Out-of-frame mutation in neurofilament light chain gene (NEFL) does not result in CMT 2E

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Neurogenetics

© Springer-Verlag 2005

10.1007/s10048-004-0202-3

A novel out-of-frame mutation in the neurofilament light chain gene ( NEFL) does

not result in Charcot-Marie-Tooth disease type 2E

Cinzia Andrigo1, Chiara Boito1, Paola Prandini1, a Mostacciuolo2,

e Siciliano3, Corrado Angelini1 and Elena Pegoraro1

(1) Neuromuscular Center, Department of Neurosciences, University of Padua,

35128 Padua, Italy

(2) Department of Biology, University of Padua, Padua, Italy

(3) Department of Neurosciences, University of Pisa, Pisa, Italy

Published online: 15 January 2005

(Letter to the Editor)

Sirs,

Charcot Marie Tooth (CMT) disease is a clinically and genetically heterogeneous

group of hereditary motor and sensory neuropathies. Mutations in the

neurofilament light chain gene ( NEFL), that encodes a major constituent of the

intermediate filaments (IF), cause autosomal dominant CMT linked to chromosome 8

(CMT2E) [1, 2]. Neurofilament proteins play a pivotal role in filament assembly

and control the radial growth of large myelinated axons [3]. Several missense

mutations in the NEFL gene have been reported [1, 2] and are predicted to

produce alterations in the formation of a normal IF network in neurons [4].

We studied two patients from a family diagnosed with an axonal autosomal

dominant form of CMT according to the European CMT Consortium criteria, where

mutations in the most common CMT genes ( PMP22, P0 , and Cx32) had been excluded

(Fig. 1). The proband, R.C., is a 63-year-old woman who presented at age 15 with

gait difficulties and weakness in both feet. Since diagnosis, her symptoms have

progressed, and muscle weakness involved the intrinsic hand muscles at age 40.

Currently, at age 63, R.C. is still ambulant. She has pes cavus and hammertoes,

distal muscle weakness in the upper and lower limbs and no ankle reflexes. The

extensors of the feet and toes and the intrinsic foot muscles are the most

severely affected muscles. Her sensory examination is normal, but she reports

nocturnal paresthesia in her left hand.

Fig. 1A–D DNA analysis of the NEFL gene in the family with axonal CMT. In panel

A, the SSCP analysis of the PCR product for primer set NEFL 4F-4R is shown. A

unique conformer is shown in the proband (R.C.). In panel B, the alignment of

control and patient DNA NEFL sequences revealed a G insertion at position 1,576

of the NEFL coding sequence. The sequence traces corresponding to the insertion

in the NEFL gene are shown in panel C. In panel D, the 1,576insG mutation has

been confirmed in the proband genomic DNA by polyacrylamide gel fractionation of

the PCR product encompassing the insertion, and by Ear I restriction digestion.

The probands daughters did not inherit the one base pair insertion from their

mother.

The patient has two daughters, one of whom is affected by peripheral neuropathy

(B.I.). B.I. is a 39-year-old woman who presented at age 18 with painful

sensations in the lower limbs. Soon after, the patient complained of progressive

lower limb weakness, foot paresthesia and effort cramps in the quadriceps.

Currently, her neurological examination shows marked paresis on dorsiflexion of

the feet with stepwise ambulation, slight muscle hypotrophy and weakness of the

hand muscles, superficial hypoesthesia of the feet and pes cavus. B.I.s father

died at 52 years of age after an accident. He was not neurologically evaluated,

but he never complained of any muscle weakness. Electromyography studies of both

R.C. and B.I. showed a pattern of axonopathic involvement with a reduction or

absence of motor and sensory evoked potential amplitudes, and normal or slightly

reduced conduction velocity.

In the proband (R.C.) a novel, out-of-frame single base insertion in exon 4 of

the NEFL gene (1,576insG) was identified (Fig. 1). This mutation results in a

premature stop codon in the region of the gene encoding the tail domain of the

NEFL protein. The 1,576insG mutation does not co-segregate with clinical

phenotype in the family because it was not inherited by the affected daughter

(Fig. 1). Moreover, genotype analysis using 10 polymorphic microsatellite

markers mapping to 8p21, where the NEFL gene maps, showed that both sisters

share the same maternal and paternal haplotypes. The 1,576insG mutation was

absent in 700 control chromosomes and in 84 CMT2 chromosomes.

We suggest that the 1,576insG is a rare polymorphism of the NEFL gene. Indeed,

all the NEFL gene mutations to date reported are missense mutations occurring at

amino acids that are highly conserved and important for the structure or

function of the protein [1,2]. Several nucleotide changes have been reported in

the region of the gene encoding the tail domain of the NEFL protein and none so

far has been shown to be pathogenetic, most likely because the tail region is

not necessary for assembly of neurofilaments [4,5]. Furthermore, the 1,576insG

NEFL allele incorporates a premature termination codon in the last exon of the

gene, and therefore is thought to specifically escape nonsense mediated RNA

decay [6], produces a truncated protein lacking the very last 13 amino acids

which are possibly not crucial for protein function.

The current hypothesis is that mutations in the head or rod domains of NEFL

interfere with the ability of the mutated protein to assemble into filaments.

Experimental evidence derived from transgenic mouse studies [7] and from

transient transfection studies with mutant NEFL cDNAs (Q333P and P8R) has shown

that these mutations alter the formation of a normal IF network and axonal

transport in neurons, possibly contributing to the development of CMT neuropathy

[4, 5]. Moreover, targeted disruption of the NEFL gene in knock-out mice does

not result in an overt phenotype [8].

The report of this naturally occurring frame-shift mutation of the NEFL gene,

which does not result in CMT in humans implies that haploinsufficiency is not

the molecular basis in CMT2E. Our findings, taken together with the evidence in

the literature, clearly suggest that a dominant negative mechanism may underlie

CMT2E.

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