Detection of lamivudine resistance-associated hepatitis B virus mutations by multi-analyte suspension array / 中华检验医学杂志

significantly save the time of diagnosis and facilitate the clinical application of large samples.

In vitro Efficacy of mda-7 Gene for Hepatocellular Carcinoma Gene Therapy Mediated by Human Ribosomal DNA Targeting Vector / 生物化学与生物物理进展

Human ribosomal DNA (hrDNA) targeting vector pHr is a homologous recombinant plasmid for human genome which developed in the State Key Laboratory of Medical Genetics. pHr was used to construct a recombinant plasmid pHr-CMG expressing mda-7/GFP fusion gene and its efficacy in the hepatocellular carcinoma cell line Bel-7402 was investigated. The expression of mda-7/GFP fusion gene was detected by fluorescent microscope, RT-PCR and Western blotting, and its function was detected by cell-cycle analyses, MTT assay and Hoechst33258 staining. The results demonstrated that pHr-CMG vector could express MDA-7/GFP fusion protein effectually and the mda-7 gene could induce cell apoptosis and proliferation suppression in Bel-7402 cell line, which might be caused by the G2/M cell cycle arrest. These results also suggested that human ribosomal DNA targeting vector system and the pHr-CMG vector may be applied in further gene therapy researches for hepatocellular carcinoma.

Transcriptional Regulation of The Alzheimer's Disease-related Gene,Nicastrin / 生物化学与生物物理进展

The gamma-secretase complex mediates the final cleavage of APP to generate the principal component of amyloid plaques in the brains of Alzheimer's disease patients.Four integral membrane proteins (PS,NCT,PEN-2 and APH-1) are essential and sufficient for gamma-secretase activity.To identify the promoter of human nicastrin gene (NCT),its 5' -flanking region has been characterized and a 270 bp fragment containing the TSS (transcription start site) for the promoter activity has been identified.EMSA assays confirmed that all four AP-1 binding sites and two NFAT sites in the NCT promoter region were able to bind relative transcription factors in vitro.Mutations,as well as treatment with PDTC,which adjust the regulatory effect of AP-1 and NFAT,altered NCT promoter activity in both HeLa cells and rat cortical neurons.The results demonstrated that AP-1 and NFAT are involved in the regulation of hNCT transcription and suggest that balanced activation of AP-1 and NFAT ensures a strict temporal and tissue-specific control of NCT transcription.

Mutation screening of the dystrophin gene in 14 Chinese Duchenne/Becker muscular dystrophy patients without gross deletions / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To search for the dystrophin gene mutations of Duchenne muscular dystrophy (DMD) patients without gross deletions, in order to offer accurate genetic counseling and prenatal diagnosis for those families.</p><p><b>METHODS</b>All 79 exons of the dystrophin gene as well as its 5'-UTR and 3'-UTR of 14 Chinese DMD/Becker muscular dystrphy (BMD) patients without detectable gross deletions were screened by denaturing high performance liquid chromatography (DHPLC) and heteroduplex fragments were identified by subsequent sequencing.</p><p><b>RESULTS</b>Seven causative point mutations, including two novel ones, were detected in 7 patients. Fourteen known polymorphisms and 7 unknown intronic variations were also detected. Five mothers of the patients were obligate carriers.</p><p><b>CONCLUSION</b>DHPLC is an efficient way of identifying point mutations and the female carriers in DMD families.</p>

Rapid prenatal diagnosis of chromosome aneuploidies in 60 uncultured amniotic fluid samples by fluorescence in situ hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the feasibility of rapid prenatal diagnosis of chromosome aneuploidies by interphase fluorescence in situ hybridization (FISH) using uncultured amniotic fluid.</p><p><b>METHODS</b>Bacterial artificial chromosome (BAC) DNA probes were prepared and validated by using cultured peripheral blood. Interphase FISH for chromosomes 13, 18, 21, X and Y was performed in 60 amniotic fluid samples for the rapid prenatal diagnosis of chromosome aneuploidies, and the results were compared with the karyotypes from conventional cytogenetic analysis.</p><p><b>RESULTS</b>Of all 60 cases, 58 were concordant with their karyotypes, and 1 case of inv(9) and another case of t(2,12) were identified by karyotyping. Two cases of trisomy 21 and 1 case of trisomy 18 were detected by FISH and confirmed with conventional cytogenetics (sensitivity=100%). There were no false-positive or false-negative results.</p><p><b>CONCLUSION</b>This evaluation demonstrated that FISH employing BAC DNA probes could accurately and rapidly detect aneuploidies involving the above 5 chromosomes. However, as it does not identify structural chromosome aberrations and aneuploidies involving other chromosomes, it is not a substitute for conventional chromosome analysis, and the negative FISH result should be carefully interpreted.</p>

Screening of LRRK2 interactants by yeast 2-hybrid analysis / 中南大学学报(医学版)

Objective To isolate and identify the potential binding partners of LRRK2,a gene linked to both dominant familial form and sporadic form of Parkinson's disease,thus to further our knowledge of its function.Methods We used a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait.The bait amplified by polymerase chain reaction(PCR) was then cloned into a yeast expression plasmid pGBKT7.After being sequenced and analyzed,pGBKT7-bait was transformed into the yeast strain AH109.Western blot was performed to confirm the expression of pGBKT7-bait in AH109 yeast strain.Then human fetal brain cDNA library was trarnsformed into that yeast strain.which could express pGBKT7-bait fusion protein.The yeast strain which contained pGBKT7-bait and human fetal brain cDNA library was plated on quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade)containing X-a-gal.We retested these positive colonies using 2 independent yeast strains AH109 contained pGBKT7-bait or pGBKT7,respectively.At last,these plasmids isolated from these true positive colonies were analyzed by bioinformatics.Results We obtained 9 true positive colonies,these colonies were sequenced, and we performed sequence Blast in GenBank.Three colonies of the 9 positive colonies were not in open reading-frames.Among other 6 colonies,there were known proteins including spermatid perinuclear RNA-binding protein(STRBP)and BCL2-associated athanogene 5 isoform b(BAG5),as well as unknown proteins including tyrosine phosphatase non-receptor type(PTPN23),1(3)mbt-like 3 isoform b(L3 MBTL3),RALY RNA binding protein-like isoform 1(RALYL),and Homo sapiens mRNA for KIAA1783 protein,partial cds(KIAA 1783).Conclusion True positive colonies of LRRK2 are successfully obtained by the yeast 2-hybrid.Our screened proteins may provide a new research clue for revealing biological functions of LRRK2,pathogenesis of Parkinson's disease,and other neurodegerations.

Efficient Derivation of Mesenchymal Stem Cells and Neural Precursor Cells From Human Embryonic Stem Cells Through Teratoma Formation / 生物化学与生物物理进展

Many somatic cell typos were obtained by in vitro differentiation or teratoma formation of human embryonic stem ceLls (hESCs). However, it is unclear whether specific cell types can be obtained from hESCs-derived teratoma. It was reported that many kinds of cells, including neural progetfitor/precursor cells (NPCs) and mesenchymal stem cells (MSCs) were isolated efficiently from the teratoma of hESCs through in vitro selection. The teratoma-derived NPCs and MSCs showed specific characteristics of molecular markers similar to the primary NPCs and MSCs. Moreover, these teratoma-induced NPCs and MSCs can be further induced to differentiate into neurons, astrocytes, or adipose and bone cells, reflecting their inherent multi-potencies. Given that teratoma normally contains a mixture of ectoderm, mesodenn, and endoderm lineage cells at different differentiation stage, it was suggested that hESCs-derived teratoma could be an alternative source to generate a variety of uncommitted progenitor cells or terminally differentiated somatic cells, which may be otherwise difficult to obtain through direct in vitro differentiation.

p53 Anti-tumor Research in Bel-7402 by Using Human-derived Vector / 生物化学与生物物理进展

In order to study the tumor suppression effect of p53 with CMV enhancer and hTERT promoter mediated by human-derived vector pHrn in liver cancer cell Bel-7402, report plasmid pchEGFP, tumor suppressor plasmids pchp53Arg and pchp53Pro were constructed by inserting expression cassette CMVe+hTERTp+EGFP, CMVe+hTERTp+p53Arg and CMVe+hTERTp+p53Pro into pHrn respectively. 24 h after cell transfection by lipofectamine 2000, GFP expression pattern was analyzed through fluorescence microscope and flow cytometry; RT-PCR and Western blot were taken to study the p53 expression pattern. The cell apoptosis by Hoechst 33258 and Annexin V-FITC/PI staining was also studied. Results show that the expression of GFP and p53 protein in Bel-7402were detected, but apparent cell apoptosis could not be found. The recombinant p53 mediated by human-derived vector could express in Bel-7402, but no significant tumor suppression effect was detected, which might result from the down regulation effect of the wild type p53 on hTERT promoter.

Role of PITX2 R62H Mutation in Ring Dermoid of Cornea / 生物化学与生物物理进展

Ring dermoid of cornea (RDC) is an autosomal dominant disorder of cornea. The previous study identified a G185A mutation of PITX2 gene in a Chinese family with RDC. To investigate the pathological mechanism of PITX2 R62H mutation, a PITX2 prokaryotic expression plasmid were constructed and GST-PITX2 fusion protein were purified. EMSA was further conducted. The DNA-banding ability of PITX2 R62H was similar to that of the wild type PITX2 were found. The cell lines stably expressed PITX2 was also generated, and cell cycle were analyzed by flow cytometry, and the expression of ?-catenin and Cyclin D1 were detected by quantitative Real-time PCR. The results showed that proliferating ability of cells expressing PITX2 R62H was lower than that of cells expressing PITX2 WT, as well as ?-catenin and Cyclin D1 mRNA level. These findings revealed that PITX2 R62H mutation affected the Wnt/?-catenin→PITX2 pathway, promoted the genes expressing abnormally, and led to abnormal cell proliferation and the formation of RDC, which may play an important role in pathogenesis of RDC.

Mutation analysis of neurofilament-light gene in Chinese Charcot-Marie-Tooth disease / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the characteristic of the mutation of neurofilament-light (NF-L) gene in Chinese Charcot-Marie-Tooth disease (CMT) patients.</p><p><b>METHODS</b>Mutation analysis of NF-L gene was made by use of polymerase chain reaction-single strand conformation polymorphsim combined with DNA direct sequencing in 32 CMT probands from the Hans of five provinces in China who had been diagnosed by clinical feature and electromyography and/or biopsy of sural nerve.</p><p><b>RESULTS</b>In 32 CMT probands, only one sporadic case was found to display variant banding pattern, and this case was confirmed as 1329C to T (Tyr443Tyr) single nucleotide polymorphism by sequencing.</p><p><b>CONCLUSION</b>Mutation of NF-L gene may be rare in Chinese CMT patients.</p>

FBN1 mutation in Chinese patients with Marfan syndrome and its gene diagnosis using haplotype linkage analysis / 中华医学杂志(英文版)

<p><b>OBJECTIVES</b>To analyze the FBN1 mutations in Chinese patients with Marfan syndrome (MFS) and to make a genetic diagnosis based on haplotype linkage analysis for MFS.</p><p><b>METHODS</b>Nine MFS families (17 patients) were analyzed with single strand conformation polymorphism (SSCP) and sequencing. Four primers were designed for the flanking sequences of FBN1 gene and used for haplotype-segregation analysis of MFS(B).</p><p><b>RESULTS</b>SSCP band alteration was detected in the PCR products for exon 25 in MFS(A) II:1. Direct sequencing revealed a small 13 bp deletion; the deleted sequence is gccTc Tgcaccca at bases 3243-3456 of the cDNA in exon 25. This mutation was novel. MFS(B) families were analyzed using the haplotype linkage technique. The data suggested that MFS(B) families were linked to the FBN1 gene. The proband's daughter was an asymptomatic patient.</p><p><b>CONCLUSION</b>The combination of mutation detection and chromosome haplotype analysis can provide better evidence for a genetic diagnosis of MFS.</p>

A new mutation in the connexin 32 gene of a Chinese family with Charcot-Marie-Tooth disease associated with central conduction slowing / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To report a Chinese Charcot-Marie-Tooth disease (CMT) family whose proband had abnormal brainstem auditory evoked potentials (BAEPs) and to study its relationship with connexin 32 (Cx32) gene mutation.</p><p><b>METHODS</b>All family members were studied through clinical examinations, out of them, the proband was subjected to electromyography and BAEPs examination. Mutation analysis of Cx32 was screened by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) combined with DNA direct sequencing in the proband, 8 family members and 50 unrelated normal individuals.</p><p><b>RESULTS</b>The proband had highly decreased nerve conduction velocities and delayed BAEPs. Leu131Pro mutation was found in the proband and 3 family members, not found in 50 normal controls.</p><p><b>CONCLUSION</b>This mutation has not been reported previously. Central nervous system can be affected in CMT patients.</p>

A simple and convenient method for construction of gene site-directed mutagenesis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To introduce a new technique for rapid construction of gene site-directed mutagenesis.</p><p><b>METHODS</b>Three primers are synthesized. One is a primer with the needed mutation; the other two containing appropriate enzyme sites for construction of the PCR fragment into a suitable plasmid are located at the flanks of the mutation primer. After the amplification of the PCR fragment using the mutation primer and the reverse flanking primer, another PCR is performed using the previous PCR mutation segment as primer and the other flanking primer. The final PCR segment can be cloned into an appropriate plasmid by using the enzyme sites in the primers.</p><p><b>RESULTS</b>Two site-directed mutagenesis have been successfully constructed in the Parkin gene by this method.</p><p><b>CONCLUSION</b>The method is effective and simple for construction of gene site-directed mutagenesis.</p>

Molecular cloning of the human CX 58 gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To clone a novel human connexin gene and find out the relationship between this gene and hereditary deafness.</p><p><b>METHODS</b>Through the basic local alignment search tool (BLAST) analysis against the database of expressed sequence tags (dbEST) of National Center for Biotechnology Information (NCBI) using the coding sequence of mouse Cx 57 gene, 9 novel ESTs were obtained and a contig was assembled. Nested polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) were performed using primers designed on the contig. A novel gene was obtained and was mapped by homologous analysis against human genome sequence. Mutation analysis was performed in 12 autosomal dominant hereditary deafness families.</p><p><b>RESULTS</b>Nine ESTs were obtained by homologous analysis and a contig was assembled. Through nested PCR and RACE, a full length of cDNA was obtained from human liver, kidney Ready cDNA and placenta cDNA library, and was named CX 58. By comparison with human genome sequence, CX 58 was mapped at 1p32.3-p34.1. Mutation analysis of CX 58 was performed in 12 autosomal dominant hereditary deafness families, but no mutation was detected.</p><p><b>CONCLUSION</b>A novel human connexin gene named CX 58 was cloned and mapped to 1p32.3-p34.1. The mutation of CX 58 may not result in autosomal dominant hereditary deafness.</p>

Heterozygosity Microdeletion of Tbx1 Gene at 22q11.2 in Conotruncal Heart Malformation Patients / 中国医师杂志

Objective This research is to assess the heterozygosity of Tbx1 gene on 22q11 2 in the patients with conotruncal heart malformation.Methods By fluorescence in situ hybridization (FISH) with partial segment of Tbx1 gene, we examined 22 patients with conotruncal heart malformation including 5 syndromic cases and 17 isolated cases. Northern blot was performed with RNA of 50 human tissues.Results Two of 5 syndromic patients had chromosome 22q11 2 hemizygote microdeletion of the Tbx1 gene, while 17 isolated patients did not show such deletion. Northern blot showed that there were Tbx1 gene positive expression in skeletal muscle, testis, lung and fetal heart.Conclusions Our study suggests that Tbx1 gene may be one of the pathogenic gene related to CATCH22.Association of the Tbx1 gene and conotruncal heart teratogenic gene is to be further detected in gene mutation of patients without heterozygosity deletion.

Prenatal diagnosis of prelingual deafness by determination of SLC26A4 gene mutation / 中华妇产科杂志

G mutation of SLC26A4, the parents and the second child were carriers of the same mutation, while the fetus had a wild-type form. Conclusion It is feasible to identify deafness related genes by screening for GJB2 and SLC26A4 mutation, thus providing correct prenatal diagnosis and avoiding deaf delivery of baby.