A NEW APPROACH TO GENE DIAGNOSIS OF DUCHENNE／BECKER MUSCULAR DYSTROPHY AMPLIFIED FRAGMENT LENGTH POLYMORPHISMS

Four (CA), repeats, located in introns,44,45,49 and 50 of the dystrophin gene,were evaluated in Chinese.These loci are highly polymorphic,with polymorphism information contents of 0.872,0.772,0.870 and 0.718,respectively.All four loci can be easily amplified and labelled using two duplex PCR reactions with α-^32P-dCTP and can be detected by denaturing polyacrylamide gel electrophoresis.Using these four loci and the two polymorphic(CA)n repeats located at the 5′ and 3′ ends of the dystrophin gene,we have developed a new PCR-based procedure-Amp-FLP( amplified fragment length polymorphism)linkage analysis for the gene diagnosis of DMD/BMD.This method can detect intragenic recombination rapidly and efficiently and greatly improves the success rate of carrier deterction and prenatal diagnosis in non-deletion DMD/BMD families.All of the loci used in this procedure are intragenic.In addition ,the loci in introns 44,45,49 and 50 are located in the deletion-prone region of the dystrophin gene,making them valuable and usefui in the identification of deletion mutations.Here we report one case of deletion detection using these four loci.     

Exon Deletions of Parkin Gene in Patients with Parkinson Disease

Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 ( 1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.     

Relationship between tumor suppressor gene p53 and tumors of adipose tissue

Objective　To investigate the relationship between p53 gene and tumors of adipose tissue at the level of protein and gene. Methods　Immunohistochemical LSAB, PCR-SSCP and DNA sequencing were used in 82 cases. Results　p53 protein is expressed only in liposarcomas, in which the positive staining rate was 48.08% (25/52). In different subtypes of liposarcomas, the positive staining rate in well differentiated liposarcomas was 30.00% (9/30), which is much lower than that of the poorly differentiated liposarcomas (P<0.005). Abnormality in the single-stranded DNA pattern was determined in 2 samples (pleomophic liposarcomas) by PCR-SSCP analysis. Missense mutations in exon 8 codon 268 of p53 gene (AAC→ATC) were detected by DNA sequencing. Another heterozygotic cosense mutation may exist at exon 6 codon 221 of p53 gene (GAG→GAA). Conclusions　The data suggest that the p53 protein has a relationship with development, differentiation and malignancy of liposarcoma. Detecting the level of p53 protein expression may be valuable in evaluating the level of differentiation and malignancy of liposarcoma. There appear point mutation on exon 8,6 of p53 gene.     

SMN基因缺失多重连接探针扩增法检测和识别脊柱肌肉萎缩症的纯合型或杂合型SMN基因缺失

Objective: Spinal muscular atrophy(SMA), an autosomal recessive neuromuscular degen-eration of the anterior horn ceils of the spinal cord and brain stem, results in one of the most common dis-eases with muscle fatigue and atrophy. Most SMA cases including all the types are due to the homozygous deletion of at least exon 7 within the survival motor neuron 1 (SMN-1) gene. Although a ““golden stand-ard““ assay ( PCR with mismatch primer followed by enzyme digestion) is very reliable for the identifica-tion of homozygous SMN-1 deletion, the carrier detection of heterozygous SMN-1 deletion remains a chal-lenge. Methods: Some PCR-based gene dosage assays or multiplex PCR allow for the determination of the copy number of SMN-1 gene to identify heterozygous deletion, but these procedures are often time consuming and available on a limited clinical basis. Recently developed MLPA (multiplex ligation-de-to establish the copy number of the SMN gene. We performed a validation for simultaneous detection of homozygous SMN-1 deletions of SMA patients and heterozygous SMN-1 deletions of SMA carriers in a sim-ple assay using a MLPA-SMA assay specific reagent. Results: Six out of 20 patients with SMA were found to have homozygous SMN-1 deletion, confirmed by the PCR/digestion assay. All 4 parents of the children with SMA had heterozygous SMN-1 deletion, confirmed by an independent relative quantitative analysis. Conclusion: MLPA provides a simple, rapid and accurate method of simultaneously detecting homozygous deletions and heterozygous deletions in a sinzle assay for both SMN-1 and SMN-2 zenes.     

Molecular analysis of THH—induced mutations at HPRT locus in human promyelocytic leukemia cells with multiplex polymerase chain reaction

Objective: To study the genotoxicity and antitumor activity of a Chinese medicinal herb, Triptery-gium Hypoglaucum (Level) Hutch (THH). Methods: The genotoxicity and antitumor activity of THH were investigated in human promyelocytic leukemia cells on the mutation of hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene by using single cell clone culture, two-way screening counting, multiplex PCR amplification and gel electrophoresis. Results: The results showed that different mutant spectra existed between the spontaneous mutation and induced mutation by THH. Only 7. 7% (1/13) of spontaneous mutants showed deletion mutations, whereas the induced mutants included 46. 6% (27/58) deletions. Mapping of all intra-genic deletion breakpoints showed a random distribution in all 9 exons, but toward the 3'-end of the HPRT gene. Deletion of exon 1 only appeared when whole gene was deleted. Deletions of exon 7/8 and 9 often showed linkage deletions (71. 4%). Conclusion: THH can induce the mutation, mainly dele     

Identification of seven novel mutations in the factor VIII gene in 18 unrelated Chinese patients with hemophilia A

Background  Hemophilia A (HA) is an X-linked inherited bleeding disorder caused by decreased activity of factor VIII (FVIII) due to heterogenous mutations in the FVIII coding gene (F8). The type of mutation plays an important role in the FVIII inhibitor formation. To date, several studies on the spectra of F8 defects have been performed in Western populations, but similar studies in Asian races are scarce. Here, we reported the distribution of the F8 gene mutations in 18 unrelated Chinese patients with HA.
Methods  Intron 22 and intron 1 inversions in the F8 gene were screened in 158 unrelated patients with HA using a long-distance PCR and multiplex PCR method. Direct sequencing of the coding region of the F8 gene was used to identify the mutations responsible for HA in 18 unrelated Chinese HA patients who were negative for intron 22 and intron 1 inversions; sequences were compared with the HAMSTeRS database. A clotting method was used to assay the FVIII activity level and the Bethesda assay was used to detect the FVIII inhibitor.
Results  A total of 18 different HA F8 mutations were identified, seven of which were described for the first time. These novel mutations included five small deletions, one point mutation and one small insertion. One novel mutation (4382-3 AC deletion) was associated with inhibitor development.
Conclusion  These data extend our insight into the mechanisms by which novel amino acid mutations may lead to HA and how the HA patient genotypes influence the risk of FVIII inhibitor.

     

Original article ：Identification of seven novel mutations in the factor Ⅷ gene in 18 unrelated Chinese patients with hemophilia A

Background Hemophilia A （HA） is an X-linked inherited bleeding disorder caused by decreased activity of factor Ⅷ（FⅧ） due to heterogenous mutations in the FⅧ coding gene （F8）. The type of mutation plays an important role in the FVIII inhibitor formation. To date, several studies on the spectra of F8 defects have been performed in Western populations, but similar studies in Asian races are scarce. Here, we reported the distribution of the F8 gene mutations in 18 unrelated Chinese patients with HA. Methods Intron 22 and intron 1 inversions in the F8 gene were screened in 158 unrelated patients with HA using a long-distance PCR and multiplex PCR method. Direct sequencing of the coding region of the F8 gene was used to identify the mutations responsible for HA in 18 unrelated Chinese HA patients who were negative for intron 22 and intron 1 inversions; sequences were compared with the HAMSTERS database. A clotting method was used to assay the FⅧ activity level and the Bethesda assay was used to detect the FⅧ inhibitor. Results A total of 18 different HA F8 mutations were identified, seven of which were described for the first time. These novel mutations included five small deletions, one point mutation and one small insertion. One novel mutation （4382-3 AC deletion） was associated with inhibitor development. Conclusion These data extend our insight into the mechanisms by which novel amino acid mutations may lead to HA and how the HA patient genotypes influence the risk of FⅧ inhibitor.     

Reporter LacZ gene transfer into cultured ocular cells of human eyes in vitro

Objective To investigate whether a reporter LacZ gene could be transferred into cultured ocular cells of human eyes in vitro. Methods Fibroblast cells of Tenon’s capsule, trabecular meshwork cells, and muscle celms in the ciliary body of human eyes were cultured and the pcDNA3-LacZ gene was transferred into these cells using a cationic liposome delivery system.The cells were subsequently fixed with 4% paraformaldehyde, mixed with X-gal, then observed under a microscope. Results Blue stain was seen in the cytoplasm of the cultured cells under the microscope, demonstrating the successful transfer of the LacZ gene into these cells.Conclusion Reporter LacZ gene was easily transferred into the cultured ocular cells in vitro.This provides insights into the transfer of the genes into these cells to study the pathogenesis and therapy of glaucoma.     

The role of prourokinase gene in protecting vein grafts from intimal hyperplasia

Objective To study the duration of prourokinase gene expression in vein grafts and the role of the prourokinase gene in protecting vein grafts from neointimal hyperplasia.Methods Fifty-four Wistar rats were used in this study. In each rat, the jugular vein was excised and distended for 30 minutes using a solution containing either Adv5-CMV (control group) or Adv5-CMV/ Pro-UK (treatment group). Next, the jugular vein was reversed and interposed into the divided carotid artery of the same rat. On the 14th day after transfection, vein grafts of the control group were collected in order to perform a fibrinolysis test for prourokinase (Pro-UK) activity. On the 2nd, 7th, 14th, 28th, and 60th day, the vein grafts of the treatment group were likewise collected in order to detect prourokinase activity. On the 28th day, the vein grafts of both groups were explanted to evaluate the 3H-TDR incorporation so that pathologic analysis could be performed.Results Pro-UK activity could not be detected in the control group     

Mutation analysis in a Chinese family with multiple endocrine neoplasia type 1

Background Multiple endocrine neoplasia type 1 （MEN1） by germline mutations of the tumor suppressor gene MEN1. with MEN1. Methods A large Chinese family with MEN1 was collected MEN1 gene were amplified and sequenced. is an autosomal dominant cancer syndrome which is caused This study aimed to identify mutations in a Chinese pedigree All of the coded regions and their adjacent sequences of the Results In this family, a heterozygous cytosine insertion in exon 10 （c.1546_1547insC） inducing a frame shift mutation of MEN1 was found in the proband and the other two suffering members of his family. This mutation was linked to a novel single nucleotide polymorphism （SNP）in intron 3 （IVS3＋18C〉T）. Conclusions The mutation in exon 10 of MEN1 gene might induce development of parathyroid hyperplasia and pituitary adenoma and cosegregate with MEN1 syndrome. The significance of the new found IVS3＋18C〉T of MEN1 needs a further investigation.     

Characterization of hemagglutinin gene of influenza A virus subtype H9N2

Objective　To determine the origin of human influenza A (H9N2) virus and the relationship among H9N2 strains isolated from different hosts, on the basis of molecular biology. Methods　Viruses were passed in embryonated hen eggs, and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (version 1.03) and Editseg (version 3.69) softwares. Results　The amino acid sequences at the cleavage site between HA1 and HA2 domains of H9N2 viruses isolated in China are R-S-S-R. One pigeon strain contains seven potential glycosylation sites on the HA protein molecule, while all others have eight. There are 2 to 15 differences of amino acid sequences distributed at 24 different positions on the HA protein molecules among six H9N2 viruses. The H9N2 viruses with multiple lineages of HA genes were co-circulating in China recently. Conclusion　The highest possibility is that human influenza A (H9N2) virus was derived from Chicken H9N2 virus, and not derived from pigeon H9N2 virus. However, it is still unknown whether the H9N2 virus could transmit from person to person. The H9N2 viruses with multiple lineages of HA genes are co-circulating in China.     

Systematic examination of DNA variants in the parkin gene in patients with Parkinson’s disease

Increasing evidence suggests that genetic factors play an important role in the pathogenesis of Parkinson‘s disease(PD). Three genes, namely α-synuclein, parkin, and UCH-L1, have been implicated in familial PD. An exon deletion in the parkin gene is the mutation most frequentlymentioned in published data. The parkin gene was first identified by Japanese researchers, and, since fragment deletions in coding exons of this gene have been proven     

毛细管电泳PCR-SSCP分析p16基因突变

Lung cancer is one of the most common cancers in the world. Some genetic alterations such as p53 gene and ras gene mutations, have been identified in this disease. Recently, a putative tumor suppressor gene, the p16/CDKN2/MTS1 gene containing 3 extrons and 2 introns, located in the chromosome p21 region, was cloned independently by three research groups. Traditionally, gene mutation analysis was performed by slab polyacrylamide gel electrophoresis. However, this method is laborious, time-consuming, low sensitivity and harmful to human health. Capillary electrophoresis (CE) with the characteristics of rapidity and high performance has numerous advantages over conventional slab polyacrylamide gel electrophoresis. An important advantage of CE is that the commercially available system is automation.     

Novel mutation of the cyclin-dependent kinase 4 gene in a Chinese patient with intimal sarcoma of the pulmonary artery

The long arm of human chromosome 12 contains a region that has been found to be amplified in a number of different tumors, including osteosarcomas and soft tissue sarcomas. There are more than 5 genes located in this area such as CDK2, CDK4, WNT1, MDM2 and WNTIOb. CDK4 gene consists of eight exons, of which the start codon is located in the beginning of exon 2 and the stop codon in the a member of the Ser-Thr catalytic domain extends beginning of exon 8. CDK4 is protein kinase family and its from amino acid 6 to 295.     

Identification of E. coli K12 chromosomal insertion sites of bacteriophage φ297

Objective：To identify the specific integration site of prophage φ297 in the host of E. coli K12 chromosome. Methods：Using molecular techniques such as Siebert PCR for walking from the int gene of prophage 297, which is similar to that of phage 933W to an unknown region in genomic DNA. A special adaptor is ligated to the ends of DNA fragments generated by digestion of genomic DNA with restriction enzymes that generates blunt ended fragments. Clone and subclone of PCR products, DNA sequencing and data analysis were used in this study. Results：The attL, attR and the core sequences were determined. The bacterial attachment site of phage φ297 was located in the yecE gene of E. coli K12. Conclusion：The phage φ297 integrates into the yecE gene of the E. coli K12 genome.