Heterogeneous Clinical Phenotypes of dHMN Caused by Mutation in HSPB1 Gene: A Case Series

Abstract

Mutations in HSPB1 are known to cause Charcot-Marie-Tooth disease type 2F (CMT2F) and distal hereditary motor neuropathy (dHMN). In this study, we presented three patients with mutation in HSPB1 who were diagnosed with dHMN. Proband 1 was a 14-year-old male with progressive bilateral lower limb weakness and walking difficulty for four years. Proband 2 was a 65-year-old male with chronic lower limb weakness and restless legs syndrome from the age of 51. Proband 3 was a 50-year-old female with progressive weakness, lower limbs atrophy from the age of 44. The nerve conduction studies (NCS) suggested axonal degeneration of the peripheral motor nerves and needle electromyography (EMG) revealed chronic neurogenic changes in probands. Open sural nerve biopsy for proband 2 and the mother of proband 1 showed mild to moderate loss of myelinated nerve fibers with some nerve fiber regeneration. A novel p.V97L in HSPB1 was identified in proband 3, the other two variants (p.P182A and p.R127W) in HSPB1 have been reported previously. The functional studies showed that expressing mutant p.V97L HSPB1 in SH-SY5Y cells displayed a decreased cell activity and increased apoptosis under stress condition. Our study expands the clinical phenotypic spectrum and etiological spectrum of HSPB1 mutation.

Keywords: CMT2F; HSPB1; dHMN; heat shock protein; restless legs syndrome.

Figure 1

The family tree of three families (A,C,E), the Sanger sequencing verification results of probands (B,D,F), and clinical characterizations (G–I). (G) Tibialis anterior muscle and first dorsal interosseous muscle (H) atrophy of F1−II−2. (I). Bilateral proximal and distal lower limbs atrophy of proband 3. An arrow indicates the proband; square, male; circle, female; a filled symbol indicates affected; and an arrow through a symbol indicates the individual is deceased; +/− heterozygous, −/− homozygous normal for the HSPB1 mutation.

Figure 2

Sural nerve biopsy of F1-II-2 (A) and F2-II-7 (B). (A) Mild to moderate decrease of large myelinated fibers, clusters of regeneration (thick arrow) and demyelination (thin arrow) (bar = 5 μm). (B) Decrease of myelinated nerve fibers with some nerve fiber regeneration (bar = 10 μm).

Figure 3

Effect of HSPB1-p.V97L mutation on H₂O₂-treated SH-SY5Y cells. (A) Effect of HSPB1-p.V97L mutation on cell survival using CCK-8 cell viability assay. SH-SY5Y cells were transiently transfected with plasmid vector expressing p.V97L mutant HSPB1 or wild-type HSPB1 for 24 h or 48 h and then incubated with H₂O₂ (300 μM) for 24 h; (B) cell morphology was observed with an inverted phase-contrast microscope. Control = SH-SY5Y cells. Data are presented as the means ± SEM from three independent experiments performed in triplicate. ns, no significance; * p < 0.05; *** p < 0.001. Scale bar = 50 µm.

Figure 4

Effects of HSPB1-p.V97L mutation on H₂O₂-induced apoptosis in SH-SY5Y cells. Cells were transfected with mutated HSPB1 or wild-type HSPB1 for 48 h followed by H₂O₂ (300 µM) incubation for 24 h. (A) Representative photographs of fluorescent staining for DAPI (blue) and TUNEL (red). (B) Representative bands of western blot. (C–E) Quantitative analysis of the western blot bands. Control = SH-SY5Y cells. Data are presented as the means ± SEM of three independent experiments. ns, no significance, * p < 0.05; *** p < 0.001. Scale bar = 50 µm.

Figure 5

The schematic diagram of HSPB1 structure with mutations. Full length of HSPB1 (NM_001540.5) in brown consists of 205 amino acids. * stop codons; red, reported here; yellow, AR inheritance; purple, UMN involvement; underline, ALS-like phenotype.