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Biomedical subjects

Kun Xia

Publications and source records attributed to Kun Xia.

At least 91 records · Page 5Linked to original sources
PubMed2004

[Mutation analysis of ganglioside-induced differentiation associated protein-1 gene in Chinese Charcot-Marie-Tooth disease].

OBJECTIVE: To study the mutation feature of ganglioside-induced differentiation associated protein-1 (GDAP1) gene in Chinese Charcot-Marie-Tooth disease(CMT) patients. METHODS: Mutation analysis was carried out by use of polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) combined with DNA direct sequencing of the six exons and their flanking regions of GDAP1 gene in twenty-three CMT patients, including 8 probands of autosomal recessive CMT families and 15 sporadic patients. RESULTS: A compound heterozygous mutation A533G and A767G were unveiled in one autosomal recessive CMT kindred. The homozygous and heterozygous T507G were common SNPs in Chinese population. CONCLUSION: A533G and A767G of GDAP1 gene were new mutations firstly reported.

Adolescent↗
PubMed2004

[Subunit of voltage-dependent calcium channels in the murine spiral ganglion cells].

OBJECTIVE: To investigate subunit type of voltage-dependent calcium channels in the spiral ganglion cells of the mouse. METHODS: The spiral ganglion cells were dissected from cochleae of neonatal mice and cultured for 24 h. Total RNA was extracted from cultured spiral ganglion cells. After reverse transcription, resulting cDNA was amplified by polymerase chain reaction (PCR) with primers targeted to nucleotide sequences corresponding to 7 different calcium channel subunits. The types of calcium channel subunits were identified by PCR analysis and nucleotide sequencing. RESULTS: Reverse transcription (RT)-PCR products representing subunit gene expression were strongly and consistently amplified for alpha1 D, alpha1 E, alpha2/delta, beta1 and beta3. Nucleotide sequencing confirmed the identity of mouse cochlear subunit cDNAs. CONCLUSIONS: alpha1D, alpha1E, alpha2/delta, beta1 and beta3 subunits are expressed in spiral ganglion cells. And the coexpression of alpha1D and alpha1 E demonstrate the presence of L-type and R-type calcium channels in mammalian spiral ganglion cells.

Animals↗
PubMed2004

[A novel oligonucleotide arrays-based multiplex amplifiable probe hybridization technology].

Multiplex amplifiable probe hybridization (MAPH) has recently been developed to detect gene copy number changes in total genome in several genetic disease. Here we reported a novel oligonucleotide arrays-based multiplex amplifiable probe hybridization technology for DNA fragment copy number measurement. A set of amplifiable probes were prepared by locus-specific forward and reverse primers synthesized with the T7 and T3 promoter sites at their respective 5'-ends to the interest DNA fragments and purified by Qiagen PCR products purification kit. The set of probes were then hybridized with genome DNA immobilized on the nylon membrane. The selective probes after MAPH were collected with streptavidin coated magnetic beads and amplified by a biotin labeled universal primers. The biotinylated PCR products were then intended for hybridization to the corresponding oligonucleotide arrays consisted of 10 exons of DMD gene probes and positive,negative control probes immobilized on glass slides. After hybridization, the slides were washed by hybridization buffer and stained by streptavidin-Cy3. The hybridization fluorescence images were scanned by the ScanArray software and further quantitatively analyzed by the ImageJ software. Here we used oligonucleotide array technology to replace the agarose gel analysis for detecting the biotinylated PCR products so that extendable ability of parallel assay in our method had greatly been improved. One control female, one control male and one DMD patient were tested in this study. The results demonstrate the feasibility of high-throughput detection and relative quantification of DNA fragment copy numbers changes in total genome DNA combination of MAPH and high-density oligonucleotide arrays in single reaction.

Gene Dosage↗
PubMed2004

[Mutation analysis of keratin 9 gene in a pedigree with epidermolytic palmoplantar keratoderma].

OBJECTIVE: To identify mutations of keratin 9 (KRT9) gene in a big Chinese family with epidermolytic palmoplantar keratoderma(EPPK) combined with knuckle-pad-like lesions and nail lesions. METHODS: Genomic DNA from peripheral blood of all available members in this family and 50 unrelated healthy individuals was used for amplification of the whole coding sequence and the intron-exon boundaries of KRT9 gene by PCR; The mutation was detected by direct sequence analysis and identified by restriction endonuclease Dde I. RESULTS: A mutation of AAT>AGT at codon 160 (N160S) was found in all patients but not in unaffected family members and 50 controls. CONCLUSION: The mutation of AAT>AGT at codon 160 (N160S) is the disease-causing mutation in this Chinese pedigree with EPPK.

Asian People↗
PubMed2004

[Mutation screening in selected exons of myosin 7a gene in prelingual non-syndromic hearing impairment patients].

OBJECTIVE: To ascertain the frequency and characteristics of myosin 7a gene mutations in Chinese with prelingual nonsyndromic hearing impairment. METHODS: Most of cases were collected within Hunan province, including 31 sporadic congenital deaf patients and 65 patients from 34 hereditary prelingual deafness families, and 100 health individuals were used as control. Genomic DNA was extracted from the patients and subjected to the PCR to amplify selected exons of myosin 7a gene, and then the amplified products were screened for base variations by single strand conformational polymorphismanalysis (SSCP). The bands with abnormal conformation were sequenced to confirm the mutation. RESULTS: G to A substitution was detected at nucleotide 617 in exon 7 as hetrozygous state in two patients and was not found in unaffected members in their family. This mutation caused Arg206Gln within a highly conserved heptapeptide sequence of myosin 7a protein, and was close relevant to the prelingual nonsyndromic deafness. CONCLUSIONS: The Arg206Gln mutation in exon 7 of myosin 7a is possibly a novel mutation to cause prelingual nonsyndromic hearing impairment. Our results provide the evidence that exon 7 of Myosin 7a is a mutational hotspot region in genetic deafness.

Adolescent↗
PubMed2004

[Mapping of a pedigree with congenital nystagmus].

To investigate the relationship between X chromosome and obligatory gene of a pedigree with congenital nystagmus,we used the following markers: DXS1214, DXS1068, DXS993, DXS8035, DXS1047, DXS8033, DXS1192 and DXS1232. Genome screening and genotyping were conducted in this pedigree of congenital nystagmus, and linkage analysis by LINKAGE package was used to determine the potential location. The linkage was not found on the Xp (All LOD score <-1) but on Xq (the maximum LOD score=2). The related gene of this pedigree was located on the long arm of X chromosome. We demonstrate that Xq26-Xq28 is a common locus for CMN. It bring us closer to the identification of a gene responsible for X-linked CMN.

Chromosome Mapping↗
PubMed2004

[Interaction of connexin 26 with the C-terminal of neuroendocrine specific protein].

OBJECTIVE: To screen and identify the interactive proteins with connexin 26 (Cx26) by the yeast two hybrid technique. METHODS: The whole coding region of Cx26 (GJB2) gene was amplified from normal human genomic DNA by polymerase chain reaction (PCR). The "bait" Cx26 was then subcloned into the vector pGBKT7 plasmid of the MatchMaker Gal4 Two-Hybrid System 3 as a target to screen its interactive proteins ("prey") from the human fetal brain eDNA library by the yeast two hybrid technique. The false positive clones were discarded from the preys by one to one yeast two hybrid method between Cx26 and the preys. The DNAs of the preys were sequenced and BLAST analyzed against the GenBank, and also underwent other bioinformatics analysis. RESULTS: The insert of one positive clone contained 145 amino acids residues that was identical to the C-terminal of the neuroendocrine specific protein (NSP) and the open reading frame of the insert was correct. CONCLUSION: Cx26 is interacted with the C-terminal of NSP. NSP may participate in the process of Cx26 transportation, assembling the connexon, or influencing the functions of its connexons.

Alcohol Oxidoreductases↗
PubMed2003

In vitro chondrogenesis of human bone marrow-derived mesenchymal progenitor cells in monolayer culture: activation by transfection with TGF-beta2.

Bone marrow-derived mesenchymal progenitor cells are capable of chondrogenesis, making them a possible source of cells for cartilage tissue engineering. Because of this, we studied the effect of human transforming growth factor beta2 (TGF-beta2) on mesenchymal progenitor cell chondrogenesis in monolayer culture using gene transfection technology. A recombinant pcDNA3.1(+)/TGF-beta2 construct containing a full-length TGF-beta2 from a human placental cDNA library was created through gene cloning and DNA recombination. The construct was then lipofected into mesenchymal progenitor cells isolated from human bone marrow. RT-PCR, Western blotting, and immunohistochemistry analyses were performed to identify the expression of TGF-beta2 and cartilage-associated genes and proteins. The results showed that TGF-beta2 was expressed throughout the culture period. The transfected progenitor cells expressed and produced collagen type II and aggrecan 48 h after transfection, and the expression and synthesis were upregulated after 4 weeks. In contrast, the tested genes and proteins were not detected in non-transfected cells. This shows that transfection of pcDNA3.1(+)/TGF-beta2 into mesenchymal progenitor cells is able to provide transient and persistent expression of cartilage-specific genes and proteins, and suggests that the differentiation of human marrow-derived mesenchymal progenitor cells into chondrocytes in monolayer culture is feasible and may be induced by TGF-beta2.

Aggrecans↗
PubMed2003

Sodium chloride modified silica nanoparticles as a non-viral vector with a high efficiency of DNA transfer into cells.

Development of reliable vectors is a major challenge in gene therapy. Previous gene transfer methods using non-viral vectors, such as liposomes or nanoparticles, have resulted in relatively low levels (35 to approximately 50%) of gene expression. We have developed a silicon nanoparticle (SNAP) system, a novel non-viral vector, for DNA transfer into cells. SNAP was synthesized chemically and modified with sodium chloride or sodium iodide. Electronmicroscopy of SNAP and fluorescence microscopy of fluorescence-labeled SNAP revealed that they were generated uniformly, had diameters of 10-100 nm, and showed a better efficiency (about 70%) of DNA transfection into cells as well as protection of DNA against degradation. The microscopy also demonstrated the adhesion of SNAP with HT1080 cell surface and entry of SNAP into the cells without cytotoxicity. Intravenous and/or intra-abdominal administration of the SNAP to mice revealed the accumulation of SNAP in the cells of the brain, liver, spleen, lung, kidney, intestine, prostate and the testis without any pathological cell changes or mortality, suggesting that they passed through the blood-brain, blood-prostate, and blood-testis barriers. These findings indicate that the SNAP generated has good biological characteristics as a potential promising vector for gene transfer, gene therapy and drug delivery.

Animals↗
PubMed2003

[Spastin gene mutation in Chinese patients with hereditary spastic paraplegia].

OBJECTIVE: To investigate the mutation characteristics of spastin gene in Chinese patients with hereditary spastic paraplegia (HSP) and thus provide a basis for the gene diagnosis of HSP. METHODS: Mutation of spastin gene was screened by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) combined with DNA direct sequencing in 31 unrelated affected HSP individuals in China, of whom 22 were from autosomal dominant families and 9 were sporadic HSP patients. Co-segregation analysis was carried out after the finding of abnormal SSCP bands. RESULTS: Six cases were found to have abnormal SCP bands, and among them, two missense mutations (T1258A, A1293G in exon 8) and one deletion mutation (1667delACT or 1668delCTA or 1669delTAC in exon 14) were found and all of them were not reported previously. They were all co-segregated with the disease and were localized within the functional domain of spastin gene. Besides, T1258A was seen in two unrelated families. CONCLUSION: The mutation rate (18.2%) in autosomal dominant HSP in Chinese patients is comparatively low. Point mutation is the major mutation type and exon 8 may be the mutation hot spot.

Adenosine Triphosphatases↗
PubMed2003

[In silicon cloning of the human TECTB gene].

The coding sequence of the mouse Tectb and chick Tectb gene were subjected to Blastn searching against the human dbEST and Htgs in NCBI. One BAC clone sequence(GenBank: AL157786) was obtained, which shows high homology to the two genes. We predicted the exons and introns in the homologous region of AL157786 using GENSCAN, MZEF and Blast 2 sequence program, and then assembled the predicted exons into the coding sequence of the human TECTB. The open reading frame of human TECTB gene is 990 bp composed of ten exons, which encodes a protein of 329 amino acids. Human TECTB gene shows 88.1% identity in 990 bp overlap with that of the mouse Tectb gene and the predicted polypeptide shows 94.2% identity in 329 as with the mouse beta-tectorin. The TECTB gene was mapped to human chromosome 10q25 by electronic-PCR.

Amino Acid Sequence↗
PubMed2003

[Exclusive gene mapping on retinitis pigmentosa with markers on chromosomes 3 in a Chinese kindred].

OBJECTIVE: To study the relationship between the rhodopsin gene on chromosome 3 and autosomal dominant retinitis pigmentosa (ADRP) in a Chinese kindred. METHODS: Sixteen normal persons and 18 RP patients in a ADRP family were recruited. Genome scan method based on fluorescence labeled (using 3 different labels: 6-FAM, HEX, and NED) microsatellite markers with multiplex PCR system was used to identify loci influencing susceptibility to ADRP. Fourteen microsatellites (D3S1297, D3S1263, D3S1266, D3S1289, D3S1300, D3S3681, D3S1271, D3S1292, D3S1569, D3S1279, D3S1614, D3S1262, D3S1580 and D3S1311) on chromosome 3 were used as genetic markers. Linkage analysis (using Genescan3.0, GeneScan Analysis 2.1, Genotyper 2.1 and Designer sofe system) was performed using these markers. RESULTS: The LOD value was <or= -2 in all 14 microsatellites in chromosome 3, indicating that there was no linkage between these markers and RP related gene in this kindred. CONCLUSION: The RP related gene in this kindred is located on chromosomes other than chromosome 3 and is not caused by the mutation of rhodopsin gene.

Asian People↗
PubMed2003

[Silica nanoparticles modified as carriers for gene transfection].

The silica nanoparticles are modified by sodium iodine or sodium chloride with different concentration. Their ability to bind and protect plasmid DNA was demonstrated by agarose gel electrophoresis. The EGFP-N1 plasmid was transfected into HT1080 by combing silica nanoparticle. Eletromicroscope examine revealed that nanoparticle-DNA could enter into cells, these silica nanoparticles protect DNA against nuclease digestion and the GFP has an effective expression. In conclusion, these nanoparticles might be used as DNA carriers for gene transfection.

Animals↗
PubMed2003

[Genetic diagnosis and prenatal genetic diagnosis of fragile X syndrome].

In order to obtain a simple,fast,accurate and low-cost diagnosis method of fragile X syndrome, cytogenetic tests and molecular genetic tests were carried out with direct amplification of (CGG)(n) repeat sequence in 5' terminal of FMR1 gene by PCR and the cDNA sequence of FMR1 by RT-PCR from six mental retardation pedigrees. The proband of pedigree A with high expression of fragile X chromosome(35/273) was detected to be a full mutation patient of fragile X syndrome by the molecular genetic test. There is no expression of fragile X chromosome in the proband and his mother of pedigree B, which was further confirmed as a non-fragile X pedigree by the molecular genetic test. A male foetus of the pedigree C has fragile X chromosome(5/93), but the proband and his mother has no fragile X chromosome. By further detection using molecular genetic test, the male foetus is a full mutation patient of fragile X syndrome, his mother is a permutated carrier, and his brother is a mosaic patient. The proband of pedigree D has high expression of fragile X chromosome (17%), his sister also has expression of fragile X chromosome (5%). By further detection with molecular genetic test, the proband is a full mutation patient of fragile X syndrome,and his sister is a mosaic patient. The probands of pedigrees E and F of the mother were found with suspicions fragile X chromosome, being confirmed as the non-fragile X pedigrees by the molecular genetic test. The conclusion is that the analysis test with direct amplification of 5'j(CGG)n repeat sequence and cDNA sequence in FMR1 gene is simple,fast,low-cost and can be applied in screening, diagnosis and prenatal diagnosis of fragile X syndrome.

English Abstract↗
PubMed2002

A novel locus for autosomal dominant nonsyndromic hearing loss identified at 5q31.1-32 in a Chinese pedigree.

Hearing impairment is an extremely heterogeneous disorder. A total of 35 loci and 17 related genes for autosomal dominant nonsyndromic hearing loss have been identified. In a Chinese pedigree characterized by autosomal dominant inheritance with bilateral, postlingual, progressive, and sensorineural nonsyndromic hearing impairment, the putative disease gene locus was localized to chromosome 5q31.1-32 by a genome-wide scan. Fine mapping indicated that the disease gene was located within an 8.8-cM region between markers D5S2056 and D5S638, with a maximum two-point logarithm of differences (LOD) score of 6.89 (theta = 0) at D5S2017. By the candidate gene approach, mutation screening of the DIAPH1 and POU4F3 genes at 5q31 was performed. No mutation was found, suggesting that this is a novel deafness locus, which has been named DFNA42.

China↗
PubMed2002

[Molecular cloning of the human CX 58 gene].

OBJECTIVE: To clone a novel human connexin gene and find out the relationship between this gene and hereditary deafness. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Animals↗
PubMed2002

[A simple and convenient method for construction of gene site-directed mutagenesis].

OBJECTIVE: To introduce a new technique for rapid construction of gene site-directed mutagenesis. METHODS: 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. RESULTS: Two site-directed mutagenesis have been successfully constructed in the Parkin gene by this method. CONCLUSION: The method is effective and simple for construction of gene site-directed mutagenesis.

Base Sequence↗