Autosomal dominant Charcot-Marie Tooth Disease type 2 …



MS # 200202414

Charcot-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: 9966

Paper word count: 14681219

Abstract

We report an Italian family with autosomal-dominant Charcot-Marie-Tooth disease (CMT) peripheral neuropathy fitting the clinical and electrophysiological criteria of in classical CMT forms, the nerve biopsy was characterised bywhich there were giant axons. We excluded linkage in the sural nerve biopsy. Linkage to the known CMT2 loci (CMT2A, CMT2B, CMT2D, CMT2F) and we did not detect mutations in the known CMT2 genes (Cx32, MPZ, NEFL), GAN and, NEFM, in the reported family; moreover, it was excluded the presence of the and CMT1A duplication/HNPP deletion. Our were excluded. This family with CMT and giant axons has a clinico-pathological and genetic entity distinct from classical CMT.

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 peripheral nervous system 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) (Ben Othmane et al., 1993),), 3q13-q22 (CMT2B) (Kwon et al., 1995), 7p14 (CMT2D) (Ionasescu et al., 1996), 8p21 (CMT2E) (Mersiyanova et al., 2000) and 7q11-q21 (CMT2F) (Imailov et al., 2001). Recently, mutations in the kinesin family member 1Bß gene (KIF1Bß) (Zhao et al., 2001) and the neurofilament light chain gene (NEFL) (Mersiyanova et al., 20002) were shown to underlie CMT2A and CMT2E respectively.. Mutations in the connexin 32 gene (GJB1, Cx32) (Timmerman et al., 19963) and specific mutations in the myelin protein zero gene (MPZ, P0) (De Jonghe et al., 19994) may also result in a CMT2 phenotype. Recently, mutations in gigaxonin (GAN) (Bomont et al., 20005), 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.

Methods

We performed clinical (5n=5) and conventional electrophysiological (3n=3) examinations in affected members of a multi-generation kindred from Southern Italy (Figure 1). Electromyography ().

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 (Navon et al.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) (De Jonghe et al.), 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.

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) havehad weakness and atrophy of thehands, feet and of legs, with a peroneal distribution; both had generalized hypo/areflexia, stepping gait, in the upper

extremities motor deficits are restrictedwere unable to the hands.walk on their heels and presented loss of tactile and vibration senses are in the distal area of the legs.lower limbs. The only clinical signs in patients IV-3, IV-4 and V-1 arewere hypo/areflexia and a discrete deficit in vibration sense confined to the distal aspectsarea of the the legs.lower limbs. Pes cavus was present since infancy in all patients.affected persons. Autonomic symptoms, nerve enlargement, and kinky hair were not present. None of the patients hasshowed 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) without with evidence of sporadic simplex “onion bulb” and de-remyelinationformations.

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, compatible with axonal loss.. 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 primary

predominant axonal pathologydamage likely accompanied by secondary demyelination; therefore, this disorder can be considered a unique, but rare form of CMT.

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, recently linkage to the GAN region on chromosome 16p was recently described in a family with a HMSN IICMT2-like phenotype, but the inheritance pattern in this consanguineous Algerian family was clearly autosomal-recessive (Zemmouri et al., 20008). 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.

In conclusion, our data confirm the existence of CMT with giant axons as a clinico-pathological entity. This disorder also represents a distinct genetic entity.

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.

References

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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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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)

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)

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)

GAN-10F GATGACTCACCAAGCTTGCT (5)

GAN-10R TCGTAATTGGTACCTAAGCC (5)

GAN-11F CTGTTTCCTGGTGATTCTGG (5)

GAN-11R CTTTCGGAGCTATGTTATGG (5)

Table 2 Electroneuromyographic data in affected patients

|Patients |III-6 |IV-3 (F/32) |V-1 |normal |

|(Sex/Age) |(M/66) | |(M/12) |values |

| | |MCV |30.3 |26.6 |26.4 |≥ 50.0 |

| |Median nerve | | | | | |

|Motor | | | | | | |

|ENG | | | | | | |

| | |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 |

| |Median nerve |SCV |RA |20.8 |32.5 | ≥ 44.5 |

|Sensory | | | | | | |

|ENG | | | | | | |

| | |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 | |

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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Legends

Fig.1

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

[pic]

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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