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Neuromuscular Disorders 14 (2004) 461–470 www.elsevier.com/locate/nmd Genotype –phenotype correlations in nemaline myopathy caused by mutations in the genes for nebulin and skeletal muscle a-actin Carina Wallgren-Petterssona,*, Katarina Pelina,1, Kristen J. Nowakb,c, Francesco Muntonid, Norma B. Romeroe, Hans H. Goebelf, Kathryn N. Northg, Alan H. Beggsh, Nigel G. Laingb,c, the ENMC International Consortium on Nemaline Myopathy a Department of Medical Genetics, University of Helsinki, and The Folkhälsan Institute of Genetics, University of Helsinki, Helsinki, Finland b Centre for Neuromuscular and Neurological Disorders, University of Western Australia, QEII Medical Centre, Nedlands, WA, Australia c Centre for Medical Research, West Australian Institute for Medical Research, Nedlands, WA, Australia d Department of Pediatrics, Imperial College London, Hammersmith Hospital, London, UK e Institut de Myologie, INSERM U.523, Hopital de la Salpetriere, 47 Boulevard de l’Hopital, 75013 Paris, France f Department of Neuropathology, Johannes Gutenberg University Medical Centre, Mainz, Germany g Neurogenetics Research Unit, Children’s Hospital at Westmead, Faculty of Medicine, University of Sydney, Sydney, NSW, Australia h Genetics Division, Children’s Hospital Boston and Harvard Medical School, Boston, MA, USA Received 22 September 2003; received in revised form 24 February 2004; accepted 23 March 2004 Abstract We present comparisons of the clinical pictures in a series of 60 patients with nemaline myopathy in whom mutations had been identified in the genes for nebulin or skeletal muscle alpha-actin. In the patients with nebulin mutations, the typical form of nemaline myopathy predominated, while severe, mild or intermediate forms were less frequent. Autosomal recessive inheritance had been verified or appeared likely in all nebulin cases. In the patients with actin mutations, the severe form of nemaline myopathy was the most common, but some had the mild or typical form, and a few showed other associated features such as intranuclear rods or actin accumulation. Most cases were sporadic, but in addition there were instances of both autosomal dominant and autosomal recessive inheritance, while two families showed mosaicism for dominant mutations. Although no specific phenotype was found to be associated with mutations in either gene, clinical and histological features together with pedigree data may be used in guiding mutation detection. Finding the causative mutation(s) determines the mode of inheritance and permits prenatal diagnosis if requested, but will not as such permit prognostication. q 2004 Elsevier B.V. All rights reserved. Keywords: Nemaline (rod) myopathy; Mutation; Mosaicism 1. Introduction Nemaline myopathy is a clinically and genetically heterogeneous congenital myopathy identified in 1958 by Dr R.D.K. Reye [1] and then described five years later in two separate, original reports [2,3]. This myopathy is defined on the basis of muscle weakness and the presence in the muscle fibres of nemaline bodies. For a review, see North and co-workers [4]. * Corresponding author. Tel.: þ 358-9-315-5521; fax: þ358-9-315-5106. E-mail address: [email protected] (C. Wallgren-Pettersson). 1 Present address: Department of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland. 0960-8966/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2004.03.006 The International Database on Nemaline Myopathy was established to answer clinical questions about nemaline myopathy, to facilitate gene discovery and to serve as a basis for genotype – phenotype correlations. Clinical and histological data have been contributed by members of the European Neuromuscular Centre (ENMC) International Consortium on Nemaline Myopathy and other clinicians and pathologists from various countries around the world. To date, disease-associated mutations have been detected in the genes for nebulin (NEB) or skeletal muscle alphaactin (ACTA1) in 60 of the more than 270 patients whose clinical data have been entered into the database. Mutations in these two genes appear to be the most common causes of 462 C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470 nemaline myopathy [5]; in one series of 97 patients, 23 had nebulin mutations and 13 had actin mutations. The result is based on mutation analysis of 25 –50% of the giant nebulin gene, which has a transcript of 21 kb and a total of 183 exons [6,7,8], and on sequencing of the six coding exons of ACTA1 [9,10]. Mutations in genes encoding the other thin filament proteins: tropomyosin 2 and 3 and troponin T1 appear to be rare causes of nemaline myopathy. Mutations in the gene for tropomyosin 3 (TPM3) have been identified in four kindreds [11 –15], and mutations in the gene for tropomyosin 2 (TPM2) in three patients in two unrelated families [16]. A severe form of nemaline myopathy with unusual associated features due to a recessive mutation in the gene for fast troponin T (TNNT1) has been described in children of a Pennsylvanian Amish community [17]. In addition to the genes currently known to be associated with nemaline myopathy [18,19], on the basis of genetic linkage results, at least one further gene is believed to exist. It also remains to be elucidated whether some cases of the severe form associated with the fetal akinesia sequence are caused by mutations in a separate, hitherto unidentified gene [20]. These genes and the associated, rarer forms of nemaline myopathy are beyond the scope of the present study. Here we report on an extensive clinical comparison between patients with nebulin mutations and those with actin mutations, defining the range of clinical presentations and the degree of overlap between phenotypes associated with mutations in either gene. 2. Patients and methods The 60 patients with NEB or ACTA1 mutations came from 16 different countries. Clinical and histological data were entered into the international database and each patient’s nemaline myopathy was categorised according to the classification approved by the ENMC International Consortium on Nemaline Myopathy [21] (Table 1). The present series comprised 26 patients with nebulin mutations from 22 families and 34 patients with actin mutations from Table 1 Summary of clinical classification of nemaline myopathy into six different categories [18,21] 1. Severe congenital nemaline myopathy. Patients lacking spontaneous movements or respiration at birth, or with severe contractures or fractures at birth 2. Intermediate congenital nemaline myopathy. Patients breathing and moving at birth but later unable to achieve respiratory independence or ambulation 3. Typical congenital nemaline myopathy. Patients with onset in early childhood, typical distribution of muscle weakness, milestones delayed but reached, course slowly progressive or non-progressive 4. Mild childhood-onset nemaline myopathy 5. Adult-onset nemaline myopathy 6. Other forms of nemaline myopathy, with unusual associated features 30 families (for family history, clinical and histological details, see Table 2). Fifteen of the nebulin mutations have been reported previously [7,8], as have all of the actin mutations [9,10,22– 24]. Based on detailed comparisons of up to 166 clinical items per patient recorded in the international database, we summarise in the present paper the clinical correlates of these mutations. 3. Results Among singleton patients with a very severe clinical picture at birth, actin mutations dominated (20/30 probands) over nebulin mutations (6/22 probands) ðP ¼ 0:0005Þ: Among probands with the typical or mild clinical form, mutations in the nebulin gene (17/22) were more common than mutations in the actin gene (11/30) ðP ¼ 0:004Þ (Table 2). There were altogether nine multiplex families with nebulin mutations and five with actin mutations. The nonfamilial cases with identical mutations included two sharing the same actin mutation, and one patient found to have the same nebulin mutation as an affected sib pair in another family. Among the patients sharing the same mutation in either gene, clinical severity did show some variability, but no fundamental differences were noted (Tables 2 and 3). In both groups, muscle weakness was generalised, usually symmetrical, and most pronounced in the neck flexors (Figs. 1 and 2). In the nebulin group, the ankle dorsiflexors were very weak also, while the extensors of the knees were well preserved in comparison with the knee flexors. In the actin group, on the other hand, the knee extensors were weaker than the flexors, and there was better preservation of the ankle dorsiflexors (Table 2). Histologically, in both patient groups, predominance of type 1 fibres and variability of fibre size was common. There were also no apparent differences in the quantity and distribution of nemaline bodies. Accumulations of actin filaments, in the presence or absence of nemaline bodies, were seen in a few patients with actin mutations, but none were reported in the nebulin group. Three patients in the actin group had intranuclear nemaline bodies while none were observed in the nebulin group. In the nebulin group, immunohistochemical studies of nebulin yielded inconsistent patterns [25]. Structural abnormalities of the heart were encountered in one patient in each group. There were no instances of cardiomyopathy. 4. Patients with nebulin mutations Table 3A lists nine different hitherto unpublished mutations in the nebulin gene, and 16 previously published ones [7,8]. Most of the mutations found in the nebulin gene are predicted to lead to premature truncation of the nebulin C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470 463 Table 2 Clinical and histological details, and family history of altogether 60 patients with nemaline myopathy Group Nebulin Actin No. of families No. of patients Category of nemaline myopathy Family history: no. of families 22 26 (M:F ¼ 12:14) Severe 6, intermediate 3, typical 12, mild 5 Singleton pt 13, AR 9 Consanguinity Patients deceased, ages at death Patients alive, (no.), age range (median age) 4/22 Couples 5/26, 1 day–19 mo 30 34 (M:F ¼ 21:13) Severe 18, typical 8, mild 3, other 5 Singleton pt 25, AR 2, AD 1, mosaic parent 2 2/30 Couples 16/34, 1 day–16 yrs (median 4 mo) Achieved walking Course of the disease Polyhydramnios Fetal movements Respiration neonatally Antigravity movements neonatally Hypotonia neonatally Feeding difficulties neonatally Major contractures neonatally Apgar scores Cardiac status EMG findings Muscle weakness, distribution Muscle weakness, if not formally tested Asymmetry of muscle power Muscle biopsy findings Fibre type predominance Nemaline bodies Quantity Location Severe, (1), 0.6 yr; Intermed., (3), 9–30 yr; Typical, (12), 1.3– 37 yr (median 7 yrs); Mild, (5), 23–42 yr 15/16 (Latest 3.7 yr) (one not walking at 9 yr) Improving 3/21, stable 11/21, deteriorating 4/21 4/18 Normal 13/21, weak and/or infrequent 8/21, absent 0/21 Normal 16/22, insufficient 5/22, absent 1/22 Present 15/19, none 4/19 Severe 3/18, moderate 11/18, none 4/18 13/22 7/20 1 min: Mean 6.9, range 1– 10; 5 min: Mean 6.9, range 1–10 Normal 16/18, structural abn. 1/18, right overload 1/18, cardiomyopathy none Neurogenic 1/14, myogenic 1/14, mixed 6/14, normal 4/14 Neck flexors and ankle dorsiflexors especially weak Knee extensors , knee flexors Proximal 8/9, distal 1/9 Severe, (7), 4 mo– 10 yr (median 1.5 yr); Typical, (6), 1–42 yr (median 8 yr); mild, (3), 18–45 yr; other, (1), 7.5 yr 9/9 (Latest 5 yrs) Improving 5/18, stable 2/18, deteriorating 6/18 8/31 Normal 14/30, weak and/or infrequent 14/30, absent 2/30 Normal 13/30, insufficient 11/30, absent 6/30 Present 15/30, none 15/30 Severe 20/29, moderate 7/29, none 2/29 26/31 8/25 1 min: Mean 5.6, range 1–9; 5 min: Mean 7.5, range 5–10 Normal 17/19, structural abn. 1/19, right overload 1/19, cardiomyopathy none Neurogenic 7/10, myogenic 3/10 No 18/19, yes 1/19 No 19/20, yes 1/20 Type 1 14/16, type 2 2/16 Type 1 12/12, type 2 0 (þ þþ ) 8/16, (þ þ) 6/16, (þ ) 2/16 Internal 5/11, subsarc. 2/11, both 4/11 (þþ þ) 9/14, (þþ ) 3/14, (þ) 2/14 Internal 4/11, subsarc. 4/11, both 3/11, intranuclear 2/10 Neck flexors especially weak Knee extensors . knee flexors Proximal 10/11, distal 1/11 M:F, males:females; category, form of nemaline myopathy as defined on the basis of the criteria outlined by the ENMC International Consortium on Nemaline Myopathy [18,21], singleton pt, singleton patient, no other family members affected; AR, autosomal recessive; AD, autosomal dominant; structural abn., structural abnormality. Nemaline bodies, quantity: (þ) small quantity of nemaline bodies in a small proportion of muscle fibres; (þþ ) nemaline bodies present in more than one fourth of the fibres; (þ þ þ) numerous nemaline bodies in most fibres. Nemaline bodies, location: Subsarc., subsarcolemmal. Full clinical details were not available on all patients. Thus, for the separate items, the number of patients in the relevant age group for whom data were known is indicated. For example, children who died before they had reached the age at which they would normally have walked are not included in the numbers of the section ‘Achieved walking’. protein [7,8]. Thus, it appeared likely that a majority of the patients, homozygous or compound heterozygous for such mutations, would lack the carboxy terminus of the nebulin protein, preventing its anchoring into the Z discs. The patients’ muscle biopsies should also then show no immunohistochemical labelling with antibodies to the C-terminal end of nebulin. Clinically, the patients might have been expected to have an early-lethal form of nemaline myopathy. Nevertheless, the typical form was the most common (Fig. 1), and histologically, in most of the patients studied using antibodies against nebulin, the C-terminal end of the protein was detected [25]. Although the predominant clinical presentation of these 26 patients with nebulin mutations was the typical, congenital-onset form of nemaline myopathy, there were six severe cases [26] (Fig. 2) as well as three intermediate and five mild cases (Table 2). Autosomal recessive inheritance was either verified by the identification of mutations on both alleles of the nebulin gene, or appeared likely because one mutation detected in the patient(s) was also found in one of the unaffected parents. In the latter families, the search for the other mutation is ongoing in the giant nebulin gene. It is to be noted that while the patients in three consanguineous families showed homozygosity for 464 C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470 Table 3A Patients with mutations in the nebulin (NEB) gene Patient no. Category Type of mutation Exona Amino acid substitution Homozygous/heterozygous 1.1 1.2 2 3.1 3.2 4 5 6 7 8.1 8.2 9.1 9.2 10 11 12 13 14 15 16 17 18 19 20 21 22 Typical Typical Typical Severe Severe ?Typical Typical Severe ?Typical Mild Mild Typical Intermediate Typical ?Typical Severe Severe Typical Severe ?Typical Intermediate ?Typical ?Mild Mild Mild Intermediate Frameshift Frameshift Frameshift Missense Frameshift Frameshift Frameshift Frameshift Splicing Nonsense Frameshift Frameshift Frameshift Frameshift Missense Frameshift Missense Frameshift Missense Frameshift Nonsense Nonsense Frameshift Frameshift Splicing Nonsense Splicing Nonsense Missense Missense Splice site Missense Nonsense Frameshift Missense 156 (165) 156 (165) 163 (172) 122 163 (172) 177 (181)b 163 (172) 177 (181)b 154 (163) 181 (185) 180 (184) 171 (177D)b 171 (177D)b 171 (177D)b 151 (160) 122 151 (160) 122 151 (160) 61 152 (161) 180 (184) 180 (184) 171 (177D)b Intron 147 (156) 148 (157) Intron 156 (165) 163 (172) 122 142 (152) Intron 148 (157) 147 (156) 170 (177C)b 7 167 (176)b Stop at 58507 Stop at 58507 Stop at 61237 Ser4665Ile Stop at 61237 Stop at 63917 Stop at 61237 Stop at 63917 Heterozygous Heterozygous Compound heterozygote Compound heterozygote Compound heterozygote Homozygous Homozygous Homozygous Homozygous Homozygous Homozygous Compound heterozygote Compound heterozygote Compound heterozygote Heterozygous Heterozygous Heterozygous Heterozygous Compound heterozygote Heterozygous Compound heterozygote Heterozygous Heterozygous Heterozygous Compound heterozygote Heterozygous 7 Glu6536Stop7 Stop at 65088 Stop in exon 1718 Stop in exon 1718 Stop in exon 1718 Thr5681Pro8 Stop at 4655 Thr5681Pro8 Stop at 4655 Thr5681Pro8 Stop at 2801 Tyr5689Stop8 Leu6523Stop8 Stop at 65128 Stop in exon 1718 Arg5564Stop8 8 Gln6105Stop Ser4665Ile Pro5389Ala Asp5528His Stop in exon 170 Tyr6216Ser Mutations found in the nebulin gene of 26 patients with nemaline myopathy. Category: form of nemaline myopathy as defined on the basis of the criteria outlined by the ENMC International Consortium on Nemaline Myopathy [18,21]. a Previous exon nomenclature, based on protein repeat domain number [43], in parentheses. b Alternatively spliced exon. a mutation in the nebulin gene, compound heterozygosity for nebulin mutations was found in a fourth consanguineous family. The majority of the nebulin mutations found to date reside in the 30 end of the gene (Table 3A), encoding the C-terminus of the protein, which is where the search for mutations has initially concentrated. The C-terminal end of the protein extends into the Z disc. Due to the extensive alternative splicing of nebulin exons in this region, several different nebulin isoforms are expressed. In five patients with mild or typical phenotypes, mutations were identified in exons 170 and 171 (previously named 177C and 177D), which are known to be differentially expressed [7,27,28]. Patient 21, with a mild phenotype, is a compound heterozygote for a nonsense mutation in exon 170 (former 177C) and a frameshift mutation in exon 7. The mutation in exon 7 is expected to cause truncation of all nebulin isoforms at amino acid 227 in the N-terminal end of the protein. The nonsense mutation in exon 170 (177C) is expected to affect only isoforms expressing exon 170 (177C). Such isoforms seem to be rare [6]. In five other patients with typical or intermediate phenotypes, compound heterozygosity was determined, with one of the mutations in each patient being a missense mutation, and the other a truncating mutation in a constitutively expressed exon (Table 3A, patients 2, 9.1, 9.2, 10 and 17). Patients 2 and 17 are both compound heterozygotes for mutations in exon 163 (172) expected to cause truncation of the C-terminal end of nebulin, and a missense mutation, Ser46651Ile, which changes a conserved SDXXYK-actin-binding motif. Patient 10, with a typical phenotype, was found to be a compound heterozygote for a frameshift mutation in exon 61 and a missense mutation, Thr5681Pro, in exon 151 (160). The frameshift mutation is expected to cause truncation of all nebulin isoforms at amino acid 2801. Exon 151 is expected to be expressed in all nebulin isoforms, and the missense mutation is predicted to cause a conformational change in the protein [8]. The same missense mutation was also found in an affected sib-pair (patients 9.1 and 9.2), who carried a frameshift mutation in exon 122 on their other allele. The splice-site mutations in patients 4, 16 and 19 are predicted to generate transcripts otherwise normal, but lacking one exon. In half the typical cases, only one heterozygous mutation is known, precluding predictions regarding what nebulin isoforms might be present in C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470 465 Table 3B Patients with mutations in the actin (ACTA1) gene Patient no. Clinical category Type of mutation Exon/codon change Amino acid substitution Homozygous/heterozygous 1 2 3 4 5 6.1 6.2 6.3 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25.1 25.2 25.3 26 27 28 29 30 Other form Other form Other form Other form Severe Typical ?Typical ?Mild Mild Mild Typical Severe Severe Severe Severe Severe ?Typical Typical Severe Severe Other form Severe Typical Severe Typical Severe Severe Severe Severe Severe Severe Severe Typical Severe Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Splice site Splice site Splice site Missense Missense Missense Missense Missense 2/GGC-CGC 4/GTG-CTG 4/GTG-TTG 2/CAC-TAC 2/CTT-CCT 5/GAG-GTG 3/AAC-AGC 3/AAC-AGC 3/AAC-AGC 3/ATG-GTG 3/ATC-ATG 4/GGC-GAC 4/CGC-TGC 4/CGC-GGC 5/ATG-ACG 5/CGC-CAC 5/CAG-CTG 5/GGT-TGT 6/ATG-AGG 6/AAC-AAG 6/GAC-GGC 7/ATC-CTC 7/GTC-TTC 6/ATG-AAG 7/CGC-AGC 6/GCG-GAG 5/GAG-CAG Intron 5 ag/GTAT-at/GTAT Intron 5 ag/GTAT-at/GTAT Intron 5 ag/GTAT-at/GTAT 4/GCG-GGG 3/GTG-GCG 2/GTG-CTG 7/AAA-CAA 3/GTC-TTC Gly15Arg8 Val163Leu8 Val163Leu8 His40Tyr8 Leu94Pro8 Glu259Val8 Asn115Ser8,9 Asn115Ser8,9 Asn115Ser8,9 Met132Val8,21 Ile136Met9 Gly182Asp8 Arg183Cys8 Arg183Gly9 Met227Thr22 Arg256His8 Gln263Leu8,23 Gly268Cys9 Met269Arg22 Asn280Lys8 Asp286Gly8 Ile357Leu9 Val370Phe8 Met283Lys22 Arg372Ser22 Ala272Glu22 Glu224Gln22 – 22 – 22 – 22 Ala170Gly22 Val134Ala22 Val35Leu22 Lys373Gln22 Val43Phe22 Heterozygous Heterozygous Heterozygous Heterozygous Compound heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous, father mosaic Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous, mother mosaic Heterozygous Heterozygous Homozygous Homozygous Homozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Mutations identified in the actin gene of 34 patients with nemaline myopathy. Category: form of nemaline myopathy as defined on the basis of the criteria outlined by the ENMC International Consortium on Nemaline Myopathy [18,21]. the patients’ muscles. In most of these cases, the known mutation is expected to result in a truncated protein. In three out of four patients with a severe clinical picture in whom both mutations were known, the mutations were either frameshift or nonsense mutations. One patient with a severe phenotype showed compound heterozygosity for a frameshift mutation in the differentially expressed exon 177 (181), and another frameshift mutation in exon 163 (172). Both mutations are expected to cause truncation of the protein molecules, but due to the alternative usage of exon 177 (181), this allele is expected to result in the expression of normal nebulin isoforms as well. A somewhat similar situation was observed in another patient, also with severe nemaline myopathy (Patient 15, Table 3A), found to be a compound heterozygote for a nonsense mutation in exon 148 (157) and a splice-site mutation in intron 147 (156). The nonsense mutation is predicted to cause the synthesis of a truncated protein lacking all the simple repeats and the unique C-terminal domains of nebulin, whereas the splice- site mutation should allow the formation of both normal and abnormal transcripts. The remaining severe cases showed homozygous or heterozygous truncating mutations in exon 180 (184), which are expected to cause absence of the serine-rich domain and the SH3 domain in the C-terminus [26]. 5. Patients with actin mutations Thirty-four patients had ACTA1 mutations. Most of these mutations were de novo dominant, missense mutations, predicted to result in the formation of an altered actin molecule (Table 3B). The severe form of nemaline myopathy was the most common clinical presentation (Fig. 2), but eight out of the 34 patients had the typical form (Table 2, Fig. 1) and three were mild cases [22]. Five had unusual associated features, including three with intranuclear rods and three whose histological picture was 466 C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470 Fig. 2. Two patients with severe nemaline myopathy, in one caused by a mutation in the nebulin gene, in the other by a mutation in the actin gene. Fig. 1. Two patients with the typical form of nemaline myopathy, one with a mutation in the nebulin gene, the other with a mutation in the actin gene. Reprinted from Journal of the Neurological Sciences, Volume 89, C. Wallgren-Pettersson, Congenital nemaline myopathy: a clinical follow-up study of twelve patients, 1–14, 1989, with permission from Elsevier. dominated by an excess of thin filaments, i.e. actin accumulation [9,29]. The majority of the cases with actin mutations were sporadic, but it is to be noted that there were five multiplex families. One of these families showed autosomal dominant and two autosomal recessive inheritance, while in each of the two other families, one of the parents was found to have somatic mosaicism for the mutation. These families are the only two showing mosaicism out of the total of nearly 80 families in which ACTA1 mutations have been identified [23]. The mosaic parents had not sought medical attention for any muscle complaint but neither did well in sports. Two families with actin mutations were consanguineous. In one of these, the nemaline myopathy was recessively inherited, while in the other, the disorder was caused by a new dominant mutation that had occurred in the patient. 6. Discussion Although initial studies suggested that nebulin mutations were associated with the typical form of nemaline myopathy while actin mutations were found preferentially in severe and mild cases [21], the present data clearly demonstrate a significant overlap as mutations in either gene may be associated with a wide range of severity (Figs. 1 and 2). Nevertheless, in general, the severity and the course of the disease appear milder in the group of patients with nebulin mutations than in the group with actin mutations (Tables 2 and 3). Familial cases showing an autosomal recessive inheritance pattern point in the direction of the nebulin gene, although actin mutations are not a priori ruled out as the cause of the disease. An autosomal dominant pattern is likely to be due to a mutation in the actin gene rather than in the nebulin gene, as to our knowledge no dominant mutations have hitherto been identified in the nebulin gene. Among sporadic cases, a singleton patient with a very severe clinical picture at birth is more likely to have a de novo dominant actin mutation (20/30 probands) than two recessive nebulin mutations (6/22 probands) ðP ¼ 0:0005Þ: Conversely, probands with the typical or mild clinical form are more likely to have mutations in the nebulin gene (17/22) than in the actin gene (11/30) ðP ¼ 0:004Þ: As most new cases of nemaline myopathy are sporadic, the family history often does not provide a clear indication of the mode of inheritance. Thus, determining whether the cause is a new dominant mutation, or two inherited recessive mutations, requires identification of the causative mutation(s) [18]. Although highly desirable for confirming diagnosis and mode of inheritance in individual families, mutation detection in nemaline myopathy is currently not available as a service, with the exception of analysis of the actin gene. For the giant nebulin gene, substantial efforts are needed to establish practically feasible methods for routine mutation detection. Possibly, this can be achieved using denaturing high performance liquid chromatography (Lehtokari et al. in preparation). Both patient groups included neonatally severely affected patients. Mortality was greater in the group with actin mutations. In a number of these cases, active support was withdrawn as it failed to lead to improvement over a period of days or weeks, and prognosis was deemed hopeless. In each group, those who improved and lived into late childhood usually achieved walking. Detailed clinical comparisons, however, did not identify any definite prognostic indicators. Thus, from these data, it does not appear justified in individual cases to base predictions of prognosis on which gene the mutation resides in. In other words, the finding of a mutation in either of the two genes will not as such facilitate decision-making in questions of active versus C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470 passive treatment of severely affected infants. These difficult situations thus need to be managed along the same lines as for any neonate suffering from a severe disorder. In both the nebulin group and the actin group, muscle weakness was generalised and usually symmetrical, with a predilection for the neck flexors. In the nebulin group, the dorsiflexors of the ankles were especially weak also, and the knee extensors were less severely affected than the knee flexors, confirming previous findings [30,31]. In the actin group, in contrast, the extensors of the knee were weaker than the flexors. The reason for these subtle differences in patterns of muscle involvement between the two groups is not clear at present. Detailed histological comparisons of the two groups with mutations detected are in preparation. Those compiled in the present paper are based on biopsy findings reported by the respective pathologists involved in the diagnostic procedure, and are thus heterogeneous. Details such as the presence or absence of intranuclear nemaline bodies had not always been assessed. However, in summary, if the histological picture is dominated by actin accumulations, or if intranuclear nemaline bodies are present, it is worth searching for a mutation in the actin gene [9], and where labelling with different nebulin antibodies yields inconsistent patterns, the nebulin gene would be first priority [25]. Thus, the clinical and histological features of individual patients may give a rough indication of where to start molecular genetic analysis. Disruption of the myofilamentous structure as seen on muscle biopsy, and so-called neurogenic findings seen at electromyography were more common in the actin group than in the nebulin group, in which mixed patterns of both ‘myogenic’ and ‘neurogenic’ patterns are known from a previous study to be common in older patients [32]. However, purely ‘neurogenic’ findings were mainly seen in patients with the severe form, and as no patients with the severe form due to nebulin mutations had undergone electromyography, it is unclear whether these differences are simply related to severity of the disease or whether they are related to genetic etiology. A better understanding of the disease pathogenesis from mutations in the nebulin gene resulting in the various phenotypes awaits a detailed characterisation of the numerous human nebulin isoforms and their expression patterns. Z discs vary in structure and width in different muscle and fibre types, and during development. A positive correlation has been shown between Z-disc width and the number of nebulin C-terminal repeats [27]. It has been suggested that differential expression of the nebulin C-terminal repeats, together with the differentially expressed titin N-terminal repeats, account for the full range of Z-disc widths and structures [27]. Thus, a wide nebulin isoform diversity appears to be required for normal muscle development [6 –8]. The typical form of nemaline myopathy, clinically milder than the severe form, appears to be associated with 467 the maintenance of a broad, albeit not full, isoform diversity. Mutations in the alternatively spliced exons 170 (177C) and 171 (177D) appear to correlate with a typical or even mild phenotype, suggesting that exon 171 (177D) is expressed in a few nebulin isoforms only. Our results from RT-PCR studies of RNA isolated from different leg muscles indicate that this is the case [6]. The majority of isoforms would thus be normal in these patients. Other patients with the typical form have homozygous or heterozygous splice-site mutations leading to skipping of one exon, normally not alternatively spliced out. Yet others show the presence of a missense mutation in addition to a truncating one, permitting from that allele the expression of full-length nebulin molecules. One patient (patient 5, Table 3A) with typical nemaline myopathy was found to be homozygous for a nonsense mutation in exon 181 (185) of the nebulin gene, expected to truncate the last 134 amino acids of the protein [7]. Unlike such mutations in many other genes, the nebulin mutations do not always lead to a severe form of the disorder. The presence of the carboxy terminus of the protein despite homozygous mutations predicted to cause truncation of the protein is likely to be due to the expression only of isoforms not containing the exon harbouring the mutation, or to skipping of this exon to produce an internally deleted protein. It is also possible that some of the mutations affect exonic splicing enhancers, thus inducing exon skipping [33]. In addition, immunohistochemical labelling with antibodies specific to the C-terminal end of nebulin indicate that truncated proteins are expressed in a subset of fibres [7,25]. Apparently, this alternative nebulin molecule is sufficient to maintain a muscle function compatible with life, in many cases leading only to a relatively mild muscle disorder. Skipping of an exon harbouring a disease-causing mutation to produce milder disease is a phenomenon previously well documented. Among the muscle disorders the evidence comes mainly from patients with out-of-frame deletions of certain regions of the dystrophin gene causing muscular dystrophy of the milder, Becker type instead of the expected Duchenne-type severity [34]. In one family segregating a mutation in the dystrophin gene the patients exhibited a range of clinical pictures, the variation in severity being explained by differences in levels of exon skipping. The phenotypes varied from that of a healthy adult man with mildly elevated serum creatine kinase activity through a mildly affected young adult to an overtly affected middle-aged man with cardiomyopathy and Becker-type muscular dystrophy [35]. Alternative splicing of the AMP deaminase gene apparently rescues the phenotype in some persons with a common homozygous nonsense mutation [36]. These examples might indicate hope for therapeutic approaches such as inducing skipping of the exon harbouring the disease-causing mutation, e.g. through the use of antisense oligonucleotides [37] or through enhanced expression of alternative isoforms to maintain a broad isoform diversity. 468 C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470 In half the typical cases and in one of the intermediate cases with nebulin mutations, to date only one heterozygous mutation has been identified. In most of these cases, the mutation is predicted to lead to truncation of the nebulin molecule. We therefore speculate that the other as yet unidentified mutation is likely to be less severe, e.g. of missense or splice-site nature or present in differentially expressed exons. Some patients with severe nemaline myopathy were found to have mutations in exon 180 (184) of the nebulin gene, or truncating mutations in exons expressed in the majority of isoforms. The mutations in exon 180 (184) are predicted to cause absence of the C-terminal serine-rich and SH3 domains. These two domains are highly conserved between species, and they are present in the Z discs of all types of skeletal muscle, suggesting that they play important roles in the structure of vertebrate Z discs [27,38]. These domains do not however appear to be necessary for the incorporation of nebulin C-terminal modules into the Z discs [39]. This observation does not rule out the importance of the nebulin serine-rich and SH3 domains in the regulation and maintenance of the highly ordered structure of the Zdisc. SH3 domains closely related to the nebulin SH3 domain, such as those of the yeast actin-binding protein ABP1 and cortactin, reside in proteins involved in the regulation of the assembly of the actin cytoskeleton [38]. A recently described protein, myopalladin, has been reported to bind to the SH3-domain of nebulin and hence link nebulin to a-actinin in the Z disc [40]. Furthermore, the nebulin SH3-domain has been shown to have high binding affinity for a stretch of titin known to colocalise with nebulin in the Z disc [41]. The mutations in the actin gene are spread through all six of the coding exons, with some being located within known functional sites of the actin protein [10,42]. There appears to be some clustering of mutations causing certain histological phenotypes, e.g. that of actin myopathy with accumulation of thin filaments [40]. The majority of mutations are missense and cause dominant disease, suggesting that they do so by producing a poison peptide, i.e. by a gain of function. This is supported by two ACTA1 mutations that effectively lead to a null allele resulting in recessive disease [23]. The presence of these two mutations in unaffected heterozygotes suggest that normal function can be achieved in some cases by having one wild-type ACTA1 allele. In each of two families with actin mutations, one parent showed somatic mosaicism for the mutation, a phenomenon not hitherto encountered in families with mutations in the nebulin gene. This needs to be taken into account in the genetic counselling of families in whom actin mutations have been identified. Somatic mosaicism has been observed in two out of nearly 80 families in total in which ACTA1 mutations have been identified [23]. Hitherto, there have been no reports of proven gonadal mosaicism, but the observed existence of somatic mosaicism makes gonadal mosaicism likely. A further aspect is notable in the genetic counselling of families with nemaline myopathy. The occurrence in a consanguineous family of a de novo dominant mutation in the actin gene, and the compound heterozygosity in one of the four consanguineous families with nebulin mutations show that caution is warranted in drawing conclusions regarding the mode of inheritance from pedigree data only. In summary, it is possible that the nature of the mutation and its effect on the protein determines the phenotype to a greater extent than the gene in which the mutation has occurred. Naturally, other factors also, both genetic and non-genetic, contribute to the diversity of clinical pictures. Extended clinical series, further experimental studies and wider knowledge of the normal isoform diversity and structure – function relationships of the sarcomeric proteins in question are needed to answer remaining questions regarding genotype– phenotype correlations. Acknowledgements We thank the following colleagues for contributing data and samples on one or two patients each: Dr Janice Anderson, Prof. Corrado Angelini, Dr Emilia Bijlsma, Prof. Peter Van den Bergh, Prof. Kate Bushby, Prof. Carsten Bönnemann, Prof. Angus Clarke, Drs Basil Darras and Martin Eswara, Prof. Michel Fardeau, Drs Maria Luisa Giovanucci Uzielli, Nathalie Goemans, Claudio Graziano, Carolyn Green and Margaret Grunnet, Prof. Folker Hanefeld, Drs Arvid Heiberg, Jens Michael Hertz, Marc D’Hooghe and Imelda Hughes, Prof. Christoph Hübner, Prof. Susan Iannaccone, Prof. Constantin von Kaisenberg, Drs Martin Lammens, Carol Leicher, Hanns Lochmüller, Meriel McEntagart, Julie McGaughran, Thor Ruud-Hansen, Rolf Schlösser and Gudrun Schreiber, Prof. Rebecca Sutphen, Drs Kathryn Swoboda and Tal Thomas, Prof. Haluk Topaloglu, Prof. Andoni Urtizberea, Drs Christophe Vial, Jaqueline Vigneron and Sheila Wallace. We are grateful to the other members of the ENMC International Consortium (Drs Anthony Akkari, Olli Carpén and Avril Castagna, Prof. Victor Dubowitz, Drs Baziel van Engelen, Marc Fiszman, Claudio Graziano and Edna Hardeman, Prof. Susan T. Iannaccone, Dr Heinz Jungbluth, Prof. Siegfried Labeit, Drs Martin Lammens and Carmen Navarro, Prof. Ikuya Nonaka, Drs Berardino Porfirio and Norma B. Romero, Prof. Caroline Sewry, Prof. Lars-Eric Thornell, Dr Mariz Vainzof) for continuing inspiring collaboration, to the same persons and to clinicians around the world for contributing data and samples not used in this study, and to the European Neuromuscular Centre (ENMC) for organisational support. We thank Patricia Zilliacus, MSc, for assistance with the international database on nemaline myopathy, and Dr Pankaj B. Agrawal for statistical calculations. KP was supported by grants to CWP from the University of Helsinki, the Association Francaise contre les Myopathies, the Sigrid Jusélius Foundation, the Finska C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470 Läkaresällskapet and the Medicinska understödsföreningen Liv och Hälsa, AHB by NIH AR44345, the Muscular Dystrophy Association of the USA and the Joshua Frase Foundation. NGL is supported by the Australian National Health and Medical Research Council (Fellowship 139170, Project grants 139039 and 110242) and the Association Francaise contre les Myopathies. KJN is a CJ Martin Fellow (ID No. 212086) with the Australian National Health and Medical Research Council. References [1] Schnell C, Kan A, North KN. An artefact gone awry: identification of the first case of nemaline myopathy by Dr R.D.K. Reye. Neuromuscul Disord 2000;10:307 –12. [2] Shy GM, Engel WK, Somers JE, Wanko T. Nemaline myopathy: a new congenital myopathy. Brain 1963;86:793– 810. [3] Conen PE, Murphy EG, Donohue WL. Light and electron microscopic studies of myogranules in a child with hypotonia and muscle weakness. Can Med Assoc J 1963;89:983–6. [4] North KN, Laing NG, Wallgren-Pettersson C, the ENMC International Consortium on Nemaline Myopathy. Nemaline myopathy: current concepts. J Med Genet 1997;34:705–13. [5] Wallgren-Pettersson C, Laing NG. 83rd ENMC International Workshop: Fourth Workshop on Nemaline Myopathy, 22–24 September, 2000, Naarden, The Netherlands. Neuromuscul Disord 2001;11: 589–95. [6] Donner K, Sandbacka M, Lehtokari V-L, Wallgren-Pettersson C, Pelin K. Complete genomic structure of the human nebulin gene and identification of alternatively spliced isoforms. Eur J Hum Genet, in press. [7] 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;96:2305–10. [8] Pelin K, Donner K, Holmberg M, Jungbluth H, Muntoni F, WallgrenPettersson C. Nebulin mutations in autosomal recessive nemaline myopathy: an update. Neuromuscul Disord 2002;12:680 –6. [9] 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;23:208–12. [10] Ilkovski B, Cooper ST, Nowak K, et al. Nemaline myopathy caused by mutations in the muscle alpha-skeletal-actin gene. Am J Hum Genet 2001;68:1333 –43. [11] Laing NG, Majda BT, Akkari PA, et al. Assignment of a gene (NEM1) for autosomal dominant nemaline myopathy to chromosome 1. Am J Hum Genet 1992;50:576–83. [12] Laing NG, Wilton SD, Akkari PA, et al. A mutation in the alphatropomyosin gene TPM3 associated with autosomal dominant nemaline myopathy NEM1. Nat Genet 1995;9:75–9. [13] Tan P, Briner J, Boltshauser E, et al. Homozygosity for a nonsense mutation in the alpha-tropomyosin slow gene TPM3 in a patient with severe infantile nemaline myopathy. Neuromuscul Disord 1999;9: 573–9. [14] Wattanasirichaigoon D, Swoboda KJ, Takada F, et al. Mutations of the slow muscle alpha-tropomyosin gene, TPM3, are a rare cause of nemaline myopathy. Neurology 2002;59:613–7. [15] Durling HJ, Reilich P, Müller-Höcker J, et al. De novo missense mutation in a constitutively expressed exon of the slow alpha-tropomyosin gene TPM3 associated with atypical, sporadic case of nemaline myopathy. Neuromuscul Disord 2002; 12:947–51. 469 [16] Donner K, Ollikainen M, Ridanpää M, et al. Mutations in the btropomyosin (TPM2) gene in two families diagnosed with nemaline myopathy. Neuromuscul Disord 2002;12:151–8. [17] Johnston JJ, Kelley RI, Crawford TO, et al. A novel nemaline myopathy in the Amish caused by a mutation in troponin T1. Am J Hum Genet 2000;67:814–21. [18] Wallgren-Pettersson C, Hilpelä P, Donner K, et al. Clinical and genetic heterogeneity in autosomal recessive nemaline myopathy. Neuromuscul Disord 1999;9:564– 72. [19] Wallgren-Pettersson C. Gene table: the congenital myopathies. Eur J Paed Neurol 2001;5:87–8. [20] Lammens M, Moerman PH, Fryns JP, et al. Fetal akinesia sequence caused by nemaline myopathy. Neuropediatrics 1997;28: 116–9. [21] Wallgren-Pettersson C, Laing NG. Report of the 70th ENMC International Workshop: nemaline myopathy. Neuromuscul Disord 2000;10:299–306. [22] Jungbluth H, Sewry CA, Nowak KJ, et al. Mild phenotype of nemaline myopathy with sleep hypoventilation due to a mutation in the skeletal muscle actin (ACTA1) gene. Neuromuscul Disord 2001;11:35 –40. [23] Sparrow JC, Nowak KJ, Durling HJ, et al. Muscle disease caused by mutations in the skeletal muscle alpha-actin gene (ACTA1). Neuromuscul Disord 2003;13:519 –31. [24] Buxmann H, Schlosser R, Schlote W, et al. Congenital nemaline myopathy due to ACTA1-gene mutation and carnitine insufficiency: a case report. Neuropediatrics 2001;32:267 –70. [25] Sewry C, Brown SC, Pelin K, et al. Abnormalities in the expression of nebulin in chromosome-2 linked nemaline myopathy. Neuromuscul Disord 2001;11:146–53. [26] Wallgren-Pettersson C, Donner K, Sewry C, et al. Mutations in the nebulin gene can cause severe congenital nemaline myopathy. Neuromuscul Disord 2002;12:674 –9. [27] Millevoi S, Trombitas K, Kolmerer B, et al. Characterization of nebulette and nebulin and emerging concepts of their roles for vertebrate Z-discs. J Mol Biol 1998;282:111 –23. [28] Pelin K, Labeit S, Wallgren-Pettersson C. Nebulin, Human. Encyclopedia of Molecular Medicine, vol. 5. Chichester: Wiley; 2002. p. 2223–5. [29] Goebel HH, Anderson JR, Hübner C, Oexle K, Warlo I. Congenital myopathy with excess of thin myofilaments. Neuromuscul Disord 1997;7:160 –8. [30] Wallgren-Pettersson C. Congenital nemaline myopathy: a clinical follow-up study of twelve patients. J Neurol Sci 1989;89:1–14. [31] Wallgren-Pettersson C, Kivisaari L, Jääskeläinen J, Lamminen A, Holmberg C. Ultrasonography, CT and MRI of muscles in congenital nemaline myopathy. Pediatr Neurol 1990;6:20–8. [32] Wallgren-Pettersson C, Sainio K, Salmi T. Electromyography in congenital nemaline myopathy. Muscle Nerve 1989;12:587–93. [33] Cartegni L, Chew SL, Krainer AR. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 2002;3:285–98. [34] Chelly J, Gilgenkrantz H, Lambert M, et al. Effect of dystrophin gene deletions on mRNA levels and processing in Duchenne and Becker muscular dystrophies. Cell 1990;63:1239 –48. [35] Ginjaar IE, Kneppers ALJ, vd Meulen J-DM, et al. Dystrophin nonsense mutation induces different levels of exon 29 skipping and leads to variable phenotypes within one BMD family. Eur J Hum Genet 2000;8:793 –6. [36] Morisaki H, Morisaki T, Newby LK, Holmes EW. Alternative splicing: a mechanism for phenotypic rescue of a common inherited defect. J Clin Invest 1993;91:2275 –80. [37] van Deutekom JCT, Bremmer-Bout M, Janson AAM, et al. Anitsense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells. Hum Mol Genet 2001;10: 1547– 54. 470 C. Wallgren-Pettersson et al. / Neuromuscular Disorders 14 (2004) 461–470 [38] Politou AS, Millevoi S, Gautel M, Kolmerer B, Pastore A. SH3 in muscles: solution structure of the SH3 domain from nebulin. J Mol Biol 1998;276:189–202. [39] Ojima K, Lin ZX, Bang M-L, et al. Distinct families of Z-line targeting modules in the COOH-terminal region of nebulin. J Cell Biol 2000;150:553–66. [40] Bang M-L, Mudry RE, McElhinny AS, et al. Myopalladin: a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and Iband protein assemblies. J Cell Biol 2001;153:413–27. [41] Politou AS, Spadaccini R, Joseph C, et al. The SH3 domain of nebulin binds selectively to type II peptides: theoretical prediction and experimental validation. J Mol Biol 2002;316: 305 –15. [42] Sheterline P, Clayton J, Sparrow JC. Actin. Oxford: Oxford University Press; 1999. [43] Pfuhl M, Winder SJ, Castiglione Morelli MA, Labeit S, Pastore A. Correlation between conformational and binding properties of nebulin repeats. J Mol Biol 1996;257:367– 84.