Targeted PMP22 TATA-box editing by CRISPR/Cas9 reduces demyelinating neuropathy of Charcot-Marie-Tooth disease type 1A in mice.

Charcot-Marie-Tooth 1A (CMT1A) is the most common inherited neuropathy without a known therapy, which is caused by a 1.4 Mb duplication on human chromosome 17, which includes the gene encoding the peripheral myelin protein of 22 kDa (PMP22). Overexpressed PMP22 protein from its gene duplication is thought to cause demyelination and subsequently axonal degeneration in the peripheral nervous system (PNS). Here, we targeted TATA-box of human PMP22 promoter to normalize overexpressed PMP22 level in C22 mice, a mouse model of CMT1A harboring multiple copies of human PMP22. Direct local intraneural delivery of CRISPR/Cas9 designed to target TATA-box of PMP22 before the onset of disease, downregulates gene expression of PMP22 and preserves both myelin and axons. Notably, the same approach was effective in partial rescue of demyelination even after the onset of disease. Collectively, our data present a proof-of-concept that CRISPR/Cas9-mediated targeting of TATA-box can be utilized to treat CMT1A.

Small heat shock protein B3 (HSPB3) mutation in an axonal Charcot-Marie-Tooth disease family.

Heat shock protein B3 (HSPB3) gene encodes a small heat-shock protein 27-like protein which has a high sequence homology with HSPB1. A mutation in the HSPB3 was reported as the putative underlying cause of distal hereditary motor neuropathy 2C (dHMN2C) in 2010. We identified a heterozygous mutation (c.352T>C, p.Tyr118His) in the HSPB3 from a Charcot-Marie-Tooth disease type 2 (CMT2) family by the method of targeted next generation sequencing. The mutation was located in the well conserved alpha-crystalline domain, and several in silico predictions indicated a pathogenic effect of the mutation. Clinical and electrophysiological features of the patients indicated the axonal type of CMT. Clinical symptoms without sensory involvements were similar between the present family and the previous family. Mutations in the HSPB1 and HSPB8 genes have been reported to be relevant with both types of CMT2 and dHMN. Our findings will help in the molecular diagnosis of CMT2 by expanding the phenotypic range due to the HSPB3 mutations.

Clinical characterization and genetic analysis of Korean patients with X-linked Charcot-Marie-Tooth disease type 1.

Mutations in the gap junction protein beta 1 gene (GJB1) cause X-linked Charcot-Marie-Tooth disease type 1 (CMTX1). CMTX1 is representative of the intermediate type of CMT, having both demyelinating and axonal neuropathic features. We analyzed the clinical and genetic characterization of 128 patients with CMTX1 from 63 unrelated families. Genetic analysis revealed a total of 43 mutations including 6 novel mutations. Ten mutations were found from two or more unrelated families. p.V95M was most frequently observed. The frequency of CMTX1 was 9.6% of total Korean CMT family and was 14.8% when calculated within genetically identified cases. Among 67 male and 61 female patients, 22 females were asymptomatic. A high-arched foot, ataxia, and tremor were observed in 87%, 41%, and 35% of the patients, respectively. In the male patients, functional disability scale, CMT neuropathy score, and compound muscle action potential of the median/ulnar nerves were more severely affected than in the female patients. This study provides a comprehensive summary of the clinical features and spectrum of GJB1 gene mutations in Korean CMTX1 patients.

X-linked Charcot-Marie-Tooth disease type 6 (CMTX6) patients with a p.R158H mutation in the pyruvate dehydrogenase kinase isoenzyme 3 gene.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. Mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene have been found to cause X-linked dominant CMT type 6 (CMTX6). This study identified the p.R158H PDK3 mutation after screening 67 probable X-linked CMT families. The mutation fully segregated with the phenotype, and genotyping the family indicated the mutation arose on a different haplotype compared with the original Australian CMTX6 family. Results of bisulphite sequencing suggest that methylated deamination of a CpG dinucleotide may cause the recurrent p.R158H mutation. The frequency of the p.R158H PDK3 mutation in Koreans is very rare. Magnetic resonance imaging revealed fatty infiltration involving distal muscles in the lower extremities. In addition, fatty infiltrations were predominantly observed in the soleus muscles, with a lesser extent in tibialis anterior muscles. This differs from demyelinating CMT1A patients and is similar to axonal CMT2A patients. The clinical, neuroimaging, and electrophysiological findings from a second CMTX6 family with the p.R158H PDK3 mutation were similar to the axonal neuropathy reported in the Australian family.

A novel INF2 mutation in a Korean family with autosomal dominant intermediate Charcot-Marie-Tooth disease and focal segmental glomerulosclerosis.

Mutations in the inverted formin-2 (INF2) gene were recently identified in patients with autosomal dominant intermediate Charcot-Marie-Tooth (DI-CMT) disease and focal segmental glomerulosclerosis (FSGS). Here, we identified a novel p.L132P INF2 mutation in a Korean family with DI-CMT and FSGS by whole-exome sequencing. This mutation was cosegregated with affected individuals in the family and was not found in the 300 controls. The two affected members exhibited juvenile onset sensorimotor polyneuropathy and FSGS. Nerve conduction studies showed an intermediate range of motor nerve conduction velocities. We report a novel INF2 mutation in a family with DI-CMT and FSGS as the first case in Koreans. The INF2 mutation appears to be a major cause of CMT with FSGS.

Inhibition of thrombin-induced microglial activation and NADPH oxidase by minocycline protects dopaminergic neurons in the substantia nigra in vivo.

The present study shows that activation of microglial NADPH oxidase and production of reactive oxygen species (ROS) is associated with thrombin-induced degeneration of nigral dopaminergic neurons in vivo. Seven days after thrombin injection in the rat substantia nigra (SN), tyrosine hydroxylase immunocytochemistry showed a significant loss of nigral dopaminergic neurons. This cell death was accompanied by localization of terminal deoxynucleotidyl transferase-mediated fluorecein UTP nick-end labelling (TUNEL) staining within dopaminergic neurons. This neurotoxicity was antagonized by the semisynthetic tetracycline derivative, minocycline, and the observed neuroprotective effects were associated with the ability of minocycline to suppress NADPH oxidase-derived ROS production and pro-inflammatory cytokine expression, including interleukin-1beta and inducible nitric oxide synthase, from activated microglia. These results suggest that microglial NADPH oxidase may be a viable target for neuroprotection against oxidative damage.