DNAJB2-related Charcot-Marie-Tooth disease type 2: Pathomechanism insights and phenotypic spectrum widening

Abstract

Background and purpose: Mutations in DNAJB2 are associated with autosomal recessive hereditary motor neuropathies/ Charcot-Marie-Tooth disease type 2 (CMT2). We describe an Italian family with CMT2 due to a homozygous DNAJB2 mutation and provide insight into the pathomechanisms.

Methods: Patients with DNAJB2 mutations were characterized clinically, electrophysiologically and by means of skin biopsy. mRNA and protein levels were studied in lymphoblastoid cells (LCLs) from patients and controls.

Results: Three affected siblings were found to carry a homozygous DNAJB2 null mutation segregating with the disease. The disease manifested in the second to third decade of life. Clinical examination showed severe weakness of the thigh muscles and complete loss of movement in the foot and leg muscles. Sensation was reduced in the lower limbs. All patients had severe hearing loss and the proband also had Parkinson's disease (PD). Nerve conduction studies showed an axonal motor and sensory length-dependent polyneuropathy. DNAJB2 expression studies revealed reduced mRNA levels and the absence of the protein in the homozygous subject in both LCLs and skin biopsy. Interestingly, we detected phospho-alpha-synuclein deposits in the proband, as already seen in PD patients, and demonstrated TDP-43 accumulation in patients' skin.

Conclusions: Our results broaden the clinical spectrum of DNAJB2-related neuropathies and provide evidence that DNAJB2 mutations should be taken into account as another causative gene of CMT2 with hearing loss and parkinsonism. The mutation likely acts through a loss-of-function mechanism, leading to toxic protein aggregation such as TDP-43. The associated parkinsonism resembles the classic PD form with the addition of abnormal accumulation of phospho-alpha-synuclein.

Keywords: Parkinson's disease; genetic and inherited disorders; neuropathology; peripheral neuropathies.

FIGURE 1

Pedigree, DNA sequencing and schematic representation of DNAJB2 protein domain structure. (a) Family pedigree. (b) Sanger chromatograms of patient II.4 with the homozygous (HOM) c.145delG variant in the DNAJB2 gene, patient II.3 (heterozygous [HET]) and II.5 (wild‐type [WT]). (c) DNAJB2 protein domains, the alternatively spliced C‐terminal parts of the two isoforms and the residue harboring the disease mutation (p. Val49TrpfsTer25) are indicated. The J, G/F common region, and C‐terminal domains (CTD), the Ser/Thr‐rich region, two ubiquitin interacting motifs (UIM) and the C‐terminal geranylgeranyl (GG) anchor of DNAJB2b are shown. Partly modified from Sarparanta et al., Int J Mol Sci. 2020; 21: 1409

FIGURE 2

Dat‐Scan, muscle computed tomography (CT) and sural nerve biopsy. (a) Muscle CT of patient II.2 showing severe fatty replacement of thigh and leg muscles, in both the anterior and posterior compartments. (b) Dat‐Scan of patient II.2 showing a severe, bilateral but asymmetric reduction of tracer uptake indicative of presynaptic dopaminergic terminal degeneration. (c) Sural nerve biopsy of patient II.1, taken at age 36 years. Semithin sections, stained with toluidine blue, disclosing a mild‐to‐moderate reduction of nerve fiber density, mainly involving the fibers of large caliber. A few fibers had thin myelin in relation to the axon diameter (arrow). Some fibers showed evidence of acute axonal degeneration and there were rare regeneration clusters. Scale bar = 15 μm. CTRL, control

FIGURE 3

DNAJB2 mRNA and protein levels, expression of DNAJB2 protein in skin. (a) DNAJB2 mRNA and protein expression analysis in lymphoblastoid cell lines (LCLs) from an affected patient (II.4), an unaffected carrier (III.1) and control subjects. It shows in the homozygous subject residual mRNA level of 33% and the absence of the protein, and in the heterozygous one 65% of mRNA level and of 40% of the protein. DNAJB2 mRNA level was normalized to GAPDH mRNA level. Bars and vertical lines indicate mean and ±1 SD of three independent experiments, respectively. Asterisks indicate a statistically significant (***p ≤ 0.001) difference from controls, as determined by Student's t‐test. (b) Immunoblot analysis from LCLs and skin. DNAJB2 protein expression was missing in both LCLs and skin from the homozygous patients (II.1 and II.4) and significantly reduced in the LCLs from the heterozygous carrier (III.1). Beta‐actin expression was normal in all the samples. (c) Immunohistochemical staining on skin biopsy sections using antibodies direct to neurofilament‐200 (in green) and DNAJB2 protein (in red) in both patient II.4 and control. Sections from control (upper panel) demonstrated many axons labeled by the axonal marker Neurofilament heavy chain (NF‐H) and DNAJB2 antibodies, while in the patient (lower panel) axons were labeled with antibodies to NF‐H but not by DNAJB2 antibodies. Scale bar = 10 μm. CTRL, control

FIGURE 4

TDP‐43 expression and phospho‐alpha‐synuclein aggregates in patients’ skin. (a) Western blot analysis of phospho‐TDP43 in the skin tissue of patients II.1, II.2 and control. TDP‐43 is expressed in patients’ skin and absent in the control. Beta‐actin was used as loading control. (b) Immunohistochemical staining on skin biopsy sections using antibodies direct to phospho‐alpha‐synuclein (p‐alpha‐syn; in red), PGP 9.5 (pan‐axonal marker; in green) and P0 (myelinated fibers; in blue) in patient II.4, patient II.2, who had also Parkinson disease, and control. The analysis showed in patient II.2 aggregates of p‐alpha‐syn which were absent in both patient II.4 and control. In the true intraneural deposition, the p‐alpha‐syn and PGP 9.5 signals are colocalized (arrowhead) and continuous along the nerve fiber; otherwise, p‐alpha‐syn signal without colocalized PGP 9.5 staining (asterisk) is a non‐specifc signal due to fuorophore precipitates that often presents a dot‐like staining distribution. Scale bar = 10 μm