with mutations scattered all along its length and the fact that most patients have two different mutations, more precise genotype–phenotype correlations can only be defined after the identification of a very great number of mutations.
The seven patients presented here, with a muscle disorder clinically and histologically different from all known forms of NM, are the only ones in whom we have identified two missense mutations. This corroborates the conclusion that NM, with more generalized muscle weakness and abundant nemaline body formation, is the result of more disruptive mutations in NEB, while missense mutations cause this milder entity with so small or even no nemaline bodies that they are easily overlooked or not detectable at routine light microscopy.
Based on these findings we suggest that in distal myopathy of early onset preferentially affecting the anterior muscles of the lower leg, nebulin mutations may be suspected, and in these cases, high resolution light microscopy of semithin epon sections and electron microscopy may be helpful in demonstrating the presence, if any, of nemaline bodies.
It remains to be seen whether compound heterozygosity for two different missense mutations in NEB can cause a similar phenotype. Currently however molecular genetic identification of mutations in NEB is very cumbersome because of the enormous size of the gene (Lehtokari et al., 2006), and work is ongoing to allow more efficient screening.
There are currently 18 families with NM known in Finland. In 14 of these families, we have identified ten different NEB mutations. Three of these mutations have been found in more than one family. The most common Finnish mutation is the g.171944G>T mutation in exon 122 (p.Ser4665Ile), seen in compound heterozygous form in nine families with NM (Lehtokari et al., 2006). Seven of these families have the typical form and one has the intermediate form of NM. The missense mutation g.207181A>C (p.Thr5681Pro) in exon 151 has previously been identified in compound heterozygous form together with a frameshift mutation in three families with the typical form of NM. The exon 151 and 122 missense mutations both change a conserved amino acid from a polar, uncharged one to a non-polar, hydrophobic amino acid. The missense mutation in exon 122 is at a highly conserved actin binding site and the missense mutation in exon 151 is very close to another actin binding site. The missense mutation in exon 122 has previously been identified in altogether nine healthy carriers and the exon 151 mutation in five, corroborating the recessive nature of these mutations (Pelin et al., 2002; Lehtokari et al., 2006, unpublished observations).
We conclude that homozygosity for missense mutations in NEB causes a novel type of recessively inherited distal myopathy. This observation provides one explanation for the enigma of why NM caused by mutations in a gene as large as NEB is relatively rare. Based on knowledge of mutation frequency one would expect a disorder caused by mutations all along the length of a very big gene to be a common entity, but NM caused by mutations in NEB is not. The discovery of this distal nebulin myopathy provides at least a partial solution; a different type of mutation in the same gene causes a different muscle disorder.
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The authors thank the patients for participating in the study. Ms. Leena Vantunen, Paula Merilahti, Liisa Lempiäinen and Svetlana Zueva are acknowledged for excellent laboratory work, Jaakko Sarparanta, MSc, for technical assistance. The Helsinki groups were supported by grants from the Sigrid Jusélius Foundation, the Finska Läkaresällskapet, the Association Francaise contre les Myopathies, the Liv och Hälsa Foundation and the Academy of Finland (CWP and BU). KP was supported by grants from the University of Helsinki Research Funds, the Association Francaise contre les Myopathies and the Oskar Öflund Foundation.
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