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Original Contribution |

Clinical and Electrophysiological Features in Charcot-Marie-Tooth Disease With Mutations in the NEFL Gene FREE

Gabriel Miltenberger-Miltenyi, MD; Andreas R. Janecke, MD; Julia V. Wanschitz, MD; Vincent Timmerman, PhD; Christian Windpassinger, PhD; Michaela Auer-Grumbach, MD, PhD; Wolfgang N. Löscher, MD, PhD
[+] Author Affiliations

Author Affiliations: Section of Clinical Genetics (Drs Miltenberger-Miltenyi and Janecke) and Clinical Department of Neurology (Drs Wanschitz and Löscher), Innsbruck Medical University, Innsbruck, Austria; Peripheral Neuropathy Group, Molecular Genetics Department, Flanders Interuniversity Institute for Biotechnology, Institute Born Bunge, University of Antwerp, Antwerp, Belgium (Dr Timmerman); Neurogenetics Section, Centre for Addiction and Mental Health, Toronto, Ontario (Dr Windpassinger); and Institute of Medical Biology and Human Genetics, Medical-University Graz, Graz, Austria (Drs Windpassinger and Auer-Grumbach).


Arch Neurol. 2007;64(7):966-970. doi:10.1001/archneur.64.7.966.
Text Size: A A A
Published online

Background  To date, 13 different neurofilament light-chain polypeptide gene (NEFL) mutations have been identified in 55 patients with Charcot-Marie-Tooth disease (CMT) from 16 families. NEFL mutations were found to be associated with axonal and demyelinating variants of CMT.

Objectives  To describe the clinical features of 11 patients with CMT and NEFL mutations and to explore possible genotype-phenotype correlations.

Design  Standardized neuromuscular and nerve conduction studies were performed, and the coding regions of the peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ), gap junction β-1 protein (GJB1), and NEFL genes were analyzed by direct DNA sequencing.

Setting  Two university hospitals in Austria (referral centers for neuromuscular disorders).

Patients  Eleven patients with CMT and NEFL mutations.

Main Outcome Measure  We genotyped NEFL in all of the patients and healthy relatives and correlated the genotype with the phenotype.

Results  A novel NEFL mutation (p.L93P) was detected in 1 family with 4 affected individuals exhibiting a severe CMT phenotype. Nerve conduction velocities were intermediately slowed to a range of 35 to 39 m/s. In a second family and in a sporadic patient, a p.P8R mutation was identified with intermediate and severe nerve conduction slowing.

Conclusion  The results argue against an obvious genotype-phenotype correlation regarding disease onset, degree of muscle weakness, and nerve conduction slowing caused by NEFL mutations.

Figures in this Article

Charcot-Marie-Tooth disease (CMT) (Online Mendelian Inheritance in Man #118220), also called hereditary motor and sensory neuropathy, has been subdivided into 2 main groups based on the underlying pathological findings.15 The demyelinating variant, CMT1, is electrophysiologically characterized by reduced motor nerve conduction velocities (NCVs) of the median nerve (NCV < 38 m/s) as well as segmental demyelination, segmental remyelination, and hyperplasia of Schwann cells causing onion bulb formations on histopathological examination. In the axonal variant, CMT2, NCVs are almost normal, but amplitudes of compound motor evoked potentials are reduced and nerve pathological findings show axonal loss and regenerative sprouting.The additional classification of intermediate CMT has been suggested in cases with motor NCVs between 25 and 45 m/s.6,7

Charcot-Marie-Tooth disease is genetically very heterogeneous, and mutations in 36 genes have been described so far (Inherited Peripheral Neuropathies Mutation Database, http://www.molgen.ua.ac.be/CMTMutations/). A large study of 323 patients with CMT by Jordanova et al8 found neurofilament light-chain polypeptide gene (NEFL) mutations in 2% of the cases. Thirteen disease-causing NEFL mutations and clinical features of 55 patients have been described, providing a limited set of data for genotype-phenotype analysis.817

Here we describe the results of clinical, electrophysiological, and genetic studies in 2 Austrian families and 1 single patient exhibiting a severe clinical CMT phenotype and a novel NEFL mutation.

PATIENTS

Two Austrian families (family 1 and family 2) and 1 sporadic patient with classical CMT were studied. Neurological examinations and standard nerve conduction studies were performed in 9 individuals (4 affected and 5 at risk) of family 1, in 10 individuals (6 affected and 4 at risk) of family 2, and in the sporadic patient. Written informed consent was obtained from all of the participants according to the Declaration of Helsinki, and the study was approved by the local ethics committee.

MUTATION ANALYSIS

Participants' DNA was extracted from peripheral blood samples using an automated extractor according to the manufacturer's protocols (GenoM 48; Qiagen, Vienna, Austria). Mutations in the most common genes (peripheral myelin protein 22 gene [PMP22] duplication and deletion, PMP22 point mutations, and mutations in myelin protein zero [MPZ] and gap junction β-1 protein [GJB1] genes) were excluded first. Intronic primers were designed to flank each of the 4 NEFL (GenBank accession number NM_006158) exons encoding 542 amino acids. Each polymerase chain reaction fragment was bidirectionally sequenced in each index case (ABI 3100 DNA sequencer with BigDye terminator mix and SeqScape Software version 2.0; Applied Biosystems, Vienna, Austria). Eight further individuals from family 1 and 9 individuals from family 2 were investigated for the presence of the respective sequence change identified in the index case. Sequencing of these 2 positions was performed in 200 normal chromosomes derived from a reference set of ethnically matched individuals.

CLINICAL FEATURES

In family 1, 56 individuals were identified (Figure 1). In all of the affected individuals, disease onset was during the second decade of life related to plantar extensor weakness and foot deformity, ie, pes cavus, that resulted in ankle arthrodesis in 2 patients at ages 14 (V:10) and 17 (IV:18) years. Distal lower limb atrophy and weakness progressed over years and involved distal upper limbs at older ages. Two patients became wheelchair bound. Sensory loss predominantly affected the lower limbs, and large fiber function and tendon reflexes were absent in the lower limbs (Table 1).

Place holder to copy figure label and caption
Figure 1.

Pedigree of family 1. Circles indicate females; squares, males; filled symbols, affected individuals; open symbols, unaffected individuals; slash, deceased; L/w, heterozygous p.L93P mutation; and w/w, absence of mutation (w indicates wild-type allele).

Graphic Jump Location
Table Graphic Jump LocationTable 1. Clinical and Electrophysiological Features of Patients With Charcot-Marie-Tooth Disease Investigated in This Study

In family 2, disease onset was before age 15 years in all of the patients except for 1 in whom gait problems were not reported until age 25 years. Symptoms were slowly progressive, resulting in a severe and disabling CMT phenotype. Interestingly, the oldest patient was initially diagnosed with spinocerebellar ataxia, as she presented with markedly dysarthric speech, nystagmus, and ataxia (Table 1).

A severe CMT phenotype without signs of ataxia was observed in the single patient who had no reported family history (Table 1).

ELECTROPHYSIOLOGICAL RESULTS

Electrophysiological studies (Table 1) of families 1 and 2 demonstrated intermediate motor NCVs and absent median and sural nerve sensory action potentials. Compound motor action potentials were reduced to variable degrees. All of the clinically unaffected individuals who were tested had normal nerve conduction study results. The sporadic patient (patient 33) had severely reduced NCVs and compound motor action potentials.

MOLECULAR RESULTS

The index patient of family 1 (IV:25) showed a heterozygous single nucleotide exchange (c.278T>C) resulting in a leucine-to-proline amino acid change at codon 93 (p.L93P) in the coil 1a domain of the NEFL protein. The leucine-93 residue is highly conserved (Figure 2). No other nucleotide changes were detected. All of the affected family members but none of the 5 unaffected family members carried the mutation (Figure 1), which was also absent in 100 controls.

Place holder to copy figure label and caption
Figure 2.

Compilation, sequence homology, and localization of NEFL mutations with respect to protein domains. Mutation sites are indicated in gray. Amino acid changes and associated phenotypes (according to the original publications) are indicated below the mutation sites. CMT1 indicates Charcot-Marie-Tooth disease, demyelinating variant; CMT2, Charcot-Marie-Tooth disease, axonal variant; N, N terminus; C, C terminus; AA, amino acid.

Graphic Jump Location

In the affected patients of family 2 as well as in the sporadic patient, we found a heterozygous C-to-G nucleotide change in the first coding exon of NEFL (c.23C>G) resulting in a proline-to-arginine substitution at codon 8 (p.P8R) located in the head domain of the NEFL protein. The p.P8R change was absent in clinically unaffected individuals of family 2. It was absent in the unaffected parents of the sporadic case, suggesting that this mutation arose de novo (Figure 2 and Table 1).

To date, 13 different NEFL mutations have been identified in 55 patients with CMT from 16 families.We describe phenotypic features of 11 further patients with CMT from 3 families with NEFL mutations, and we also describe a novel NEFL mutation, p.L93P. Nerve conduction studies suggested that this mutation resulted in an axonopathy with secondary demyelination in all of the affected individuals, which was also seen in family 2 associated with a p.P8R mutation. Indeed, neuropathological studies in 2 cases demonstrated predominantly axonal atrophy in intermediate CMT, but axonal swelling, paranodal abnormalities, and onion bulb formations have also been observed.12,15

So far, 22 patients with a mutation involving the proline residue at codon 88,10 have been described and at least 5 different mutational events can be inferred, suggesting that codon 8 is a hot spot for NEFL mutations. Of 13 patients with this mutation, 1 was classified as having intermediate CMT and the others were classified as having CMT1, with NCVs ranging from 13 to 39 m/s. However, the classification based on nerve conduction studies alone becomes difficult in cases where the compound motor action potential amplitude is reduced, as in our single patient. Despite this, phenotypic variability can be seen with codon 8 mutations not only with NCVs but also with disease onset, which varied between ages 2 and 25 years.8,10

The phenotypic variability found for codon 8 mutations can also be observed for mutations at codon 22 in the head domain of the NEFL protein1315 or at codon 397 in the coil 2b domain11,12 (Figure 2).

This indicates that no simple genotype-phenotype correlation exists and suggests the existence of modifiers in NEFL-related CMT. However, it appears that mutations localizing to the NEFL protein head domain can cause more severe motor and sensory NCV slowing than mutations in the coil 2B domain (Figure 2 and Table 2),8,12 while the intrafamilial phenotype appears to “run true.”

Table Graphic Jump LocationTable 2. Clinical and Electrophysiological Features of Patients With Charcot-Marie-Tooth Disease With Mutations in the NEFL Gene Described in the Literature

In addition, the phenotypic variability of the NEFL mutation seems to also include possible central nervous system involvement, as 1 patient of family 2 showed signs of cerebellar dysfunction and was initially diagnosed with spinocerebellar ataxia. Of previously described patients with NEFL mutations, 1 was initially diagnosed with Friedreich ataxia12 and another had a complex phenotype with nystagmus and global developmental delay.8 Although the pathophysiological abnormalities underlying central nervous system dysfunction remain unclear, their occurrence is suggested by the widespread presence of neurofilament light-chain polypeptide in the central nervous system. However, concomitant mutations in NEFL and other genes cannot be excluded.18

Since the manuscript was accepted for publication, 3 additional mutations in the NEFL gene have been reported, highlighting the phenotypic variability in CMT with NEFL mutations.19,20 The NEFL reference sequence has recently been changed; therefore, the new mutation reported in this article is now described as c. 281T>C, p.L94P.

Correspondence: Wolfgang N. Löscher, MD, PhD, Clinical Department of Neurology, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria (wolfgang.loescher@i-med.ac.at).

Accepted for Publication: August 9, 2006.

Author Contributions:Study concept and design: Janecke, Wanschitz, and Löscher. Acquisition of data: Miltenberger-Miltenyi, Janecke, Wanschitz, Timmerman, Windpassinger, Auer-Grumbach, and Löscher. Analysis and interpretation of data: Miltenberger-Miltenyi, Janecke, Timmerman, Windpassinger, Auer-Grumbach, and Löscher. Drafting of the manuscript: Miltenberger-Miltenyi, Janecke, Windpassinger, Auer-Grumbach, and Löscher. Critical revision of the manuscript for important intellectual content: Janecke, Wanschitz, Timmerman, Windpassinger, Auer-Grumbach, and Löscher. Obtained funding: Janecke and Timmerman. Administrative, technical, and material support: Miltenberger-Miltenyi, Janecke, Windpassinger, Auer-Grumbach, and Löscher. Study supervision: Wanschitz.

Financial Disclosure: None reported.

Funding/Support: This work was supported in part by the Fund for Scientific Research–Flanders, a special research fund of the University of Antwerp, the Medical Foundation Queen Elisabeth, the Association Belge contre les maladies Neuromusculaires, the Interuniversity Attraction Poles Program P5/19 of the Belgian Federal Science Office, and grant P18470-B5 from the Austrian Science Fund.

Additional Contributions: Els De Vriendt and Veerle Van Gerwen provided technical assistance with the mutation analyses in some patients. We thank the families for their participation in this study.

Harding  AEThomas  PK Genetic aspects of hereditary motor and sensory neuropathy (types I and II). J Med Genet 1980;17 (5) 329- 336
PubMed Link to Article
Dyck  PJLambert  EH Lower motor and primary sensory neuron diseases with peroneal muscular atrophy, I: neurologic, genetic, and electrophysiologic findings in hereditary polyneuropathies. Arch Neurol 1968;18 (6) 603- 618
PubMed Link to Article
Dyck  PJLambert  EH Lower motor and primary sensory neuron diseases with peroneal muscular atrophy, II: neurologic, genetic, and electrophysiologic findings in various neuronal degenerations. Arch Neurol 1968;18 (6) 619- 625
PubMed Link to Article
Kaku  DAParry  GJMalamut  RLupski  JRGarcia  CA Nerve conduction studies in Charcot-Marie-Tooth polyneuropathy associated with a segmental duplication of chromosome 17. Neurology 1993;43 (9) 1806- 1808
PubMed Link to Article
Kaku  DAParry  GJMalamut  RLupski  JRGarcia  CA Uniform slowing of conduction velocities in Charcot-Marie-Tooth polyneuropathy type 1. Neurology 1993;43 (12) 2664- 2667
PubMed Link to Article
Davis  CJBradley  WGMadrid  R The peroneal muscular atrophy syndrome: clinical, genetic, electrophysiological and nerve biopsy studies, I: clinical, genetic and electrophysiological findings and classification. J Genet Hum 1978;26 (4) 311- 349
PubMed
Acsadi  ASMichael  EKrajewski  KLewis  RA Electrophysiologic criteria defining Charcot-Marie-Tooth disease with intermediate conduction velocities. Neurology 2004;62(suppl 5)A415
Jordanova  ADe Jonghe  PBoerkoel  CF  et al.  Mutations in the neurofilament light chain gene (NEFL) cause early onset severe Charcot-Marie-Tooth disease. Brain 2003;126 (pt 3) 590- 597
PubMed Link to Article
Mersiyanova  IVPerepelov  AVPolyakov  AV  et al.  A new variant of Charcot-Marie-Tooth disease type 2 is probably the result of a mutation in the neurofilament-light gene. Am J Hum Genet 2000;67 (1) 37- 46
PubMed Link to Article
De Jonghe  PMersivanova  INelis  E  et al.  Further evidence that neurofilament light chain gene mutations can cause Charcot-Marie-Tooth disease type 2E. Ann Neurol 2001;49 (2) 245- 249
PubMed Link to Article
Choi  BOLee  MSShin  SH  et al.  Mutational analysis of PMP22, MPZ, GJB1, EGR2 and NEFL in Korean Charcot-Marie-Tooth neuropathy patients. Hum Mutat 2004;24 (2) 185- 186
PubMed Link to Article
Züchner  SVorgerd  MSindern  ESchroder  JM The novel neurofilament light (NEFL) mutation Glu397Lys is associated with a clinically and morphologically heterogeneous type of Charcot-Marie-Tooth neuropathy. Neuromuscul Disord 2004;14 (2) 147- 157
PubMed Link to Article
Yoshihara  TYamamoto  MHattori  N  et al.  Identification of novel sequence variants in the neurofilament-light gene in a Japanese population: analysis of Charcot-Marie-Tooth disease patients and normal individuals. J Peripher Nerv Syst 2002;7 (4) 221- 224
PubMed Link to Article
Georgiou  DMZidar  JKorosec  MMiddleton  LTKyriakides  TChristodoulou  K A novel NF-L mutation Pro22Ser is associated with CMT2 in a large Slovenian family. Neurogenetics 2002;4 (2) 93- 96
PubMed Link to Article
Fabrizi  GMCavallaro  TAngiari  C  et al.  Giant axon and neurofilament accumulation in Charcot-Marie-Tooth disease type 2E. Neurology 2004;62 (8) 1429- 1431
PubMed Link to Article
Yamamoto  MYoshihara  THattori  NSobue  G Glu528del in NEFL is a polymorphic variant rather than a disease-causing mutation for Charcot-Marie-Tooth disease in Japan. Neurogenetics 2004;5 (1) 75- 77
PubMed Link to Article
Leung  CLNagan  NGraham  THLiem  RK A novel duplication/insertion mutation of NEFL in a patient with Charcot-Marie-Tooth disease. Am J Med Genet A 2006;140 (9) 1021- 1025
PubMed Link to Article
Hodapp  JACarter  GTLipe  HPMichelson  SJKraft  GHBird  TD Double trouble in hereditary neuropathy: concomitant mutations in the PMP-22 gene and another gene produce novel phenotypes. Arch Neurol 2006;63 (1) 112- 117
PubMed Link to Article
Fabrizi  GMCavallaro  TAngiari  C  et al.  Charcot-Marie-Tooth disease type 2E, a disorder of the cytoskeleton. Brain 2007;130 (pt 2) 394- 403
PubMed Link to Article
Kabzinska  DPerez-Olle  RGoryunov  D  et al.  Is a novel I214M substitution in the NEFL gene a cause of Charcot-Marie-Tooth disease? functional analysis using cell culture models. J Peripher Nerv Syst 2006;11 (3) 225- 231
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.

Pedigree of family 1. Circles indicate females; squares, males; filled symbols, affected individuals; open symbols, unaffected individuals; slash, deceased; L/w, heterozygous p.L93P mutation; and w/w, absence of mutation (w indicates wild-type allele).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Compilation, sequence homology, and localization of NEFL mutations with respect to protein domains. Mutation sites are indicated in gray. Amino acid changes and associated phenotypes (according to the original publications) are indicated below the mutation sites. CMT1 indicates Charcot-Marie-Tooth disease, demyelinating variant; CMT2, Charcot-Marie-Tooth disease, axonal variant; N, N terminus; C, C terminus; AA, amino acid.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Clinical and Electrophysiological Features of Patients With Charcot-Marie-Tooth Disease Investigated in This Study
Table Graphic Jump LocationTable 2. Clinical and Electrophysiological Features of Patients With Charcot-Marie-Tooth Disease With Mutations in the NEFL Gene Described in the Literature

References

Harding  AEThomas  PK Genetic aspects of hereditary motor and sensory neuropathy (types I and II). J Med Genet 1980;17 (5) 329- 336
PubMed Link to Article
Dyck  PJLambert  EH Lower motor and primary sensory neuron diseases with peroneal muscular atrophy, I: neurologic, genetic, and electrophysiologic findings in hereditary polyneuropathies. Arch Neurol 1968;18 (6) 603- 618
PubMed Link to Article
Dyck  PJLambert  EH Lower motor and primary sensory neuron diseases with peroneal muscular atrophy, II: neurologic, genetic, and electrophysiologic findings in various neuronal degenerations. Arch Neurol 1968;18 (6) 619- 625
PubMed Link to Article
Kaku  DAParry  GJMalamut  RLupski  JRGarcia  CA Nerve conduction studies in Charcot-Marie-Tooth polyneuropathy associated with a segmental duplication of chromosome 17. Neurology 1993;43 (9) 1806- 1808
PubMed Link to Article
Kaku  DAParry  GJMalamut  RLupski  JRGarcia  CA Uniform slowing of conduction velocities in Charcot-Marie-Tooth polyneuropathy type 1. Neurology 1993;43 (12) 2664- 2667
PubMed Link to Article
Davis  CJBradley  WGMadrid  R The peroneal muscular atrophy syndrome: clinical, genetic, electrophysiological and nerve biopsy studies, I: clinical, genetic and electrophysiological findings and classification. J Genet Hum 1978;26 (4) 311- 349
PubMed
Acsadi  ASMichael  EKrajewski  KLewis  RA Electrophysiologic criteria defining Charcot-Marie-Tooth disease with intermediate conduction velocities. Neurology 2004;62(suppl 5)A415
Jordanova  ADe Jonghe  PBoerkoel  CF  et al.  Mutations in the neurofilament light chain gene (NEFL) cause early onset severe Charcot-Marie-Tooth disease. Brain 2003;126 (pt 3) 590- 597
PubMed Link to Article
Mersiyanova  IVPerepelov  AVPolyakov  AV  et al.  A new variant of Charcot-Marie-Tooth disease type 2 is probably the result of a mutation in the neurofilament-light gene. Am J Hum Genet 2000;67 (1) 37- 46
PubMed Link to Article
De Jonghe  PMersivanova  INelis  E  et al.  Further evidence that neurofilament light chain gene mutations can cause Charcot-Marie-Tooth disease type 2E. Ann Neurol 2001;49 (2) 245- 249
PubMed Link to Article
Choi  BOLee  MSShin  SH  et al.  Mutational analysis of PMP22, MPZ, GJB1, EGR2 and NEFL in Korean Charcot-Marie-Tooth neuropathy patients. Hum Mutat 2004;24 (2) 185- 186
PubMed Link to Article
Züchner  SVorgerd  MSindern  ESchroder  JM The novel neurofilament light (NEFL) mutation Glu397Lys is associated with a clinically and morphologically heterogeneous type of Charcot-Marie-Tooth neuropathy. Neuromuscul Disord 2004;14 (2) 147- 157
PubMed Link to Article
Yoshihara  TYamamoto  MHattori  N  et al.  Identification of novel sequence variants in the neurofilament-light gene in a Japanese population: analysis of Charcot-Marie-Tooth disease patients and normal individuals. J Peripher Nerv Syst 2002;7 (4) 221- 224
PubMed Link to Article
Georgiou  DMZidar  JKorosec  MMiddleton  LTKyriakides  TChristodoulou  K A novel NF-L mutation Pro22Ser is associated with CMT2 in a large Slovenian family. Neurogenetics 2002;4 (2) 93- 96
PubMed Link to Article
Fabrizi  GMCavallaro  TAngiari  C  et al.  Giant axon and neurofilament accumulation in Charcot-Marie-Tooth disease type 2E. Neurology 2004;62 (8) 1429- 1431
PubMed Link to Article
Yamamoto  MYoshihara  THattori  NSobue  G Glu528del in NEFL is a polymorphic variant rather than a disease-causing mutation for Charcot-Marie-Tooth disease in Japan. Neurogenetics 2004;5 (1) 75- 77
PubMed Link to Article
Leung  CLNagan  NGraham  THLiem  RK A novel duplication/insertion mutation of NEFL in a patient with Charcot-Marie-Tooth disease. Am J Med Genet A 2006;140 (9) 1021- 1025
PubMed Link to Article
Hodapp  JACarter  GTLipe  HPMichelson  SJKraft  GHBird  TD Double trouble in hereditary neuropathy: concomitant mutations in the PMP-22 gene and another gene produce novel phenotypes. Arch Neurol 2006;63 (1) 112- 117
PubMed Link to Article
Fabrizi  GMCavallaro  TAngiari  C  et al.  Charcot-Marie-Tooth disease type 2E, a disorder of the cytoskeleton. Brain 2007;130 (pt 2) 394- 403
PubMed Link to Article
Kabzinska  DPerez-Olle  RGoryunov  D  et al.  Is a novel I214M substitution in the NEFL gene a cause of Charcot-Marie-Tooth disease? functional analysis using cell culture models. J Peripher Nerv Syst 2006;11 (3) 225- 231
PubMed Link to Article

Correspondence

CME


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