In Response to Cluster Analysis of Patients With Empty Nose Syndrome and Its Relation to Surgical Outcome.
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Letter↗
Biomedical subjects
Yun-Shien Lee
Publications and source records attributed to Yun-Shien Lee.
Promoter polymorphisms of DNMT3B and the risk of head and neck squamous cell carcinoma in Taiwan: a case-control study.
Three single nucleotide polymorphisms (SNPs) of the DNMT3B promoter region, C46359T (-149C>T), -283T>C, and -579G>T might be a cancer susceptible factor for several cancers. In this study, we genotyped 226 head-and-neck squamous-cell carcinoma (HNSCC) patients and 249 controls to examine the association between three SNPs of the DNMT3B promoter and the associated risk of the development and/or metastasizing tendency of HNSCC for the population of Taiwan. We observed that only the T/T genotype (C46359T) was found to be present in both patient and control groups (100% frequency). The alleles frequency of -283CC, -283CT and -283TT among patients and controls was, respectively, 88.1% versus 84.3%, 11.9% versus 15.3%, and 0% versus 0.4%. The allele -597GG was not found in both groups, whereas the allele frequency of -597TT and -597GT for patients and controls was, respectively, 88.1% versus 85.5%, and 11.9% versus 14.5%. For both DNMT3B SNPs, inter-group comparison of the allele frequency between patients and controls and distribution of SNPs among cancer patients either featuring or not featuring cervical metastasis did not reveal any significant difference. In conclusion, the relative distribution of three DNMT3B SNPs among a Taiwanese population can not be used as a stratification marker to predict either an individual's susceptibility to HNSCC and/or the likelihood of cervical metastasis of HNSCC.
Adult↗
Molecular characterization of adenocarcinoma and squamous carcinoma of the uterine cervix using microarray analysis of gene expression.
In an attempt to understand the molecular mechanisms for the different clinical features between adenocarcinoma/adenosquamous carcinoma (AC/ASC) and squamous carcinoma (SC) of the uterine cervix, we analyzed gene expression profiles of different histological subtypes of cervical cancer. Cancer specimens and the surrounding normal tissue counterparts were separately collected from cervical cancer patients undergoing type III radical hysterectomy. Paired total RNA (cancer and normal tissues) was isolated and analyzed with cDNA microarrays containing duplicate spots of 7 334 sequence-verified human cDNA clones. Selected differentially expressed genes specific for AC or SC were further verified using real-time quantitative polymerase chain reaction (RTQ-PCR) and immunohistochemistry. Genes, including CEACAM5, TACSTD1, S100P and MSLN were upregulated in AC. Contrarily, genes involved in epidermal differentiation complex such as S100A9 and ANXA8 were upregulated in SC. Cross-validation of the results using an independent but comparable group of patients with known long-term outcomes (n = 63, median follow-up 70.3 months; range, 4-208 months) showed that the correlation between the selected 6 differentially expressed genes and histology was highly significant. CEACAM5 (p < 0.0001) and TACSTD1 (p = 0.009) were significant prognostic factors by multivariate Cox proportional hazards regression analysis. The combination of cDNA microarray, RTQ-PCR and immunohistochemical results of this study showed that it is possible to define different gene profiles for AC and SC. Moreover, TACSTD1 expression may be a novel poor prognostic factor.
Adenocarcinoma↗
Interleukin-1 receptor antagonist (IL-1RA) prevents apoptosis in ex vivo expansion of human limbal epithelial cells cultivated on human amniotic membrane.
Stem cells of the corneal epithelium have been found to be located exclusively at the anatomical junction between the cornea and the conjunctiva, the limbus. Ex vivo expanded limbal epithelial cells on amniotic membrane (AM) are capable of restoring the corneal surface with limbal stem cell deficiency. Recent studies indicate that intact AM preserves the limbal epithelial phenotype and that distinct epithelial morphology is noted among various culture matrix. However, the factors in response to the interaction between limbal epithelial cells and AM were not well understood. Using Annexin V-fluorescein isothiocyanate staining, we found that human limbal epithelial cells expanded on intact human AM demonstrated fewer apoptotic cells as compared with those on plastic dishes. To identify the anti-apoptotic factors, we performed cDNA microarray analysis and showed that interleukin-1 receptor antagonist (IL-1RA) was overexpressed in cultures on intact AM, which was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (Q-PCR) and enzyme-linked immunosorbent assay. In addition, we also noted that the phenomenon of apoptosis detected in cultures on plastic dishes could be reversed by adding recombinant IL-1RA protein into the media, whereas apoptosis of limbal epithelial cells cultivated on intact AM could be induced by exogenous neutralizing IL-1RA neutralizing antibody. These results demonstrated that intact human AM may prevent cultured human limbal epithelial cells from undergoing apoptosis. IL-1RA might be a candidate mediator to exert as an anti-apoptotic molecule during the interaction between human limbal epithelial cells and intact human AM.
Adolescent↗
Differentially profiling the low-expression transcriptomes of human hepatoma using a novel SSH/microarray approach.
BACKGROUND: The main limitation in performing genome-wide gene-expression profiling is the assay of low-expression genes. Approaches with high throughput and high sensitivity for assaying low-expression transcripts are urgently needed for functional genomic studies. Combination of the suppressive subtractive hybridization (SSH) and cDNA microarray techniques using the subtracted cDNA clones as probes printed on chips has greatly improved the efficiency for fishing out the differentially expressed clones and has been used before. However, it remains tedious and inefficient sequencing works for identifying genes including the great number of redundancy in the subtracted amplicons, and sacrifices the original advantages of high sensitivity of SSH in profiling low-expression transcriptomes. RESULTS: We modified the previous combination of SSH and microarray methods by directly using the subtracted amplicons as targets to hybridize the pre-made cDNA microarrays (named as "SSH/microarray"). mRNA prepared from three pairs of hepatoma and non-hepatoma liver tissues was subjected to the SSH/microarray assays, as well as directly to regular cDNA microarray assays for comparison. As compared to the original SSH and microarray combination assays, the modified SSH/microarray assays allowed for much easier inspection of the subtraction efficiency and identification of genes in the subtracted amplicons without tedious and inefficient sequencing work. On the other hand, 5015 of the 9376 genes originally filtered out by the regular cDNA microarray assays because of low expression became analyzable by the SSH/microarray assays. Moreover, the SSH/microarray assays detected about ten times more (701 vs. 69) HCC differentially expressed genes (at least a two-fold difference and P < 0.01), particularly for those with rare transcripts, than did the regular cDNA microarray assays. The differential expression was validated in 9 randomly selected genes in 18 pairs of hepatoma/non-hepatoma liver tissues using quantitative RT-PCR. The SSH/microarray approaches resulted in identifying many differentially expressed genes implicated in the regulation of cell cycle, cell death, signal transduction and cell morphogenesis, suggesting the involvement of multi-biological processes in hepato-carcinogenesis. CONCLUSION: The modified SSH/microarray approach is a simple but high-sensitive and high-efficient tool for differentially profiling the low-expression transcriptomes. It is most adequate for applying to functional genomic studies.
Carcinoma, Hepatocellular↗
Microarray-based comparative genomic hybridization analysis of Wolf-Hirschhorn syndrome in a fetus with deletion of 4p15.3 to 4pter.
BACKGROUND: Wolf-Hirschhorn syndrome (WHS), caused by the deletion of a segment in chromosome 4, is characterized by mental and developmental defects. Clinical manifestations of WHS include intrauterine growth restriction, failure to thrive in the neonatal period that is present simultaneously with hypotonia, typical "Greek helmet" facial appearance, cleft lip and palate, mental deficiency, and seizures. CASE: We present a case of WHS with prenatal conventional cytogenetics of 46,XY,der(4)t(4;13)(p15.3;p11.2)pat. High-resolution mapping using microarray-based comparative genomic hybridization (array-CGH), including Affymetrix 10K arrays and cDNA microarrays, confirmed the loss of genes in the deleted region. CONCLUSIONS: The correlation between these candidate genes and the phenotypes of WHS may expand our understanding of the defective development caused by 4p deletion.
Abnormalities, Multiple↗
Identification of differential gene expression between intestinal and diffuse gastric cancer using cDNA microarray.
To compare the gene expression profiling between intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC), cDNA microarray containing 7334 gene elements was performed on 12 paired IGC specimens/its' normal epithelial tissue and 11 paired DGC specimens/its' normal epithelial tissue. Twenty-seven genes were co-overexpressed in IGC and DGC. These overexpressed genes were related to transcription and translation, DNA replications and mitosis, calcium binding, apoptosis and mitochondria protein. Twelve genes were co-underexpressed in IGC and DGC. These underexpressed genes were associated with cell adhesion and migration, organelle movement and intracellular transport, and matrix metalloproteinase. A clustering dendrogram of IGC and DGC with 27 genes significantly differed between IGC and DGC. Nineteen genes were more overexpressed in DGC than in IGC, including annexin A1 (ANXA1), chemokine ligand 7 (CCL7), and chemokine ligand 8 (CCL8). Eight genes were more overexpressed in IGC than in DGC, including claudin 4 (CLDN4). The results of quantitative real-time PCR and immunohistochemical staining confirmed the microarray finding. The gene expression profiling between IGC and DGC suggested that they might have unique genetic pathways which share some of the same and some different genetic alterations.
Adult↗
Molecular signature of clinical severity in recovering patients with severe acute respiratory syndrome coronavirus (SARS-CoV).
BACKGROUND: Severe acute respiratory syndrome (SARS), a recent epidemic human disease, is caused by a novel coronavirus (SARS-CoV). First reported in Asia, SARS quickly spread worldwide through international travelling. As of July 2003, the World Health Organization reported a total of 8,437 people afflicted with SARS with a 9.6% mortality rate. Although immunopathological damages may account for the severity of respiratory distress, little is known about how the genome-wide gene expression of the host changes under the attack of SARS-CoV. RESULTS: Based on changes in gene expression of peripheral blood, we identified 52 signature genes that accurately discriminated acute SARS patients from non-SARS controls. While a general suppression of gene expression predominated in SARS-infected blood, several genes including those involved in innate immunity, such as defensins and eosinophil-derived neurotoxin, were upregulated. Instead of employing clustering methods, we ranked the severity of recovering SARS patients by generalized associate plots (GAP) according to the expression profiles of 52 signature genes. Through this method, we discovered a smooth transition pattern of severity from normal controls to acute SARS patients. The rank of SARS severity was significantly correlated with the recovery period (in days) and with the clinical pulmonary infection score. CONCLUSION: The use of the GAP approach has proved useful in analyzing the complexity and continuity of biological systems. The severity rank derived from the global expression profile of significantly regulated genes in patients may be useful for further elucidating the pathophysiology of their disease.
Algorithms↗
Touchdown thermocycling program enables a robust single nucleotide polymorphism typing method based on allele-specific real-time polymerase chain reaction.
Different methods have been developed for single nucleotide polymorphism (SNP) typing during recent years. Allele-specific polymerase chain reaction (ASPCR) is a cost-saving method that scores SNPs by difference of the PCR efficiency of allele-specific primers. However, ASPCR for SNP typing is notoriously confounded for its locus-specific unpredictability and the laborious gel electrophoresis. In the current study, we investigated the real-time kinetics of ASPCR and found that a simple touchdown thermocycling protocol improved its specificity significantly. Combined with real-time PCR, we developed a homogeneous genotyping method and scored more than 1000 genotypes, including all transition and transversion SNPs. A clear genotyping result was identified and validated the robustness of the method. Optimization of reactions and intrinsic modification of allele-specific primers, a laborious process but one that is repeatedly reported to be inevitable for successful ASPCR, was proved to be unnecessary with our method. Accuracy was confirmed with mass spectrometry. These characters enabled real-time ASPCR with the touchdown thermocycling protocol being very competitive among various SNP typing methods for large-scale genetic studies.
Alleles↗
Linear allele-specific long-range amplification: a novel method of long-range molecular haplotyping.
Haplotypes have been repeatedly shown to be more powerful than collections of single-locus markers in gene-mapping studies. Various haplotyping methods including statistical estimation are employed but molecular haplotyping, the acquisition of information directly on physical DNA sequences, has been in demand for its accuracy and independence from family pedigrees. We investigated the allelic specificity of long-range PCR, which was successful for long-range haplotyping in recent reports, and found problems of initial mispriming and crossover amplification significantly confounded its application. Based on these observations, we designed a novel method based on linear amplification of a hemizygous DNA segment with a single phosphorothioate-modified oligonucleotide. Our results revealed, with a single nucleotide polymorphism as the discriminative marker, downstream haplotypes of 14-15 kb DNA segment could be confidently scored. With two rounds of the method and five single nucleotide polymorphisms, molecular haplotypes of 29.3 kb spanning the HCR and CDSN genes, two genes associated with the susceptibility of psoriasis, of 11 members, belonging to a CEPH family, were revealed. Clear Mendelian segregation of 35 highly heterozygous SNPs confirmed the accuracy of the method. Problems of low specificity associated with long-range PCR were not observed. The simplicity, along with long-sequence accessibility and feasibility of a single nucleotide difference as the discriminative marker indicated our method holds promise for future gene-mapping studies.
Adenomatous Polyposis Coli↗
Thyroid hormone receptor-dependent transcriptional regulation of fibrinogen and coagulation proteins.
Thyroid hormone (T(3)) regulates growth, development, and differentiation. These activities are mediated by the nuclear thyroid hormone receptors (TRs), which belong to the steroid/TR superfamily of ligand-dependent transcription factors. The effect of T(3) treatment on target gene regulation was investigated in a TRalpha-overexpressing hepatoma cell line (HepG2-TRalpha), by performing cDNA microarrays. We demonstrate that 148 of the 7597 genes represented were up-regulated by T(3), including fibrinogen and several other components of the coagulation factor system. To confirm the microarray results, fibrinogen and a small number of the blood clotting components were further investigated using quantitative RT-PCR. The T(3)-induction ratios observed with quantitative RT-PCR for factors such as thrombin (8-fold), coagulation factor X (4.9-fold), and hepatoglobin (30-fold) were similar to those observed by the cDNA microarray analysis. Further investigation, using HepG2-TRalpha (cell lines, revealed a 2- to 3-fold induction of fibrinogen transcription after 24 h of T(3) treatment. In addition, T(3) treatment increased the level of fibrinogen protein expression 2.5- to 6-fold at 48 h. The protein synthesis inhibitor, cycloheximide, did not inhibit the induction of fibrinogen by T(3), indicating that this regulation was direct. Furthermore, transcription run-on experiments indicate that the induction of fibrinogen by T(3) is regulated largely at the level of transcription. Similar observations were made on the regulation of fibrinogen by T(3) using rats that received surgical thyroidectomy (TX) as an in vivo model. These results suggest that T(3) plays an important role in the process of blood coagulation and inflammation and may contribute to the understanding of the association between thyroid diseases and the misregulation of the inflammatory and clotting profile evident in the circulatory system of these patients.
Animals↗
Establishment of cDNA microarray analysis at the Genomic Medicine Research Core Laboratory (GMRCL) of Chang Gung Memorial Hospital.
BACKGROUND: Advances in molecular and computational biology have led to the development of powerful, high-throughput methods for analysis of differential gene expression, which are opening up new opportunities in genomic medicine. DNA microarray technology has been enthusiastically integrated into basic biomedical research and will eventually become a molecular monitoring tool for various clinical courses. METHODS: As a core research facility of Chang Gung University (CGU) and Chang Gung Memorial Hospital (CGMH), the Genomic Medicine Research Core Laboratory (GMRCL) welcomes investigators from every discipline to employ DNA microarray technology in the quest for knowledge of genomic medicine. The first tasks for GMRCL are to optimize the standard operating procedures (SOP) for each instrument and to assure the quality of every procedure. RESULTS: During the first year after the establishment of the GMRCL at the CGMH, we tested and adopted procedures that were satisfactory for our purposes. These procedures included: replication of bacterial stocks, amplification of human DNA clones, annotation of each DNA clone, production of cDNA microarrays, validation of RNA quality and quantity, labeling of target specimens, competitive hybridization, scanning of slides, data analysis, and post-microarray validation of results. We present a summarization of the materials and procedures used at the GMRCL and discuss the reasons for using them. CONCLUSIONS: The information about the cDNA microarray analysis system at the GMRCL is compliant with the minimal information about a microarray experiment (MIAME) format. The information may be useful to both the investigators who are using this core facility and researchers at other institutes, who will establish their own in-house cDNA microarray systems.
Humans↗