Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China.

Objective: Hemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. A wide range of mutations, showing extensive molecular heterogeneity, have been described in hemophilia B patients.

[Mechanism of GLI3 gene transcription regulation in idiopathic congenital talipes equinovarus].

Objective: To investigate the mechanism of transcription regulation of GLI3 gene in idiopathic congenital talipes equinovarus.

Methods: pGL3-Gli3 luciferase report vectors were constructed, and the activity of Gli3 promoter was explored. A P-Match software was used to analyze the sequence upstream of the transcription start site of rat Gli3 gene, which was subsequently verified with chromatin immunoprecipitation assay (CHIP) and electrophoretic mobility shift assay (EMSA).

[Possible role of GLI3 gene in the pathogenesis of idiopathic congenital talipes equinovarus].

Objective: To investigate the relationship between GLI3 gene and pathogenesis of idiopathic congenital talipes equinovarus (ICTEV).

Method: Potential mutations in the coding region of GLI3 were detected among 84 patients with ICTEV by denaturing gradient electrophoresis. Expression of GLI3 in the ICTEV patients' disease tissues was assessed by reverse transcription PCR.

[Expression of COL9A1 gene and its polymorphism in children with idiopathic congenital talipes equinovarus].

Objective: COL9A1 gene is located in the susceptibility region of idiopathic congenital talipes equinovarus (ICTEV) (6q12-13). This study aimed to investigate the expression of the COL9A1 gene and the distribution of single nucleotide polymorphism (SNP) of COL9A1 gene in patients with ICTEV and normal controls.

Methods: Immunohistochemistry was used to detect the expression of COL9A1 in 25 children with ICTEV and 5 normal controls.

[The regulation of UTROPHIN expression by EN1].

To investigate possible factors up-regulating the expression of UTROPHIN, potential regulatory elements in the promoter of the human UTROPHIN was predicted by P-match software and verified by EMSA and ChIP. The mechanism of EN1 regulation of the human UTROPHIN expression was evaluated by RNA interference and real-time PCR analyses. Two potential EN1 binding sites in UTROPHIN promoter region were predicted by P-Match software but only the second site was verified to interact directly with EN1 by EMSA and ChIP.

[Molecular and prenatal diagnosis of a pedigree with spinocerebellar ataxia].

Objective: To identify the type of a pedigree with spinocerebellar ataxia, and carry out asymptomatic carrier detection and prenatal diagnosis.

Methods: The blood samples of two patients in the spinocerebellar ataxia pedigree were collected. Based on the clinical characteristics of the pedigree and the disease incidence in China, the regions containing the CAG repeat of the SCA1, SCA2 and SCA3/MJD genes were amplified by polymerase chain reaction (PCR).

[Teaching experience in integrated course of human development and genetics].

Establishment of integrated course system in human development and genetics is an important part of course reformation, and the improvement of this system is achieved by integrating the content of course, stabilizing teaching force, building teaching materials and applying problem-based learning. Integrity-PBL teaching model is founded and proved to be feasible and effective by teaching practice. Therefore, it maybe play an important role in improving teaching effect and cultivating ability of students to analyse and solve problems.

[The functions of GLI3 and EN1 genes in idiopathic congenital talipes equinovarus].

To investigate the role of gene Gli3 in idiopathic congenital talipes equinovarus (ICTEV), we constructed the Gli3 luciferase reporter gene expression vectors to analyze the promoter activity of the rat gene Gli3. The regulatory element in the promoter region of the rat Gli3 was predicted using P-Match software and further verified by ChIP experiment. Meanwhile, the correlation between the rat En1 and ICTEV was evaluated by RT-PCR, immunohistochemistry, and Western blotting analyses.

The expression of Gli3, regulated by HOXD13, may play a role in idiopathic congenital talipes equinovarus.

Background: Idiopathic congenital talipes equinovarus (ICTEV) is a congenital limb deformity. Based on extended transmission disequilibrium testing, Gli-Kruppel family member 3 (Gli3) has been identified as a candidate gene for ICTEV. Here, we verify the role of Gli3 in ICTEV development.

[Prenatal diagnosis of Duchenne and Becker muscular dystrophy by multiplex ligation-dependent probe amplification].

Duchenne/Becker muscular dystrophy (DMD/BMD) is an X-linked lethal recessive disease caused by mutation in the DMD gene. There is no efficient treatment for this serious and disabling disease. We established a combination method to detect carriers and performed prenatal diagnosis.

[Association study between mutations of transcription regulator sequences of COL1A1 gene and idiopathic con-genital talipes equinovarus].

RT-PCR was used to detect the expressions of COL1A1 mRNA in 20 patients with idiopathic congenital talipes equinovarus (ICTEV). The primers were designed by Primer 5 according to sequences of -1 031 bp~ +30 bp and the first intron of COL1A1. PCR-DGGE was used to screen the mutations in COL1A1 gene.

[Detection of new mutations in the dystrophin gene by denaturing high-performance liquid chromatography].

Objective: Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by dystrophin gene mutations; 55%-65% of these pathogenic mutations are large deletion and duplication mutations that can be detected by multiplexed polymerase chain reaction. However, finding the remaining micro-mutations (substitutions, deletions or insertions of one or several nucleotides) cannot be achieved in this way. The aim of the present study was to detect mutations of the dystrophin gene in individuals with Duchenne muscular dystrophy (DMD) by denaturing high-performance liquid chromatography (DHPLC) and to establish a rapid and sensitive screening platform for micro-mutations leading to DMD.

Array-MLPA: comprehensive detection of deletions and duplications and its application to DMD patients.

Multiplex ligation-dependent probe amplification (MLPA) is widely used to screen genes of interest for deletions and duplications. Since MLPA is usually based on size-separation of the amplification products, the maximum number of target sequences that can be screened in parallel is usually limited to approximately 40. We report the design of a robust array-based MLPA format that uses amplification products of essentially uniform size (100-120 bp) and distinguishes between them by virtue of incorporated tag sequences.

[Application study on inversion diagnosis of F8 gene in hemophilia A].

Objective: To establish an effective method of genetic diagnosis on hemophilia A (HA) by detecting the inversion mutation in intron 22 of F8 gene.

Methods: Intron 22 inversion mutation in F8 gene was detected by using long distance-polymerase chain reaction (LD-PCR) and inversion-PCR (I-PCR) in 31 HA patients. The mothers of HA patients with intron 22 inversion mutation were selected to carrier diagnosis and amniotic fluid of the pregnant women with inversion mutation was collected at intermediate stage of gestation, and used to prenatal genetic diagnosis.

[Analysis of association between COL9A1 gene and idiopathic congenital talipes equinovarus].

Genotypes of 2 SNPs(rs592121 and rs1135056) within COL9A1 gene in 84 ICTEV nuclear pedigrees were analyzed by restriction fragment length polymorphism and DNA sequencing. Analysis of association between SNP locus and ICTEV was performed using ETDT software. Haplotypes and their frequencies in 84 nuclear pedigrees were established and analyzed by TRANSMIT software.

[Non-invasive prenatal genetic diagnosis using multiple displacement amplification].

Objective: To investigate the feasibility of multiple displacement amplification (MDA) to apply in the non-invasive prenatal genetic diagnosis of Duchenne muscular dystrophy (DMD).

Methods: Maternal blood was obtained from 20 pregnant women at 7 to 25 weeks of gestation. After the discontinuous density gradient centrifugation with Percoll, the fetal nucleated red blood cells (NRBCs) were stained with Kleihauer test.

[Detection of fetal nucleated red blood cells in the maternal circulation by Kleihauer test].

Maternal blood was obtained from 18 pregnant women at 7 to 25 weeks of gestation. After Percoll discontinuous density gradient centifugation, the fetal nucleated red blood cells (NRBCs) were stained with Kleihauer test. Positive fetal cells appeared with an intense red cytoplasmic staining while maternal cells with adult haemoglobin were colourless.

[Proteomic analysis of the ankle joint bone, ankle joint tissue and spinal cord of clubfoot-like deformity in rat fetuses].

Objective: To explore the etiology of idiopathic talipes equinovarus (ITEV) in all-trans retinoic acid (ATRA) induced clubfoot-like deformity in rat fetuses with two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS).

Methods: Clubfoot-like deformity model in rat fetuses was induced with ATRA (135 mg/kg) in gestation day (GD10) pregnant Wistar rats. 2-DE was applied to separate the total proteins of ankle joint tissue, ankle joint bone and spinal cord of the animal models.

[Association and mutation analysis of GLI3 gene in idiopathic congenital talipes equinovarus].

Objective: To explore the association and mutation of GLI3 gene in idiopathic congenital talipes equinovarus(ICTEV).

Methods: (1) Genotype of 2 single nucleotide polymorphism (SNP) in 84 idiopathic congenital talipes equinovarus nuclear pedigree were analyzed by restriction fragment length polymorphism. Association analysis was directed between single SNP locus and ICTEV through ETDT software, respectively.

[Relationship of phenotype with type of deletion of dystrophin gene].

Objective: To detect the distribution characteristics of dystrophin gene deletions in the northeastern of China and the relationship of severity with type of deletion.

Methods: To screen deletion distribution of 124 DMD/BMD patients via multiplex PCR, male high-risk fetuses were detected deletion by the same method.

Results: The deletion frequency was 49%.

[Analysis of association between 5' HOXD gene and idiopathic congenital talipes equinovarus].

Objective: Four single nucleotide polymorphisms (SNP) in HOXD10, HOXD12 and HOXD13 genes were chosen to investigate SNP and haplotypes distribution in idiopathic congenital talipes equinovarus nuclear pedigrees.

Methods: Genotypes of 4 SNPs in 84 idiopathic congenital talipes equinovarus nuclear pedigrees were analyzed by restriction fragment length polymorphism and DNA sequencing. Analysis of association between SNP locus and idiopathic congenital talipes equinovarus was performed using ETDT software.

[DMDUse of fetal specific antibody-HbF to detect fetal erythroblasts for non-invasive prenatal diagnosis of DMD].

Maternal blood was obtained at 8-26 weeks of gestation. After discontinuous density gradient centrifugation with Percoll, HbF antibody was used to identify fetal NRBC. The precise differentiation between fetal and maternal NRBC is based on the constitutional difference between fetal and adult hemoglobin (Hb).

[Studying dystrophin gene deletion in the northeast of China and applicating].

To detect the distribution characteristics of dystrophin gene deletions of the DMD/BMD patients in the northeast of China and apply for prenatal gene diagnosis, we have analyzed the distribution of the breakpoints of the deleted-patients and the optimized primer-assembly after screening deletions of 120 DMD/BMD patients via multiplex PCR with 12-pair primers and male high-risk fetuses have been detected deletion by multiplex PCR. Results indicated the deletion frequency was 49.2%, about 66.

[Analysis and application of SCA1 and SCA3/MJD gene CAG repeats in Han population in Northeastern China].

Objective: To investigate the normal range of (CAG)n in spinocerebellar ataxia type 1 (SCA1) gene and spinocerebellar ataxia type 3 (SCA3/MJD) gene in 110 normal subjects of Han population in Northeastern China, to assess the genotypes for clinically diagnosed spinocerebellar ataxia(SCA) individuals including 25 patients from 8 families and 6 sporadic patients, and to make presymptomatic and prenatal diagnosis.

Methods: DNA fragments from the normal subjects and the patients were detected by fluorescence-PCR. Homozygosities were selected for DNA sequencing.