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A novel 9-bp insertion in the GJB1 gene causing a mild form of X-linked CMT with late onset
Neuromuscular Disorders 16 (2006) 878–881 www.elsevier.com/locate/nmd
Case report
A novel 9-bp insertion in the GJB1 gene causing a mild form of X-linked CMT with late onset G. Vazza a, L. Merlini b, C. Bertolin a, M. Zortea a, M.L. Mostacciuolo b
a,*
a Department of Biology, University of Padova, Italy Neuromuscular Unit, Division of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy
Received 8 May 2006; received in revised form 26 July 2006; accepted 6 September 2006
Abstract X-linked Charcot-Marie-Tooth disease is the second most common variant of CMT. CMTX1 is caused by mutations in the GJB1 gene encoding for connexin 32. We describe an Italian family with an intermediate CMTX phenotype with late onset. Mutation screening of the GJB1 gene revealed a 9-bp duplication leading to the insertion of three aminoacids (Thr-Val-Phe) between the end of the second extracellular domain and the beginning of the fourth transmembrane domain. This is the third in-frame insertion in the GJB1 gene identified so far and, like the previous ones, it consists in the duplication of the flanking sequence which is repeated in tandem in the wild-type gene. Ó 2006 Elsevier B.V. All rights reserved. Keywords: CMT; Mutation screening; GJB1 (connexin 32)
1. Introduction Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of peripheral neuropathies with an overall prevalence of 1/2500 [1]. Clinical manifestations of CMT include a progressive sensory-motor neuropathy, weakness and atrophy of the distal muscles in the feet and/or hands, sensory impairment, hyporeflexia and foot deformity. On the basis of motor nerve conduction velocity (MCV), CMT is classified into the two major forms: demyelinating or type 1 (median MCV < 38 m/s) and axonal or type 2 (median MCV > 38 m/s) [2]. CMT is genetically heterogeneous and, to date, several chromosomal loci for autosomal dominant, autosomal recessive and X-linked forms have been reported [3]. * Corresponding author. Tel.: +39 049 827 6213; fax: +39 049 827 6209. E-mail address: [email protected] (M.L. Mostacciuolo).
0960-8966/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2006.09.002
X-linked CMT is the second most frequent variant of CMT. CMTX1 locus (OMIM 302800) on chromosome Xq13.1 accounts for about 90% of the X-linked cases and at least 10% of all CMT cases [4]. CMTX1 is caused by mutations in the GJB1 gene, which encodes for connexin 32 (Cx32), an integral transmembrane protein expressed in several tissues, especially in the peripheral nervous system (Schwann’s cells) [5]. In the peripheral nerves, Cx32 is located in the paranodal regions and in the Schmidt-Lantermann incisures, in which six Cx32 subunits constitute a transmembrane hemichannel (connexon). The association of connexons with those of other cells form the functional channels of gap junctions [6]. Until now, over 280 different mutations responsible for CMTX1 have been detected in GJB1 (www.molgen.ua.ac.be/CMTMutations/). Missense mutations are the most common, whereas only two in-frame insertions/duplications have been described so far. Clinical CMTX1 manifestations are highly variable, even in affected individuals of the same family.
G. Vazza et al. / Neuromuscular Disorders 16 (2006) 878–881
However, affected males usually show a progressive motor and sensory neuropathy with variable electrophysiological changes and a MCV which suggests either demyelinating neuropathy (MCV < 38 m/s) or mixed forms (MCV 30–40 m/s) [7]. Onset varies between 5 and 25 years of age, but in males the first symptoms commonly develop within the first decade of life. Here, we present a new GJB1 in-frame insertion/duplication found in an Italian family, leading to an intermediate CMT phenotype with late onset. 2. Case report A 42-year-old male from the center-south of Italy was clinically evaluated for CMT by one of our team (L.M.). At the age of 39, the subject first noticed atrophy of the thumb abductor and, shortly afterwards, leg muscle atrophy. At presentation, he had hand and leg muscle atrophy, bilateral pes cavus, areflexia and apallesthesia. In addition, the subject’s hand showed complete atrophy of the thenar muscles, and mild atrophy of the dorsal interossei and hypothenar muscles. Median and ulnar MCV were 36 and 37 m/s, respectively, distal latencies were at the upper limit of normality, while the M amplitudes were markedly reduced, particularly in the abductor pollicis brevis muscle (0.3 mV). Median SNCV was 36 with a SAP of 1.4 uV. Sural SAP was absent. At the time of the first neurological evaluation, no other relatives were reported as affected. A more detailed counseling highlighted the presence of other affected relatives, both amongst siblings and across different generations (Fig. 1). A subsequent clinical evaluation showed a similar mild CMT phenotype in the elder brother. He first
879
noticed foot deformity at the age of 22 and hand weakness at the age of 40. The hand muscles were atrophic with the thenar compartment more affected than the hypothenar, leg atrophy and pes cavus were also present. The achilles tendon reflexes were absent. There was a discrete sensory loss in the legs with apallesthesia. Median MCV was 31 m/s, distal latency was 5.9 ms (normal < 4.0 ms), and the M amplitude 1.3 mV. Peroneal MCV was 28 m/s with a distal latency of 8 ms and M amplitude of 2.3 mV. Sural SAP was absent. Their sister, aged 45, had not noticed gait difficulties, but on clinical examination, she showed mild cavus of the feet without detectable muscle wasting or weakness in the limbs. Median MCV was 38 m/s, distal latency 4.1 ms, and M amplitude 8 mV. Median SCV in the arm was 50 m/s with a SAP of 16 uV. Sural SAP was 3 uV. The family tree suggests that the disease segregates as a typical X-linked trait, since two cousins on the proband’s maternal side (III-1 and III-2), aged 69 and 70 years, respectively, showed a CMT phenotype with late onset (after the age of 35) similar to that of the proband. No clinical data were available for the obligate carriers II-2 and III-4 who died at the age of 70 and 64, respectively. 3. Molecular analysis After obtaining signed informed consent, blood samples were collected from the proband (IV-3), his brother (IV-1), and from his sister (IV-2). Total DNA was extracted from leucocytes by a standard ‘‘salting out’’ method. The 1.5 Mb CMT1A duplication on chromosome 17p11.2 was first excluded using restriction enzymes [8]
Fig. 1. Pedigree of the family with CMTX. The arrow indicates the proband (IV-3), the solid square, affected subjects; the circle with a spot, obligated carriers; the grey circle, carriers with positive EMG; the cross-hatched symbol, deceased subjects.
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G. Vazza et al. / Neuromuscular Disorders 16 (2006) 878–881
and three microsatellite markers (RM11GT, D17S1358, D17S921). The GJB1 gene was then analyzed by PCR amplification and direct sequencing of coding exon 2 using an ABI 3100 DNA sequencer (Applied Biosystems). Sequence analysis in the proband revealed a 9-bp insertion at nucleotide position 580 from the translation start site (RefSeq NM_000166). Considering the flanking sequences, the mutation can be best described as an in-frame duplication (c.571_579dupACCGT CTTC) of one of the two 9-bp direct repeats which are located before the duplicated sequence (Fig. 2A). At the protein level this mutation results in the duplication of three residues (p.Thr191_Phe193dup), which, as in the nucleotide sequence, is a double repeat of the amino acid motif (Thr-Val-Phe). The same GJB1 inframe duplication was identified in the proband’s brother (IV-1), and, in the heterozygous state, in the proband’s sister (IV-2). The mutation was confirmed by SSCP analysis, which showed an abnormal migration pattern for both the patients when compared to control samples (Fig. 2B). The same analysis was then performed on 200 healthy subjects (100 males and 100 females) ethnically matched in order to rule out the possibility of a polymorphism. No altered migration pattern was observed in the 300 control X chromosomes analyzed.
Fig. 2. Sequence analysis of GJB1 gene. (A) Electropherogram of the GJB1 exon 2 showing the 9-bp duplication in the proband. Open boxes indicate the wild-type nucleotide and amino acid tandem repeat, the grey box indicates the duplicated sequence. (B) SSCP analysis. PCR products were separated on a 8% acrylamide (29:1 acrylamide/ bisacrylamide) gel and then silver stained. C, control; IV-1 and IV-3, affected subjects.
4. Discussion In this study we describe a case of CMTX caused by an in-frame 9-bp duplication in the GJB1 gene. GJB1 insertions/duplications are rare and only 10 insertiontype mutations, accounting for about 3% of all GJB1 mutations, have been described to date (www.molgen.ua.ac.be/CMTMutations/). Of these mutations, 8 are single nucleotide insertions, causing frame-shift and/or anticipated stop codons, and only 2 are in-frame insertions which determine the inclusion of 2 and 7 new amino acids respectively. Interestingly, a finer analysis of both in-frame insertions revealed that they consist of wild-type sequence duplications involving short direct or tandem repeats. In particular the former (c.94_95insTCTTCA) [9] is the duplication of a 6 nucleotide flanking tandem repeat whereas the latter (c.184_185insCACTCCAGCCTG GCTGCAACA) involves a 21 nucleotide segment flanked by two direct repeats [10]. The mutation reported here is the third in-frame insertion in the GJB1 gene identified so far and, like the previous ones, it consists in the duplication of a 9bp flanking sequence which is repeated twice in tandem in the wild-type gene. Considering these data, it is reasonable to hypothesize that all these mutations originated through a molecular mechanism involving direct interaction between these repeats, such as unequal meiotic crossing-over (UCO). UCO consists of a non-reciprocal exchange of genetic material to produce duplications and deletions with the same frequency. In the light of these considerations, it is interesting to note that the reciprocal deletion of the same sequence, which is duplicated in our family, has already been reported among GJB1 mutations (c.572_580delCCGTCTTCA), thus supporting the hypothesis that UCO could be the molecular mechanism involved in the generation of such mutations [6]. The comparison of the CMTX phenotypes caused by the duplication and the deletion of the same 9-bp motif would be of great interest, but unfortunately no clinical data are provided in the original paper reporting the 9bp deletion [6]. The c.571_579dupACCGTCTTC duplication leads to an insertion of three amino acids (Thr-Val-Phe) in the linker region between the second extracellular domain and the fourth transmembrane domain of the Cx32 protein. This duplication produces a weak perturbation of the Cx32 hydrophobic profile, as predicted by the in silico analysis performed using SPLIT4 (http:// split.pmfst.hr/split/4/) and TMHMM (http://www. cbs.dtu.dk/services/TMHMM/) software. We suggest that this mutation probably does not significantly compromise the overall structure of Cx32, which should not, therefore, show major functional alterations. This hypothesis would agree with the CMTX phenotype
G. Vazza et al. / Neuromuscular Disorders 16 (2006) 878–881
observed in this family which is characterized by milder neurological manifestations and a late onset as compared to the typical clinical descriptions currently available for CMTX.
Acknowledgements We thank all the members of the family for participating in this study. We are grateful to Prof. M. Zordan for critical help in manuscript editing.
References [1] Vance JM. Hereditary motor and sensory neuropathies. J Med Genet 1991;28(1):1–5. [2] Harding AE, Thomas PK. The clinical features of hereditary motor and sensory neuropathy types I and II. Brain 1980;103(2):259–80. [3] Berger P, Young P, Suter U. Molecular cell biology of CharcotMarie-Tooth disease. Neurogenetics 2002;4(1):1–15.
881
[4] Ionasescu VV. Charcot-Marie-Tooth neuropathies: from clinical description to molecular genetics. Muscle Nerve 1995;18(3):267–75. [5] Bergoffen J, Scherer SS, Wang S, et al. Connexin mutations in Xlinked Charcot-Marie-Tooth disease. Science 1993;262(5142):2039–42. [6] Bone LJ, Deschenes SM, Balice-Gordon RJ, Fischbeck KH, Scherer SS. Connexin32 and X-linked Charcot-Marie-Tooth disease. Neurobiol Dis 1997;4(3-4):221–30. [7] Nicholson G, Nash J. Intermediate nerve conduction velocities define X-linked Charcot-Marie-Tooth neuropathy families. Neurology 1993;43(12):2558–64. [8] Stronach EA, Clark C, Bell C, et al. Novel PCR-based diagnostic tools for Charcot-Marie-Tooth type 1A and hereditary neuropathy with liability to pressure palsies. J Peripher Nerv Syst 1999;4(2):117–22. [9] Haites NE, Nelis E, Van Broeckhoven C. 3rd workshop of the European CMT consortium: 54th ENMC International Workshop on genotype/phenotype correlations in Charcot-MarieTooth type 1 and hereditary neuropathy with liability to pressure palsies 28–30 November 1997, Naarden, The Netherlands. Neuromuscul Disord 1998;8(8):591–603. [10] Kawakami H, Inoue K, Sakakihara I, Nakamura S. Novel mutation in X-linked Charcot-Marie-Tooth disease associated with CNS impairment. Neurology 2002;59(6):923–6.
Case report
A novel 9-bp insertion in the GJB1 gene causing a mild form of X-linked CMT with late onset G. Vazza a, L. Merlini b, C. Bertolin a, M. Zortea a, M.L. Mostacciuolo b
a,*
a Department of Biology, University of Padova, Italy Neuromuscular Unit, Division of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy
Received 8 May 2006; received in revised form 26 July 2006; accepted 6 September 2006
Abstract X-linked Charcot-Marie-Tooth disease is the second most common variant of CMT. CMTX1 is caused by mutations in the GJB1 gene encoding for connexin 32. We describe an Italian family with an intermediate CMTX phenotype with late onset. Mutation screening of the GJB1 gene revealed a 9-bp duplication leading to the insertion of three aminoacids (Thr-Val-Phe) between the end of the second extracellular domain and the beginning of the fourth transmembrane domain. This is the third in-frame insertion in the GJB1 gene identified so far and, like the previous ones, it consists in the duplication of the flanking sequence which is repeated in tandem in the wild-type gene. Ó 2006 Elsevier B.V. All rights reserved. Keywords: CMT; Mutation screening; GJB1 (connexin 32)
1. Introduction Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of peripheral neuropathies with an overall prevalence of 1/2500 [1]. Clinical manifestations of CMT include a progressive sensory-motor neuropathy, weakness and atrophy of the distal muscles in the feet and/or hands, sensory impairment, hyporeflexia and foot deformity. On the basis of motor nerve conduction velocity (MCV), CMT is classified into the two major forms: demyelinating or type 1 (median MCV < 38 m/s) and axonal or type 2 (median MCV > 38 m/s) [2]. CMT is genetically heterogeneous and, to date, several chromosomal loci for autosomal dominant, autosomal recessive and X-linked forms have been reported [3]. * Corresponding author. Tel.: +39 049 827 6213; fax: +39 049 827 6209. E-mail address: [email protected] (M.L. Mostacciuolo).
0960-8966/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2006.09.002
X-linked CMT is the second most frequent variant of CMT. CMTX1 locus (OMIM 302800) on chromosome Xq13.1 accounts for about 90% of the X-linked cases and at least 10% of all CMT cases [4]. CMTX1 is caused by mutations in the GJB1 gene, which encodes for connexin 32 (Cx32), an integral transmembrane protein expressed in several tissues, especially in the peripheral nervous system (Schwann’s cells) [5]. In the peripheral nerves, Cx32 is located in the paranodal regions and in the Schmidt-Lantermann incisures, in which six Cx32 subunits constitute a transmembrane hemichannel (connexon). The association of connexons with those of other cells form the functional channels of gap junctions [6]. Until now, over 280 different mutations responsible for CMTX1 have been detected in GJB1 (www.molgen.ua.ac.be/CMTMutations/). Missense mutations are the most common, whereas only two in-frame insertions/duplications have been described so far. Clinical CMTX1 manifestations are highly variable, even in affected individuals of the same family.
G. Vazza et al. / Neuromuscular Disorders 16 (2006) 878–881
However, affected males usually show a progressive motor and sensory neuropathy with variable electrophysiological changes and a MCV which suggests either demyelinating neuropathy (MCV < 38 m/s) or mixed forms (MCV 30–40 m/s) [7]. Onset varies between 5 and 25 years of age, but in males the first symptoms commonly develop within the first decade of life. Here, we present a new GJB1 in-frame insertion/duplication found in an Italian family, leading to an intermediate CMT phenotype with late onset. 2. Case report A 42-year-old male from the center-south of Italy was clinically evaluated for CMT by one of our team (L.M.). At the age of 39, the subject first noticed atrophy of the thumb abductor and, shortly afterwards, leg muscle atrophy. At presentation, he had hand and leg muscle atrophy, bilateral pes cavus, areflexia and apallesthesia. In addition, the subject’s hand showed complete atrophy of the thenar muscles, and mild atrophy of the dorsal interossei and hypothenar muscles. Median and ulnar MCV were 36 and 37 m/s, respectively, distal latencies were at the upper limit of normality, while the M amplitudes were markedly reduced, particularly in the abductor pollicis brevis muscle (0.3 mV). Median SNCV was 36 with a SAP of 1.4 uV. Sural SAP was absent. At the time of the first neurological evaluation, no other relatives were reported as affected. A more detailed counseling highlighted the presence of other affected relatives, both amongst siblings and across different generations (Fig. 1). A subsequent clinical evaluation showed a similar mild CMT phenotype in the elder brother. He first
879
noticed foot deformity at the age of 22 and hand weakness at the age of 40. The hand muscles were atrophic with the thenar compartment more affected than the hypothenar, leg atrophy and pes cavus were also present. The achilles tendon reflexes were absent. There was a discrete sensory loss in the legs with apallesthesia. Median MCV was 31 m/s, distal latency was 5.9 ms (normal < 4.0 ms), and the M amplitude 1.3 mV. Peroneal MCV was 28 m/s with a distal latency of 8 ms and M amplitude of 2.3 mV. Sural SAP was absent. Their sister, aged 45, had not noticed gait difficulties, but on clinical examination, she showed mild cavus of the feet without detectable muscle wasting or weakness in the limbs. Median MCV was 38 m/s, distal latency 4.1 ms, and M amplitude 8 mV. Median SCV in the arm was 50 m/s with a SAP of 16 uV. Sural SAP was 3 uV. The family tree suggests that the disease segregates as a typical X-linked trait, since two cousins on the proband’s maternal side (III-1 and III-2), aged 69 and 70 years, respectively, showed a CMT phenotype with late onset (after the age of 35) similar to that of the proband. No clinical data were available for the obligate carriers II-2 and III-4 who died at the age of 70 and 64, respectively. 3. Molecular analysis After obtaining signed informed consent, blood samples were collected from the proband (IV-3), his brother (IV-1), and from his sister (IV-2). Total DNA was extracted from leucocytes by a standard ‘‘salting out’’ method. The 1.5 Mb CMT1A duplication on chromosome 17p11.2 was first excluded using restriction enzymes [8]
Fig. 1. Pedigree of the family with CMTX. The arrow indicates the proband (IV-3), the solid square, affected subjects; the circle with a spot, obligated carriers; the grey circle, carriers with positive EMG; the cross-hatched symbol, deceased subjects.
880
G. Vazza et al. / Neuromuscular Disorders 16 (2006) 878–881
and three microsatellite markers (RM11GT, D17S1358, D17S921). The GJB1 gene was then analyzed by PCR amplification and direct sequencing of coding exon 2 using an ABI 3100 DNA sequencer (Applied Biosystems). Sequence analysis in the proband revealed a 9-bp insertion at nucleotide position 580 from the translation start site (RefSeq NM_000166). Considering the flanking sequences, the mutation can be best described as an in-frame duplication (c.571_579dupACCGT CTTC) of one of the two 9-bp direct repeats which are located before the duplicated sequence (Fig. 2A). At the protein level this mutation results in the duplication of three residues (p.Thr191_Phe193dup), which, as in the nucleotide sequence, is a double repeat of the amino acid motif (Thr-Val-Phe). The same GJB1 inframe duplication was identified in the proband’s brother (IV-1), and, in the heterozygous state, in the proband’s sister (IV-2). The mutation was confirmed by SSCP analysis, which showed an abnormal migration pattern for both the patients when compared to control samples (Fig. 2B). The same analysis was then performed on 200 healthy subjects (100 males and 100 females) ethnically matched in order to rule out the possibility of a polymorphism. No altered migration pattern was observed in the 300 control X chromosomes analyzed.
Fig. 2. Sequence analysis of GJB1 gene. (A) Electropherogram of the GJB1 exon 2 showing the 9-bp duplication in the proband. Open boxes indicate the wild-type nucleotide and amino acid tandem repeat, the grey box indicates the duplicated sequence. (B) SSCP analysis. PCR products were separated on a 8% acrylamide (29:1 acrylamide/ bisacrylamide) gel and then silver stained. C, control; IV-1 and IV-3, affected subjects.
4. Discussion In this study we describe a case of CMTX caused by an in-frame 9-bp duplication in the GJB1 gene. GJB1 insertions/duplications are rare and only 10 insertiontype mutations, accounting for about 3% of all GJB1 mutations, have been described to date (www.molgen.ua.ac.be/CMTMutations/). Of these mutations, 8 are single nucleotide insertions, causing frame-shift and/or anticipated stop codons, and only 2 are in-frame insertions which determine the inclusion of 2 and 7 new amino acids respectively. Interestingly, a finer analysis of both in-frame insertions revealed that they consist of wild-type sequence duplications involving short direct or tandem repeats. In particular the former (c.94_95insTCTTCA) [9] is the duplication of a 6 nucleotide flanking tandem repeat whereas the latter (c.184_185insCACTCCAGCCTG GCTGCAACA) involves a 21 nucleotide segment flanked by two direct repeats [10]. The mutation reported here is the third in-frame insertion in the GJB1 gene identified so far and, like the previous ones, it consists in the duplication of a 9bp flanking sequence which is repeated twice in tandem in the wild-type gene. Considering these data, it is reasonable to hypothesize that all these mutations originated through a molecular mechanism involving direct interaction between these repeats, such as unequal meiotic crossing-over (UCO). UCO consists of a non-reciprocal exchange of genetic material to produce duplications and deletions with the same frequency. In the light of these considerations, it is interesting to note that the reciprocal deletion of the same sequence, which is duplicated in our family, has already been reported among GJB1 mutations (c.572_580delCCGTCTTCA), thus supporting the hypothesis that UCO could be the molecular mechanism involved in the generation of such mutations [6]. The comparison of the CMTX phenotypes caused by the duplication and the deletion of the same 9-bp motif would be of great interest, but unfortunately no clinical data are provided in the original paper reporting the 9bp deletion [6]. The c.571_579dupACCGTCTTC duplication leads to an insertion of three amino acids (Thr-Val-Phe) in the linker region between the second extracellular domain and the fourth transmembrane domain of the Cx32 protein. This duplication produces a weak perturbation of the Cx32 hydrophobic profile, as predicted by the in silico analysis performed using SPLIT4 (http:// split.pmfst.hr/split/4/) and TMHMM (http://www. cbs.dtu.dk/services/TMHMM/) software. We suggest that this mutation probably does not significantly compromise the overall structure of Cx32, which should not, therefore, show major functional alterations. This hypothesis would agree with the CMTX phenotype
G. Vazza et al. / Neuromuscular Disorders 16 (2006) 878–881
observed in this family which is characterized by milder neurological manifestations and a late onset as compared to the typical clinical descriptions currently available for CMTX.
Acknowledgements We thank all the members of the family for participating in this study. We are grateful to Prof. M. Zordan for critical help in manuscript editing.
References [1] Vance JM. Hereditary motor and sensory neuropathies. J Med Genet 1991;28(1):1–5. [2] Harding AE, Thomas PK. The clinical features of hereditary motor and sensory neuropathy types I and II. Brain 1980;103(2):259–80. [3] Berger P, Young P, Suter U. Molecular cell biology of CharcotMarie-Tooth disease. Neurogenetics 2002;4(1):1–15.
881
[4] Ionasescu VV. Charcot-Marie-Tooth neuropathies: from clinical description to molecular genetics. Muscle Nerve 1995;18(3):267–75. [5] Bergoffen J, Scherer SS, Wang S, et al. Connexin mutations in Xlinked Charcot-Marie-Tooth disease. Science 1993;262(5142):2039–42. [6] Bone LJ, Deschenes SM, Balice-Gordon RJ, Fischbeck KH, Scherer SS. Connexin32 and X-linked Charcot-Marie-Tooth disease. Neurobiol Dis 1997;4(3-4):221–30. [7] Nicholson G, Nash J. Intermediate nerve conduction velocities define X-linked Charcot-Marie-Tooth neuropathy families. Neurology 1993;43(12):2558–64. [8] Stronach EA, Clark C, Bell C, et al. Novel PCR-based diagnostic tools for Charcot-Marie-Tooth type 1A and hereditary neuropathy with liability to pressure palsies. J Peripher Nerv Syst 1999;4(2):117–22. [9] Haites NE, Nelis E, Van Broeckhoven C. 3rd workshop of the European CMT consortium: 54th ENMC International Workshop on genotype/phenotype correlations in Charcot-MarieTooth type 1 and hereditary neuropathy with liability to pressure palsies 28–30 November 1997, Naarden, The Netherlands. Neuromuscul Disord 1998;8(8):591–603. [10] Kawakami H, Inoue K, Sakakihara I, Nakamura S. Novel mutation in X-linked Charcot-Marie-Tooth disease associated with CNS impairment. Neurology 2002;59(6):923–6.