The term hereditary motor neuropathy, synonymous with distal spinal muscular atrophy, is used to describe a group of slowly progressive conditions in which the hallmark is weakness and wasting of the feet and hands. The distinction from axonal forms of Charcot–Marie–Tooth neuropathy is generally made because of the absence of sensory involvement but overlaps, such as seen with mutations in the gene for glycyl tRNA synthetase, exist. Different clinical patterns of hereditary motor neuropathy, with varying degrees of upper and lower limb predominance, are described.1 Mutations in the gene encoding the small heat shock protein HSPB1 have been associated with a subtype of hereditary motor neuropathy (HMN) characterised by the development of symmetrical, progressive weakness and wasting of the distal limbs, most notable in the peroneal muscles, which first manifests as difficulty with running and walking.2^–4 Onset of symptoms occurs in adolescence or early adulthood in most cases, but progression is typically slow, with assisted mobility becoming necessary only after an average of more than a decade. The presence of minor sensory symptoms has varied even between families affected by the same mutation, leading to the classification of the condition as either HMN-II or Charcot-Marie-Tooth type-2D (CMT2D).

Here we describe a novel mutation in HSPB1 leading to strikingly asymmetrical and unusually late onset lower limb weakness.

METHODS

Mutation detection

Blood was collected with informed consent as part of a systematic study of the genetic basis of lower motor neuron disorders which was conducted with full local ethics committee approval.

Oligonucleotide primers were designed to amplify the coding sequence and intron–exon boundaries of the HSPB1 gene by PCR performed on DNA extracted from the patient’s blood lymphocytes. Products were denatured by heating to 96°C and allowed to cool by 1°C per minute to maximise heteroduplex formation in the presence of any sequence variation. The re-annealed products were screened by denaturing high performance liquid chromatography and any variants were characterised by direct sequencing using an ABI prism 3700 sequencer.

Expression of mutant protein in cell culture

A plasmid construct consisting of the full length HSPB1 cDNA was cloned into the mammalian expression vector pEGFP and used as a template for site directed mutagenesis. The presence of the mutation was confirmed by sequencing. The same procedure was carried out for a range of previously described HSPB1 mutants (S135F, P182L and R136W).2^–4

The human HeLa cell line was grown in Dulbecco’s modified Eagle’s medium with the addition of 10% fetal calf serum, 2 mM glutamine and 0.1 mg/ml penicillin and streptomycin, as a monolayer at 37°C in a humidified incubator with 5% CO₂/95% O₂. Cells were transfected using FuGENE-6 (Roche, Burgess Hill, UK), seeded on to sterile coverslips in 12 well plates at a density of 1×105 per well and grown in a monolayer for approximately 24 h until 70–80% confluent. At 24–48 h post transfection, coverslips were washed in phosphate buffered saline at room temperature and fixed in 4% paraformaldehyde for 20 min. Coverslips were mounted with Vectasheild mounting medium containing 4′,6-diamidino-2-phenylindole. Images were recorded using Axiovision software (Carl Zeiss, Welwyn Garden City, UK).

RESULTS

Clinical report

The proband is a 72-year-old male who was healthy until the age of 65 years when he first became aware of difficulty in standing on the tips of his toes and his walking became unsteady. He was noted to have weakness of the right ankle plantar flexion and wasting of the right calf. The condition progressed slowly over a 3 year period to complete foot drop on the right, with less severe involvement of the left leg. Tendon reflexes were reduced at the knees and absent at the ankles. The upper limbs and proximal lower limbs remain unaffected and no sensory involvement or other neurological abnormality was detected on examination. Nerve conduction studies demonstrated normal motor conduction velocities with reduced amplitude of compound motor action potentials in the lower limbs, and electromyography was consistent with chronic denervation. A biopsy from the right vastus lateralis muscle showed changes consistent with denervation. There is a limited family history of a similar condition in the proband’s mother who required assistance with her mobility because of a right sided foot drop from her mid-sixties until her death at the age of 85 years from cancer. The proband has no siblings and his only offspring has no symptoms at the age of 32 years and was not available for examination or genetic testing.

Molecular and cellular analysis

Mutation screening revealed a guanine to cytosine transversion in the first exon (cDNA 349g→c) (fig 1A), leading to the substitution of the small non-polar amino acid, glycine, with a large polar, positively charged and hydrophilic amino acid, arginine, at residue 84 (G84R). This residue is highly conserved and lies adjacent, but just outside, the consensus alpha-crystallin domain in which all other mutations, with the exception of P182L, are found (fig 1B). The mutation was not found in 200 ethnically matched control patients and does not appear as a rare variant in EST or SNP databases.

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Figure 1 Sequencing from the proband revealed a missense mutation resulting in the substitution G84R (A). Evolutionary comparison (B) shows that the affected residue is highly conserved down to C intestinalis but lies outside the consensus sequence of the α-crystallin domain (black), and adjacent to a MAP kinase recognition domain (grey). Expression of the G84R mutant in HeLa cells (C) results in the accumulation of the mutant protein in dense cytoplasmic aggregates (arrow). In comparison with known mutations in HSPB1 and HSPB8, the G84R mutation resulted in aggregate formation in a similar fraction of cells to mutations in the α-crystallin domain but a lower fraction than associated with the mutation in the N terminal I-V-I motif (D).

Expression of HSPB1 G84R

To examine the effect of the G84R mutation, the mutant protein was expressed in HeLa cells and compared with a selection of previously described HSPB1 mutants. Occasional cytoplasmic protein aggregates were observed in cells transfected with wild-type protein but the aggregates associated with the mutant protein were both more extensive and present in a significantly greater number of cells (G84R 36.0% (95% confidence interval (CI) 25.2 to 46.8) vs wild-type 12.4% (95% CI 2.8 to 22.0); p<0.001) (fig 1C). Abnormal aggregation has been described in association with all previously described pathological mutations in both HSPB1 and HSPB8, another small heat shock protein in which mutations result in a distal hereditary motor neuropathy. The level of aggregate formation associated with the G84R mutation was compared with the previously described mutations by expressing each of the variants in HeLa cells and was found to be similar (fig 1D).

DISCUSSION

HSPB1 is the best characterised of the 10 human small heat shock proteins, collectively defined by the presence of the highly conserved α-crystallin domain. This domain is central to the function of these proteins as molecular chaperones that form oligomeric complexes capable of binding a wide range of target proteins under conditions of cellular stress and protecting them from being denatured.5 6 A number of additional functions have also been described specifically for HSPB1, including a direct antiapoptotic effect,7 resistance to oxidative stress8 and a role in development, cell differentiation9 and axonal outgrowth in the CNS.10 The mechanism by which mutation of this gene results in a selective form of neuropathy remains unknown.

Five mutations in HSPB1 have been reported in association with the HMN phenotype in 11 families, including a founder mutation in four Chinese families.2^–4 All of these mutations involve functionally significant residues, in either the α-crystallin domain or a short C terminal I-X-I motif, that are predicted to affect the formation of small HSP dimers. A number of studies have indicated that the mutations result in an abnormally increased binding affinity between the mutant protein and wild-type HSPB1 or the monomeric forms of the other small HSP proteins that participate in the functional oligomeric complexes.2 11 12 This effect can be observed when the mutant proteins are expressed in cell culture by the formation of insoluble aggregates. Aggregate formation is also a feature of the neuropathy associated mutations in the related chaperone HSPB813 but with both genes the degree of aggregate formation does not correlate with the clinical severity.

The G84R mutation is unique in occurring outside the consensus sequence of the α-crystallin domain in a highly conserved region at the 5′ end of this domain and immediately adjacent to a MAP kinase recognition sequence required for the phosphorylation and activation of HSPB1 (fig 1B). In cell culture, expression of the G84R mutant resulted in HSPB1 aggregation at a similar level to the S135F and R136W mutations which appeared morphologically identical (fig 1C, D). In comparison with these previously described mutations, the clinical condition associated with G84R mutation was unusual for its late onset and marked asymmetry in both the proband and his apparently affected mother, thus extending the phenotypic spectrum associated with HSPB1 mutations.