Humans ; Neoplasms/genetics* ; Nuclear Proteins ; Co-Repressor Proteins ; Cell Line, Tumor ; Molecular Chaperones

OBJECTIVE: To analyze the distribution characteristics of hereditary peripheral neuropathy (HPN) pathogenic genes in Chinese Han population, and to explore the potential pathogenesis and treatment prospects of HPN and related diseases. METHODS: Six hundred and fifty-six index patients with HPN were enrolled in Peking University Third Hospital and China-Japan Friendship Hospital from January 2007 to May 2022. The PMP22 duplication and deletion mutations were screened and validated by multiplex ligation probe amplification technique. The next-generation sequencing gene panel or whole exome sequencing was used, and the suspected genes were validated by Sanger sequencing. RESULTS: Charcot-Marie-Tooth (CMT) accounted for 74.3% (495/666) of the patients with HPN, of whom 69.1% (342/495) were genetically confirmed. The most common genes of CMT were PMP22 duplication, MFN2 and GJB1 mutations, which accounted for 71.3% (244/342) of the patients with genetically confirmed CMT. Hereditary motor neuropathy (HMN) accounted for 16.1% (107/666) of HPN, and 43% (46/107) of HPN was genetically confirmed. The most common genes of HMN were HSPB1, aminoacyl tRNA synthetases and SORD mutations, which accounted for 56.5% (26/46) of the patients with genetically confirmed HMN. Most genes associated with HMN could cause different phenotypes. HMN and CMT shared many genes (e.g. HSPB1, GARS, IGHMBP2). Some genes associated with dHMN-plus shared genes associated with amyotrophic lateral sclerosis (KIF5A, FIG4, DCTN1, SETX, VRK1), hereditary spastic paraplegia (KIF5A, ZFYVE26, BSCL2) and spinal muscular atrophy (MORC2, IGHMBP, DNAJB2), suggesting that HMN was a continuum rather than a distinct entity. Hereditary sensor and autosomal neuropathy (HSAN) accounted for a small proportion of 2.6% (17/666) in HPN. The most common pathogenic gene was SPTLC1 mutation. TTR was the main gene causing hereditary amyloid peripheral neuropathy. The most common types of gene mutations were p.A117S and p.V50M. The symptoms were characterized by late-onset and prominent autonomic nerve involvement. CONCLUSION CMT and HMN are the most common diseases of HPN. There is a large overlap between HMN and motor-CMT2 pathogenic genes, and some HMN pathogenic genes overlap with amyotrophic lateral sclerosis, hereditary spastic hemiplegia and spinal muscular atrophy, suggesting that there may be a potential common pathogenic pathway between different diseases.
Amyotrophic Lateral Sclerosis ; Charcot-Marie-Tooth Disease/genetics* ; DNA Helicases/genetics* ; DNA-Binding Proteins/genetics* ; Flavoproteins ; HSP40 Heat-Shock Proteins ; Humans ; Intracellular Signaling Peptides and Proteins/genetics* ; Kinesins ; Ligases/genetics* ; Molecular Chaperones ; Multifunctional Enzymes ; Muscular Atrophy, Spinal/genetics* ; Mutation ; Phosphoric Monoester Hydrolases ; Protein Serine-Threonine Kinases ; RNA Helicases/genetics* ; RNA, Transfer ; Transcription Factors/genetics*

Apoptosis ; Human Umbilical Vein Endothelial Cells ; Humans ; Lipoproteins, LDL/adverse effects* ; MicroRNAs/genetics* ; Molecular Chaperones/genetics*

OBJECTIVE: To explore the association between rare HSPB1 variants and amyotrophic lateral sclerosis (ALS). METHODS: We performed next-generation sequencing for 166 Chinese ALS patients to screen for possible pathogenic rare variants of HSPB1. The control individuals were obtained from 1000 Genome Project and an in-house whole-exome sequencing database. The Sequence Kernel Association Test (SKAT) and the SKAT-optimal test (SKAT-O) were used to identify the association between rare HSPB1 variants and ALS. RESULTS: We identified 3 possible pathogenic rare variants of HSPB1 (all were missenses), including c.379C>T (p.R127W), c.446A>C (p.D149A) and c.451A>C (p.T151P). Compared with 1000 Genome Project, SKAT p=3.61×10 CONCLUSIONS Rare variants of HSPB1 are probably associated with the pathogenesis of ALS.
Amyotrophic Lateral Sclerosis/genetics* ; Asian Continental Ancestry Group ; Heat-Shock Proteins ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Humans ; Molecular Chaperones ; Phenotype

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.
Bacterial Proteins ; Diphtheria Toxin/genetics* ; Escherichia coli/genetics* ; Humans ; Molecular Chaperones/genetics* ; Recombinant Proteins/genetics*

OBJECTIVETo explore the feasibility of using PCR-based capillary electrophoresis method to analysis mutation of the TOR1A gene in a family affected with primary torsion dystonia (PTD).

METHODSPeripheral blood sample was collected from proband and amnionic fluid from her fetus for the extraction of DNA. The 5th exon of the TOR1A gene and its flanking sequences were amplified with PCR and analyzed with agarose electrophoresis, fluorescence labeled fragment analysis and Sanger sequencing.

RESULTSFluorescence labeled fragment analysis was performed through capillary electrophoresis, which showed that the proband carried a c.907_909delGAG (p.Glu303del) deletional mutation of the TOR1A gene. The result was verified by Sanger sequencing. The fetus DNA was also found with the same mutation by capillary electrophoresis, inferring that the fetus was probably affected with the disease.

CONCLUSIONThe mutation of c.907_909delGAG of the TOR1A gene was speculated as pathologic cause of proband in this family. Fragment analysis by capillary electrophoresis combined with DNA sequencing is an efficient test for small deletional mutations and feasible for its prenatal diagnosis.

Adult ; Dystonia ; diagnosis ; genetics ; Electrophoresis, Capillary ; Female ; Humans ; Molecular Chaperones ; genetics ; Mutation ; Prenatal Diagnosis ; Sequence Analysis, DNA

Objective: To investigate the expression characteristic of the Daxx gene in the mouse testis and its role in spermatogenesis. METHODS: Real-time PCR, Western blot and immunofluorescence were used in examining the expression characteristics of DAXX in the testis tissue from wild-type, Sertoli cell-specific androgen receptor knockout (SCARKO) and androgen receptor knockout (ARKO) mice at different postnatal weeks . RESULTS: The Daxx gene was highly expressed in the testis tissue and mainly in the nuclei of the wild-type mice at 4 postnatal weeks. Compared with the wild-type, the ARKO mice showed a markedly decreased expression of DAXX (0.299±0.026), which displayed a polar distribution in the spermatogenic cells (0.853±0.058) and exhibited no significant difference in the SCARKO mice (1.000±0.015). CONCLUSIONS The Daxx gene expression is the highest in the middle-stage development of the mouse testis, significantly decreased in ARKO mice as compared with the wild-type, and its location influenced by specific AR knockout in Sertoli cells. DAXX may be involved in the regulation of spermatogenesis in mice.
Animals ; Carrier Proteins ; genetics ; metabolism ; Cell Nucleus ; genetics ; metabolism ; Gene Expression ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Knockout ; Molecular Chaperones ; Nuclear Proteins ; genetics ; metabolism ; Receptors, Androgen ; genetics ; Sertoli Cells ; Spermatogenesis ; genetics ; Testis ; metabolism

OBJECTIVETo investigate the presence of Cosmc gene mutation in children with Henoch-Schönlein purpura (HSP) and the association between Cosmc gene mutation and the susceptibility to HSP.

MESULTSEighty-four children who were diagnosed with HSP between March 2014 and December 2015 were selected as the HSP group. Fifty-eight healthy volunteers matched for age and sex were enrolled as the control group. Fasting venous blood (5 mL) from the two groups was collected in EDTA anticoagulated tubes, followed by the isolation of peripheral blood mononuclear cells (PBMCs) through density gradient centrifugation. Genomic DNA was extracted from PBMCs according to the manufacturer's protocol, and the whole exon region of Cosmc gene was amplified by touch-down polymerase chain reaction (touch-down PCR). The PCR products were identified by 1% agarose gel and sequenced in order to further examine the association between Cosmc gene mutation and the susceptibility to HSP.

RESULTSSequencing results showed two mutations (c.393T>A and c.72A>G) of Cosmc gene in children with HSP. There were no significant differences in the genotype and allele frequencies at the two loci between the HSP and control groups, and this distribution was not associated with sex.

CONCLUSIONSThe mutations c.393T>A and c.72A>G in the exon region of Cosmc gene in children with HSP are not associated with the onset of HSP.

Child ; Child, Preschool ; Female ; Genetic Predisposition to Disease ; Humans ; Male ; Molecular Chaperones ; genetics ; Mutation ; Purpura, Schoenlein-Henoch ; etiology ; genetics

To analyze the differentially expressed proteins which interacted with NF-kappaB in the uterine lower segment smooth muscle tissues under different status of labor onset, and to provide a new foundation on the mechanisms for labor onset.
 Methods: NF-κB P65 protein expression in smooth muscle tissues from the term non-labor group, natural term labor group and drug-induced term labor group was analyzed by Western blot. Co-immunoprecipitation and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) were performed to detect the proteins interacting with NF-κB p65 in the NF-κB p65 complexes. The components of the complex were identified by LC-ESI-MS/MS (liquid chromatography-tandem electrospray mass spectrometry) and database analysis. The identified differentially expressed proteins were confirmed by Western blot.
 Results: Positive expression of NF-κB was detected in all of the three groups. 10 differentially expressed proteins were identified by LC-ESI-MS/MS in human lower segment myometrium tissues in the term non-labor group and natural term labor group, mean while, 5 differentially expressed proteins were identified in the term non-labor group and the drug-induced labor group. 3 differential expression proteins were detected in all of the 3 groups, including Heat shock 70, Annexin A6 and Desmin, which were verified by Western blot. These proteins were mainly involved in chaperone, signal transduction, cell structure, and energy metabolism process, respectively.
 Conclusion: NF-κB expressed in uterine smooth muscle cells is involved in the process of initiation and regulation of labor onset through a number of proteins relevant to signal transduction, cell structure and energy metabolism.
Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Energy Metabolism ; genetics ; Female ; Humans ; Immunoprecipitation ; Labor, Obstetric ; genetics ; Molecular Chaperones ; genetics ; Myocytes, Smooth Muscle ; Myometrium ; physiology ; NF-kappa B ; genetics ; physiology ; Pregnancy ; Protein Interaction Mapping ; Proteomics ; Signal Transduction ; genetics ; Tandem Mass Spectrometry ; Transcription Factor RelA

OBJECTIVETo identify the expression characteristics of the 1700001022RIK (RIKEN cDNA 1700001022) gene in mice and explore its function by bioinformatic analysis.

METHODSUsing the expression profile of gene microarray, we detected the expression of a new testis-specific gene, 1700001022RIK, in mice. We analyzed its expression characteristics in the testis tissue and their changes in different developmental stages of the testis by RT-PCR, real-time RT-PCR, Western blot, and immunohistochemistry. We performed bioinformatic analysis using a bioinformatic software.

RESULTSThe 1700001022RIK gene was specifically expressed in the mouse testis in an age-dependent manner, most highly in the adult mice. The 1700001022RIK protein was mainly expressed in the spermatogonia, spermatocytes, and round spermatids of the adult mice. Bioinformatic analysis showed that the 1700001022RIK protein amino acid sequence had a high similarity in human and mice, which indicated that this gene was highly conserved in mammals.

CONCLUSION1700001022RIK is a testis-specific gene mainly expressed in the spermatogonia, spermatocytes, and round spermatids of seminiferous tubules, which might be involved in the regulation of spermatogenesis.

Age Factors ; Animals ; Blotting, Western ; Computational Biology ; DNA, Complementary ; Gene Expression ; Genomics ; Male ; Mice ; Molecular Chaperones ; genetics ; Seminiferous Tubules ; Spermatids ; Spermatocytes ; Spermatogenesis ; genetics ; Spermatogonia ; Testis