Brain Advance Access originally published online on April 17, 2007
Brain 2007 130(6):1497-1506; doi:10.1093/brain/awm068
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Phenotypical spectrum of DOK7 mutations in congenital myasthenic syndromes
1Friedrich-Baur-Institute, Department of Neurology, Ludwig-Maximilians-University, Munich, Germany, 2Department of Neurology, Bethesda Children's Hospital, Budapest, Hungary, 3Servicio de Neurologia, Hospital Universitari La Fe, Valencia, Spain, 4Unitat de Patologia Neuromuscular, Servei de Neurologia, Hospital Sant Joan de Déu, Esplugues (Barcelona), Spain, 5Department of Cancer Genetics, Unit 1010, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA, 6Hospital de Criancas Maria Pia, Porto, Portugal, 7Department of Neuropediatrics; Staedtische Kliniken, Neuss, Germany, 8Department of Neurology, Martin-Luther-Universität Halle-Wittenberg, Halle/Saale, Germany, 9Departments of Neurology/Neurosurgery & Pediatrics, Montreal Children's Hospital, McGill University, Montreal, Quebec, Canada, 10Department of Neuropediatrics, Hospital D. Estefania, Lisbon, Portugal, 11Department of Medical Genetics, University of Turku and Turku University Hospital, Turku, Finland, 12Department of Pediatric Neurology, Turku University Hospital, Turku, Finland, 13National Center for Public Health, National Institute of Environmental Health, Department of Molecular Genetics and Diagnostics, Budapest, Hungary, 14Children's Hospital, Technical University Dresden, Germany and 15Neurosciences Group, Weatherall Institute of Molecular Medicine, Department of Clinical Neurology, University of Oxford, UK
Corresponding to: Hanns Lochmüller, MD, Friedrich-Baur-Institute, Molecular Myology Lab, Marchioninistrasse 17, 81377 München, Germany E-mail: hanns.lochmueller{at}med.uni-muenchen.de
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Dok (‘downstream-of-kinase’) family of cytoplasmic proteins play a role in signalling downstream of receptor and non-receptor phosphotyrosine kinases. Recently, a skeletal muscle receptor tyrosine kinase (MuSK)-interacting cytoplasmic protein termed Dok-7 has been identified. Subsequently, we and others identified mutations in DOK7 as a cause of congenital myasthenic syndromes (CMS), providing evidence for a crucial role of Dok-7 in maintaining synaptic structure. Here we present clinical and molecular genetic data of 14 patients from 12 independent kinships with 13 different mutations in the DOK7 gene. The clinical picture of CMS with DOK7 mutations is highly variable. The age of onset may vary between birth and the third decade. However, most of the patients display a characteristic ‘limb-girdle’ pattern of weakness with a waddling gait and ptosis, but without ophthalmoparesis. Respiratory problems were frequent. Patients did not benefit from long-term therapy with esterase inhibitors; some of the patients even worsened. DOK7 mutations have emerged as one of the major genetic defects in CMS. The clinical picture differs significantly from CMS caused by mutations in other genes, such as the acetylcholine receptor (AChR) subunit genes. None of the patients with DOK7 mutations had tubular aggregates in the muscle biopsy, implying that ‘limb-girdle myasthenia (LGM) with tubular aggregates’ previously described in literature may be a pathogenic entity distinct from CMS caused by DOK7 mutations.
Key Words: congenital myasthenic syndromes; DOK7; neuromuscular junction; limb-girdle myasthenia (LGM)
Abbreviations: AChR, acetylcholine receptor; CMAP, compound muscle action potential; CMS, congenital myasthenic syndromes; DOK7, downstream-of-kinase 7; LGM, limb-girdle myasthenia; MuSK, skeletal muscle receptor tyrosine kinase; NMJ, neuromuscular junction; PH, pleckstrin homology; PTB, phosphotyrosine-binding domain
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Received November 17, 2006. Revised February 21, 2007. Accepted March 12, 2007.
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Introduction |
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The neuromuscular junction (NMJ) is a complex structure that serves to efficiently communicate signals from the motor neuron to the skeletal muscle and to induce muscle contraction [for an overview see (Hughes et al., 2006
)]. Congenital myasthenic syndromes (CMS) are a group of inherited disorders in which function of this highly specialized structure is impaired (Engel and Sine, 2005). Although the common feature of all CMS is an exercise-induced weakness of skeletal muscle, the clinical picture of CMS is very diverse. Weakness and fatigue usually present from birth or appear in the first 2 years of life. Symptoms at onset may be hypotonia, respiratory distress or joint contractures at birth. The disease can also manifest in the first year of life with bilateral ptosis, ophthalmoparesis and facial weakness or in early childhood with walking difficulties and frequent falls. Late onset of muscle weakness in adolescence or early adulthood has been reported as well.
Defects in the presynaptic nerve terminal, the synaptic cleft and the postsynaptic apparatus have been identified during the last decade as the molecular causes for CMS (reviewed in: Hantai et al., 2004; Beeson et al., 2005; Engel and Sine, 2005). However, the underlying molecular mechanisms are still unknown for a number of CMS forms (Beeson et al., 2005). One of these forms, CMS with predominantly proximal limb weakness, has recently been associated with recessive mutations of the DOK (‘downstream-of-kinase’) 7 gene (Beeson et al., 2006). Here we present clinical and genetic data of 14 patients from 12 CMS kinships with mutations in the DOK7 gene. Furthermore, we emphasize that patients with DOK7 mutations have characteristic features that place them in a distinct subgroup of the patients previously termed ‘limb-girdle’ CMS in literature.
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DNA samples
Venous blood samples were obtained from the patients as well as from their unaffected relatives, if available. All studies were carried out with informed consent of the patients or their parents and were approved by the institutional ethics review board. Genomic DNA was isolated using a blood and tissue culture DNA extraction kit according to the manufacturer's recommendations (Wizard Genomic DNA Purification Kit, Promega, Mannheim, Germany).
Genotyping, sequence analysis, RFLPs
PCR primers for analysis of the DOK7 gene were designed according to the published genomic structure [GenBank accession numbers gi:27652626 (genomic sequence), mRNA: AB220918
[GenBank]
] of the DOK7 gene. In the patients, all seven exons and flanking intronic regions of the DOK7 gene were amplified by PCR and sequenced. PCR-amplified fragments were purified with the NucleoSpin Extract Kit (Macherey-Nagel, Düren, Germany) and sequenced using an Applied Biosystems model 3100 Avant DNA sequencer and fluorescence-labelled dideoxy terminators (Perkin-Elmer, Foster City, CA, USA).
Patients’ relatives (where available) and control individuals were screened for each of the observed missense mutations by restriction digest or sequencing of PCR products.
Screening for the mutation A33V was performed by restriction analysis using BfuAI, screening for S45L was done using Hpy188I and H132Q using PstI. Screening for P469H in exon 7 was done by direct sequencing. Sixty healthy control individuals were tested for the mutations A33V and H132Q, 100 control DNAs for S45L and 200 control individuals were screened for the mutation P469H.
Cloning procedures
To analyse potential splicing aberrations of the intronic base alteration (IVS2-1G>T), we introduced exon 3 and flanking intronic sequences into the Exontrap-vector (MoBiTec, Göttigen, Germany). We constructed two DOK7 minigenes containing genomic sequence from intron 2, exon 3 and intron 3 and two different nucleotides at position 1 in intron 2: DOK7-1G (wild-type), DOK7-1T (potential splice mutation). A 569 bp fragment of the genomic DNA of patient 6 and a healthy control donor was amplified using the primer pair 5'-AGGTCAGTCGACCAGCCCGGGTCTCTGCACT GTCACG and 5'-CACCCTGCGGCCGCCATGAATGTCCCATCTTCCTGCACG (introducing SalI and NotI sites for cloning) and cloned into the SalI and NotI site of the pET01 Exontrap vector (MoBiTec, Göttingen, Germany). Absence of PCR-induced mutations was verified by direct sequencing.
RNA analysis
For transfection of the constructs into HEK 293 cells, 3 x 105 cells were seeded into a six-well plate 24 h before transfection. To analyse the splice mutation (IVS2-1G>T), cells were transfected with 2.5 µg of plasmid DNA 24 h after plating using the calcium phosphate method. The cells were harvested 48 h post-transfection and total RNA was isolated using the Trizol reagent (Invitrogen, Karlsruhe, Germany). RT-PCR was performed with 5 µg of total RNA with the primer pair 5'-GATCGATCCGCTTCCTGCCCC (in the Exontrap 5' exon) sense and 5'-CCACGCACACATGGCCTCGTGGCT (in exon 3 of DOK7) or 5'-CTGCCGGG CCACCTCCAGTGCC (in the Exontrap 3' exon).
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Genetic analysis
The index patient (Tables 1 and 2, patient 1, Fig. 1A, C and D), a now 16-year-old boy, originates from a large Hungarian kindred. The parents observed walking difficulties around the age of 2.5 years as first symptom. At neurological examination, he presented with ptosis, facial weakness, swallowing and chewing difficulties, combined with a generalized muscle weakness especially of the proximal limb muscles. The boy suffered from several episodic crises with respiratory insufficiency. He developed scoliosis and slight muscle atrophy. Repetitive stimulation of distal motor nerves revealed a pathological decremental response of the compound muscle action potential (CMAP) at rest. Muscle biopsy showed an unspecific type II fibre atrophy and anti-AChR antibodies were negative. An intravenous Edrophonium test was positive, a long-term therapy with Mestinon® was only partially effective, however. Thymectomy and therapy with immunosuppressants were not beneficial. A similarly affected sister died of respiratory failure at the age of 13 years. An older brother is mildly affected, presenting with ptosis and facial weakness (Fig. 1B). The two patients have nine healthy siblings and healthy parents. DNA of the deceased sister was not available for genetic analysis. In this family, all previously known CMS genes (AChR subunits, COLQ, RAPSN, CHAT, MUSK, SCN4A) have been excluded by direct sequencing or by haplotype analysis using loci-specific microsatellite DNA markers (von der Hagen et al., 2006
) (data not shown). Subsequently, a genome-wide screen for the disease locus was performed using a GeneChip® Mapping 10K Array Set. Assuming a recessive trait at 100% penetrance, parametric linkage analysis of family 1 identified a LOD score suggestive of linkage in chromosome 4p16.3-p16.1 (data not shown). The DOK7 gene recently identified by Okada et al. (2006) is located within this region. Sequence analysis of the DOK7 gene in family 1 revealed the presence of two compound heterozygous mutations, H132Q in exon 4 and 1124_1127dupTGCC in exon 7.
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Subsequent screening of the DOK7 gene in our patient cohort of about 200 so far unsolved CMS cases revealed the presence of mutations in 11 additional CMS kinships adding up to a total of 14 patients. This corresponds to 5% of the patients referred to our centre for genetic analysis for CMS. DOK7 mutations are the third most frequent genetic cause for CMS in our patient cohort next to CHRNE and RAPSN mutations and at similar frequency to synaptic CMS caused by COLQ mutations.
All patients with DOK7 mutations are of Caucasian descent and originate from Germany (3 patients), Spain (4), Portugal (2), Quebec (Canada) (1), Hungary (2), South America (1) and Finland (1). Patients 3–10 have been reported in brief by Beeson et al. (2006), corresponding there to patients 15–21.
Clinical data
Onset of symptoms
Typically, onset of symptoms was in the second year of life when the patients started walking. Gait was often awkward or waddling, the parents noted frequent falls or difficulties in running. There were some exceptions, though: in five cases, symptoms were apparent at birth or in the first months of life. Four patients were hypotonic at birth (patients 3, 8, 12 and 14); two patients (patients 13 and 14) had delayed motor milestones.
In contrast, late onset of symptoms in the twenties was reported for two Spanish siblings (patients 9 and 10), and patient 7 noted abnormal fatigue and a reduced endurance level at 12 years of age.
Clinical phenotype
The majority of our patients with DOK7 mutations show a bilateral ptosis (12 of 14 patients), but no restriction of eye movements (Fig. 1A). However, temporary diplopia was reported in some cases (patients 10 and 11). Facial and bulbar weakness was frequent (9 of 14 patients), several patients complained of chewing and swallowing difficulties. Patient 5 has a very prominent weakness of jaw muscles. Slight atrophy of the tongue was noted in two patients (Fig. 1E).
Respiratory function is impaired in 10 patients; lung function seemed to deteriorate during the course of the disease. Adult patients show a reduced vital capacity, several became dependent on nocturnal assisted ventilation (patients 3, 6, 7, 8, 11 and 13). Four patients experienced crises with weakness and respiratory insufficiency which made admission to the ICU necessary. A similarly affected sister of patients 1 and 2 (not listed in Table 2) deceased in hospital during a respiratory crises precipitated by pneumonia. Two patients with disease onset at birth needed assisted ventilation during their first months of life (patients 3 and 14).
Most patients developed scoliosis and/or lordosis in the course of the disease, general muscle atrophy or atrophy of selective muscles is apparent in some patients (patients 1, 6, 8 and 14, Fig. 1C, D, G, H, K and L). All but one patient (exception: patient 2, Fig. 1B) suffer from a moderate to severe muscle weakness manifesting preferentially in proximal muscles of upper and lower limbs. Patients are unable to raise the arms above the head or to get up from the floor without support. Patient 14, a 24-month-old girl (Fig. 1G), was not able to get up or turn her body in the lying position; her motor milestones were delayed. Gait is waddling in most cases; in four cases, the waddling is accompanied by an additional inward rotation of knees [termed ‘sinuous gait’ in (Slater et al., 2006). (Fig. 1F and L, supplementary video material). This sinuous gait seems characteristic for CMS patients with DOK7 mutations, and has not been noticed in other CMS patients.
In general, weakness is exercise-dependent. Daytime-dependent fluctuation of symptoms with worsening in the evening—as it has been described for other CMS forms—was observed only in two patients. Interestingly, nine patients report fluctuation of symptoms on longer periods (good or bad weeks), sometimes dependent on emotional stress or weather conditions.
In some patients disease course was progressive, leading to loss of ambulance in adolescence or adulthood (patients 6, 8 and 13). Respiratory function deteriorated in several patients, making nocturnal ventilation necessary. In other cases, severity of symptoms remained nearly constant.
Laboratory tests, electrophysiology, muscle biopsy
AChR antibody testing was negative in all patients. Repetitive nerve stimulation was performed in 12 of the 14 patients; in 10 of them, a pathological decremental response was found. None of the patients showed a repetitive CMAP upon a single nerve stimulus. Creatine kinase levels were normal or only slightly elevated. Conventional muscle biopsies displayed only unspecific mild myopathic changes, e.g. fibre type I predominance or central nuclei (Supplementary Fig. 1). Tubular aggregates were not observed. No endplate biopsies were available for analysis.
Response to therapy
For five of the patients an intravenous Edrophonium test was positive. No clear effect upon intravenous administration of Edrophonium was seen in three patients. However, long-term therapy with esterase inhibitors over several years was not successful in any patient. Different medications were tried for patient 1 over the course of several years: Mestinon® (up to 60 mg 10 times a day) was efficient only for a short period; subsequently, neither Ambenonium chlorate (10 mg/day) nor chinidine sulfate (450 mg/day) led to any change. Patient 8 received Mestinon® for 3 weeks at the age of 16 years; he felt an improvement first, but then symptoms worsened and treatment was discontinued. Patient 13—at 10 years of age—first improved significantly with Mestinon® 60 mg four times a day; he was able to walk longer distances without using his wheelchair, before he started deteriorating continuously after 1 year of treatment. Patients 4 (at the age of 3 years) and 7 (at the age of 38 years) were treated for several weeks with 60 and 210 mg Mestinon® per day, respectively, but in both cases no effect was noticed, neither by the patients themselves, nor by their physicians. At present, only patient 14 is still treated with Mestinon® with some positive effects. Since the age of 1 year, she receives 60 mg four times a day, she became more active and movement of extremities improved. She is now able to sit without support, stand and walk a few steps. However, she has been followed only for about 1 year, so far.
In some cases, e.g. patient 5, esterase inhibitors even resulted in immediate deterioration of symptoms and were discontinued. Similarly, patient 12 received up to 60 mg/dose four times a day for about a week, then she become unable to perform activities of daily life any more, but recovered strength after the treatment was stopped. Combination of esterase inhibitors and 3,4-diaminopyridine was tried in three cases (patients 6, 9 and 10), but without any substantial benefit. The three patients received Pyridostigmine 30–60 mg and 3,4-diaminopyridine 10–15 mg four times a day. The medication showed a good effect first in patients 6 (improvement of strengths of the legs for about 1 year) and 10 (for about 3 months) upon subjective and objective physical examination, but then lost its effect. The combination of esterase inhibitors with diaminopyridine had no effect at all in patient 9.
DOK7 mutations
Thirteen different DOK7 mutations have been identified (Table 1 and Fig. 2): four missense mutations, one nonsense, one splice-site and seven frameshift mutations. The majority of the mutations are located in exon 7 of the gene, especially the frameshift mutations. Only one frameshift mutation, 555delC, is located in exon 5. The missense mutations are located in the N-terminal exons of the gene, except mutation P469H in exon 7; this is the first missense mutation in exon 7 published so far. The mutations A33V in exon 2 and S45L in exon 3 are located in the pleckstrin homology (PH) domain of the protein; S45L has been identified in two independent patients from Spain and Portugal. The mutation H132Q in exon 4 identified in family 1 is located in the phosphotyrosine binding (PTB) domain of Dok-7.
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The intronic nucleotide exchange IVS2-1G>T modifies the acceptor splice site of exon 3. Minigene experiments in a cell-culture system showed that due to this mutation, the regular acceptor splice site of exon 3 is not recognized by the splicing machinery. Several aberrant splice products have been detected, the most abundant being transcripts completely lacking exon 3. In other transcripts alternative cryptic acceptor splice sites have been used leading to transcripts lacking 26 or 155 bp of exon 3 (Supplementary Fig. 2). All transcripts result in a frameshift and premature termination of translation.
All but three patients (patients 3, 11 and 14) carry the frameshift mutation 1124_1127dupTGCC in exon 7 at least heterozygously. Three patients from two families are homozygous for this particular mutation. Interestingly, at least one of the mutations for all patients is located in exon 7 of the DOK7 gene. No patient with both DOK7 mutations located in exons 1–6 was observed.
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Pathogenic mechanisms of DOK7 mutations
Here, we present the clinical features of 14 patients with mutations in the recently described gene DOK7. The pathogenic mechanisms of DOK7 mutations have so far not been characterized well. Some of the patients with DOK7 mutations described by Beeson et al. were reported to have small endplates (Beeson et al., 2006
; Slater et al., 2006). However, it is not yet clear how exactly Dok-7 malfunction may lead to decreased endplate size.
One of the mutations, the frequent C-terminal domain mutation 1124_1127dupTGCC, has been studied in cell culture. Interestingly, all but six patients of the 28 DOK7 patients reported so far carry at least one copy of this frameshift mutation. Furthermore, we identified five additional CMS patients (not listed here) in our cohort who are heterozygous carriers of 1124_1127dupTGCC. Although their clinical features are compatible with LGM, no second heteroallelic mutation has yet been detected in the coding regions of the DOK7 gene. Analysis of AChR clusters induced by Dok-7 harbouring the mutation 1124_1127dupTGCC showed a significant reduction in the number of branched-type AChR plaques compared to wild-type Dok-7 in fully differentiated transfected C2C12 myotubes, suggesting that the C-terminal domain might play a key role in the maturation of the synaptic structure (Beeson et al., 2006). The mutations identified by us in the C-terminal domain of Dok-7 (Fig. 2) might have similar effects in myotubes.
Known Dok-family proteins from Dok-1 to Dok-6 play a role in signalling downstream of receptor and non-receptor phosphotyrosine kinases. Like other Dok proteins, Dok-7 has a PH domain and a PTB domain in the N-terminal moiety and multiple tyrosine residues in the C-terminal region. Okada et al. (2006) demonstrated that Dok-7 can induce the aneural activation of MuSK and subsequent clustering of AChR in cultured myotubes. The PH domain of Dok-7, as well as the PTB domain, have been shown to be indispensable for the activation and phosphorylation of MuSK in heterologous cells as well as in C2 myotubes (Okada et al., 2006). Missense mutations in these two domains—as observed in five patients in this study—might impair the interaction of Dok-7 with MuSK. So far, no obvious correlation of clinical symptoms and the location of the mutations could be established. Remarkably, our patients with late onset of symptoms are all homozygous for the common frameshift mutation 1124_1127dupTGCC. Interestingly, no patient with both DOK7 mutations located in exons 1–6 was observed. Complete lack of Dok-7 is probably not compatible with postnatal life, since Dok7 deficient mice die shortly after birth (Okada et al., 2006).
Comparison of clinical phenotypes
Slater et al. (2006) described a group of patients suffering from inherited myasthenia with predominant limb-girdle weakness. Impaired neuromuscular transmission in these patients resulted from structural abnormalities of the endplates (reduced endplate size and postsynaptic folding), rather than from changes in AChR channel kinetics. Mutations in the DOK7 gene have been identified in six out of seven available DNA samples from these patients (Beeson et al., 2006). Features typically seen in our patients with DOK7 mutations mainly correspond to the major symptoms of the patients described by (Slater et al., 2006): onset of symptoms is usually in the second year of life, when walking difficulties or frequent falls become apparent. Limb-girdle weakness is predominant; gait is mostly waddling or swaying accompanied sometimes by an inwards rotation of hips and knees. The course of disease is progressive, sometimes leading to loss of ambulation. Symptoms might fluctuate over longer time periods; no daytime-dependent fluctuations have been observed as for other CMS forms. Similarly, our patients did not benefit from a therapy with esterase inhibitors or 3,4-diaminopyridine. However, most of our DOK7 patients had ptosis and facial weakness. Many suffered from deterioration of respiratory function during the course of disease or experienced respiratory crises. Onset of symptoms at birth or late onset in early adulthood broaden the phenotype in our cohort of DOK7 patients.
Although the clinical features of our patients with DOK7 mutations vary considerably, the patients can be distinguished from CMS patients with mutations in other genes: ocular and facial muscles are frequently affected in other CMS forms, especially in CMS caused by CHRNE or RAPSN mutations (Table 3). In contrast to CMS with CHRNE mutations, eye movements are generally unaffected in patients with DOK7 mutations. However, transient diplopia—as reported for RAPSN (Müller et al., 2003)—might be present.
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Patients with CHAT or RAPSN mutations often present with episodic apneas at birth or in the first months of life, whereas those with DOK7 mutations predominantly experience a progressive deterioration of their respiratory function and may be dependent on night ventilation later in life. However, four DOK7 patients presented with hypotonia and respiratory distress at birth, as it has been described for other CMS forms [e.g. CHAT, RAPSN or COLQ mutations, (Ohno et al., 2000
, 2001, 2002; Müller et al., 2003; Barisic et al., 2005)]. Congenital joint contractures or other congenital deformities were not detected.
Late-onset of symptoms in adolescence or early adulthood (as in patients 7, 9 and 10) may also occur in Slow-Channel CMS (Sine et al., 1995) or in CMS caused by RAPSN mutations (Burke et al., 2003; Müller et al., 2003). Severity of weakness and walking disability is comparable to patients with synaptic CMS caused by COLQ mutations or Slow-Channel CMS. CMS caused by COLQ mutations might manifest as LGM as well, as it has already been reported [e.g. (Müller et al., 2004), patient 3]. However, in many cases, though not all, these patients may be distinguished by electrophysiolgical characteristics e.g. the repetitive CMAP upon single nerve stimulus.
Comparison to other LGM patients
Our patients with DOK7 mutations differ from other patients previously reported as LGM with predominantly proximal limb weakness (Table 4). Some of the reported LGM patients had serum antibodies to AChR; these individuals were not considered in Table 4. Symptoms at onset were mainly walking difficulties, but onset was often later in childhood or in adolescence. None of the patients described displayed ptosis or respiratory distress, facial and bulbar muscles were mostly spared. Weakness usually affected just limb and trunk muscles. In contrast, all but two of our DOK7 patients had bilateral ptosis, and 9 out of 14 had facial weakness; this may be an important criterion to distinguish among the two groups of LGM patients. Tubular aggregates were detected in the muscle biopsies of the majority of these patients, but in none of the DOK7 patients. LGM patients benefited from long-term therapy with esterase inhibitors which is in sharp contrast to DOK7 patients.
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Compared to the rather homogeneous group of LGM patients previously reported, the group of LGM cases negative for DOK7 mutations in our patient cohort is heterogeneous (Table 4, third row). Still, only one-third (5 out of 17 patients) have ptosis or facial weakness and only three patients experienced respiratory distress. Fluctuation over longer periods was observed only in two patients. Moreover, most patients with normal DOK7 are successfully treated with esterase inhibitors. Tubular aggregates were detected in five of them, four being the Sicilian patients with inherited LGM reported by (Rodolico et al., 2002
). However, tubular aggregates have been observed in several muscle disorders and are generally considered to be a secondary and non-specific phenomenon [overview in (Chevessier et al., 2005)]. Evaluation of clinical data of our patient cohort and of previously published cases revealed that inherited myasthenic syndromes with limb-girdle weakness are neither clinically nor genetically homogeneous. Therefore, additional genetic defects leading to LGM remain to be determined. Moreover, clinical diagnosis of a CMS might be hampered by the unusual clinical phenotype of the patients with DOK7 mutations and the failure to respond to esterase inhibitors. Due to the frequency of DOK7 mutations and their high concentration in exon 7 of the gene, we suggest that sequencing of this exon may constitute an efficient first genetic screen for myasthenia patients, similar to the test for the RAPSN mutation N88K.
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Supplementary Data |
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Supplementary Data is available at BRAIN online.
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* Present address: Department of Neuropediatrics, Children's University Hospital, University of Essen, Germany
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Acknowledgements |
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We wish to thank the patients and their families for participating in this study. We thank Ursula Klutzny and Mandy Heiliger for technical assistance and Petra Mitzscherling for technical assistance in haplotype analyses of CMS loci. LLB and RK thank Baili Zhang and Tamer Ahmed for assistance with SNP and microsatellite genotyping. This work was supported by grants from the Association Francaise contre les Myopathies (AFM) to HL, AA and JSM and by a German–Hungarian project for joint research from the German Research Foundation DFG and Hungarian Academy of Sciences (grant 436UNG113/153) to HL, AA, JSM, AH and VK. AA, HL, US, MD and AH are members of the German Muscular Dystrophy Network (MD-NET 01GM0601) funded by the German Ministry of Education and Research (BMBF, Bonn, Germany); www.md-net.org. MD-NET is a partner of TREAT-NMD(EC, 6th FP, proposal #036825; www.treat-nmd.eu. MS receives support from the MCH Foundation. JJV is member of the Spanish Neuromuscular Network funded by Spanish Health Ministry (FIS-PI051622). VM receives a BAYHOST fellowship from the Bavarian state. DB is supported by MRC and Myasthenia Gravis Association/Muscular Dystrophy Campaign, UK.
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