Giacomo Lus et al.

MS # 200202414Charcot-Marie-Tooth disease with giant axons: a clinico-pathological and genetic entity.

G. Lus, MD, E. Nelis1, PhD, A. Jordanova, PhD1, A. Löfgren, MSc1, T. Cavallaro3, MD, A.

Ammendola, MD, M.A.B. Melone, MD, N. Rizzuto3, MD, V. Timmerman1, PhD, R. Cotrufo,

MD and P. De Jonghe1,2, MD, PhD

Affiliations:

Department of Neurological Sciences, First Division of Clinical Neurology, Faculty of

Medicine, Second University of Naples and Interuniversity Center for Research in

Neuroscience, Naples (Italy), 1Molecular Genetics Department, Peripheral Neuropathy Group,

Flanders Interuniversity Institute for Biotechnology (VIB), Born-Bunge Foundation (BBS),

University of Antwerp (UIA), Antwerpen (Belgium), 2Division of Neurology, University

Hospital Antwerp (UZA), Antwerpen (Belgium), 3Department of Neurological and Visual

Sciences, Section of Clinical Neurology, University of Verona, Verona (Italy).

Supplementary contents

Running title:

Charcot Marie Tooth with giant axons

Keywords:

CMT, giant axons, novel entity

Address all correspondence to:

Dr. G. Lus, M.D., Department of Neurological Sciences, First Division of Clinical Neurology,

Faculty of Medicine, Second University of Naples, v. Pansini, 5, ed. 10, 80131 Naples, Italy;

e-mail: giacomo.lus@unina2.it

Title character count: 88

Abstract word count: 66

Paper word count: 1219

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Giacomo Lus et al.

Abstract

We report an Italian family with autosomal-dominant Charcot-Marie-Tooth disease

(CMT) in which there were giant axons in the sural nerve biopsy. Linkage to the known

CMT2 loci (CMT2A, CMT2B, CMT2D, CMT2F) and mutations in the known CMT2 genes

(Cx32, MPZ, NEFL GAN, NEFM, and CMT1A duplication/HNPP deletion were excluded.

This family with CMT and giant axons has a pathological and genetic entity distinct from

classical CMT.

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Introduction

Charcot-Marie-Tooth disease (CMT) can be: autosomal-dominant, autosomal-recessive,

or X-linked. The current classification schema for CMT divides the disorders into two

categories: 1) those having apparent Schwann-cell dysfunction leading to loss of myelin with

onion bulbs and reduced motor and sensory conduction velocities (demyelinating CMT or

CMT1); and 2) those with axonal degeneration as well as normal or slightly reduced nerve

conduction velocity (NCV) (axonal CMT or CMT2). Molecular genetic studies of CMT1 have

demonstrated, in most cases, duplication/point mutation in the PMP-22 gene (CMT1A), or

point mutation in the P0 gene (CMT1B). These studies have also demonstrated that CMT2 is

a heterogeneous disorder with autosomal-dominant disease loci at chromosomes 1p35-p36

(CMT2A), 3q13-q22 (CMT2B), 7p14 (CMT2D), 8p21 (CMT2E) and 7q11-q21 (CMT2F).

Recently, mutations in the kinesin family member 1Bß gene (KIF1Bß) (1) and the

neurofilament light chain gene (NEFL) (2) were shown to underlie CMT2A and CMT2E.

Mutations in the connexin 32 gene (GJB1, Cx32) (3) and specific mutations in the myelin

protein zero gene (MPZ, P0) (4) may also result in a CMT2 phenotype. Recently, mutations

in gigaxonin (GAN) (5), a novel cytoskeletal protein, were shown to cause giant axonal

neuropathy (GAN).

The clinical and electrophysiological features of CMT2 with giant axons noted on

nerve biopsy were first reported in 1985 in a German kinship (6). In this report, we describe

findings from another kinship with CMT and giant axons; molecular genetic analyses

excluded all previously reported mutations associated with CMT.

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Methods

We performed clinical (n=5) and conventional electrophysiological (n=3) examinations in

affected members of a multi-generation kindred from Southern Italy (Figure 1).

The DNA of patients III-6, III-9, IV-3 was screened for the presence of CMT1A

duplication/HNPP deletion using seven microsatellite markers: 4A, 9A and 9B {1338},

D17S2216, D17S2220, D17S2224 and D17S2230 {1339}. Mutation screening of PMP22,

MPZ, Cx32, NEFL, GAN and the neurofilament medium chain gene (NEFM) was also

performed. Single-strand conformation polymorphism (SSCP) analysis of the coding region

of MPZ and Cx32 was performed as described previously (7). The coding regions of NEFL,

NEFM and GAN were amplified using primer sets reported in Table 1.

NEFL was analysed by denaturing high-performance liquid chromatography (DHPLC) using

the WAVE automated instrument (Transgenomics, Santa Clara, CA), while PMP22, NEFM

and GAN were analysed by direct DNA sequencing using the BigDye Terminator Cycle

Sequencing kit with AmpliTaq DNA Polymerase, FS (ABI PRISM, Applied Biosystem Inc.,

Foster City, CA). Data were collected and analysed using the ABI DNA sequencing analysis

software, version 3.6.

Short tandem repeat (STR) markers were used to cover the CMT2A (D1S2667, D1S434,

D1S228, D1S170), CMT2B (D3S1267, D3S1290, D3S1549), CMT2D (D7S2496, D7S526)

and CMT2F (D7S634, D7S1797, D7S802) regions. Genomic DNA was amplified by PCR

with standard techniques using fluorophore-labeled forward primers. Fragment analysis was

performed on an ABI3700 automated DNA sequencer. Allele calling was performed with the

ABI GENESCAN 2.2 and GENOTYPER 2.0 software.

Sural nerve biopsy was only performed in patient III-6 and was processed according to

standard procedures for light and electron microscopy examination.

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Results

The family history was obtained from the propositus (patient III-6), s a 60-year-old

man, having progressive weakness and hypotrophy of the legs, began at age 40. Patient III-9

has a similar clinical phenotype, while the other patients, IV-3, IV-4 and V-1, have only

minor signs and symptoms. The two most severely affected patients (III-6, III-9) had

weakness and atrophy of hands, feet and of legs, with a peroneal distribution; both had

generalized hypo/areflexia, stepping gait, were unable to walk on their heels and presented

loss of tactile and vibration senses in the distal area of the lower limbs. The only clinical signs

in patients IV-3, IV-4 and V-1 were hypo/areflexia and a discrete deficit in vibration sense

confined to the distal area of the lower limbs. Pes cavus was present since infancy in all

affected persons. Autonomic symptoms, nerve enlargement, and kinky hair were not present.

None of the patients showed clinical or ECG signs of cardiac involvement. In the three

patients examined (table 2), EMG of the tibialis anterior and abductor digiti quinti muscles

showed motor unit potentials with increased amplitude and duration, and decreased

recruitment. These abnormalities were more pronounced in the muscles of the lower limbs.

NCV studies showed a moderate to severe reduction of motor and sensory NCVs in

combination with a severe reduction of compound motor action potential (CMAP) and

sensory nerve action potential (SNAP) amplitudes. In subject III-6, motor NCVs were slowed,

while SNAPs of the median and sural nerves could not be elicited.

A sural nerve biopsy of patient III-6 showed nerve fascicles consisting mainly of small-to-

medium calibre fibres and numerous giant axons wrapped in a very thin myelin sheath (Figure

2) with evidence of sporadic simplex “onion bulb” formations.

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The ultrastructural study excluded demyelinating axons and revealed an accumulation of

neurofilaments with segregation of the organelles in the axoplasm of the giant axons (Figure

3). STR analysis excluded the presence of CMT1A duplication or HNPP deletion in patients’

DNA. Mutation screening of the coding regions of the PMP22, MPZ, Cx32, NEFL, NEFM

and GAN genes did not reveal a pathogenic mutation. Genotype analysis of STR markers

from the CMT2A, CMT2B, CMT2D and CMT2F regions did not show a disease-associated

haplotype that was shared by all patients, indicating that the disease is not linked to the known

CMT1 and CMT2 loci.

Discussion

Patients in this family presented with a typical CMT phenotype: muscle weakness and

atrophy with an initial peroneal distribution; involvement of sensory functions;

hypo/areflexia; slow disease course with normal life expectancy; and variable expression

within the kinship. Motor and sensory NCVs were moderately to severely reduced and the

amplitudes of the CMAPs and SNAPs were always clearly reduced. Nerve biopsy showed a

loss of large-diameter fibers in the absence of demyelination and hypertrophic changes. The

presence of giant axons with neurofilament accumulation in the axoplasm is a very unusual

finding. Unfortunately, only one nerve biopsy could be performed in this family. However,

we believe that these unusual neuropathological findings are representative of this particular

CMT variant since the clinical and electrophysiological phenotype of the biopsied patient did

not differ from that of the other affected family members. Combined evidence from the

electrophysiological and neuropathological examinations suggest predominant axonal damage

likely accompanied by secondary demyelination; therefore, this disorder can be considered a

unique, but rare form of CMT.

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This is the second report of a CMT family with giant axons. Compared with the first-

reported kinship (6), the family members of the present study had no cardiac involvement.

Giant axons are a characteristic feature of diseases such as GAN and some toxic neuropathies.

However, there was no history of exposure to neurotoxins that could explain the presence of

giant axons in this family. The different inheritance pattern and age of onset, and the different

clinical expression including the absence of kinky hair and central nervous system

involvement, argue against a diagnosis of GAN. However, linkage to the GAN region on

chromosome 16p was recently described in a family with a CMT2-like phenotype, but the

inheritance pattern in this consanguineous Algerian family was clearly autosomal-recessive

(8). In our family, we formally excluded a GAN mutation. In addition, we excluded mutations

in all HMSN II-related genes/loci either by direct mutation analysis (Cx32, MPZ/P0, NEFL)

or by linkage analysis (CMT2A, CMT2B, CMT2D, CMT2F). NEFM and PMP22 mutations

were also excluded.

Acknowledgments

This work is funded through grants from the Fund for Scientific Research-Flanders (FWO,

Belgium) and the University of Antwerp. E. Nelis and V. Timmerman are postdoctoral

fellows of the FWO.

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References

1. Zhao C, Takita J, Tanaka Y et al. Charcot-Marie-Tooth disease type 2A caused by

mutation in a microtubule motor KIF1B. Cell 2001; 105: 587-597.

2. Mersiyanova IV, Perepelov AV, Polyakov AV et al. A new variant of Charcot-Marie-

Tooth disease type 2 (CMT2E) is probably the result of a mutation in the

neurofilament light gene. Am J Hum Genet 2000; 67: 37-46.

3. Timmerman V, De Jonghe P, Spoelders P et al. Linkage and mutation analysis of

Charcot-Marie-Tooth neuropathy type 2 families with chromosomes 1p35-p36 and

Xq13. Neurology 1996; 46: 1311-1318.

4. De Jonghe P, Timmerman V, Ceuterick C et al. The Thr124Met mutation in the

peripheral myelin protein zero (MPZ) gene is associated with a clinically distinct

Charcot-Marie-Tooth phenotype. Brain 1999; 122: 281-290.

5. Bomont P, Cavalier L, Blondeau F et al. The gene mutated in giant axonal neuropathy

encodes for gigaxonin, a novel member of the cytoskeletal BTB/Kelch repeat family.

Nat Genet 2000; 26: 370-374.

6. Vogel P, Gabriel M, Goebel HH, Dyck PJ. Hereditary motor sensory neuropathy type

II with neurofilament accumulation: new finding or new disorder? Ann Neurol 1985;

17: 455-461.

7. Navon R, Timmerman V, Löfgren A, Liang P, Nelis E, Zeitune M et al. Prenatal

diagnosis of Charcot-Marie-Tooth disease type 1A (CMT1A) using molecular

genetic techniques. Prenatal Diagnosis 1995; 15: 633-640.

8. Zemmouri R, Azzedine H, Assami S et al. Charcot-Marie-Tooth 2-like presentation of

an Algerian family with giant axonal neuropathy. Neuromuscul Disord 2000; 10:

592-598.

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WEB SITE ONLY

Table 1: Primer sequences for amplification of the coding region of NEFL, NEFM and GAN

Primer Name Primer Sequence Reference

NEFL1-1F CAGAATCCTCGCCTTGGCT personal data

NEFL1-1R GATCCAGAGCTGGAGGAGTA personal data

NEFL1-2F GCTTACTCAAGCTACTCGGC personal data

NEFL1-2R GCTCGTACAGCGCCCGGAAGC personal data

NEFL1-3F CAGCAACGACCTCAAGTCCA personal data

NEFL1-3R CCTCGGCGTCCTCGCGGCTC personal data

NEFL1-4F GGAGGAGACCCTGCGCAACC personal data

NEFL1-4R GTTCTGCATGTTCTTGGCGG personal data

NEFL1-5F CAGATCCAGTACGCGCAGAT personal data

NEFL1-5R GATTTCCAGGGTCTTGGCCT personal data

NEFL1-6F GAGAGCGCCGCCAAGAACACC personal data

NEFL1-6R ACCCCTGGTTTCGCTTTCTG personal data

NEFL2F CTAGGCCTTTGCAACTACACTAC personal data

NEFL2R CCTAAGGTTTAATGGCTGCTG personal data

NEFL3-1F GGGTACTCAGAGCAAGTTGTG personal data

NEFL3-1R TTCGGTCTGCTCCTCTTGGAC (2)

NEFL3-2F CAGCTCCTATCTGATGTCCACC (2)

NEFL3-2R CACCCAGTTTACACTTGAAGTTGC (2)

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NEFL4F ACTGGACTTACCCTGGATTTGC (2)

NEFL4R CCTGATTTCGGGAGAATTATTCC (2)

NEFM1F1 ACGCTGTGACAGCCACACGCC (2)

NEFM1R1 TCTGCTGCTCCAGGTAGTGCAC (2)

NEFM1F2 GGGCTGAACGACCGCTTTGCC (2)

NEFM1R2 TGCGATGCCTGGATCTGGGCC (2)

NEFM1F3 CGAGGAGGAGGTGGCCGACC (2)

NEFM1R3 CTGGCCGAGGCCGCGGTTCC (2)

NEFM2F CTGTTTGCAAGGATGAGTCTGG (2)

NEFM2R CCACGCACGTAGTAAGCATCG (2)

NEFM3F1 ATGTAATGAAGCTCAGAAGGCC (2)

NEFM3R1 GCCTTCTTCTTCCTCCTTTTCC (2)

NEFM3-2F GAAGAGGAACCCGAAGCTGAAG personal data

NEFM3-2R GTGACTTGGGCACAGGAGACTT personal data

NEFM3F3 AGGAGCTGGTGGCAGATGCC (2)

NEFM3R3 TCCTCTTTCTGTTCACCTTTCC (2)

NEFM3F4 CACCAGTGGAAGAGGCAAAGTC (2)

NEFM3R4 CTCAAGTCTAGGCCATTGGTGAC (2)

NEFM3F5 GGAGGGAAGAGGAGAAAGGC (2)

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NEFM3R5 GCTTAACCTTTTGCAATGGACTC (2)

GAN-1F GGAGGAAGGAGGCTTCTGAT personal data

GAN-1R GGACAGGGGACAGGGTCT personal data

GAN-2F ATAGCTATTTCTGTTCTTTCATA (5)

GAN-2R TATAATGGATGAAAGGAGACC (5)

GAN-3F GTTTGGGTTTTAAATGTACA (5)

GAN-3R CAACTAAAATTTGAATTAAAAAGAAA (5)

GAN-4F CCCTCTTCTGCAGGTCCAC (5)

GAN-4R TGGAACTACCTCTCCCATACAC (5)

GAN-5F TAAACTAAAACTAGTGTGGCTACT (5)

GAN-5R GTATCTTTAAAAGGCTCTGAGTC (5)

GAN-6F TCTTCAGATGCTGTTTCTATATATG (5)

GAN-6R GCTCCGTTTCTTCCCTGAAC (5)

GAN-7F CAGCTTTCAATATGATATTGGC (5)

GAN-7R CACCATCAGTTATATTAAAGGTTT (5)

GAN-8F ACAGTTTAATATCTGTTCACCT (5)

GAN-8R AAAAGCCAGGCAGGGTAA (5)

GAN-9F TGCTGCAGAGTTAAACCAG (5)

GAN-9R CAAAACTAAACAAAGCTAAAATA (5)

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GAN-10F GATGACTCACCAAGCTTGCT (5)

GAN-10R TCGTAATTGGTACCTAAGCC (5)

GAN-11F CTGTTTCCTGGTGATTCTGG (5)

GAN-11R CTTTCGGAGCTATGTTATGG (5)

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Table 2

Electroneuromyographic data in affected patients

Patients

(Sex/Age)

III-6

(M/66)

IV-3

(F/32)

V-1

(M/12)

normal

values

MOTOR

ENG

Median

nerve

MCV 30.3 26.6 26.4 ≥ 50.0

MDL 6.0 7.9 5.5 ≤ 4.5

AMP* 4.9 3.5 4.3 ≥ 8.0

Peroneal

nerve

MCV 32.6 31.4 27.6 ≥ 41.2

MDL 6.4 6.2 7.0 ≤ 6.0

AMP* 0.4 1.2 1.4 ≥ 5.0

SENSORY

ENG

Median

nerve

SCV RA 20.8 32.5 ≥ 44.5

AMP RA 2.3 3.9 ≥ 9.0

Sural

nerve

SCV RA 25.1 29.5 ≥ 45.5

AMP RA 0.4 0.9 ≥ 3.0

EMG CD CD CD

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ENG = electroneurography;*distal stimulation; MCV = motor nerve conduction velocity( m/sec) ;

MDL = motor distal latency (m/sec); SCV = sensory nerve conduction velocity(m/sec);

AMP = amplitude(μV); RA= response absent; CD = chronic denervation.

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fig.1

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fig.2

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fig.3

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Legends

Fig.1

Pedigree of the family: males are indicated by squares, female by circle. Autosomal dominant

inheritance is evident

Fig.2

Nerve biopsy of sural nerve, patient III-6. Giant axons evidenced within fibers of small and

medium caliber. (Toluidine blue) Scale bar = 130 μm.

Fig. 3

Nerve biopsy of sural nerve, patient III-6. One giant axon with neurofilaments accumulation

and segregation of the organelles in the axoplasm (more evident in the higher power view in

the window). Scale bar = 8 μm.

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