Molecular analysis in Japanese patients with Charcot-Marie-Tooth disease: DGGE analysis for PMP22, MPZ,and Cx32/GJB1 mutations

Charcot-Marie-Tooth disease (CMT) is a heterogeneous disorder and is traditionally classified into two major types, CMT type 1 (CMT1) and CMT type 2 (CMT2). Most CMT1 patients are associated with the duplication of 17p11.2-p12 (CMT1A duplication) and small numbers of patients have mutations of the peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ), connexin 32 (Cx32/GJB1), and early growth response 2 (EGR2) genes. Some mutations of MPZ and Cx32 were also associated with the clinical CMT2 phenotype. We constructed denaturing gradient gel electrophoresis (DGGE) analysis as a screening method for PMP22, MPZ, and Cx32 mutations and studied 161 CMT patients without CMT1A duplication. We detected 27 mutations of three genes including 15 novel mutations; six of PMP22, three of MPZ, and six of Cx32. We finally identified 21 causative mutations in 22 unrelated patients and five polymorphic mutations. Eighteen of 22 patients carrying PMP22, MPZ, or Cx32 mutations presented with CMT1 and four of them with MPZ or Cx32 mutations presented with the CMT2 phenotype. DGGE analysis was sensitive for screening for those gene mutations, but causative gene mutation was not identified in many of the Japanese patients with CMT, especially with CMT1. Other candidate genes should be studied to elucidate the genetic basis of Japanese CMT patients.     

Postural stabilization and balance assessment in Charcot–Marie–Tooth 1A subjects

The aim of the present study was to assess postural stabilization skill in adult subjects affected by Charcot–Marie–Tooth disease (CMT) type 1A. For this purpose ground reaction force (GRF) was measured by means of a piezoelectric force platform during the sit-to-stand (STS) movement, until a steady state erect posture was achieved. Specific indexes to quantify Centre of Mass acceleration, both during postural stabilization and during quiet standing, were computed using a mathematical model. Forty-seven CMT1A subjects were recruited for the study, and the control group was formed by forty-one age- and sex-matched healthy subjects.The results show that CMT1A subjects are less stable than controls during the quiet stance. Greater difficulty (high values of Y_inf, the final instability rate) to maintain erect posture appears to be mainly associated with plantar-flexor muscle weakness, rather than to damage of the proprioceptive system. The worst performances shown by CMT1A subjects in the stabilization phase (high values of I, the global index of postural stabilization performance) seem to be associated with reduced muscle strength and the loss of large sensory nerve fibres.Distal muscle weakness appears to affect both postural stabilization and quiet erect posture. The presented protocol and the analysis of postural stabilization parameters provide useful information on CMT1A balance disorders.     

Effects of magnesium and lactose supplementation on bone metabolism in the X-linked hypophosphatemic mouse

Besides rickets and osteomalacia, the X-linked hypophosphatemic male mouse ($\text{Hyp}\text{Y}$) presents with low serum calcium (Ca) and increased urinary hydroxyproline (OH-Pro) excretion, suggesting a parathyroid hormone (PTH)-stimulated bone resorption despite reduced magnesium (Mg) bone content. In this study, we have investigated by histochemical methods the state of bone resorption in 50-day-old untreated $\text{Hyp}\text{Y}$ mice and the effects of 4 wk of Mg therapy or dietary lactose supplementation on bone formation and resorption. Mineral and skeletal changes were evaluated on serum, urinary and bone ash concentrations of Ca, phosphorus (P) and Mg, and by histomorphometric analysis of tetracycline double labeled undecalcified caudal vertebrae. The number of acid phosphatase stained chondroclasts and osteoclasts was lower than normal in untreated $\text{Hyp}\text{Y}$ and was restored after Mg therapy while the osteoclastic surface was increased above normal. Accordingly, serum P and urinary Ca, P, Mg, cAMP and OH-Pro were increased while $\text{TmP}\text{GFR}$ was unchanged. On the other hand, dietary lactose corrected serum Ca which probably suppressed PTH secretion since the renal P conservation was improved and the osteoclast number and the osteoclastic surface were decreased. Both treatments reduced the growthplate and osteoid seam thickness and increased the bone calcification rate. The results indicate that the low skeletal Mg present in $\text{Hyp}\text{Y}$ partially impairs bone responsiveness to PTH since Mg therapy restored the osteoclastic bone resorption which secondarily provided new minerals for bone mineralization. The greater than normal bone resorption found in Mg $\text{treated}\text{-}\text{Hyp}\text{Y}$ and the decreased bone resorption observed in lactose treated animals indicate that the chronic hypocalcemia induces secondary hyperparathyroidism in $\text{Hyp}\text{Y}$ mice.     

Aberrant trafficking of a Leu89Pro connexin32 mutant associated with X-linked dominant Charcot–Marie–Tooth disease

Objective: To determine the functional abnormalities of the Leu89Pro mutation in connexin32 (CX32), which we have previously reported is present within an X-linked dominant Charcot–Marie–Tooth disease family. In this family, male patients were moderately to severely affected.

Methods: We performed immunofluorescence to investigate whether the Leu89Pro CX32 protein was transported to the cell membrane in HeLa and Schwann cells. First, we constructed the eukaryotic express plasmids expressing CX32 (wild-type or Leu89Pro) and enhanced green fluorescent protein by the gene recombination technology. Then the recombinant plasmids were transiently transfected into communication-incompetent HeLa cells and human Schwann cells by the lipofectamine method. Later, we double-labeled cells for both CX32 and markers of the ER (calnexin) or the Golgi (58-kDa protein) at 24 h or 48 h. The images were collected using a Leica TCS SP5 II confocal microscope.

Results: The mutant CX32 protein was localized in the endoplasmic reticulum and failed to reach the cell membrane to form gap junctions.

Conclusion: Our results indicated that the Leu89Pro substitution in the second transmembrane domain of CX32 disrupts the trafficking of the protein, inhibiting the assembly of CX32 gap junctions, which in turn may result in peripheral neuropathy. This functional abnormality may explain the moderate to severe phenotype seen in Leu89Pro patients, and as such represents a promising therapeutic target in the treatment of this subset of CMTX patients.     

Neurofilament light mutation causes hereditary motor and sensory neuropathy with pyramidal signs

To identify novel mutations causing hereditary motor and sensory neuropathy (HMSN) with pyramidal signs, a variant of Charcot‐Marie‐Tooth disease (CMT), we screened 28 CMT and related genes in four members of an affected Japanese family. Clinical features included weakness of distal lower limb muscles, foot deformity, and mild sensory loss, then late onset of progressive spasticity. Electrophysiological studies revealed widespread neuropathy. Electron microscopic analysis showed abnormal mitochondria and mitochondrial accumulation in the neurons and Schwann cells. Brain magnetic resonance imaging (MRI) revealed an abnormally thin corpus callosum. In all four, microarrays detected a novel heterozygous missense mutation c.1166A>G (p.Y389C) in the gene encoding the light‐chain neurofilament protein (NEFL), indicating that NEFL mutations can result in a HMSN with pyramidal signs phenotype.     

Characterization of the mitofusin 2 R94W mutation in a knock‐in mouse model

Charcot‐Marie‐Tooth disease (CMT) comprises a group of heterogeneous peripheral axonopathies affecting 1 in 2,500 individuals. As mutations in several genes cause axonal degeneration in CMT type 2, mutations in mitofusin 2 (MFN2) account for approximately 90% of the most severe cases, making it the most common cause of inherited peripheral axonal degeneration. MFN2 is an integral mitochondrial outer membrane protein that plays a major role in mitochondrial fusion and motility; yet the mechanism by which dominant mutations in this protein lead to neurodegeneration is still not fully understood. Furthermore, future pre‐clinical drug trials will be in need of validated rodent models. We have generated a Mfn2 knock‐in mouse model expressing Mfn2^R94W, which was originally identified in CMT patients. We have performed behavioral, morphological, and biochemical studies to investigate the consequences of this mutation. Homozygous inheritance leads to premature death at P1, as well as mitochondrial dysfunction, including increased mitochondrial fragmentation in mouse embryonic fibroblasts and decreased ATP levels in newborn brains. Mfn2^R94W heterozygous mice show histopathology and age‐dependent open‐field test abnormalities, which support a mild peripheral neuropathy. Although behavior does not mimic the severity of the human disease phenotype, this mouse can provide useful tissues for studying molecular pathways associated with MFN2 point mutations.     

Psychometrics evaluation of Charcot‐Marie‐Tooth Neuropathy Score (CMTNSv2) second version,using Rasch analysis

Charcot‐Marie‐Tooth Neuropathy Score second version (CMTNSv2) is a validated clinical outcome measure developed for use in clinical trials to monitor disease impairment and progression in affected CMT patients. Currently, all items of CMTNSv2 have identical contribution to the total score. We used Rasch analysis to further explore psychometric properties of CMTNSv2, and in particular, category response functioning, and their weight on the overall disease progression. Weighted category responses represent a more accurate estimate of actual values measuring disease severity and therefore could potentially be used in improving the current version. Copyright © 2014 John Wiley & Sons, Ltd.