Brain Advance Access originally published online on March 5, 2008
Brain 2008 131(5):1217-1227; doi:10.1093/brain/awn029
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Relative contribution of mutations in genes for autosomal dominant distal hereditary motor neuropathies: a genotype–phenotype correlation study
1Peripheral Neuropathy Group, VIB Department of Molecular Genetics, 2Neurogenetics laboratory, Institute Born-Bunge, 3Neurogenetics Group, VIB Department of Molecular Genetics, University of Antwerp, Antwerpen, Belgium, 4Muscle Unit, Division of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy, 5Service de Neurologie, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium, 6Clinic of Child Neurology and Psychiatry, University of Belgrade, Belgrade, Serbia, 7Department of Neurology, University Hospital Gasthuisberg, Leuven, Belgium, 8Muskelzentrum St Gallen, Kantonsspital St Gallen, St Gallen, 9Department of Neurology, University Hospital of Basel, Basel, Switzerland, 10Neurology Department, University Hospital of Montpellier, Montpellier, France, 11National Center for Neuromuscular Diseases, Department of Neurology, University Hospital Center Zagreb, Zagreb, Croatia, 12Department of Child Neurology, Charles University, 2nd School of Medicine Prague, 13Department of Neurology, Charles University, 2nd School of Medicine and University Hospital Motol, Prague, Czech Republic, 14Neuromuscular Unit, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland, 15Laboratory of Molecular Pathology, Sofia Medical University, Sofia, Bulgaria, 16Institute of Medical Biology and Department of Internal Medicine, Diabetes and Metabolism, Medical University Graz, Austria, 17Department of Clinical Genetics, Leiden University Medical Center, Leiden, 18Department of Neurology, University Medical Center Utrecht, Utrecht, The Netherlands and 19Division of Neurology, University Hospital Antwerpen, Antwerpen, Belgium
Correspondence to: Prof. Dr Vincent Timmerman, PhD, Peripheral Neuropathy Group, VIB - Department of Molecular Genetics, University of Antwerp, Universiteitsplein 1, B-2610 Antwerpen, Belgium E-mail: vincent.timmerman{at}ua.ac.be
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Summary |
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Distal hereditary motor neuropathy (HMN) is a clinically and genetically heterogeneous group of disorders affecting spinal
-motor neurons. Since 2001, mutations in six different genes have been identified for autosomal dominant distal HMN; glycyl-tRNA synthetase (GARS), dynactin 1 (DCTN1), small heat shock 27 kDa protein 1 (HSPB1), small heat shock 22 kDa protein 8 (HSPB8), Berardinelli-Seip congenital lipodystrophy (BSCL2) and senataxin (SETX). In addition a mutation in the (VAMP)-associated protein B and C (VAPB) was found in several Brazilian families with complex and atypical forms of autosomal dominantly inherited motor neuron disease. We have investigated the distribution of mutations in these seven genes in a cohort of 112 familial and isolated patients with a diagnosis of distal motor neuropathy and found nine different disease-causing mutations in HSPB8, HSPB1, BSCL2 and SETX in 17 patients of whom 10 have been previously reported. No mutations were found in GARS, DCTN1 and VAPB. The phenotypic features of patients with mutations in HSPB8, HSPB1, BSCL2 and SETX fit within the distal HMN classification, with only one exception; a C-terminal HSPB1-mutation was associated with upper motor neuron signs. Furthermore, we provide evidence for a genetic mosaicism in transmitting an HSPB1 mutation. This study, performed in a large cohort of familial and isolated distal HMN patients, clearly confirms the genetic and phenotypic heterogeneity of distal HMN and provides a basis for the development of algorithms for diagnostic mutation screening in this group of disorders.
Key Words: distal HMN; BSCL2; HSPB1; HSPB8; SETX
Abbreviations: AD, autosomal dominant; AR, autosomal recessive; CMAP, compound muscle action potential; CMT, Charcot–Marie–Tooth disease; EMG, electromyography; HMN, hereditary motor neuropathy; HMSN, hereditary motor and sensory neuropathy; NCV, nerve conduction velocity; SMA, spinal muscular atrophy; SNAP, sensory nerve action potentials; STRs, short tandem repeats
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Received October 12, 2007. Revised January 25, 2008. Accepted February 8, 2008.
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Introduction |
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TopSummaryIntroductionPatients and MethodsResultsDiscussionSupplementary materialReferences |
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Distal hereditary motor neuropathy (HMN), also known as distal spinal muscular atrophy (SMA) or the spinal form of Charcot–Marie–Tooth disease (CMT), covers a spectrum of clinically and genetically heterogeneous diseases characterized by the selective involvement of motor neurons in the peripheral nervous system. In contrast to proximal SMA, distal HMN initially and predominantly affects the distal limb muscles suggesting a length-dependent disease mechanism affecting the longest motor axons first. The vast majority of patients present a classical distal muscular atrophy syndrome in the legs without clinical sensory loss. The disease usually begins in childhood or adolescence and starts with weakness and wasting of distal muscles of the anterior tibial and peroneal compartments of the legs. Later on, weakness and atrophy may expand to the proximal muscles of the lower limbs and/or to the distal upper limbs. However, in some patients and families the disease starts or even predominates in the hands. Foot deformities (e.g. pes cavus) are sometimes present. Based on clinical and electrophysiological evidence, Dyck and Lambert (1968a
, b) postulated that distal HMN was a separate clinical and genetic entity. Subsequently, a classification has been proposed subdividing distal HMN in seven subtypes based on age at onset, mode of inheritance, distribution of muscle weakness and additional clinical features (Harding, 1993). These subtypes were based on the description of a limited number of patients in small families. Currently, with detailed reports of a larger number of distal HMN families including several large pedigrees and the identification of gene defects, the phenotypes initially defined in this classification could be confirmed at the molecular genetic level. However, some clinical features were not included in the original classification (e.g. pyramidal signs, congenital onset and X-linked inheritance) and have resulted into further diversification of the clinical spectrum of distal HMN (Irobi et al., 2006).
Since 2001, eight genes have been identified for distal HMN. Six genes have been associated with autosomal dominant (AD) distal HMN: mutations in heat shock 27 kDa protein 1 (HSPB1) (Evgrafov et al., 2004) and heat shock 22 kDa protein 8 (HSPB8) (Irobi et al., 2004b) are found in families with distal HMN type II, mutations in glycyl-tRNA synthetase (GARS) (Antonellis et al., 2003) are associated with distal HMN type V, mutations in Bernardinelli-Seip congenital lipodystrophy 2 (BSCL2) (Windpassinger et al., 2004) cause distal HMN type V and Silver syndrome, a single dynactin 1 (DCTN1) (Puls et al., 2003) mutation was found in a family with adult-onset motor neuron disease with breathing difficulties due to vocal cord paralysis, and mutations in senataxin (SETX) (Chen et al., 2004) are reported in families with an early-onset distal HMN with pyramidal tract signs (juvenile amyotrophic lateral sclerosis; ALS4). Furthermore, a mutation was found in the VAMP-associated protein B and C (VAPB) gene in Brazilian families with clinically diverse motor neuron disorders, including atypical ALS with a slow progression, typical ALS and SMA affecting mainly proximal muscles (Nishimura et al., 2004). Currently, no mutations in VAPB were reported in distal HMN patients and families. Mutations causing autosomal recessive (AR) distal SMA with respiratory distress (SMARD1; distal HMN type VI) were described in the immunoglobulin µ-binding protein 2a (IGHMBP2) (Grohmann et al., 2001) gene. In addition, a mutation was recently found in the pleckstrin homology domain-containing, family G member 5 gene (PLEKHG5) in an autosomal recessive African family with childhood onset and severe evolution (Maystadt et al., 2007). The mutations in genes for distal HMN are listed in the Inherited Peripheral Neuropathy Mutation Database (www.molgen.ua.ac.be/-CMTMutations). Interestingly, most of these genes encode for ubiquitously expressed proteins with diverse cellular functions: protein translation and synthesis (GARS, BSCL2), RNA/DNA metabolism (SETX, IGHMBP2), axonal guidance and trafficking (HSP27, DCTN1, PLEKHG5), cellular protection (HSP22, HSP27) and apoptosis (HSP27) (Van Den Bosch and Timmerman, 2006). More insights into the role of these proteins in motor neuron function will increase our knowledge of the pathophysiological mechanisms underlying distal HMN.
With the identification of mutations in these genes it also became apparent that mutations in the same gene are associated with clinically separate disease entities, as mutations in GARS (Sivakumar et al., 2005), HSPB1 (Evgrafov et al., 2004; Tang et al., 2005a) and HSPB8 (Irobi et al., 2004b; Tang et al., 2005b) have been found both in familial and isolated patients with distal HMN and hereditary motor and sensory neuropathy (HMSN; CMT) phenotypes. However, there is scarce information about the definite contribution of mutations in these genes in distal HMN patients and about possible genotype–phenotype correlations.
In this study, we investigated a cohort of 112 familial and isolated patients with a diagnosis of distal motor neuropathy and determined the distribution of mutations in the known genes associated with AD distal HMN. The cohort included 10 index patients of families that have previously been reported in manuscripts describing novel genes and related phenotypes (Timmerman et al., 1992; De Jonghe et al., 2002; Evgrafov et al., 2004; Irobi et al., 2004a, b). In addition, we included the analysis of VAPB to investigate whether the phenotype associated with mutations in VAPB could be expanded to distal HMN.
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Patients and Methods |
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Selection criteria
We selected in our patient database all distal HMN patients using a pre-defined set of criteria. The clinical criteria for distal HMN were previously designed by the European CMT Consortium (De Jonghe et al., 1998
) and include: slowly progressive muscle wasting and weakness of predominantly the distal parts of the extremities with variable severity. In contrast to the published criteria, we included patients with pyramidal tract signs such as spastic gait, brisk reflexes and Babinski signs since in the mean time it has been demonstrated that these signs are common in distal HMN patients with SETX and BSCL2 mutations. We excluded patients presenting with predominant proximal muscle weakness, sensory symptoms and signs, and significant central nervous system involvement other than pyramidal tract signs. The clinical diagnosis of distal HMN was confirmed by nerve conduction studies and concentric needle electromyography (EMG) i.e. normal sensory nerve conduction velocities (NCVs), normal or slightly reduced motor NCVs and neurogenic alterations on needle EMG examination.
Patient cohort
The cohort encompassed 112 index patients who were initially referred for molecular genetic testing. Genomic DNA samples were provided through Neurology Departments and Neuromuscular Centres worldwide, with the majority from European origin. For 57 out of 112 index patients, a dominant inheritance pattern, based on a parent to child transmission, could be determined. Twenty-one patients were referred as ‘isolated’ since they did not have a familial history of neuropathy in first and second degree relatives. No family history was available for the remaining 34 patients. Of 74 patients, we had information on age at onset of the disease; in six patients the first symptoms presented before the first year, in 19 patients the disease started in the first decade of life, in 23 index patients the onset of disease was within the second decade of life and in 26 patients the first symptoms presented after the second decade of life. In 21 index patients, pyramidal tract signs were present. Eight patients presented bulbar symptoms including facial weakness, dysarthria, dysphagia, vocal cord paralysis or breathing difficulties. We obtained detailed electrophysiological information on 63 index patients. The remaining patients had an electrophysiological evaluation by their referring neurologist confirming a diagnosis of distal HMN but no detailed data were available. All referring neurologists were active in neuromuscular centres and were familiar with the clinical and electrophysiological phenotype of distal HMN. From all patients included in this cohort informed consent was obtained by the referring physician.
Molecular genetic analysis
The coding regions and exon–intron boundaries of HSPB1, HSPB8, BSCL2, GARS, SETX, DCTN1 and VAPB were PCR-amplified using primer oligonucleotides designed with the Primer3 and SNPbox software tools (Supplementary Table 1) (Rozen and Skaletsky, 2000; Weckx et al., 2004). PCR conditions are available upon request. PCR products were cleaned up using the Exonuclease I–Shrimp Alkaline Phosphatase enzyme (USB, Cleveland, Ohio). Mutation screening was performed by direct sequencing of the purified PCR fragments using the BigDye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Foster city, CA). Fragments were separated on an ABI 3730 automated capillary DNA sequencer (Applied Biosystems). The resulting sequences were aligned and analyzed with the SeqManII (DNAstar Inc., Madison, WI) and the novoSNP program (Weckx et al., 2005). The nucleotide numbering of the genes is relative to the ATG translation initiation site with A as +1 of the corresponding cDNA sequences (HSPB8: NM_014365
[GenBank]
.2; HSPB1: NM_001540
[GenBank]
.2; BSCL2: NM_032667
[GenBank]
.7; GARS: NM_002047
[GenBank]
.2; SETX: NM_015046
[GenBank]
.5; DCTN1: NM_004082
[GenBank]
.2; VAPB: NM_004738
[GenBank]
.3), according to the nomenclature of DNA sequence variants (http://www.hgvs.org/mutnomen). Sequence variants were confirmed by repeated PCR and bidirectional sequencing. Where possible, segregation of the mutation with the disease phenotype in the family was analysed.
Genotyping and paternity testing
Pyrosequencing with the PSQTMHS 96A System (Biotage AB, Uppsala, Sweden) was performed to genotype the Pro182Leu HSPB1-mutation. Primers for pyrosequencing were designed using the SNP Primer Design software version 1.0.1 (Biotage AB available upon request). Six informative short tandem repeats (STRs), D7S2435, D7S2490 or D7S2518, D7S2470, D7S2421, D7S669 and D7S2204, were used for segregation analysis at the 7q11–q21 locus (HSPB1). Genotype analysis of the 11q12–q14 locus (BSCL2) was performed with 13 STR markers: D11S1765, D11S4076, D11S480, D11S4205, D11S1883, D11S987, CA9, CA10 (Magre et al., 2001), WP1, WP3, WP7, WP8 and WP9 (Windpassinger et al., 2004). Paternity was tested using 15 highly informative STRs distributed throughout the genome (ATA38A05, D1S1646, D1S1653, D1S1360, D2S2256, D3S3037, D4S2382, D4S3240, D7S509, D8S1759, D9S1118, D12S1056, D12S2082, D16S2619 and GATA152H04). STRs were PCR-amplified and PCR fragments were resolved on an ABI 3730 automated DNA sequencer. Genotypes were analysed using the ABI Prism Genescan software (Applied Biosystems) and Trace Inspector, an in-house developed software program (http://www.vibgeneticservicefacility.be/).
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Results |
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TopSummaryIntroductionPatients and MethodsResultsDiscussionSupplementary materialReferences |
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In 17 out of 112 index patients, we found sequence variations causing pathogenic missense mutations in four distal HMN genes: BSCL2, HSPB8, HSPB1 and SETX (Table 1). These mutations were not detected in a panel of 400 control chromosomes. No mutations were found in DCTN1, GARS and VAPB. Clinical and electrophysiological data of the 17 index patients are summarized in Tables 1 and 2. The 17 index patients have an HMN phenotype with distal weakness and atrophy. In seven patients with a BSCL2 mutation, the weakness was more pronounced in the hands than legs. Age at onset was variable and upper motor neuron signs were observed in eight patients. In most patients, normal or slightly reduced motor NCVs were found with reduced compound muscle action potential (CMAP) amplitudes. Sensory NCVs were in the normal range or at the lower limit and sensory nerve action potentials (SNAP) had normal amplitudes. Two patients; CMT-705:II.1 and CMT-M:IV.12 had a slightly decreased sensory median NCV of 39.7 and 40.0 m/s respectively, due to a concomitant Carpal Tunnel Syndrome. Recent re-evaluation showed a reduced Sural nerve NCV of 36.2 m/s in CMT-263:IV.1, suggesting a transition of distal HMN to HMSN-II, although the sensory NCVs in the upper limbs remained normal (Table 2). The absence of sensory response in the Sural nerve in patient CMT-206:IV.1 may suggest a shift in diagnosis to HMSN-II as well, which was recently described in the context of BSCL2 mutations (Table 2) (Bienfait et al., 2007
). Although we do not have evidence for a superimposed acquired sensory-motor neuropathy, this observation, based on a single patient, should be interpreted with caution. Of interest is that several secondary patients in this family CMT-206 had a pure motor neuropathy phenotype (Irobi et al., 2004a).
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Mutations in BSCL2
The previously reported c.263A>G (Asn88Ser) and c.269C>T (Ser90Leu) mutations were found in probands of three and five distal HMN families, respectively (Table 1). The segregation of the Asn88Ser mutation in the family from Italy could be confirmed (Supplementary Fig. 1) (Irobi et al., 2004a
). In the family of Serbia, however, the segregation of the pathogenic Asn88Ser mutation could not be verified since the son CMT-644:IV.1 was clinically and electrophysiologically normal. This is possibly related to his relatively young age (27 years). In family CMT-745, no additional DNA samples of the relatives were available to confirm the segregation of the mutation. Comparison of the chromosome 11q haplotypes, defining the BSCL2 locus of families with the Asn88Ser mutation, i.e. CMT-206:IV.1 and CMT-644:IV.3, and of families CMT-I:III.14, CMT-705:II.1 and CMT-539:III.2 with the Ser90Leu mutation suggested that these patients are unlikely to be related (Fig. 1A). The Ser90Leu mutation was absent in the healthy parents and sister of patient CMT-734:II.1 (Supplementary Fig. 1). The paternity of this patient was confirmed (data not shown), pointing that the BSCL2-Ser90Leu mutation occurred de novo.
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Mutations in HSPB8
Two mutations targeting the same lysine residue in HSPB8 were identified in three index patients (Table 1). The same c.423G>C (Lys141Asn) mutation segregated in the unrelated Belgian and Czech families of patients CMT-M:IV.12 and CMT-196:VI.9, and the c.421A>G (Lys141Glu) mutation was found in the Bulgarian patient CMT-278:III.1 (Irobi et al., 2004b
). Segregation of this latter mutation was confirmed in the affected son of the index patient (Supplementary Fig. 1). The phenotype of family CMT-M has previously been described in detail (Timmerman et al., 1992, 1999).
Mutations in HSPB1
In this cohort, three different missense mutations in HSPB1 were found in four index patients (Table 1) (Evgrafov et al., 2004). The unrelated index patients PN-474:II.1 and CMT-751:III.1 belong to a Belgian and French family and share the same missense mutation, c.379C>T (Arg127Trp), targeting the highly conserved -crystallin domain of HSPB1 (Figs. 1B and 2). Segregation of the c.452C>T (Thr151Ile) mutation targeting the same -crystallin domain in the Croatian family CMT-263 could be confirmed in the index patient's mother, an uncle and a cousin (Supplementary Fig. 1). In the previously reported patient CMT-391:II.1, belonging to an Austrian family, a missense mutation (c.545C>T; Pro182Leu) was found in the C-terminal IXI/V domain (Fig. 2). The younger affected sib (CMT-391:II.3) showed a very similar disease course. Remarkably, none of the asymptomatic parents of CMT-391:II.1 did show the heterozygous HSPB1-mutation. False paternity in this family was excluded (data not shown). Interestingly, we detected very low levels of the mutant allele in the electropherogram of one of the parents (CMT-391:I.2) (Fig. 3A). The same low amount of mutant allele was also observed using pyrosequencing, suggesting that a fraction of this parent's lymphocytes was heterozygous for the mutation (Fig. 3B). By genotyping the five family members with six STRs at the HSPB1 locus, we could confirm the segregation of the mutant allele from the parent to the affected children (Fig. 3C). These results demonstrated that the parent CMT-391:I.2, who is clinically unaffected, is most likely mosaic for the Pro182Leu-mutation.
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Mutations in SETX
In two index patients, we identified a heterozygous missense mutation in SETX: in the Austrian patient CMT-106:III.4 we detected an N-terminal mutation, c.8C>T (Thr3Ile), and in the Belgian patient CMT-61:III.4 a missense mutation, c.6407G>A (Arg2136His), was found in the DNA/RNA helicase domain of the protein (Table 1, Fig. 2). The families to which the patients belong are described in De Jonghe et al. (2002
) and in Chen et al. (2004). |
Discussion |
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TopSummaryIntroductionPatients and MethodsResultsDiscussionSupplementary materialReferences |
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Over the last 6 years, eight genes have been identified in which mutations are associated with distal HMN. In this study, we investigated the relative contribution of mutations in the six genes associated with AD distal HMN: HSPB1, HSPB8, BSCL2, SETX, GARS, DCTN1 as well as mutations in VAPB in a cohort of 112 index distal HMN patients. In 17 index patients, we found disease-causing mutations in four genes, i.e. three patients had an HSPB8 mutation, four patients showed an HSPB1 mutation, BSCL2 mutations were found in eight probands and two families had a SETX mutation. The 17 mutations represent a mutation detection rate of 15% in the total cohort. The fact that no mutations were identified in 40 patients with a family history of distal HMN suggesting a dominant or autosomal dominant inheritance pattern indicates that additional genes must be involved in the pathogenesis of the disease. However, when we only focus on patients with a dominant inheritance the mutation yield increases to 30%. Moreover, if we exclusively consider patients with pyramidal tract signs the mutation rate rises to 38% (24% BSCL2, 9% SETX, 5% HSP27). This finding underscores the involvement of central motor tracts as a prominent aspect in several forms of distal HMN. Pyramidal tract signs were also observed in several other families with mutations in BSCL2 (Silver Syndrome) (Auer-Grumbach et al., 2005
).
Interestingly, the BSCL2 gene was found to be mutated in 7% of our distal HMN patient cohort suggesting that BSCL2 mutations are the most common causes of distal HMN. The major contribution of BSCL2 mutations to the aetiology of distal HMN was recently demonstrated in a cohort of 33 patients diagnosed with distal HMN, HMSN-II or Silver syndrome (Rohkamm et al., 2007) in which 12% of patients had a mutation. The slightly higher diagnostic yield of BSCL2 mutations found in that study may be explained by the selective inclusion of distal HMN patients with prominent hand muscle weakness. Intriguingly, only two different BSCL2 mutations have been reported so far. It is clear from our and previous studies that both mutations represent mutational hot-spots since they can arise independently in families and can occur de novo. A broad spectrum of phenotypes is associated with mutations in BSCL2 (Irobi et al., 2004a; Auer-Grumbach et al., 2005). The detailed clinical and electrophysiological observations within a single large Austrian family with the Asn88Ser mutation enabled Auer-Grumbach et al. (2005) to delineate six endophenotypes ranging from asymptomatic individuals to Silver syndrome and spastic paraplegia. A similar broad variability was seen in the clinical presentation of our patients with BSCL2 mutations. The disease onset varied from infancy in patients CMT-206:IV.1, CMT-716:I.2 (8 years) and CMT-734:II.1 (5 years) to late-onset in patient CMT-644:IV.3 (45 years), and the common presenting symptoms were atrophy and weakness of hand muscles and/or gait difficulties. The clinical presentation of the index patients with an Asn88Ser mutation consists of prominent distal muscle atrophy in the upper limbs and to a variable degree in the lower limbs without clear pyramidal tract signs. This is compatible with the distal HMN type V phenotype. The phenotype of four of the five index patients with a Ser90Leu mutation was dominated by prominent amyotrophy of the intrinsic hand muscles in association with marked spasticity in the lower limbs and the presence of additional pyramidal tract signs. This corresponds to the Silver syndrome phenotype. Based on these interfamilial phenotypic features, it seems that the BSCL2-Ser90Leu mutation is correlated with a pronounced spasticity in the lower limbs and additional upper motor neuron signs. This marked phenotypic variation between the two BSCL2 mutations has also been described in earlier reports (Silver 1966a, b; Auer-Grumbach et al., 2005; van de Warrenburg et al., 2006; Bienfait et al., 2007). Why distinct mutations in BSCL2 tend to result in different clinical outcomes remains unclear, moreover since both mutations target the same N-glycosylation motif of the seipin protein (Windpassinger et al., 2004).
Pyramidal tract signs and an early disease onset were also variably present in the two index patients (CMT-61:III.1 and CMT-106:III.1) with a SETX mutation, which underscores the involvement of the central nervous system in this distal HMN subtype. Currently, only three heterozygous mutations in SETX have been reported so far to be associated with motor neuron disease (Chen et al., 2004).
A similar phenotype was found in the patient with the C-terminal HSPB1-Pro182Leu mutation. The phenotype associated with this mutation consisted of foot deformity and gait abnormalities in early infancy (5 years) and the presence of brisk knee tendon reflexes. With disease progression the patient CMT-391:II.1 showed marked distal muscle weakness in upper and lower limbs. A mutation targeting the same C-terminal amino acid, Pro182Ser, was previously reported in a Japanese patient with distal HMN (Kijima et al., 2005). Similar to our patient, the disease in this patient started in early childhood; however, no upper motor neuron signs were reported. Mutations in the C-terminal part of HSPB1 seem to cause a more severe early-onset neuropathy than mutations in the conserved -crystallin domain of the HSPB1 protein (Arg127Trp and Thr151Ile) found in three unrelated index patients with a classic adult-onset distal motor neuropathy. The phenotype in these three patients clearly resembles distal HMN type II.
The same homogeneous juvenile to adult-onset distal HMN type II phenotype was found in the three patients with an HSPB8-mutation targeting an identical lysine residue. The HSPB8-Lys141Asn and the HSPB1-Arg127Trp mutations were also reported in Chinese families presenting mild sensory symptoms that were diagnosed with HMSN-II (Tang et al., 2005b). It is likely that the disease in these HMSN-II families represents a phenotypic variant of the clinical continuum associated with -crystallin mutations and that a diagnosis of HMSN-II rather than distal HMN was prompted by the presence of more pronounced sensory symptoms. Except for an impaired vibration sense in some elderly patients, no sensory losses were noted in the studied patients with an HSPB8 mutation. However, the presence of mild sensory deficits during a more recent re-evaluation in patient CMT-263:IV.1 (with the Thr151Ile mutation in HSPB1) illustrates the clinical overlap between distal HMN and HMSN-II (Table 2).
Another peculiar finding in this study was the evidence of a parental mosaicism transmitting the HSPB1-Pro182Leu mutation. A possible mosaicism was suspected since the affected proband's sib carried the same mutation, while the unaffected parents were negative for the mutation on standard sequencing. The absence of the disease in the mosaic parent is likely the result of the very low amount of mutant protein in the peripheral nervous system. The occurrence of gonadal mosaicism was also considered, but not examined, for a ‘de novo’ BSCL2 mutation in two sibs of a Korean family with Silver syndrome and distal HMN (Cho et al., 2007). These findings prove for the first time the occurrence of genetic mosaicism in distal HMN. The possibility of mosaicism even in sporadic distal HMN patients has important implications for genetic counselling.
We did not detect disease-causing mutations in GARS, which was somewhat unexpected since recently within a cohort of 100 patients presenting inherited or sporadic lower motor neuron degeneration, three different mutations were found in one distal HMN type V family and two families presenting distal HMN with lower limb predominance (James et al., 2006).
No mutations in DCTN1 were detected. Until now, only one mutation in DCTN1 is described in a family with distal spinal and bulbar muscular atrophy (Puls et al., 2003, 2005). No patients with a similar phenotype were included in the cohort, probably explaining the absence of DCTN1 mutations and indicating that mutations in DCTN1 are associated with a distinct and unique form of motor neuron disease.
In this study, we also included the analysis of VAPB to investigate whether the phenotype associated with the Pro56Ser mutation in VAPB, found in several Brazilian families with diverse forms of motor neuron disorders, could be expanded to distal HMN. We did not detect the Pro56Ser or other VAPB mutations in the cohort which mainly consisted of European patients, suggesting that mutations in this gene are probably not associated with distal HMN.
In summary, we examined the distribution of mutations in genes associated with AD distal HMN in a large group of familial and sporadic patients. Our data highlight the genetic and clinical heterogeneity of the disease. The mutation rate in the studied genes was relatively low in the total cohort, suggesting that other genes must be involved in the pathomechanism of motor neuropathies. We suggest that genetic testing of patients showing prominent distal muscle atrophy in the upper limbs with a variable degree of upper motor neuron signs should start with BSCL2, since the yield of mutations in BSCL2 within our distal HMN cohort is 7%. Overall, the clinical phenotype of patients with a known gene defect fit in the proposed distal HMN classification by Irobi (2004), which is based on the initial clinical distal HMN classification system of Harding (1993) (Irobi et al., 2006). This study underscores the involvement of central motor tracts in distal HMN associated with mutations in BSCL2, SETX as well as HSPB1. In addition, we provide evidence for a genetic mosaicism in transmitting an HSPB1 mutation. In genetic counselling, it might be important to consider parental mosaicism even in sporadic distal HMN patients. Additional descriptions of distal HMN families and patients with a known genetic defect are needed to further organize and refine the existing classification and to get more insight into the molecular basis of these disorders.
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Supplementary material |
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TopSummaryIntroductionPatients and MethodsResultsDiscussionSupplementary materialReferences |
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Supplementary material is available at Brain online.
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Footnotes |
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*These authors contributed equally to this work.
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Acknowledgements |
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We are grateful to the patients and their families for their cooperation in our research project. We also wish to thank E. Claesen and S. Felix for technical assistance and the Genetic Service Facility (VIB) for the sequencing support (http://www.vibgeneticservicefacility.be/). We thank Dr C. Windpassinger for providing us with primer sequences of CA9, CA10 and WP1-9. We thank Dr K.G. Claeys for assisting in the collection of clinical data. The study was supported by the University of Antwerp, the Fund for Scientific Research (FWO-Flanders), the American Muscular Dystrophy Association (MDA), the Medical Foundation Queen Elisabeth (GSKE), the ‘Association Belge contre les Maladies Neuromusculaires’ (ABMM), and the Interuniversity Attraction Poles P6/43 program of the Belgian Federal Science Policy Office (BELSPO). I.D. and J.B. are supported by PhD fellowships of the Institute for Science and Technology (IWT) and FWO-Flanders, respectively. J.I. is a postdoctoral researcher of the FWO-Flanders. PS and RM were supported by grant IGA NR/9517-3 and VZ 00064203/6506.
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References |
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TopSummaryIntroductionPatients and MethodsResultsDiscussionSupplementary materialReferences |
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