Biomedical subjects

Qing K Wang

Publications and source records attributed to Qing K Wang.

At least 19 recordsLinked to original sources

PubMed2006

Optical mapping of ventricular arrhythmias in LQTS mice with SCN5A mutation N1325S.

Transgenic expression of SCN5A mutation N1325S creates a mouse model for type-3 long QT syndrome (LQT3), TG-NS/LQT3. Optical mapping is a high temporal and spatial resolution fluorescence mapping system that records 256 action potentials simultaneously in a Langendorff-perfused heart. Here for the first-time, we provide a spatial view of VT in a genetic LQT3 model using optical mapping. Spontaneous VT was detected in TG-NS/LQT3 hearts, but not in littermate control hearts. VT was initiated primarily by activation of a new firing focus as well as functional conduction block of new activation waves. New firing was initiated at many different Loci in the heart, suggesting that "increased automaticity" is a key mechanism for initiation of VT. The sustained VT was maintained by a reentry mechanism. Nifedipine, an L-type calcium channel blocker, decreased the frequency of VT, indicating the involvement of abnormalities of the calcium homeostasis in the genesis of VT in TG-NS/LQT3 mice.

Animals↗

PubMed2006

Characterization of the cardiac sodium channel SCN5A mutation, N1325S, in single murine ventricular myocytes.

The N(1325)S mutation in the cardiac sodium channel gene SCN5A causes the type-3 long-QT syndrome but the arrhythmogenic trigger associated with N(1325)S has not been characterized. In this study, we investigated the triggers for cardiac events in the expanded N(1325)S family. Among 11 symptomatic patients with document triggers, six died suddenly during sleep or while sitting (bradycardia-induced trigger), three died suddenly, and two developed syncope due to stress and excitement (non-bradycardia-induced). Patch-clamping studies revealed that the late sodium current (I(Na,L)) generated by mutation N(1325)S in ventricular myocytes from TG-NS/LQT3 mice was reduced with increased pacing, which explains bradycardia-induced mortalities in the family. The non-bradycardic triggers are related to the finding that APD became prolonged and unstable at increasing rates, often with alternating repolarization phases which was corrected with verapamil. This implies that Ca2+ influx and intracellular Ca2+ ([Ca2+]i) ions are involved and that [Ca2+]i inhomogeneity may be the underlying mechanisms behind non-bradycardia LQT3 arrhythmogenesis associated with mutation N(1325)S.

Animals↗

PubMed2006

A novel de novo frame-shift mutation of the EDA gene in a Chinese Han family with hypohidrotic ectodermal dysplasia.

Hypohidrotic ectodermal dysplasia (HED) is characterized by severe hypohidrosis, hypotrichosis, and hypodontia. It can be inherited in autosomal dominant, autosomal recessive, or X-linked patterns. Mutations in the EDA gene, which encodes ectodysplasin-A, are responsible for X-linked HED (XLHED). In the present study, we identified a Chinese Han family with XLHED. Direct DNA sequence analysis of the entire coding region and exon-intron boundaries of EDA identified a novel de novo mutation, c.573_574insT, in two affected males and one carrier female. Restriction fragment length polymorphism (RFLP) analysis showed that the mutation was not present in 200 controls. The 1-bp insertion mutation resulted in a frameshift, which causes premature termination of EDA polypeptide and truncation of the EDA protein. These results suggest that the c.573_574insT mutation of the EDA gene is a cause for XLHED in the family. To the best of our knowledge, this is the first de novo insertion mutation of EDA described for XLHED.

Adult↗

PubMed2006

Genetic susceptibility to myocardial infarction and coronary artery disease.

Atherosclerotic involvement in the coronary arteries, which can result in heart attack and sudden death, is a common disease and prototypic of a complex human trait. To understand its genomic basis, eight linkage studies of sibling pairs have been performed. Although there was limited inter-study concordance of important loci, two gene variants in the leukotriene pathway (ALOX5AP and LTA4) have emerged as susceptibility factors for myocardial infarction (MI). Genome-wide association studies have also been undertaken, and the pro-inflammatory cytokine lymphotoxin-alpha (LTA), and its key ligand galectin-2 (LGALS2) have been identified as genes implicated in predisposition for heart attack. By cueing into the genomic basis for low serum LDL cholesterol levels, much work has been done to advance the importance of the serine protease PCSK9, which modulates LDL receptor function. Lifelong lowered LDL cholesterol associated with PCSK9 point mutations in 2-3% of individuals have been shown to provide marked protection from coronary artery disease (CAD). Most of the success in this field has been with the phenotype of MI, which is considerably more restrictive than CAD. Four principal and interdependent processes--lipoprotein handling, endothelial integrity, arterial inflammation, and thrombosis--have been supported as important via the clustering of genes, thus far implicated in CAD susceptibility. Of note, connecting genes in a single pathway (leukotriene), of a protein and its ligand (LTAalpha) or from one disease to another [age-related macular degeneration (AMD); complement factor H (CFH)], or even three disease characterized by inflammation (MHC2) have now been reported. Although the population attributable risk for any of the genes identified to date is limited, such discovery is likely to be accelerated in the future.

5-Lipoxygenase-Activating Proteins↗

PubMed2006

A novel mutation in GDF5 causes autosomal dominant symphalangism in two Chinese families.

Proximal symphalangism (SYM1) is an autosomal dominant disorder characterized by ankylosis of the proximal interphalangeal joints and fusion of carpal and tarsal bones. We identified and characterized two five-generation Chinese families with SYM1. The two families share some similarities (e.g., osseous fusion of interphalangeal joints of the 2-4 fingers) with SYM1 families with mutations in the NOG gene or the family with mutation R438L recently reported in the GDF5 gene (encoding a bone morphogenetic protein family member). However, they show some unique features including the absence of cuboid bone, the lack of shortness of the first and fifth metacarpal bones, and manifestation of flat feet. Genome-wide linkage analysis of the two families mapped the disease gene to marker D20S112 with a combined LOD score of 4.32. Mutational analysis revealed a novel E491K mutation in the GDF5 gene in both families. The mutation occurs at a highly conserved residue in the TGF-beta domain of GDF5 and represents the second GDF5 mutation identified for SYM1 to date. The E491K mutation co-segregated with the affected individuals in the two families, and did not exist in unaffected family members or 200 normal controls. These results indicate that defects in GDF5 can cause SYM1 in the Chinese population, and expand the spectrum of clinical phenotypes associated with mutant GDF5.

Amino Acid Sequence↗

PubMed2006

A novel heterozygous mutation in the Indian hedgehog gene (IHH) is associated with brachydactyly type A1 in a Chinese family.

Brachydactyly type A1 (BDA1) is caused by mutations in the Indian hedgehog gene, IHH, on chromosome 2q35-36. In this study, a large five-generation Chinese family with BDA1 was identified and characterized. All affected family members demonstrated significant homogeneous phenotype and some unique clinical features different from those associated with the reported BDA1 mutations in IHH. Linkage analysis showed that the BDA1 gene in the family was linked to marker D2S126 close to IHH with a LOD score of 4.74 at a recombination fraction of 0. DNA sequence analysis revealed a heterozygous C to T transition at nucleotide 461 of IHH, resulting in a novel T154I substitution. The T154I mutation co-segregated with all affected individuals in the family, and was not present in normal family members or 200 normal controls. These results expand the spectrum of clinical phenotype associated with IHH mutations.

Amino Acid Sequence↗

PubMed2006

Loss of heterozygosity in human aberrant crypt foci (ACF), a putative precursor of colon cancer.

Aberrant crypt foci (ACF), the earliest neoplastic lesions of the colon, have genetic and epigenetic alterations. Loss of heterozygosity (LOH) of tumor suppressor gene loci is seen in most colon cancers, but it is not known how early in tumorigenesis this takes place. Nine microsatellite markers close to specific genes, that is, APC (5q21), PTPRJ (11p11), p53 (17p13) and DCC (18q21), were analyzed in 32 ACF and samples of normal crypts from the same 28 patients. Six losses of heterozygosity were found in 5 of 32 ACF: 4 losses of heterozygosity were at 11p11, the location of the gene for protein tyrosine phosphatase receptor type J (PTPRJ) and of a second independent region of deletion; the others were at 5q21 and 18q21. Microsatellite instability (MSI) with markers for a single locus was found in 4 of 32 ACF. All the observed allelic alterations (LOH and MSI) were in 8 of 32 ACF. The finding of LOH in ACF with normal expressions of adenomatous polyposis coli (APC) and beta-catenin proteins suggests that LOH can occur very early in colon neoplasia and perhaps even before APC mutations. The finding of 3 of 4 of the losses of heterozygosity at 11p11 for PTPRJ and half of all the losses of heterozygosity in this study at PTPRJ suggest that this gene plays a role early in colon neoplasia.

Adenomatous Polyposis Coli↗

PubMed2006

Discovery of Time-Delayed Gene Regulatory Networks based on temporal gene expression profiling.

BACKGROUND: It is one of the ultimate goals for modern biological research to fully elucidate the intricate interplays and the regulations of the molecular determinants that propel and characterize the progression of versatile life phenomena, to name a few, cell cycling, developmental biology, aging, and the progressive and recurrent pathogenesis of complex diseases. The vast amount of large-scale and genome-wide time-resolved data is becoming increasing available, which provides the golden opportunity to unravel the challenging reverse-engineering problem of time-delayed gene regulatory networks. RESULTS: In particular, this methodological paper aims to reconstruct regulatory networks from temporal gene expression data by using delayed correlations between genes, i.e., pairwise overlaps of expression levels shifted in time relative each other. We have thus developed a novel model-free computational toolbox termed TdGRN (Time-delayed Gene Regulatory Network) to address the underlying regulations of genes that can span any unit(s) of time intervals. This bioinformatics toolbox has provided a unified approach to uncovering time trends of gene regulations through decision analysis of the newly designed time-delayed gene expression matrix. We have applied the proposed method to yeast cell cycling and human HeLa cell cycling and have discovered most of the underlying time-delayed regulations that are supported by multiple lines of experimental evidence and that are remarkably consistent with the current knowledge on phase characteristics for the cell cyclings. CONCLUSION: We established a usable and powerful model-free approach to dissecting high-order dynamic trends of gene-gene interactions. We have carefully validated the proposed algorithm by applying it to two publicly available cell cycling datasets. In addition to uncovering the time trends of gene regulations for cell cycling, this unified approach can also be used to study the complex gene regulations related to the development, aging and progressive pathogenesis of a complex disease where potential dependences between different experiment units might occurs.

Algorithms↗

PubMed2006

High-throughput single-nucleotide polymorphisms genotyping: TaqMan assay and pyrosequencing assay.

Single-nucleotide polymorphisms (SNPs) are DNA sequence variations that occur at a single base in the genome sequence. SNPs are valuable markers for identifying genes responsible for susceptibility to common diseases, and in some cases, they are the causes of human diseases. A genetic study of a complex disease usually involves a case-control association study that requires genotyping of a large number of SNPs in hundreds of patients (cases) and matched controls. A significant difference of the allele frequency or genotypic frequency of a SNP between the two populations is considered to be the evidence for the association between the SNP and disease. A key to a fast and effective case-control association study requires high-throughput genotyping of SNPs. Two assays-the TaqMan SNP genotyping assay and the pyrosequencing assay-have been developed for this purpose and proven to be particularly useful. Here, we present the operative protocol, clarify the key technical issues, and highlight certain cautionary notes for high throughput SNP genotyping using TaqMan and pyrosequencing assays.

Gene Frequency↗

PubMed2006

Fluorescence in situ hybridization in cardiovascular disease.

Many human diseases are associated with cytogenetic abnormalities or chromosomal disorders including translocations, deletions, duplications, inversions, and other complicated chromosomal changes. Fluorescence in situ hybridization (FISH), a technique involving hybridization of labeled probes to chromosomes and detection of hybridization via fluorochromes, has become a popular method for identification and characterization of cytogenetic abnormalities. For FISH analysis, metaphase chromosomes are prepared by mitotic arrest and hypotonic shock, and denatured. Hybridization of digoxigenin- or biotin-labeled probes to these chromosomes is visualized using fluorochromes like fluorescein isothiocyanate and Texas Red. We have successfully applied FISH technology to the characterization of chromosome breakpoints involved in disease-associated cytogenetic abnormalities to identify candidate gene(s) for the disease. FISH is also widely used in clinical diagnosis of chromosomal disorders.

Animals↗

PubMed2006

Novel HSF4 mutation causes congenital total white cataract in a Chinese family.

PURPOSE: To identify the disease-causing gene (mutation) in a Chinese family affected with autosomal dominant congenital total white cataract. DESIGN: Observational case series. METHODS: Genotyping and linkage analyses were used to identify the linkage of the disease-causing gene in the Chinese family to the HSF4 gene encoding a member of the family of heat shock transcription factors (HSFs). Direct DNA sequence analysis was used to identify the disease-causing mutation. Polymerase chain reaction/restriction fragment length polymorphism analysis was used to demonstrate cosegregation of the HSF4 mutation with the cataract and the absence of the mutation in the normal controls. RESULTS: The cataract gene in the Chinese family was linked to marker D16S3043, and further haplotype analysis defined the causative gene between D16S515 and D16S415 within which HSF4 is located. A novel mutation c.221G>A was identified in HSF4, which results in substitution of a highly conserved arginine residue by histidine at codon 74 (p.R74H). The R74H mutation cosegregated with the affected individuals in the family and did not exist in unaffected family members and 150 unrelated normal controls. CONCLUSIONS: These results identified a novel missense mutation R74H in the transcription factor gene HSF4 in a Chinese cataract family and expand the spectrum of HSF4 mutations causing cataract.

Age of Onset↗

PubMed2006

Construction of somatic cell hybrid lines: fusion of mouse thymidine kinase-deficient 3T3 fibroblasts and human lymphoblastoid cells.

Somatic cell hybrids are generated by fusion of two different parental cells. This technology has been used extensively in the production of monoclonal antibodies and has made significant contributions to the field of human genetics through its applications in gene expression, gene mapping, and positional cloning of human disease genes. In our laboratory, we have employed this technique in the positional cloning of several genes for human diseases associated with cytogenetic abnormalities (chromosomal disorders), including translocations. Somatic cell hybrids are constructed by fusing mouse thymidine kinase-deficient 3T3 fibroblasts with human lymphoblastoid cells, as a result of which specific hybrid cells containing only cytogenetically abnormal human chromosomes involved in a chromosomal disorder can be successfully isolated and cloned. These hybrid cells serve as an excellent tool with which to define the exact chromosomal breakpoints involved in a cytogenetic abnormality and to identify genes at the breakpoints.

3T3 Cells↗

PubMed2006

LINKAGE programs: linkage analysis for monogenic cardiovascular diseases.

Identification of the genes for a human disease provides significant insights into the molecular mechanism underlying the pathogenesis of the disease. A human disease gene can be identified by its chromosomal location (positional cloning). Linkage analysis is a key step in positional cloning. For monogenic disorders with a known inheritance pattern, model-based linkage analysis is effective in mapping the disease location. Therefore, model-based linkage analysis can provide a powerful tool to positional cloning of some specific molecular determinants that co-segregate with disease phenotypes in the isolated samples (e.g., large and multiplex impaired pedigrees). This chapter describes model-based human genetic linkage analysis as implemented in the LINKAGE computer package. First, we introduce the basic concepts and principles for genetic analysis of monogenic disorders. Then, we demonstrate the usages of the programs by analyzing several examples of hypothetical pedigrees with the inheritance modes of autosomal-dominant, autosomal-recessive, and genetic heterogeneity.

Cardiovascular Diseases↗

PubMed2006

SAGE programs: model-free linkage analysis for complex cardiovascular phenotypes.

A complex disease trait refers to a phenotype that does not follow simple Mendelian segregation attributable to a single gene locus, but instead may be caused by multiple disease loci, their interactions, polygenic inheritance, and environmental effects. Most cardiovascular disorders are thought to have a polygenic basis with complex interactions with environmental factors. A gene that increases or decreases the risk to a complex cardiovascular disease (susceptibility gene) can now be mapped to a specific chromosomal region by model-free linkage analysis, and follow-up molecular genetic studies can identify the specific gene at the locus. This chapter describes a protocol for model-free linkage analysis of a complex trait, as implemented in the popular genetic analysis software-SAGE. In particular, the Haseman-Elston sib-pair regression method is introduced and implemented with examples to demonstrate how to identify susceptibility loci for complex traits.

Cardiovascular Diseases↗

PubMed2006

Microarray analysis of cardiovascular diseases.

Microarray analysis is a powerful technique for high-throughput, global transcriptonomic profiling of gene expression. It holds great promise for analyzing the genetic and molecular bases of cardiovascular diseases and various other complex diseases and permits the analysis of thousands of genes simultaneously, both in diseased and nondiseased tissues and/or cell lines. Microarrays or microchips are made by depositing spots of DNA or oligonucleotides representing thousands of genes on a solid support such as a coated glass surface, and can allow the comparison of gene expression patterns in any two samples. Total RNA is isolated from the tissue or cells of interest, converted to cDNA and then cRNA labeled with biotin, and hybridized to the chips. Hybridization signals are then quantified and compared among different samples. We used oligonucleotide microarrays to obtain an unbiased assessment of expression levels of thousands of genes simultaneously in normal and diseased coronary arteries. Fifty-six genes showed differential expression in atherosclerotic coronary artery tissues, and 49 of them represent new linked genes for coronary artery disease. These studies can generate novel hypotheses relating to the pathologies of disease and further studies with animal models, molecular biology, cell biology, and biochemistry will validate these hypotheses and provide novel insights into the pathogenesis of disease.

Biotin↗

PubMed2006

Animal models for cardiac arrhythmias.

Transgenic and gene-targeted mice are now frequently used to expand the study of cardiac physiology and pathophysiology owing to the ease with which the mouse genome can be manipulated. There are many measures by which an assessment of the phenotypical expression of the transgenic mouse can be made. In the case of cardiac channelopathies and how they relate to cardiac function, telemetry is a technology that utilizes transmitters that are surgically implanted in animals for the purpose of acquiring biopotentials or physiological parameters. Electrophysiological techniques have also been used to assess cardiac function at the cellular level, by measuring whole-cell ionic currents and/or transmembrane potentials. This chapter will discuss the surgical procedures involved in successfully implanting the transmitter device in a mouse, as well as highlight the recording of and analysis of electrocardiograms. This chapter will also outline the procedures involved in isolating single-ventricular myocytes from a mouse heart. It is a protocol that was developed in our laboratory for which we have routinely and successfully isolated myocytes from both transgenic and nontransgenic mouse hearts. Although no one isolation protocol is alike, we also present our own observations that have assisted in maximizing myocyte bioavailability and yield.

Animals↗

PubMed2006

Proteomics with two-dimensional gel electrophoresis and mass spectrometry analysis in cardiovascular research.

Proteomics is a large-scale, comprehensive study of the proteins of a cell or organism. It is a unique means of characterizing proteins that are expressed in a cell or tissue at any given time-point and of identifying any modifications that they may undergo. Thus, it is a powerful technology that can detect and identify the changes of the structure and function of proteins in response to intra- and extracellular environmental signals or disease states. As proteomics can establish a link for genes and proteins with a disease, it will play an important role in defining the molecular determinants of a disease and in identifying targets for drug discoveries and diagnostics. We have carried out the first proteomics study for coronary artery disease (CAD) and found that the expression of the ferritin light chain was significantly increased in CAD tissues. In this chapter, we use the CAD study as an example to demonstrate the procedures involved in proteomics analysis. The proteome is visualized by two-dimensional gel electrophoresis, a powerful and widely used method for proteomics, and the proteins of interest are then identified by mass spectrometry. This technique should be useful in characterizing cardiovascular diseases and in defining signaling pathways for cardiovascular development and physiology.

Cardiovascular Diseases↗

PubMed2006

Generation of transgenic mice for cardiovascular research.

The transgenic mouse technology is a powerful tool that can be used for creating animal models for cardiovascular disease to identify molecular pathogenic mechanisms and for identifying the physiological functions of a novel gene. A transgenic animal can be generated by several methods, which include microinjection of a DNA fragment into the pronucleus, embryonic stem cell manipulation and injection, sperm-mediated transgenesis, and viral infection of preimplanted embryos. The microinjection method is one of the most widely used approaches. This method involves four steps: (1) collection of fertilized eggs from the superovulated female, (2) injection of DNA into the pronucleus of fertilized eggs, (3) transfer of the injected eggs back into the oviduct of a pseudopregnant foster recipient, allowing the eggs to develop into pups, and (4) identification of the transgenic founder and establishment of transgenic lines through further breeding.

Animals↗