Mkrn2 deficiency induces teratozoospermia and male infertility through p53/PERP-mediated apoptosis in testis / 亚洲男科学杂志(英文版)

The apoptosis that occurs in the immature testis under physiological conditions is necessary for male germ cell development, whereas improper activation of apoptosis can impair spermatogenesis and cause defects in reproduction. We previously demonstrated that in mice, the makorin-2 (Mkrn 2) gene is expressed exclusively in the testis and its deletion leads to male infertility. To understand the potential molecular mechanism, in this study, we found that levels of apoptosis in the testis were abnormally high in the absence of Mkrn 2. To identify specific gene(s) involved, we performed digital gene expression profiling (DGE) and pathway analysis via gene set enrichment analysis (GSEA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and we found that MKRN2 inhibits p53 apoptosis effector related to PMP22 (PERP) expression and that levels of the protein in sperm samples have an inverse correlation with infertility levels. GSEA additionally indicated that PERP is a negative regulator of spermatogenesis and that its ectopic expression induces male infertility. Further, Gene Expression Omnibus (GEO) dataset analysis showed that p53, upstream of PERP, was upregulated in oligoasthenoteratozoospermia (OAT). These observations suggest that Mkrn 2 is crucial for protecting germ cells from excessive apoptosis and implicate Mkrn 2-based suppression of the p53/PERP signaling pathway in spermatogenesis and male fertility.

miR-381 Attenuates Peripheral Neuropathic Phenotype Caused by Overexpression of PMP22

Charcot-Marie Tooth disease type 1A (CMT1A), the major type of CMT, is caused by duplication of peripheral myelin protein 22 (PMP22) gene whose overexpression causes structural and functional abnormalities in myelination. We investigated whether miRNA-mediated regulation of PMP22 expression could reduce the expression level of PMP22, thereby alleviating the demyelinating neuropathic phenotype of CMT1A. We found that several miRNAs were down-regulated in C22 mouse, a CMT1A mouse model. Among them, miR-381 could target 3′ untranslated region (3′UTR) of PMP22 in vitro based on Western botting and quantitative Real Time-PCR (qRT-PCR) results. In vivo efficacy of miR-381 was assessed by administration of LV-miR-381, an miR-381 expressing lentiviral vector, into the sciatic nerve of C22 mice by a single injection at postnatal day 6 (p6). Administration of LV-miR-381 reduced expression level of PMP22 along with elevated level of miR-381 in the sciatic nerve. Rotarod performance analysis revealed that locomotor coordination of LV-miR-381 administered C22 mice was significantly enhanced from 8 weeks post administration. Electrophysiologically, increased motor nerve conduction velocity was observed in treated mice. Histologically, toluidine blue staining and electron microscopy revealed that structural abnormalities of myelination were improved in sciatic nerves of LV-miR-381 treated mice. Therefore, delivery of miR-381 ameliorated the phenotype of peripheral neuropathy in CMT1A mouse model by down-regulating PMP22 expression. These data suggest that miRNA can be used as a potent therapeutic strategy to control diseases with copy number variations such as CMT1A.

Development of cell models for high-throughput screening system of Charcot-Marie-Tooth disease type 1

PURPOSE: Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy mainly divided into CMT type 1 (CMT1) and CMT2 according to the phenotype and genotype. Although molecular pathologies for each genetic causative have not been revealed in CMT2, the correlation between cell death and accumulation of misfolded proteins in the endoplasmic reticulum (ER) of Schwann cells is well documented in CMT1. Establishment of in vitro models of ER stress-mediated Schwann cell death might be useful in developing drug-screening systems for the treatment of CMT1. MATERIALS AND METHODS: To develop high-throughput screening (HTS) systems for CMT1, we generated cell models using transient expression of mutant proteins and chemical induction. RESULTS: Overexpression of wild type and mutant peripheral myelin protein 22 (PMP22) induced ER stress. Similar results were obtained from mutant myelin protein zero (MPZ) proteins. Protein localization revealed that expressed mutant PMP22 and MPZ proteins accumulated in the ER of Schwann cells. Overexpression of wild type and L16P mutant PMP22 also reduced cell viability, implying protein accumulation-mediated ER stress causes cell death. To develop more stable screening systems, we mimicked the ER stress-mediated cell death in Schwann cells using ER stress inducing chemicals. Thapsigargin treatment caused cell death via ER stress in a dose dependent manner, which was measured by expression of ER stress markers. CONCLUSION: We have developed genetically and chemically induced ER stress models using Schwann cells. Application of these models to HTS systems might facilitate the elucidation of molecular pathology and development of therapeutic options for CMT1.

EMP3 Overexpression in Primary Breast Carcinomas is not Associated with Epigenetic Aberrations

Epithelial membrane protein 3 (EMP3) is a trans-membrane signaling molecule with important roles in the regulation of apoptosis, differentiation and invasion of cancer cells, but the detailed is largely still unknown. We analyzed the mRNA levels and methylation statuses of EMP3 in 63 primary breast carcinomas and assessed their correlations with clinicopathologic variables. The expression of EMP3 mRNA in primary breast carcinomas was significantly higher than the expression of 20 normal breast tissues (p<10(-7)). EMP3 overexpression in breast carcinomas was significantly related to histological grade III (p=3.9X10(-7)), lymph node metastasis (p= 0.003), and strong Her-2 expression (p=3.3X10(-6)). Hypermethylation frequencies of EMP3 were detected in 36.5% of breast carcinomas by methylation-specific polymerase chain reaction. However, no significant correlations were found between methylation status of EMP3 and mRNA expression levels as well as other clinical parameters. In conclusion, EMP3 may be a novel marker of tumor aggressiveness. Overexpression of EMP3 in primary breast carcinoma is not associated with DNA methylation.

Charcot-Marie-Tooth 1A Concurrent with Schwannomas of the Spinal Cord and Median Nerve

We identified Charcot-Marie-Tooth disease type 1A (CMT1A) in a family with schwannomas in the spinal cord and median nerve. The CMT1A in this family showed an autosomal dominant pattern, like other CMT patients with PMP22 duplication, and the family also indicated a possible genetic predisposition to schwannomas by 'mother-to-son' transmission. CMT1A is mainly caused by duplication of chromosome 17p11.2-p12 (PMP22 gene duplication). A schwannoma is a benign encapsulated tumor originating from a Schwann cell. A case of hereditary neuropathy with liability to pressure palsies (HNPP) concurrent with schwannoma has been previously reported. Although it seems that the co-occurrence of CMT1A and schwannomas in a family would be the result of independent events, we could not completely ignore the possibility that the coincidence of two diseases might be due to a shared genetic background.

Clinical Phenotype of Peripheral Myelin Protein 22 Gene Duplication Abnormality in Charcot-Marie-Tooth Disease / 中国康复理论与实践

@#: Objective To analyse the characteristics of symptoms, signs and electrophysiology in Charcot-Marie-Tooth disease (CMT) with peripheral myelin protein 22 (PMP22) gene duplication abnormality.Methods 61 patients with CMT, 14 patients with family history and 47 sporadic patients were included. PMP22 gene duplication fragment was detected with PCR-double enzyme cutting assay. Medical history, signs were collected. Some of them received lumbar puncture and sural nerve pathological examination. Results The main clinical manifestation of the patients with PMP22 gene duplication abnormlity were asthenia of both lower extremities, especially dorsiflexion of foot, accompanied with distal atrophy (especially bilateral legs), some with upper extremity distal atrophy, ankle hyporeflexia or vanished and sensory disturbance. Protein in cerebrospinal fluid may increase, giant potential and conduction velocity of sensory and motor nerve decreased. Sural nerve biopsies revealed demyelination accompanied with axonal degeneration.Conclusion The main clinical manifestation of patients with PMP22 gene duplication abnormlity is charactered as the distal atrophy and asthenia of lower limbs, accompanied with sensory abnormlity. Myelin sheath and axonal alteration were found in electromyogram and peripheral nerve pathology.

A Family Harboring CMT1A Duplication and HNPP Deletion

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with duplication of chromosome 17p11.2-p12, whereas hereditary neuropathy with liability to pressure palsies (HNPP), which is an autosomal dominant neuropathy showing characteristics of recurrent pressure palsies, is associated with 17p11.2-p12 deletion. An altered gene dosage of PMP22 is believed to the main cause underlying the CMT1A and HNPP phenotypes. Although CMT1A and HNPP are associated with the same locus, there has been no report of these two mutations within a single family. We report a rare family harboring CMT1A duplication and HNPP deletion.

Charcot-Marie-Tooth type 1A Patient with a Novel Frame Shift Mutation (Ala106fs) in the PMP22 Gene

Charcot-Marie-Tooth disease (CMT) with hearing impairment is a clinically distinct rare entity described in a few families, usually with a demyelinating neuropathy. The molecular basis for this disease has not been established with certainty. Audiological evaluation has revealed auditory neuropathy in the affected individual. We report a CMT1A family with sensorineural hearing loss and a novel frame shift mutation Ala106fs (318delT) in the PMP22 gene.

Comparison of Clinical and Electrophysiological Characteristics between CMTX with Cx32 Missense Mutation and CMT1A with PMP22 Duplication

BACKGROUND: Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous disorder. Connexin32 (Cx32) gene mutations on Xq13.1 cause the X-linked form of CMT disease, and PMP22 gene duplication on 17p11.2-p12 causes CMT1A. The aim of the present study is to determine the clinical and electrophysiological characteristics between X-linked CMT patients with Cx32 missense mutations and CMT1A patients with PMP22 duplications. METHODS: We screened for 17p11.2-p12 duplication, and for point mutations in Cx32 genes of 48 Korean CMT families. Both neurological examination and nerve conduction studies were performed in all patients. RESULTS: Frequency of CMTX (6.3%) in our study was similar to Japanese, and was lower than those in European peoples. CMTX patients displayed no man-to-man transmission, and had cranial nerve involvement. CMTX patients showed more wide range of motor and sensory nerve conduction velocities than CMT1A patients. We found one family with axonal neuropathy and two families with demyelinating neuropathy in CMTX patients. CONCLUSIONS: Our findings suggest that mutations in Cx32 are probably less frequent in Asian CMT patients than European patients, and CMTX neuropathy is intermediary between CMT1 and CMT2. In addition, inheritance pattern and cranial nerve involvement are useful in differentiating CMTX from CMT1A with duplication.

A Case of Charcot-Marie-Tooth Disease type 1A with Guillain-Barre Syndrome

Charcot-Marie-Tooth disease (CMTD) is a hereditary neuropathy with slow progression, whereas Guillain-Barre syndrome is an acute acquired neuropathy of immunopathogenesis. A 17 year-old girl with hammertoes and pes cavus developed an acute quadriparesis. The duplication of PMP-22 exons in 17p12-p11.2 was confirmed by genetic study. After Intravenous ingection of immunoglobulin, she recovered to normal activity within one month with improvement of nerve conduction study. We should be concerned with the possibility of Guillain-Barre syndrome if there is rapidly progressive exacerbation on CMTD.