Objective To construct the short hairpin RNA(shRNA) expression vector of survivin and down-regulate the expression of survivin through RNA interference in neuroblastoma cell line SH-SY5Y. Methods Two pairs of oligonucleotide sequences specific for human survivin mRNA were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pBSHH1 plasmid. After being identified by restriction enzyme digestion and sequencing, the recombinant plasmids pBSHH1-S1 and pBSHH1-S2 were transfected into SH-SY5Y cells, respectively. Survivin expression in the transfected cells was assayed by both RT-PCR and western blot. Results Enzyme digestion analysis and DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pBSHH1 plasmid, and survivin expression in the transfected cells was knocked down significantly by pBSHH1-S1 or pBSHH1-S2 at both the protein and mRNA level. Conclusion The shRNA expression vectors of survivin were successfully constructed, and could down-regulate survivin expression in SH-SY5Y cells, which lay a foundation for further research on gene therapy for tumors such as neuroblastoma.

Objective To evaluate the clinical and genetic characteristics of Charcot-Marie-Tooth disease (CMT). Methods The clinical materials and hereditary histories of 110 cases in 70 families with CMT were analyzed retrospectively.Results The ratio of male to female was 2.03∶1. The age at onset was from 1 to 61 years old and the mean age was 19.1 years old. 78.2% of the patients had CMT before 30 years old.70 patients (63.6%) had positive family history and they showed mostly autosomal dominant inheritance. The rate of consanguinity was 6.9%. In the CMT group, we could find the amyotrophy of legs in 106 patients (96.4%), distal muscle weakness and atrophy of the upper limbs in 48 patients (43.6%), stork legs in 64 patients (58.2%), pes cavus in 68 patients (61.8%), decreased or diminished tendom reflexes in 108 patients (98.2%). Electromyography examination in 61 patients showed neurogenic damages. Muscle biopsy in 37 patients showed neurogenic amyotrophy. Sural nerve biopsy was performed in 25 patients. 20 patients were charactered by demyelination, Schwann cell proliferation and/or “onion bulbs” change and 5 patients were associated with axis cylinder degeneration, but tomaculous change was not found in all the 25 patients.Conclusions In the CMT group, the onset age was mostly in childhood and adolescence. The male CMT cases were more than the females. Autosomal dominant was the mostly frequent inheretance. Neuroelectrophysiology and pathological examination are important for the diagnosis and type of CMT.

Objective To study the gene mutation and clinical characteristics of hereditary spinocerebellar ataxia type 7 (SCA7).Methods The regions of SCA7 gene containing CAG repeat were amplified by means of polymerase chain reaction (PCR) and polyacrylamide gelelectrophoresis (PAGE) technique in 112 patients with autosomal dominant SCA from 92 families, 16 sporadic SCA patients, 71 family members and 60 healthy controls. The abnormal allele fragments were sequenced by ABI377 DNA sequencing machine. The correlation between clinical manifestations and CAG repeat size in SCA7 gene product was analyzed. Results 2 patients of 6 members in 1 SCA7 family carried a 71-repeat allele. The main clinic features included ataxia, hypopsia, axanthocyanopsia and retinal pigmental degeneration. Alleles from 7 to 9 repeats were seen in the other 4 healthy members. CAG repeats from 6 to 21 were found in other 126 SCA patients, 71 family members and 60 healthy controls. Conclusions Expanded triplet repeats in SCA7 gene contributes to the pathologic phenotype, and molecular genetic analysis is effective for the diagnosis of SCA7. Retinal pigmental degeneration is an important characteristic of SCA7.

Objective To investigate the mutation characteristics of atlastin gene in Chinese patients with hereditary spastic paraplegia(HSP) and establish the base of gene diagnosis of HSP.Methods Mutation analysis of atlastin gene was made by use of polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) combined with DNA direct sequencing in 30 unrelated affected HSP individuals in China in which 20 cases were from autosomal dominant families and ten cases were sporadic HSP patients.Results No abnormal SSCP bands were found in the 30 individuals and the results of DNA direct sequencing were also normal.Conclusion Mutation of atlastin gene may be rare in Chinese HSP patients.

Objective To investigate the mutation of Cx32 gene,clinical and electrophysiological features of Chinese patients with Charcot-Marie-Tooth(CMT) disease.Methods 24 CMT probands were selected for Cx32 mutation screening after the exclusion of the CMT1A 1.5 Mb duplication and male-to-male transmission.The motor and sensory nerve conduction studies were performed in all probands and most of their affected family members to establish the clinical CMT1,CMT2 or CMT intermediate diagnosis.The presence of mutations in the coding region of Cx32 was detected by single-strand conformation polymorphism analysis combined with direct sequencing.Results It was found 7 different point mutations in the coding region of Cx32 in 1 sporadic CMT1 patient and 6 X-linked inherited families,including 4 families with CMT1 diagnosis and 2 families with CMT intermediate diagnosis.There were 20 male CMTX patients,6 female CMTX patients and 12 asymptomatic female carriers among 38 family members bearing Cx32 mutation.All of the 26 patients were mildly to moderately affected clinically.Conclusions Seven different Cx32 point mutations were detected and the percentage of Chinese CMT families with Cx32 mutation is about 10% in our study.The inheritance model of CMT secondary to Cx32 mutation could be X-linked dominant,X-linked recessive or sporadic.Male patients are usually more severly affected than females with slower nerve conduction velocities.Cx32 mutation screening should be firstly performed in those CMT families without male-to-male transmission and CMT1A duplication.

Objective To explore the clinical features of autosomal recessive juvenile Parkinson disease(AR-JP).Methods The clinical materials of 28 patients from 15 families with AR-JP were analyzed retrospectively.Results The onset of all the patients was insidious and the mean age was 26.1 years old.In 23 patients(82.1%),the symptoms began at one limb or one side and progressed bilaterally in a mean time of((4.7?)3.6) years.Bradykinesia(100%),rigidity(100%),resting tremor (85.7%),postural instability(60.7%),hyperreflexia(53.6%),dystonia(32.1%) and diurnal fluctuations with sleep benefit(89.2%) were the cardinal symptoms.The mean improved Webster score was(11.2?)6.1.The mean maintenance dose of DOPA-preparation was((0.40?)0.28) g/d.The mean UPDRS motor score was(27.9?)10.3 before treatment and it decreased to(6.7?)5.4 after therapy(P

Objective To analyze the clinical features of an autosomal dominant Charcot-Marie-Tooth disease(CMT) type 2F family in china.Methods Retrospective analysis were made to show the clinical manifestations and electrophysiological data of 3 patients who come from an autosomal dominant CMT2F family. Results The clinical manifestations of 3 patients were characterized by later onset (from 37 to 60 years)and mild sensory impairments. Right hearing of one patient was lost progressively after the onset. Nerve conduction studies showed there were slow sensory and motor conduction velocities or no nerve action potentials in lower limbs but normal or mildly slow in upper limbs. Somatosensory evoked potentials on tibial nerve indicated both central and peripheral somesthesia gateway were involved. Motor evoked potentials detection found the conduction velocities in the peripheral motor gateway were slowed in lower limbs. Brainstem auditory evoked potentials showed right peripheral acoustic pathway was severely impaired but left was normal. Bilateral visual evoked potentials were normal. Conclusion Patients with CMT2F had distinct characteristics in clinical manifestations and electrophysiological data which would help clinical typing and diagnosis of CMT.

Objective To study the clinical features and genetic mutation frequency of spinocerebellar ataxia (SCA) type 6 from Chinese kindreds. Methods The SCA6 (CAG)n trinucleotide repeat mutations were detected using polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) techniques in 330 patients with autosomal dominant SCA from 160 kindreds and 77 sporadic SCA patients, and the abnormal alleles fragments were sequenced by ABI377 DNA sequencing machine. The clinical features were assessed and cranial MRI examinations were performed in these patients.Results 6 patients from 4 SCA6 Chinese kindreds had abnormal SCA6 alleles with CAG repeat expanded to 25 and 26, respectively, of which 2.5% was about the positive rate, while CAG repeat of normal SCA6 allele ranged from 5 to 17. The basic characteristics of SCA6 patients were slowly progressive cerebellar ataxia and purely cerebellar atrophy.Conclusion SCA6 is one seldom subtype of SCA in Chinese patients with its characteristics both in clinic and imaging in contrast to other subtypes.

Objective To investigate the mutation characteristics of paraplegin gene in Chinese patients with hereditary spastic paraplegia (HSP) and establish the base of the gene diagnosis of HSP.Methods Mutation analysis of paraplegin gene was carried out by use of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) combined with DNA direct sequencing in 22 unrelated affected HSP individuals in China, in which 8 probands were from autosomal recessive families and 14 cases were sporadic.Results All of the exons could be detected by PCR. 2 probands were found to have abnormal SSCP bands in exon 15 and 2 substitutes (G2063A, G2066A in exon 15) were found by DNA direct sequencing. But there were no changes in other patients of families.!The same abnormal SSCP bands and G→A substitutes were revealed in control individuals. So these changes were two polymorphisms, in which G2066A was not reported previously.Conclusion Mutations of paraplegin gene may be rare in Chinese patients with HSP. G2063A and G2066A are two polymorphisms, in which G2066A has not been reported previously.

Objectives To investigate the DRD 2 genetic susceptibility to essential tremor(ET) in Chinese.Methods Polymerase chain reaction-restricted fragment length polymorphism(PCR-RFLP) was used to determine the DRD 2 Taq ⅠA genotypes of 80 unrelated ET patients and 100 healthy controls.Results No significant differences in Taq ⅠA genotype and distributions of allele frequencies of Taq ⅠA loci at the DRD 2 gene were observed between ET and controls.Conclusions Taq ⅠA loci polymorphism at DRD 2 gene may be not associated to genetic susceptibility to ET,but the Taq ⅠA polymorphism at DRD gene could contribute the disorder of central dopaminergic neural system in ET.