Screening for, andVariants in a Cohort of Chinese Patients with Charcot-Marie-Tooth / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>SH3TC2, PMP2, and BSCL2 genes are related to autosomal recessive (AR) Charcot-Marie-Tooth (CMT) disease type 1, autosomal dominant (AD)-CMT1, and AD-CMT2, respectively. Pathogenic variants in these three genes were not well documented in Chinese CMT patients. Therefore, this study aims to detect SH3TC2, PMP2, and BSCL2 pathogenic variants in a cohort of 315 unrelated Chinese CMT families.</p><p><b>METHODS</b>A total of 315 probands from 315 unrelated Chinese CMT families were recruited from the Department of Neurology of Third Xiangya Hospital and Xiangya Hospital. We screened for SH3TC2 pathogenic variants in 84 AR or sporadic CMT probands, PMP2 pathogenic variants in 39 AD or sporadic CMT1 probands, and BSCL2 pathogenic variants in 50 AD or sporadic CMT2 probands, using polymerase chain reaction and Sanger sequencing. All these patients were out of 315 unrelated Chinese CMT families and genetically undiagnosed after exclusion of pathogenic variants of PMP22, MFN2, MPZ, GJB1, GDAP1, HSPB1, HSPB8, EGR2, NEFL, and RAB7. Candidate variants were analyzed based on the standards and guidelines of American College of Medical Genetics and Genomics (ACMG). Clinical features were reevaluated.</p><p><b>RESULTS</b>We identified three novel heterozygous variants such as p.L95V (c.283C>G), p.L1048P (c.3143T>C), and p.V1105M (c.3313G>A) of SH3TC2 gene and no pathogenic variants of PMP2 and BSCL2 genes. Although evaluation in silico and screening in the healthy control revealed that the three SH3TC2 variants were likely pathogenic, no second allele variants were discovered. According to the standards and guidelines of ACMG, the heterozygous SH3TC2 variants such as p.L95V, p.L1048P, and p.V1105M were considered to be of uncertain significance.</p><p><b>CONCLUSIONS</b>SH3TC2, PMP2, and BSCL2 pathogenic variants might be rare in Chinese CMT patients. Further studies to confirm our findings are needed.</p>

Influence of simvastatin on dopaminergic neurons of lipopolysaccharide-induced rat model of Parkinson's disease

OBJECTIVE@#To investigate the neuroprotective effects of simvastatin on lipopolysaccharide (LPS)-induced rat model of Parkinson's disease (PD) and the mechanisms involved.@*METHODS@#Hemiparkinsonian rat models were induced by stereotaxieal injection of LPS in the right substantia nigra compacta. After 2 weeks of simvastatin treatment, rotational behavior test was performed after the intraperitoneal injection of apomorphine. Expression of tyroxine hydroxylase (TH) and glial fibrillary acidic protein were analyzed through immunohistochemical staining of substantia nigra and striatum, and the level of TNF- α was evaluated using enzyme-linked immunosorbent assay.@*RESULTS@#Comparing with untreated group, behavioral symptoms of the rats were significantly less in the rats that received simvastatin treatment. The TH positive cell count in substantia nigra and striatum were significantly increased (P<0.05) and TNF- α expression was significantly decreased (P<0.05) in simvastatin group compared to untreated group.@*CONCLUSIONS@#Simvastatin could effectively inhibit the activation of astrocytes, reduce TNF- α expression, and exert anti-inflammatory effects, and thus protect the dopaminergic neurons in substantia nigra and striatum of the rat model of PD.

Influence of simvastatin on dopaminergic neurons of lipopolysaccharide-induced rat model of Parkinson's disease

Objective: To investigate the neuroprotective effects of simvastatin on lipopolysaccharide (LPS)-induced rat model of Parkinson's disease (PD) and the mechanisms involved. Methods: Hemiparkinsonian rat models were induced by stereotaxieal injection of LPS in the right substantia nigra compacta. After 2 weeks of simvastatin treatment, rotational behavior test was performed after the intraperitoneal injection of apomorphine. Expression of tyroxine hydroxylase (TH) and glial fibrillary acidic protein were analyzed through immunohistochemical staining of substantia nigra and striatum, and the level of TNF- α was evaluated using enzyme-linked immunosorbent assay. Results: Comparing with untreated group, behavioral symptoms of the rats were significantly less in the rats that received simvastatin treatment. The TH positive cell count in substantia nigra and striatum were significantly increased (. P<0.05) and TNF- α expression was significantly decreased (. P<0.05) in simvastatin group compared to untreated group. Conclusions: Simvastatin could effectively inhibit the activation of astrocytes, reduce TNF- α expression, and exert anti-inflammatory effects, and thus protect the dopaminergic neurons in substantia nigra and striatum of the rat model of PD.

Cloning and localization of A3IP -a novel protein that interacts with ataxin-3 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To clone an A3IP gene and investigate its cellular and histological localization based on previous research which has identified part of A3IP sequence interacting with carboxyl-terminal of ataxin-3.</p><p><b>METHODS</b>Bioinformatic and Northern blotting were applied to clone the A3IP gene and detect the expression of its transcripts in various human tissues and brain regions. Western blotting and immunofluorescence staining were applied to detect expression of A3IP protein in cultured cells. Immunohistochemistry staining was applied to study the expression of A3IP protein in various human tissues and brain regions.</p><p><b>RESULTS</b>cDNA cloning of A3IP gene's reading frame and its sequence assembly were completed. Three transcripts (1 kb, 1.35 kb and 6 kb, respectively) of A3IP were found to express in various human tissues and brain regions. A3IP pEGFP expresses in cytoplasm of cultured COS-7 cells and various human tissues and brain regions including cerebral cortex, cerebellum, muscle, peripheral nerve, liver and kidney.</p><p><b>CONCLUSION</b>The cloned A3IP gene encodes A3IP, a novel ataxin-3 interacting protein. Three transcripts of A3IP are expressed in various human tissues and brain regions. A3IP is a cytosolic protein.</p>

Functions of carboxyl-terminus of Hsc70 interacting protein and its role in neurodegenerative disease / 中华医学遗传学杂志

Neurodegenerative diseases are a group of chronic progressive neuronal damage disorders. The cause is unclear, most of them share a same pathological hallmark with misfold proteins accumulating in neurons. Carboxyl-terminus of Hsc70 interacting protein (CHIP) is a dual functional molecule, which has a N terminal tetratrico peptide repeat (TPR) domain that interacts with Hsc/Hsp70 complex and Hsp90 enabling CHIP to modulate the aberrant protein folding; and a C terminal U-box ubiquitin ligase domain that binds to the 26S subunit of the proteasome involved in protein degradation via ubiqutin-proteasome system. CHIP protein mediates interactions between the chaperone system and the ubiquitin-proteasome system, and plays an important role in maintaining the protein homeostasis in cells. This article reviews the molecular characteristics and physiological functions of CHIP, and its role in cellular metabolism and discusses the relationship between CHIP dysfunction and neurodegenerative diseases.

Spinal muscular atrophy mimicking myotonic dystrophy: a case report and clinical, pathological and genetic analysis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate a patient featuring a complex neuromuscular disease phenotype.</p><p><b>METHODS</b>A comprehensive analysis integrating clinical investigation, electrophysiological testing, pathological analysis and mutation screening was carried out.</p><p><b>RESULTS</b>The patient has presented clinical and pathological manifestations mimicking Duchenne muscular dystrophy. However, genetic analysis has identified no deletion in 21 exons of Dystrophin gene, no pathologic expansion of CTG repeats in DMPK gene or CCTG repeats in ZFN9 gene. Instead, a homozygous deletion of exons 7 and 8 in SMN gene was discovered.</p><p><b>CONCLUSION</b>A rare case of spinal muscular atrophy (SMA) was verified by genetic diagnosis. SMA is a group of neuromuscular disorders with great phenotypic heterogeneity and sometimes cannot be diagnosed by clinical manifestations, electrophysiological and pathological changes alone. Genetic diagnosis has become indispensable for accurate diagnosis for patients suspected to have the disease.</p>

Classification and molecular diagnostic procedure for Chacort-Marie-Tooth disease / 中华医学遗传学杂志

Charcot-Marie-Tooth disease (CMT) is the most common form of hereditary neuropathy with significant clinical and genetic heterogeneity. So far 28 genes have been cloned. The main clinical manifestations of CMT include progressive distal muscle wasting and weakness, impaired distal sensation, and diminishing or loss of tendon reflex. Patients may be classified into demyelinating type (CMT1) and axonal type (CMT2) according to electrophysiological and pathological characteristics. Establishment of a standard diagnostic procedure based on clinical, electrophysiological and pathological findings will enable accurate diagnosis in most CMT patients and provide guidance for gene consulting and prognosis.

Recent advance in genetic study of hereditary autosomal recessive cerebellar ataxia / 中华医学遗传学杂志

Autosomal recessive cerebellar ataxias (ARCA) are a highly heterogeneous group of rare neurodegenerative diseases affecting both central and peripheral nervous systems. Based on pathological mechanisms, five major types of ARCA may be distinguished, which include mitochondrial ataxia, metabolic disorder, DNA repair defect ataxia, congenital ataxias and degenerative ataxia. This review summarizes clinical features, molecular genetics and recent advances in DNA sequencing of common types of ARCA.

Fragile X-associated tremor/ataxia syndrome / 中华医学遗传学杂志

Fragile X-associated tremor/ataxia syndrome(FXTAS) is a neurodegenerative disease caused by FMR1 gene permutation(PM). The main clinical manifestations are intention tremor and/or ataxia, and the pathogenesis was related to RNA toxicity. In this paper, the research progress of clinical manifestatios, pathological characteristics, epidemiology and molecular mechanisms will be reviewed.

Cellular expression of (R127W)HSPB1 and its co-localization with neurofilament light chain / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To observe the cellular expression of (R127W) HSPB1 and its influence on neurofilament light chain (NFL) self-assembly and co-localization with NFL.</p><p><b>METHODS</b>Eukaryotic expression vectors pEGFPN1-(wt) HSPB1 and pEGFPN1- (R127W) HSPB1 were constructed. Hela cells were transiently transfected with pEGFPN1-(wt) HSPB1 or pEGFPN1- (R127W) HSPB1 and observed under a confocal microscope. Hela cells were also transiently co-transfected with Pcl-NFL and pEGFPN1-(wt)HSPB1, or pCL-NFL and pEGFPN1-(R127W)HSPB1. The self-assembly of NFL was observed and the co-localization study of HSPB1/ (R127W)HSPB1 with NFL was carried out in these two cell models by immunofluorescence technique.</p><p><b>RESULTS</b>The aggregates formed by EGFP-(R127W)HSPB1 predominantly located around the nucleus, and EGFP-(wt)HSPB1 showed diffusion pattern in Hela cells. When co expressed with EGFP-(wt)HSPB1, NFL formed homogeneous structure in cytosol. When co-expressed with EGFP-(R127W)HSPB1, however, NFL had amorphous staining pattern predominantly consisting of NFL aggregates, and NFL co-localized with (R127W)HSPB1 in these aggregates.</p><p><b>CONCLUSION</b>The R127W mutant of HSPB1 may have reduced capacity to serve as a chaperone to prevent aggregate formation, and fail to correctly organize the neurofilament network. Dysfunction of the axon cytoskeleton and axon transport may be the primary mechanism of R127W mutation of HSPB1 in the pathogenesis of Charcot-Marie-Tooth disease.</p>

The effect of HSPB8 gene mutation on cell viability in Charcot-Marie-Tooth disease type 2L / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the effect of Charcot-Marie-Tooth 2L disease causing gene K141N mutation in heat shock protein B8 gene (HSPB8) on cell viability.</p><p><b>METHODS</b>By using liposome transfection technique, (wt)HSPB8, (K141N)HSPB8 eukaryotic expression vector and green fluorescent protein (GFP) vector were transfected into SHSY-5Y cell, respectively. Twenty-four hours later, the cells were treated with 44 degree centigrade lethal heat shock for 40 minutes. The relative viability of SHSY-5Y cells in each group was tested by using tetrazole blue colorimetric method (methyl thiazolyl tetrazolium, MTT).</p><p><b>RESULTS</b>There were significant differences among the light absorption value of GFP, pEGFP-(wt)HSPB8 and pEGFP-(K141N)HSPB8 transfected groups after heat shock (P<0.05), indicating that the relative viability of cells overexpressed with (wt)HSPB8 and (K141N)HSPB8 was different from that of control cells. The viability of cells overexpressing (wt)HSPB8 was highest, followed by cells overexpressed with (K141N)HSPB8. The viability of cells tranfected with GFP only was the lowest.</p><p><b>CONCLUSION</b>HSPB8 may play an important role in the protection of cells under lethal heat shock treatment, and the K141N mutation can impair the protective effect.</p>

Research progress in roles of microRNA in polyglutamine diseases / 中华医学遗传学杂志

Polyglutamine (Poly Q) diseases are a group of neurodegenerative disorders, caused by the formation of PolyQ mutants due to trinucleotide repeats expansion in coding regions of disease-causing genes, which eventually lead to selective neuronal degeneration and death with unclarified pathogenesis. As a new type of genetic regulatory factor, microRNA (miRNA) plays an important role in modulating gene expression in eukaryote. During the recent years, more attention was paid to roles and related mechanism of miRNA involving in neurodegenerative disease, especially PolyQ diseases. This review is focused on research progress in roles of miRNA in the pathogenesis of PolyQ diseases.

Polynucleotide repeat expansion of nine spinocerebellar ataxia subtypes and dentatorubral-pallidoluysian atrophy in healthy Chinese Han population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assist the establishment of platform and provide the reference standard for mutation detection in spinocerebellar ataxia (SCA) subtypes 1, 2, 3, 6, 7, 8, 10, 12, 17 and dentatorubral-pallidoluysian atrophy (DRPLA) in Chinese Han population.</p><p><b>METHODS</b>The nucleotide repeat numbers of the 9 SCA subtypes and DRPLA were detected using fluorescence-PCR and capillary gel electrophoresis technique in 300 healthy Chinese Han individuals.</p><p><b>RESULTS</b>Among the 300 healthy controls, the range of the CAG trinucleotide repeat number was 17 to 35 in SCA1, 14-28 in SCA2, 13-41 in SCA3/MJD, 4-16 in SCA6, 5-17 in SCA7, 5-21 in SCA12, 23-41 in SCA17, and 12-33 in DRPLA; and the range of CTA/CTG trinucleotide repeat number on SCA8 locus was 12-43 and the range of ATTCT pentanucleotide repeat number on SCA10 locus was 9-32. Of which, the 12 CTA/CTG repeats of SCA8, 9 ATTCT repeats of SCA10, 23 CAG repeats of SCA17 were the shortest normal repeat number, while the 41 CAG repeats of SCA3/MJD, 32 CAG repeats of SCA10 were the largest normal number that have not been reported.</p><p><b>CONCLUSION</b>The normal ranges of polynucleotide repeats of different subtypes of SCA vary with geographical areas and ethnicities. It might be associated with the genetic and ethnic backgrounds. This is the first normal reference standard of polynucleotide repeat number of these ten SCA subtypes in Chinese Han.</p>

Establishment and application of an analytical method for PINK1 gene exon copy number / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a method for analyzing the PTEN-induced kinase 1 gene (PINK1) exon copy number and apply it to the analysis of PINK1 gene exon copy number variation (CNV) in patients with autosomal recessive early-onset Parkinsonism (AREP).</p><p><b>METHODS</b>Real-time PCR was used to analyze the exon copy number in 22 probands with AREP from unrelated Chinese Han families and 30 healthy controls.</p><p><b>RESULTS</b>Copy numbers of exons 1-8 of the PINK1 gene were analyzed, and satisfactory reaction conditions and primers for exons of the PINK1 gene were obtained. No exon CNV in the PINK1 gene was detected in this group.</p><p><b>CONCLUSION</b>An analytical method for PINK1 gene exon copy number was established. The exon CNV in the PINK1 gene was rare in Chinese patients with AREP.</p>

Detection of the CAG trinucleotide repeats of MJD1 gene by recombinant DNA technology / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a stable, accurate and intuitive method for detecting the CAG trinucleotide repeats of MJD1 gene.</p><p><b>METHODS</b>The CAG trinucleotide polymorphism of the MJD1 gene was analyzed by recombinant DNA technology and DNA sequencing in 35 spinocerebellar ataxia 3/Machado-Joseph disease (SCA3/MJD) patients from Mainland China.</p><p><b>RESULTS</b>The range of the CAG repeat of the 35 patients was 65-81 (mean = 72.96 +/- 4.24). The CAG repeats contained two CAAs and one AAG variations in the CAG motif in all the patients and majority of the healthy controls. There was a CGG/GGG polymorphism at the 3' end of the CAG repeat. The GGG allele was consistently associated with smaller CAG repeats in healthy controls. On the other hand, the CGG allele consistently existed in the patients.</p><p><b>CONCLUSION</b>Recombinant DNA technology can stably, accurately and intuitively detect the CAG trinucleotide repeat of the MJD1 gene. It should be used as a major technique to diagnose the SCA3/MJD and analyze the polymorphism of CAG sequence.</p>

Mutation analysis of ATP13A2 gene in Chinese patients with familial autosomal recessive early-onset parkinsonism / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the mutation characteristics of ATP13A2 gene in Chinese patients with familial autosomal recessive early-onset parkinsonism (AREP).</p><p><b>METHODS</b>Mutations of ATP13A2 gene were screened by polymerase chain reaction combined with DNA direct sequencing in patients with familial AREP.</p><p><b>RESULTS</b>No pathogenic mutations in ATP13A2 gene were detected in this group. Six reported polymorphisms were identified. They were IVS6+70A>G, IVS12+66A>G, m.1849C>T, IVS20-56 G>A, m2671C>T and m2824G>A.</p><p><b>CONCLUSION</b>ATP13A2 gene mutations may be rare in Chinese patients with familial autosomal recessive early-onset parkinsonism.</p>

The clinical characteristics of a pedigree with incompletely penetrated autosomal dominant hereditary spastic paraplegia and its exclusion analysis of genetic loci / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To describe the clinical features of a big family with incompletely penetrated autosomal dominant hereditary spastic paraplegia (SPG) and perform the exclusion analysis of genetic loci.</p><p><b>METHODS</b>The clinical information of this SPG family was analyzed retrospectively. Exclusion analysis of the known autosomal dominant SPG loci was performed by using multiplex fluorescence PCR, capillary electrophoresis and Linkage package.</p><p><b>RESULTS</b>There were eleven affected members available in this SPG family and the age at onset ranged from 2 to 10 years. The first symptoms were a bilateral, symmetrical, progressive lower limb weakness and spasticity. Patients presented with spasticity and hyperreflexia, positive Babinski sign and scissors gait, and the upper limbs were involved more severely than the lower limbs. No urinary inconsistence, sensory impairment, nystagmus and dementia were found. Genetic analysis showed that this family was consistent with autosomal dominant inheritance. The linkage analysis and mutation analysis revealed this family was not linked to the known autosomal dominant loci.</p><p><b>CONCLUSION</b>This SPG family had typical "pure" clinical symptoms. The age at onset was early and the signs in the upper limbs were more obvious than those in the lower limbs. The result of linkage analysis shows that this family represents a new SPG subtype.</p>

The advances in research on phosphorylation of polyglutamine disease / 中华医学遗传学杂志

Polyglutamine (polyQ) diseases are a group of hereditary neurodegenerative disorders caused by expansion of a glutamine repeat in responsible gene products. To date, the pathogenesis of polyQ diseases is still not very clear, but many researches suggest that phosphorylation of mutant proteins plays a critical role on the process of Huntington's disease, dentatorubral-pallidoluysian atrophy, spinal bulbar muscular atrophy, spinocerebellar ataxia1 and spinocerebellar ataxia 3/Machado-Joseph disease.

A novel candidate locus on chromosome 11p14.1-p11.2 for autosomal dominant hereditary spastic paraplegia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hereditary spastic paraplegia (HSP) is a group of inherited neurodegenerative disorders with the shared characteristics of slowly progressive spasticity and weakness of the lower limbs. Thirteen loci for autosomal dominant HSP have been mapped.</p><p><b>METHODS</b>A Chinese family with HSP was found in the Shandong province and Inner Mongolia Autonomous Region of China and genomic DNA of all 19 family members was isolated. After exclusion of known autosomal dominant loci, a genome wide scan and linkage analysis were performed.</p><p><b>RESULTS</b>The known autosomal dominant loci of SPG3A, SPG4, SPG6, SPG8, SPG9, SPG10, SPG12, SPG13, SPG17, SPG19, SPG29, SPG31 and SPG33 were excluded by linkage analysis. The results of a genome wide scan demonstrated candidate linkage to a locus on chromosome 11p14.1-p11.2, over an 18.88 cM interval between markers D11S1324 and D11S1933. A maximal, two point LOD score of 2.36 for marker D11S935 at a recombination fraction (theta) of 0 and a multipoint LOD score of 2.36 for markers D11S1776, D11S1751, D11S1392, D11S4203, D11S935, D11S4083, and D11S4148 at theta=0, suggest linkage to this locus.</p><p><b>CONCLUSION</b>The HSP neuropathy in this family may represent a novel genetic entity, which will facilitate discovery of this causative gene.</p>

Blockade of the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein by RNA interference / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To construct specific and effective RNA interference(RNAi) plasmid for alpha-synuclein gene and investigate RNAi blockade of the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein.</p><p><b>METHODS</b>Hairpin RNAs for four target sites were designed to construct four RNAi plasmids pSYNi-1, pSYNi-2, pSYNi-3 and pSYNi-4, using plasmid pBSHH1 vector under the control of the H1 promoter. Western blot and reverse transcription-PCR(RT-PCR) were performed to screen the most specific and effective RNAi plasmid. After confirming the sequences of the plasmids, they were co-transfected into HEK293 cells with the recombinant plasmids alpha-synuclein-pEGFP by using lipofectamin 2000. The aberrant aggregation of alpha-synuclein was measured by EGFP fluorescence and immunocytochemistry for alpha-synuclein. The inclusions in the cultured cells were identified with HE staining.</p><p><b>RESULTS</b>By Western blot and RT-PCR, pSYNi-1 showed the most effective RNAi gene silencing effect (69.6%). After transfecting the RNAi plasmid, the aberrant aggregation of alpha-synuclein in HEK293 cells induced by overexpression of wild-type alpha-synuclein was inhibited. The Lewy body-like inclusions were found in cytoplasm of cultured cells in control group, but disappeared in HEK293 cells cotransfected by pSYNi-1 and alpha-synuclein-pEGFP plasmid.</p><p><b>CONCLUSION</b>RNAi can block the aberrant aggregation and Lewy body-like inclusion formation in cytoplasm of HEK293 cell induced by overexpression of wild-type alpha-synuclein.</p>