Case study excerpted from De Jonghe et al., "Further evidence that neurofilament light chain gene mutations can cause Charcot-Marie-Tooth disease type 2E." Annals of Neurology 2001;49:245-249.

This case study provided courtesy of Vincent Timmerman, PhD (1) and Peter De Jonghe, MD, PhD (1,2), et al.

1. Flanders Interuniversity Institute for Biotechnology (VIB), Born-Bunge Foundation (BBS), University of Antwerp (UIA), Antwerpen, Belgium.
2. Division of Neurology, University Hospital Antwerpen (UZA), Antwerpen, Belgium.

The inherited peripheral neuropathies are a heterogeneous group of disorders. The most common phenotype is Charcot-Marie-Tooth disease (CMT), which is characterized by progressive weakness and atrophy, initially of the peroneal muscles and later on of the distal muscles of the arms. CMT is subdivided in CMT type 1 (CMT1) and CMT type 2 (CMT2) based on electrophysiological and neuropathological criteria. CMT1 is characterized by de- and remyelination on nerve biopsy examination and slow nerve conduction velocities (NCV) on neurophysiological testing. CMT2 is an axonal neuropathy characterized by signs of axonal regeneration in the absence of overt myelin alterations. NCVs are normal or slightly reduced in CMT2. Most CMT families can be classified as either CMT1 or CMT2 using a cut-off value of 38 m/s for the motor median nerve. However, in some CMT families, sometimes designated as intermediate CMT, patients have very variable NCVs ranging from normal to severely reduced.^1,2

Molecular genetic studies have shown that both CMT1 and CMT2 are heterogeneous. The majority of CMT1 patients have a 1.5 Mb tandem duplication in chromosome 17p11.2-p12 (CMT1A) harboring the peripheral myelin protein 22 gene (PMP22).^3,4 Point mutations in this gene may also result in CMT1. Mutations in the genes encoding myelin protein zero (MPZ, P0) (CMT1B), gap-junction protein connexin 32 (Cx32, GJB1) (CMT1X) and early growth response element 2 (EGR2) result in CMT1 or the related demyelinating neuropathies Dejerine-Sottas syndrome and congenital hypomyelination which are characterized by a more severe phenotype.⁵ Recently, mutations in the gene encoding myotubularin related protein-2 (MTMR2) have been observed in autosomal recessive demyelinating CMT linked to chromosome 11q22 (CMT4B)⁶ and mutations in the N-myc downstream-regulated gene 1 (NDRG1) underlie HMSN-Lom, a recessive demyelinating neuropathy initially observed in Bulgarian Gypsies.⁷ Molecular genetic studies have been less productive in CMT2. Mutations in Cx32 and MPZ have been observed in a subset of CMT2 patients.⁵ Genetic linkage studies have mapped three CMT2 loci i.e. CMT2A, CMT2B and CMT2D at chromosome 1p35-p36, 3q13-q22 and 7p14 respectively.^8-10 Very recently, a fourth CMT2 locus (CMT2E) was mapped to 8p21 in a single CMT2 pedigree from Mordovia, Russia. Subsequently a c.998A>C transversion mutation resulting in a Gln333Pro in the first exon of the neurofilament light (NEFL, NF-L) gene was found to show complete co-segregation with the disease phenotype.^11,12 This mutation was not observed in 160 control chromosomes. We observed another NEFL mutation in a Belgian CMT family. These data confirm that mutations in the NEFL gene are the cause of CMT2E.

We studied a Belgian multigeneration family (CMT-56) in which a CMT phenotype segregates as a dominant trait (Figure 1).

The proposita, III.5, was first seen at the age of 14 years. She had a steppage gait and paresis of distal leg muscles. Foot extensors were more severely affected than plantar flexor muscles. The hand muscles were slightly weak and atrophic. Tendon reflexes were absent. Discrete hypoesthesia for touch, pain and temperature was present in a glove/stocking distribution. Clinical examination of family members identified eight additional patients. Two additional persons elected not to participate in research studies, but were definitively affected based on information provided by close relatives. The elder patients and II.4 and II.13, examined at the age of 56 and 47 years respectively, showed an almost complete paralysis of distal muscles of the legs and a severe paresis (1/5 to 3/5 on the MRC scale) of the intrinsic hand muscles. Examination of the youngest generation confirmed a disease onset in the second decade of life.

Electrophysiological studies in patients II.13, III.5 and III.9 show severely reduced motor NCVs. The ranges of the motor NCVs are 25 m/s to 39 m/s (n = 3) for the median nerve and 30 m/s to 42 m/s (n = 2) for the ulnar nerve. The amplitudes of compound muscle action potentials (CMAP) in the upper limbs were severely reduced and ranged from 1.4 mV to 3.8 mV (normal: higher than 6.0 mV). Sensory nerve action potentials were usually absent and only one measurement of 17 m/s for a median nerve was obtained.

We screened 40 unrelated patients diagnosed as CMT2 or intermediate CMT for the presence of mutations in the NEFL gene (Genbank n¡ NM 006158). Via DHPLC we detected a heteroduplex pattern in the PCR fragment NEFL1.1 in the proband of family CMT-56 (Figure 2A). DHPLC analysis demonstrated the same heteroduplex pattern in all patients from family CMT-56, but not in their unaffected relatives. We subsequently sequenced DNA samples of three patients and found a double missense mutation at positions 22 and 23 from CC to AG in the first exon of NEFL (c.22C>A+23C>G) (Figure 2B, nucleotide numbering according to the cDNA sequence). This mutation creates an amino-acid change from Pro to Arg at codon 8 (P8R). The complete co-segregation of the heterozygous mutation with the CMT phenotype was confirmed by DHPLC. This mutation was absent in 80 normal controls suggesting that this sequence variation is not a rare polymorphism. Linkage analysis with the Pro8Arg mutation in family CMT-56 resulted in a two-point LOD score of 3.61 in the absence of recombinants.

Neurofilaments (NFs) form the cytoskeletal component of the myelinated axon and belong to the most abundant and widely expressed neuronal intermediate filament proteins. They are composed of three proteins; light (NFL), mid-sized (NFM) and heavy (NFH) chains, encoded by separate genes (reviewed in ¹³). The mouse and human NEFL gene contains 4 coding exons and the 5'UTRs are highly conserved (90% homology). The NEFL protein contains 543 amino acids with a head, rod and tail domain. The rod domain contains 4 coil sub-domains (Coil 1A, 1B, 2A and 2B) separated by 3 linker molecules. The tail of the protein has two sub-domains; A and B, of which B is acidic.¹⁴

Animal models have demonstrated that NFs are involved in determining the axon diameter. In Japanese quail (Quiverer, quv), a spontaneous recessive mutation in NEFL generates a truncated protein incapable of forming NFs.¹⁵ Homozygous mutants have no axonal NFs and exhibit mild generalized quivering. Normal radial growth of myelinated axons is severely attenuated resulting in a reduction of the axonal conduction velocity. Knockout mice lacking axonal NFs, due to a targeted disruption of the NEFL gene, have diminished axon caliber and delayed regeneration of myelinated axons following crush injury of peripheral nerve.¹⁶ When NFL is lacking, NFM and NFH can not form functional 10 nm NFs. Homozygous and heterozygous NEFL knockouts develop normally, are not lethal and do not exhibit a particular clinical phenotype. However, the transgenic mouse mutant NEFL Leu394Pro has massive degeneration of spinal motor neurons with abnormal neurofilament accumulation and severe neurogenic atrophy of skeletal muscles. At postnatal day 18, this mutant shows an abnormal gait with reduced activity and weakness of upper and lower limbs.¹⁷

These observations in spontaneous mutant and knockout animals suggest that NF abnormalities may contribute to the pathology of human neurodegenerative diseases.¹⁸ However, so far only alterations in the NEFH gene had been linked with a human disorder. A few alterations in the NEFH gene were reported in sporadic patients with amyotrophic lateral sclerosis (ALS). Most patients had a deletion in the Lys-Ser-Pro repeat region, which is a highly conserved repetitive region of the NEFH gene.¹⁹ Another ALS patient had a novel 84 bp insertion leading to an extra four Lys-Ser-Pro repeats.²⁰

Recently, a novel CMT2 locus (CMT2E) was mapped to chromosome 8p21 in a single large Russian family, obtaining a two-point LOD score of 5.93 with a short tandem repeat marker from the 5'UTR region of the NEFL gene. Subsequent mutation analysis of NEFL demonstrated the presence of a c.998A>C mutation at codon 333 (Gln333Pro) that showed complete co-segregation with the disease phenotype.¹¹ These findings and the high degree of conservation of Gln333 between distinct species lend support to the hypothesis that the NF-L mutation is indeed the disease causing mutation in this CMT2E family.

We identified a dominant double missense mutation (c.22C>A+23C>G) at codon 8 in the NEFL gene resulting in a Pro to Arg substitution in a Belgian CMT family. This mutation shows perfect co-segregation with the disease phenotype. The Pro8Arg missense mutation most likely destabilizes the head domain of the NEFL protein. Cumulative evidence from the Russian11 and Belgian families provide substantive prove that NEFL is the CMT2E gene. Both mutations occur at amino acids which are well conserved in all sequenced NEFL genes; i.e. from Xenopus to human. The patients in the Belgian family present with a classical, although rather severe CMT phenotype with a disease onset in the second decade of life. The Russian family had been diagnosed as CMT2 based in NCVs ranging from 38 m/s tot 52 m/s. In our CMT2E family, however, NVCs are sometimes severely slowed and patients could be classified as CMT1 based on NCVs alone. It is important to note that amplitudes of the CMAP are always severely reduced suggesting that the slowing is, at least partially, due to loss of fast conducting axons. Our data suggest that also patients and families that are diagnosed as CMT1 should be screened for mutations in the NEFL gene once mutations in the CMT1 genes have been excluded.

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