Genetic and Clinical Studies of Peripheral Neuropathies with Three Small Heat Shock Protein Gene Variants in Korea

Abstract

Small heat shock proteins (sHSPs) are ATP-independent chaperones that help correct the folding of denatured proteins and protect cells from stress. Mutations in HSPB1, HSPB8, and HSPB3 are implicated in inherited peripheral neuropathies (IPNs), such as Charcot-Marie-Tooth disease type 2 (CMT2) and distal hereditary motor neuropathies (dHMN). This study, using whole exome sequencing or targeted gene sequencing, identified 9 pathogenic or likely pathogenic variants in these three sHSP genes from 11 Korean IPN families. Most variants were located in the evolutionally well conserved α-crystallin domain, except for p.P182S and p.S187L in HSPB1. As an atypical case, a patient with dHMN2 showed two compound heterozygous variants of p.R127Q and p.Y142H in HSPB1, suggesting a putative case of recessive inheritance, which requires additional research to confirm. Three HSPB8 variants were located in the p.K141 residue, which seemed to be a mutational hot spot. There were no significant differences between patient groups, which divided by sHSP genes for clinical symptoms such as onset age, severity, and nerve conduction. Early-onset patients showed a tendency of slightly decreased sensory nerve conduction values compared with late-onset patients. As a first Korean IPN cohort study examining sHSP genes, these results will, we believe, be helpful for molecular diagnosis and care of patients with CMT2 and dHMN.

Keywords: Charcot-Marie-Tooth disease type 2; HSPB1; HSPB3; HSPB8; Korean; distal hereditary motor neuropathies.

Figure 1

Inherited peripheral neuropathy families with small heat shock protein gene mutations. Genotypes of the interested variants are provided at the bottom of all examined family members. Arrows indicate the proband (unfilled symbols (□, ○): unaffected individuals; black-filled symbols (■, ●): affected individuals; gray-filled symbols: unaffected individuals having corresponding mutation; ˄: twins). (A) Pedigrees with HSPB1 mutations. (B) Pedigrees with HSPB8 mutations. (C) Pedigree with HSPB3 mutation.

Figure 2

Mutations in three small heat shock protein genes. (A) Chromatograms of the mutation sites. They were obtained by Sanger sequencing. Vertical arrows indicate the mutation sites (WT: wild type allele, Mut: mutant allele). (B) Conservation of amino acids in the mutation sites (red) and vicinant sequences. Reference sequences are: NP_001531.1 (Homo sapiens), XP_519162.3 (Pan troglodytes), NP_038588.2 (Mus musculus), NP_990621.1 (Gallus gallus), NP_001072817.1 (Xenopus tropicalis), NP_001008615.2 (Danio rerio), NP_001287001.1 (Drosophila melanogaster), NP_498776.1 (Caenorhabditis elegans) for HSPB1, NP_055180.1 (H. sapiens), XP_509417.1 (P. troglodytes), NP_109629.1 (M. musculus), XP_004934466.1 (G. gallus), NP_001005658.1 (X. tropicalis), NP_001094427.2 (D. rerio), NP_001027115.1 (D. melanogaster), NP_498776.1 (C. elegans) for HSPB8, NP_006299.1 (H. sapiens), XP_517764.2 (P. troglodytes), NP_064344.1 (M. musculus), XP_001231558.1 (G. gallus), XP_002941074.1 (X. tropicalis), NP_001092922.1 (D. rerio), NP_523999.1 (D. melanogaster), NP_498776.1 (C. elegans) for HSPB3. (C) Amino acid sequence conservation at the mutation sites (red) among human sHSP paralogues. Reference amino acid sequences are: HSPB2: NP_001532.1, HSPB4: NP_000385.1, HSPB5: NP_001276736.1, and HSPB6: NP_653218.1.

Figure 3

Schemes of three small heat shock proteins and predicted secondary structure of single strand DNA region for p.N138 to p.K141 residues in HSPB8. (A) Schematic diagrams of three sHSP proteins. Pathogenic or likely pathogenic variants identified in this study are indicated at the top of the diagrams (red). Some previously reported mutations are indicated at the bottom of the diagrams (ACD: α-crystallin domain, CTD: C-terminal domain, IxI/V: IxI/V motif within CTD, NTD: N-terminal domain, and WDPF: WDPF motif within NTD). (B) Predicted secondary structure of single strand DNA region for p.N138 to p.K141 residues in HSPB8. The nucleotides corresponding to p.N138 and p.K141 are indicated in yellow. The p.K141 was distinguished from p.N138 by a red box.

Figure 4

Predicted 3D conformational changes of neighboring structures of causative (pathogenic or likely pathogenic) mutation sites in three small heat shock proteins. Wild type protein structures and their mutant structures are provided up (or left) and down (or right) in each pair. The amino acid sites with mutation are indicated in pink. Hydrogen bonds are indicated by blue dotted lines, and carbon, hydrogen, nitrogen, and oxygen are indicated in green, gray, blue, and red, respectively. Noncovalent molecular interactions (cation–π interaction) are indicated by orange dotted lines. The crystal structures of α-crystallin domains are colored in yellow; N- and C-terminal domains are depicted in gray. Wild and mutant proteins are illustrated by ribbon diagrams. (A) HSPB1, (B) HSPB8, and (C) HSPB3.

Figure 5

Comparison of clinical and electrophysiological phenotypes between two patient groups having mutations alternatively in HSPB1 and HSPB8. The patients were classified into two groups of HSPB1 and HSPB8, then their phenotypic values were compared. (A) Age of onset, (B) FDS, (C) CMTNSv2, (D) median MNCV, and (E) median SNCV.

Figure 6

Correlation of clinical phenotypes according to onset ages (OA). (A) OA vs. FDS, (B) OA vs. CMTNSv2, (C) OA vs. median MNCV, (D) OA vs. median CMAP, (E) OA vs. median SNCV, and (F) OA vs. median SNAP.