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Supplementary Table S1 from Novel Blood Biomarkers of Human Urinary Bladder Cancer

Purpose: Recent data indicate that cDNA microarray gene expression profile of blood cells can reflect disease states and thus have diagnostic value. We tested the hypothesis that blood cell gene expression can differentiate between bladder cancer and other genitourinary cancers as well as between bladder cancer and healthy controls.Experimental Design: We used Affymetrix U133 Plus 2.0 GeneChip (Affymetrix, Santa Clara, CA) to profile circulating blood total RNA from 35 patients diagnosed with one of three types of genitourinary cancer [bladder cancer (n = 16), testicular cancer (n = 10), and renal cell carcinoma (n = 9)] and compared their cDNA profiles with those of 10 healthy subjects. We then verified the expression levels of selected genes from the Affymetrix results in a larger number of bladder cancer patients (n = 40) and healthy controls (n = 27).Results: Blood gene expression profiles distinguished bladder cancer patients from healthy controls and from testicular and renal cancer patients. Differential expression of a combined set of seven gene transcripts (insulin-like growth factor–binding protein 7, sorting nexin 16, chondroitin sulfate proteoglycan 6, and cathepsin D, chromodomain helicase DNA-binding protein 2, nell-like 2, and tumor necrosis factor receptor superfamily member 7) was able to discriminate bladder cancer from control samples with a sensitivity of 83% (95% confidence interval, 67-93%) and a specificity of 93% (95% confidence interval, 76-99%).Conclusion: We have shown that the gene expression profile of circulating blood cells can distinguish bladder cancer from other types of genitourinary cancer and healthy controls and can be used to identify novel blood markers for bladder cancer.

Background Peripheral blood transcriptome profiling is a potentially important tool for disease detection. We utilize this technique in a case-control study to identify candidate transcriptomic biomarkers able to differentiate women with breast lesions from normal controls. Methods Whole blood samples were collected from 50 women with high-risk breast lesions, 57 with breast cancers and 44 controls (151 samples). Blood gene expression profiling was carried out using microarray hybridization. We identified blood gene expression signatures using AdaBoost, and constructed a predictive model differentiating breast lesions from controls. Model performance was then characterized by AUC sensitivity, specificity and accuracy. Biomarker biological processes and functions were analyzed for clues to the pathogenesis of breast lesions. Results Ten gene biomarkers were identified (YWHAQ, BCLAF1, WSB1, PBX2, DDIT4, LUC7L3, FKBP1A, APP, HERC2P2, FAM126B). A ten-gene panel predictive model showed discriminatory power in the test set (sensitivity: 100%, specificity: 84.2%, accuracy: 93.5%, AUC: 0.99). These biomarkers were involved in apoptosis, TGF-beta signaling, adaptive immune system regulation, gene transcription and post-transcriptional protein modification. Conclusion A promising method for the detection of breast lesions is reported. This study also sheds light on breast cancer/immune system interactions, providing clues to new targets for breast cancer immune therapy.

It is crucial to classify cervical lesions into high-grade squamous intraepithelial lesions (HSILs) and low-grade SILs (LSILs), as LSILs are conservatively treated by observation, based on an expectation of natural regression, whereas HSILs usually require electrosurgical excision. In the present study, peripheral blood gene expression profiles were analyzed to identify transcriptomic biomarkers distinguishing HSILs from LSILs. A total of 102 blood samples were collected from women with cervical SILs (66 HSIL and 36 LSIL) for microarray hybridization. Candidate gene signatures were identified using AdaBoost algorithms, and a predictive model was constructed using logistic regression to differentiate HSILs from LSILs. To correct for possible bias as a result of the limited sample size and to verify the stability of the predictive model, a two-fold cross validation and null set analysis was conducted over 1,000 iterations. The functions of the transcriptomic biomarkers were then analyzed to elucidate the pathogenesis of cervical SIL. A total of 10 transcriptomic genes (STMN3, TRPC4AP, DYRK2, AGK, KIAA0319L, GRPEL1, ZFC3H1, LYL1, ITGB1 and ARHGAP18) were identified. The predictive model based on the 10-gene panel exhibited well-discriminated power. A cross validation process using known disease status exhibited almost the same performance as that of the predictive model, whereas null-set analysis with randomly reassigned disease status exhibited much lower predictive performance for distinguishing HSILs from LSILs. These biomarkers were involved in the 'Rho GTPase cycle', 'mitochondrial protein import', 'oncogenic MAPK signaling', 'integrin cell surface interaction' and 'signaling by BRAF and RAF fusions'. In conclusion, peripheral blood gene expression analysis is a promising method for distinguishing HSILs from LSILs. The present study proposes 10 candidate genes that could be used in the future as diagnostic biomarkers and potential therapeutic targets for cervical SILs. A simple, non-invasive blood test would be clinically useful in the diagnosis and classification of patients with cervical SILs.

No biologically based diagnostic criteria are in clinical use today for obsessive-compulsive disorder (OCD), schizophrenia, and major depressive disorder (MDD), which are defined with reference to Diagnostic and Statistical Manual clinical symptoms alone. However, these disorders cannot always be well distinguished on clinical grounds and may also be comorbid. A biological blood-based dynamic genomic signature that can differentiate among OCD, MDD, and schizophrenia would therefore be of great utility. This study enrolled 77 patients with OCD, 67 controls with no psychiatric illness, 39 patients with MDD, and 40 with schizophrenia. An OCD-specific gene signature was identified using blood gene expression analysis to construct a predictive model of OCD that can differentiate this disorder from healthy controls, MDD, and schizophrenia using a logistic regression algorithm. To verify that the genes selected were not derived as a result of chance, the algorithm was tested twice. First, the algorithm was used to predict the cohort with true disease/control status and second, the algorithm predicted the cohort with disease/control status randomly reassigned (null set). A six-gene panel (COPS7A, FKBP1A, FIBP, TP73-AS1, SDF4, and GOLGA8A) discriminated patients with OCD from healthy controls, MDD, and schizophrenia in the training set (with an area under the receiver-operating-characteristic curve of 0.938; accuracy, 86%; sensitivity, 88%; and specificity, 85%). Our findings indicate that a blood transcriptomic signature can distinguish OCD from healthy controls, MDD, and schizophrenia. This finding further confirms the feasibility of using dynamic blood-based genomic signatures in psychiatric disorders and may provide a useful tool for clinical staff engaged in OCD diagnosis and decision making.

Gastric cancer (stomach cancer) is the fifth most common malignancy and the third leading cause of cancer-associated mortality worldwide. Identifying gastric cancer patients at an early and curable stage of the disease is essential if mortality rates for this disease are to decrease. A non-invasive blood-based test that is an indicator of gastric cancer risk would likely be of benefit in identifying gastric cancer patients at an early stage, and such a test may enhance clinical decision making. This study identified a four-gene expression signature in peripheral blood samples associated with gastric cancer. A total of 216 blood samples were collected, including those from 36 gastric cancer patients, 55 healthy controls and 125 patients with other carcinomas, and gene expression profiles were examined using an Affymetrix Gene Profiling microarray. Blood gene expression profiles were compared between patients with stomach cancer, healthy controls and patients affected with other malignancies. A four-gene panel was identified comprising purine-rich element binding protein B, structural maintenance of chromosomes 1A, DENN/MADD domain containing 1B and programmed cell death 4. The four-gene panel discriminated gastric cancer with an area under the receiver-operating-characteristic curve of 0.99, an accuracy of 95%, sensitivity of 92% and specificity of 96%. The non-invasive nature of the blood test, together with the relatively high accuracy of the four-gene panel may assist physicians in gastric cancer screening decision making.

Purpose Up to 25% of chronic hepatitis B (CHB) patients eventually develop hepatocellular carcinoma (HCC), a disease with poor prognosis unless detected early. This study identifies a blood-based RNA biomarker panel for early HCC detection in CHB. Materials and methods A genome-wide RNA expression study was performed using RNA extracted from blood samples from Malaysian patients (matched HCC, CHB, controls). Genes differentiating HCC from controls were selected for further testing using quantitative real-time polymerase chain reaction. Finally, a 6-gene biomarker panel was identified and characterized using a training set (cohort I = 126), and tested against 2 test sets (cohort II = 222; cohort III = 174). The total number of samples used for each group is: HCC + CHB = 143, CHB = 211, control = 168. Results Our gene panel displays a consistent trend distinguishing HCC from controls in our test sets, with an area under receiver-operating characteristic curve of 0.9 in cohort III. Our independent test set (cohort III) showed that the gene panel had a sensitivity of 70% with a specificity of 92%. The biomarker profile for HCC was consistently detected in a small subgroup of CHB patients, thus potentially predicting early, preclinical cases of cancer that should be screened more intensively. Conclusion The biomarkers identified in this study can be used as the basis of a blood-based test for the detection of early HCC in CHB.

Purpose: To identify candidate genomic signatures for the early detection of ovarian cancer using whole bloodbased gene expression profiles. Experimental Design: We performed Affymetrix U133Plus 2.0 GeneChip microarray analyses on whole blood RNA samples obtained from 14 ovarian cancer patients and 15 age-matched, healthy women. Genes differentially expressed were identified using a parametric Welch t-test. Real-time qRT-PCR analyses were performed on RNA prepared from 96 ovarian cancer patients and 83 age-matched healthy women, using primer sets specific for 14 genes. A Mann Whitney U test assessed individual gene significance. CA125 levels were determined in the same set of samples. We used logistic regression analyses and cross validation to assess the ability of linear combinations of specific transcripts combined with CA125 to distinguish cancer from controls. Results: Microarray analyses showed that 9583 probes were significantly different in blood gene expression profiles from healthy women as compared with those from ovarian cancer patients (p

GeneNews Limited (TSX:GEN) is a molecular diagnostics company, uniquely positioned to deliver on the promise of personalized medicine. GeneNews has developed and patented the Sentinel Principle®, an innovative approach to identify clinically actionable disease biomarkers from the blood transcriptome. Novel biomarker panels have been identified to address unmet clinical needs in a broad spectrum of malignancy, including those for oncology, cardiovascular, metabolic and neurological related disorders. Currently available products and services from GeneNews are: ColonSentry™, the world’s first blood-based molecular test for colorectal cancer, and; the SentinelGX™ Pharmacogenomic and Companion Diagnostic BloodRNA™ services, established to effectively support pharmaceutical partners in their companion diagnostic, development efforts.

Gene expression signatures in blood correlate with specific diseases. Such signatures may serve as valuable diagnostic and prognostic tools in disease management. Blood gene expression signatures associated with heart failure may be applied to predict prognosis, monitor disease progression, and optimize treatment. Blood gene expression profiles were generated for 71 subjects with heart failure and 15 controls without heart failure, using the Affymetrix GeneChip U133Plus2.0. Survival analysis identified 197 “mortality genes” that were significantly associated with patient outcome. Functional categorization showed that genes associated with T cell receptor signaling were most significantly overpresented. Cluster analysis of these T cell receptor signaling genes significantly categorized heart failure patients into three risk groups ( P = 0.031) that were distinct from the three risk groups categorized by New York Heart Association (NYHA) Classification ( P = 0.0002). By combining the analysis of clinical assessment (NYHA class) with T cell receptor signaling gene expression, we proposed a model that demonstrated an even greater differentiation of patients at risk ( P = 0.0001). In this discovery study, we identified blood expression signatures associated with heart failure patient outcomes. Characterization of these mortality genes helped identify a set of T cell receptor signaling genes that may be of utility in predicting survival of heart failure patients. These data raise the possibility of prospectively risk stratifying patients with heart failure by integrating blood gene expression signatures with current clinical assessment.

Goals: Assessment of disease severity is a frequent challenge in the management of Crohn's disease. Noninvasive, accurate markers for monitoring disease activity are urgently required. Specific gene expression patterns and molecular biomarkers associated with active Crohn's disease could serve as such markers, thereby providing a novel approach to disease activity monitoring. Background: Gene expression profiling in circulating leukocytes has shown promise in several medical conditions and blood may provide an easily accessible surrogate tissue for using gene expression profiling to assess activity of Crohn's disease. Study: In this study, we compared genome-wide transcription profiles of circulating leukocytes in patients with active and quiescent Crohn's disease. Results: We observed complex changes in blood gene expression patterns in active Crohn's disease: genes of various functional categories were differentially regulated between active and inactive Crohn's disease. We specifically identified a number of inflammatory molecules overexpressed or underexpressed in active Crohn's disease and validated a subset of these genes by real-time reverse transcription-polymerase chain reaction. Conclusions: The genes differentially regulated in peripheral leukocytes represent potential new biomarkers for assessing the activity of Crohn's disease.

Heart failure is a growing epidemic in North America, and its incidence and cost burden are increasing as the population ages. Few therapies have been shown to be clearly effective in preventing or delaying the onset of heart failure, and prognosis remains poor for this disorder. Novel genomics techniques and technologies are beginning to yield new insights into the molecular mechanisms driving progression and outcome in heart failure. In particular, gene expression profiling research is leading to better understanding of this complex condition and may improve the outlook for patients with heart failure in the future.

Background: Blood has advantages over tissue samples as a diagnostic tool, and blood mRNA transcriptomics is an exciting research field. To realize the full potential of blood transcriptomic investigations requires improved methods for gene expression measurement and data interpretation able to detect biological signatures within the “noisy” variability of whole blood. Methods: We demonstrate collection tube bias compensation during the process of identifying a liver cancer-specific gene signature. The candidate probe set list of liver cancer was filtered, based on previous repeatability performance obtained from technical replicates. We built a prediction model using differential pairs to reduce the impact of confounding factors. We compared prediction performance on an independent test set against prediction on an alternative model derived by Weka. The method was applied to an independent set of 157 blood samples collected in PAXgene tubes. Results: The model discriminated liver cancer equally well in both EDTA and PAXgene collected samples, whereas the Weka-derived model (using default settings) was not able to compensate for collection tube bias. Cross-validation results show our procedure predicted membership of each sample within the disease groups and healthy controls. Conclusion: Our versatile method for blood transcriptomic investigation overcomes several limitations hampering research in blood-based gene tests.

Background Identifying specific genes that are differentially expressed during inflammatory bowel disease flares may help stratify disease activity. The aim of this study was to identify panels of genes to be able to distinguish disease activity in Crohn's disease (CD) and ulcerative colitis (UC). Methods Patients were grouped into categories based on disease and severity determined by histological grading. Whole blood was collected by PAXgene Blood RNA collection tubes, (PreAnalytiX) and gene expression analysis using messenger RNA was conducted. Logistic regression was performed on multiple combinations of common probe sets, and data were evaluated in terms of discrimination by computing the area under the receiving operator characteristic curve (ROC-AUC). Results Nine inactive CD, 8 mild CD, 10 moderate-to-severe CD, 9 inactive UC, 8 mild UC, 10 moderate-to-severe UC, and 120 controls were hybridized to Affymetrix U133 Plus 2 microarrays. Panels of 6 individual genes discriminated the stages of disease activity: CD with mild severity {ROC-AUC, 0.89 (95% confidence interval [CI], 0.84%-0.95%)}, CD with moderate-to-severe severity (ROC-AUC 0.98 [95% CI, 0.97-1.0]), UC with mild severity (ROC-AUC 0.92 [95% CI, 0.87-0.96]), and UC with moderate-to-severe severity (ROC-AUC 0.99 [95% CI, 0.97-1.0]). Validation by real-time reverse transcription-PCR confirmed the Affymetrix microarray data. Conclusions The specific whole blood gene panels reliably distinguished CD and UC and determined the activity of disease, with high sensitivity and specificity in our cohorts of patients. This simple serological test has the potential to become a biomarker to determine the activity of disease.

Bone marrow–derived endothelial progenitor cells (EPCs) have the ability to migrate to ischemic organs. However, the signals that mediate trafficking and recruitment of these cells are not well understood. Using a functional genomics strategy, we determined the genes that were upregulated in the ischemic myocardium and might be involved in EPC recruitment. Among them, CD18 and its ligand ICAM-1 are particularly intriguing because CD18 and its heterodimer binding chains CD11a and CD11b were correspondingly expressed in ex vivo–expanded EPCs isolated from rat and murine bone marrows. To further verify the functional role of CD18 in mediating EPC recruitment and repair to the infarcted myocardium, we used neutralizing antibody to block CD18. Blockade of CD18 in EPCs significantly inhibited their attachment capacity in vitro and reduced their recruitment to the ischemic myocardium in vivo by 95%. Moreover, mice receiving EPCs that were treated with control isotype IgG exhibited significantly increased capillary density in the infarct border zone, reduced cardiac dilatation, ventricular wall thinning, and fibrosis when compared with myocardial infarction mice receiving PBS and CD18 blockade reversed the EPC-mediated improvements to the infarcted heart. Thus, our results suggest an essential role of CD18 in mediating EPC recruitment and the subsequent functional effects on the infarcted heart.

In our genome-wide survey of gene expression in human peripheral blood cells using both an expressed sequence tag (EST) and a microarray hybridization approach, we identified the expression of a large proportion (approximately 80%) of the genes encoded in the human genome. Comparison of the peripheral blood transcriptome with genes expressed in nine different human tissue types revealed that expression of over 80% was shared with any given tissue. We also sought to determine whether those gene transcripts undetected by these methods were also expressed in peripheral blood cells. Using reverse-transcriptase-polymerase chain reaction, we detected additional tissue-specific gene transcripts including beta-myosin heavy chain (heart specific) and insulin (specific to pancreatic islet beta cells), in circulating blood cells. Arguably, the detection of low levels of tissue-specific transcripts could be considered products of "illegitimate" transcription; however, our study also demonstrates that environmental conditions affect the transcriptional regulation of insulin in the peripheral blood. We thus hypothesize that blood cells can act as sentinels of disease and that we could capitalize on this property of blood for the diagnosis/prognosis of disease (the "Sentinel Principle"). Peripheral blood is an ideal surrogate tissue as it is readily obtainable, provides a large biosensor pool in the form of gene transcripts, and response to changes in the macro- and micro-environments is detectable as alterations in the levels of these gene transcripts.

Chronic Chagas' disease cardiomyopathy is a leading cause of congestive heart failure in Latin America, affecting more than 3 million people. Chagas' cardiomyopathy is more aggressive than other cardiomyopathies, but little is known of the molecular mechanisms responsible for its severity. We characterized gene expression profiles of human Chagas' cardiomyopathy and dilated cardiomyopathy to identify selective disease pathways and potential therapeutic targets. Both our customized cDNA microarray (Cardiochip) and real-time reverse transcriptase-polymerase chain reaction analysis showed that immune response, lipid metabolism, and mitochondrial oxidative phosphorylation genes were selectively up-regulated in myocardial tissue of the tested Chagas' cardiomyopathy patients. Interferon (IFN)-gamma-inducible genes represented 15% of genes specifically up-regulated in Chagas' cardiomyopathy myocardial tissue, indicating the importance of IFN-gamma signaling. To assess whether IFN-gamma can directly modulate cardio-myocyte gene expression, we exposed fetal murine cardiomyocytes to IFN-gamma and the IFN-gamma-inducible chemokine monocyte chemoattractant protein-1. Atrial natriuretic factor expression increased 15-fold in response to IFN-gamma whereas combined IFN-gamma and monocyte chemoattractant protein-1 increased atrial natriuretic factor expression 400-fold. Our results suggest IFN-gamma and chemokine signaling may directly up-regulate cardiomyocyte expression of genes involved in pathological hypertrophy, which may lead to heart failure. IFN-gamma and other cytokine pathways may thus be novel therapeutic targets in Chagas' cardiomyopathy.

Recent advances have facilitated the use of blood-derived RNA to conduct genomic analyses of human diseases. This emerging technology represents a rigorous and convenient alternative to traditional tissue biopsy-derived RNA, as it allows for larger sample sizes, better standardization of technical procedures, and the ability to non-invasively profile human subjects. In the present pilot study, we have collected RNA from blood of patients diagnosed with schizophrenia or bipolar disorder (BPD), as well as normal control subjects. Using microarray analysis, we found that each disease state exhibited a unique expressed genome signature, allowing us to discriminate between the schizophrenia, BPD, and control groups. In addition, we validated changes in several potential biomarker genes for schizophrenia and BPD by RT-PCR, and some of these were found to code to chromosomal loci previously linked to schizophrenia. Linear and non-linear combinations of eight putative biomarker genes (APOBEC3B, ADSS, ATM, CLC, CTBP1, DATF1, CXCL1, and S100A9) were able to discriminate between schizophrenia, BPD, and control samples, with an overall accuracy of 95%-97% as indicated by receiver operating characteristic (ROC) curve analysis. We therefore propose that blood cell-derived RNA may have significant value for performing diagnostic functions and identifying disease biomarkers in schizophrenia and BPD.

Adriamycin is a potent, broad-spectrum chemotherapeutic agent effective against solid tumors and malignant hematological disease. The major limiting factor for adriamycin is its cardiotoxicity. Thus, the objective of this study was to investigate the role of cardiomyocyte and endothelial cell apoptosis in adriamycin-induced cardiomyopathy, in vivo and in vitro. For in vivo study, intraperitoneal injections of adriamycin were administered to nine adult male Wistar rats and normal saline to six rats as control. Eight of the nine rats in the adriamycin group, but none in the control group, developed marked ascites and DNA ladders in agarose gel electrophoresis of genomic DNA extracted from the rat hearts (P

To obtain a genomic portrait of heart failure derived from end-stage dilated cardiomyopathy (DCM), we explored expression analysis using the CardioChip, a nonredundant 10,848-element human cardiovascular-based expressed sequence tag glass slide cDNA microarray constructed in-house. RNA was extracted from the left ventricular free wall of seven patients undergoing transplantation, and five nonfailing heart samples. Cy3- and Cy5-labeled (and reverse dye-labeled) cDNA probes were synthesized from individual diseased or nonfailing adult heart RNA, and hybridized to the array. More than 100 transcripts were consistently differentially expressed in DCM >1.5-fold (versus pooled nonfailing heart, P < 0.05). Atrial natriuretic peptide was found to be up-regulated in DCM (19-fold compared to nonfailing, P < 0.05), as well as numerous sarcomeric and cytoskeletal proteins (eg, cardiac troponin, tropomyosin), stress response proteins (eg, HSP 40, HSP 70), and transcription/translation regulators (eg, CCAAT box binding factor, eIF-1AY). Down-regulation was most prominently observed with cell-signaling channels and mediators, particularly those involved in Ca(2+) pathways (Ca(2+)/calmodulin-dependent kinase, inositol 1,4,5-trisphosphate receptor, SERCA). Most intriguing was the co-expression of several novel, cardiac-enriched expressed sequence tags. Quantitative real-time reverse transcriptase-polymerase chain reaction of a selection of these clones verified expression. Our study provides a preliminary molecular profile of DCM using the largest human heart-specific cDNA microarray to date.

Nasopharyngeal carcinoma is often diagnosed only after the disease has reached late stages and the prognosis is poor. However, when detected early, the disease is highly survivable. This review surveys molecular biomarker studies that aim to identify early, presymptomatic disease, to predict response to treatment, to indicate prognosis, to identify tumor stage, and to monitor disease progression. Blood transcriptomic studies represent a new frontier in the molecular diagnostics of nasopharyngeal carcinoma. List of Abbreviations AUC Area Under the Curve BCR B-cell Receptor CR Complete Response to Treatment CT Computed Tomography DNA Deoxyribonucleic Acid EBV Epstein-Barr Virus EGFR1 Epidermal Growth Factor Receptor 1 ELISA Enzyme-Linked Immunosorbent Assay GWAS Genome-Wide Association Study HLA Human Leukocyte Antigen IgA Immunoglobulin A IgG Immunoglobulin G MHC Major Histocompatibility Complex miRNA MicroRNA MRI Magnetic Resonance Imaging mRNA Messenger RNA NPC Nasopharyngeal Carcinoma PCR Polymerase Chain Reaction PR Partial Response to Treatment RNA Ribonucleic Acid ROC Receiver Operating Characteristic Curve SELDI-TOFMS Surface-Enhanced Laser Desorption and Ionization-Time-of-Flight Mass Spectrometry Chun Ren Lim and Michelle Lee contributed equally to this manuscript *Email: cliew@genenews.com Biomarkers in Cancer DOI 10.1007/978-94-007-7744-6_3-1 # Springer Science+Business Media Dordrecht 2014

The number of genes encoded by the human genome has long sought to be determined. With the recent publications of the complete sequence of the human genome, the number of genes encoded by the human genome has now been estimated to be approximately 32,000-38,000. Now the next step will be to determine which of these genes are expressed in a given cell, tissue or organ. Using three unique approaches taking advantage of our current cardiovascular EST database and the complete nucleotide sequence of human chromosomes 21 and 22 as well as cDNA microarray hybridization, we estimate that between 20,930-27,160 genes are expressed in the cardiovascular system.

The RING domain is a conserved zinc finger motif, which serves as a protein-protein interaction interface. Searches of a human heart expressed sequence tag data base for genes encoding the RING domain identified a novel cDNA, named striated muscle RING zinc finger protein (SMRZ). The SMRZ cDNA is 1.9 kilobase pairs in length and encodes a polypeptide of 288 amino acid residues; analysis of the peptide sequence demonstrated an N-terminal RING domain. Fluorescence in situ hybridization localized SMRZ to chromosome 1p33-34. Northern blots demonstrated that SMRZ is expressed exclusively in striated muscle. In the cardiovascular system, SMRZ is more highly expressed in the fetal heart than in the adult heart (slightly higher expression in the ventricle than in the atrium), suggesting that SMRZ is developmentally regulated. SMRZ was found to interact with SMT3b, a ubiquitin-like protein, through the SMRZ-RING domain. This interaction was abolished by mutagenesis of conserved RING domain residues. Transient transfection of SMRZ into C2C12 myoblasts showed localization of SMRZ to the nucleus. These data suggest that SMRZ may play an important role in striated muscle cell embryonic development and perhaps in cell cycle regulation.

The recent availability of the sequenced and annotated DNA sequences of chromosomes 21 and 22 has initiated the next phase in the human genome project: the application of this resource. One facet of these data is that they provide a list of ordered genes along the chromosome that can be capitalized upon to determine gene position effects. Specifically, the physical position and distribution of genes along the chromosomes may be related to gene expression in specific organs or organ systems. In this report we index the subset of genes constituting the human “cardiovascular genome” on chromosomes 21q and 22q as well as report the identification of several “cardiovascular gene” clusters. These gene clusters are suggestive of a higher order of tissue-specific gene regulation at the chromosomal level.

Identifying key genes that regulate the complex diseases of the cardiovascular system can be greatly facilitated with the use of microarrays. In an effort to obtain a global portrait of gene expression in the failing heart, we have constructed in-house a glass microscope slide cDNA microarray (termed “CardioChip”) containing 10,368 redundant and randomly-selected sequenced expressed sequence tags (representing known genes, other matched ESTs, and novel, unmatched ESTs) derived from several human heart and artery cDNA libraries. From our preliminary data with Cy3- and Cy5-labeled probes, we have identified 38 transcripts showing a minimum twofold differential expression, among which are several novel or previously-uncharacterized genes. This array—representing what we believe to be the largest cardiovascular-based cDNA array to date—establishes a practical and invaluable platform for obtaining a global genetic portrait of complex cardiovascular diseases, particularly in the failing heart.

The Human Genome Project (HGP) has amassed a vast quantity of sequencing data; over 90% of the human genes have been deposited into GenBank (June 2000). However, functional interpretation of this sequence data has proven more challenging. Much of this work has involved the study of model organisms because functional inferences based on interspecies comparison of sequences have identified implied function of many orthologous human sequences (Makalowski and Boguski 1998). Recently the zebrafish, Danio rerio, has been recognized as a useful model for the study of development biology and genetics (for review, see Driever and Fishman 1996). One significant advantage of using the zebrafish as a model organism for developmental study is the external development and transparency of the zebrafish embryo. This permits the study of subtle developmental phenotypes in vivo. The zebrafish is also well suited for studies in cardiovascular development because a beating heart is formed and functions within 1 d of fertilization. In addition, the zebrafish embryo does not require blood flow for survival during the first 2 d of development. Thus, zebrafish mutants lacking a circulatory system can still develop normally in the first 2 d (Warren and Fishman 1998) and this allows for studies of mutations that affect the development of the zebrafish heart. Despite all these advantages, the zebrafish suffers from the major drawback of being a new model organism. For example, the number of genes that have been characterized from this species is small compared with other model organisms such as mouse, Drosophila, and Caenorhabditis elegans. Expressed sequence tags (ESTs) have proven to be a powerful and rapid approach to identify new genes that are preferentially expressed in certain tissue or cell types (Hwang et al. 1997; Liew et al. 1994; Adams et al. 1995). ESTs have also been used for physical mapping, as has been demonstrated in the development of the human and mouse gene maps (Hayes et al. 1996; McCarthy et al. 1997; Deloukas et al. 1998). Currently, the number of zebrafish ESTs in the public databases is still small compared with mammalian sequences, and there are relatively few tissue-specific cDNA libraries. Mutational screens in the zebrafish have identified several thousand mutations that affect normal development of the embryo (Development, Dec. 1996), including many with essential functions during embryonic heart development (Chen et al. 1996; Stainier et al. 1996). However, the usefulness of these mutations remains limited until the genes responsible for the observed phenotypes are cloned. This is limited in part by a paucity of ordered genes on the zebrafish gene map. Linkage maps based on rapid amplified polymorphic DNAs and microsatellite markers have been produced (Postlethwait et al. 1994, 1999; Johnson et al. 1996; Knapik et al. 1996, 1998; Shimoda et al. 1999). Because linkage mapping requires polymorphic markers for map construction, radiation hybrid (RH) mapping provides a complementary approach to rapidly assign genes and ESTs on the zebrafish map because RH mapping is able to map virtually any marker. Two recent RH maps (Geisler et al. 1999; Hukriede et al. 1999) of more than 3000 markers, genes, and ESTs have dramatically increased the density of the zebrafish gene map and should facilitate the cloning of many identified mutants. Given the potential and importance of the zebrafish as a model organism for the studies of cardiac development, there is a need for development of EST resources from zebrafish heart cDNA libraries. Here we report the characterization of 5102 ESTs from a 3-d-old zebrafish embryonic heart cDNA library. We also report new map positions for 98 zebrafish ESTs identified in this cDNA library by RH mapping (Table ​(Table1)1) and identification of new synteny groups between zebrafish and human. This EST database represents a new genomic tool for studying aspects of cardiovascular development and disease in the zebrafish and a resource of genes for novel candidate gene discovery. Table 1 List of Mapped Zebrafish ESTs A total of 5102 EST sequences were processed with the TIGR Assembler to estimate the number of unique transcripts represented in the EST set. A total of 359 clusters composed of 1771 ESTs were generated, whereas the remaining 3331 ESTs did not cluster. The number of unique transcripts identified from the zebrafish embryonic heart EST set was therefore estimated at up to 3690.

Expressed sequence tag (EST) and digital Northern analyses of human fetal, adult, and hypertrophic heart cDNA libraries revealed ESTs with high homology to adenomatosis polyposis coli (APC) and its associated protein, beta-catenin, as well as their differential expression. Thus, we hypothesize that the APC/beta-catenin pathway may play a role in cardiac development and disease. Reverse transcriptase-polymerase chain reaction analysis exhibited a higher APC expression in adult compared with fetal and hypertrophic heart but no significant difference in beta-catenin mRNA level. However, beta-catenin protein level was higher in fetal and hypertrophic heart compared with adult heart, suggesting the post-translational regulation of beta-catenin by APC in the cardiovascular system. In vitro antisense inhibition of APC resulted a higher beta-catenin protein expression leading to an incomplete myotube formation, suggesting APC/beta-catenin pathway involvement in myotube development. Western blot analysis further reveals three novel isoforms, APC-F, APC-A, and APC-D, ubiquitously expressed in fetal, adult, and hypertrophic heart, respectively. Isoform switching during development and disease pathogenesis suggests functionally distinct roles for each isoform. These data (i) demonstrate the usefulness of genome-based expression analysis for rapid discovery of differentially expressed genes, (ii) implicate the APC/beta-catenin pathway in the cardiovascular development, and (iii) demonstrate APC isoform switching during cardiac development and disease.

Cardiac hypertrophy is an adaptive response to chronic hemodynamic overload. We employed a whole-genome approach using expressed sequence tags (ESTs) to characterize gene transcription and identify new genes overexpressed in cardiac hypertrophy. Analysis of general transcription patterns revealed a proportional increase in transcripts related to cell/organism defense and a decrease in transcripts related to cell structure and motility in hypertrophic hearts compared to normal hearts. Detailed comparison of individual gene expression identified 64 genes potentially overexpressed in hypertrophy, of 232 candidate genes derived from a set of 77,692 cardiac ESTs, including 47,856 ESTs generated in our laboratory. Of these, 29 were good candidates (P < 0.0002) and 35 were weaker candidates (P < 0.005). RT-PCR of a number of these candidate genes demonstrated correspondence of EST-based predictions of gene expression with in vitro levels. Consistent with an organ under various stresses, up to one-half of the good candidates predicted to exhibit differential expression were genes potentially involved in stress response. Analyses of general transcription patterns and of single-gene expression levels were also suggestive of increased protein synthesis in the hypertrophic myocardium. Overall, these results depict a scenario compatible with current understanding of cardiac hypertrophy. However, the identification of several genes not previously known to exhibit increased expression in cardiac hypertrophy (e.g., prostaglandin D synthases; CD59 antigen) also suggests a number of new avenues for further investigation. These data demonstrate the utility of genome-based resources for investigating questions of cardiovascular biology and medicine.

Telomerase replaces telomeric repeat DNA lost during the cell cycle, restoring telomere length. This enzyme is present only during cell replication and its activity reflects the extent of proliferation. Whether cardiac myocytes are terminally differentiated cells is still a highly controversial issue, and the possibility of myocyte division is frequently rejected. On this basis, telomerase was measured in pure preparations of myocytes, isolated from rats throughout their lifespan. Fetal and neonatal rat myocytes were used as positive control cells. Contrary to expectation, the authors report that telomerase activity was detectable in pure preparations of young adult, fully mature adult, and senescent ventricular myocytes, defeating the dogma that this cell population is permanent and irreplaceable. Aging decreased 31% telomerase activity in male myocytes. An opposite effect occurred in female myocytes in which this enzyme increased 72%. This differential adaptation between the two genders in the rat model may be relevant to observations in humans; myocyte loss occurs in men as a function of age, whereas myocyte number is preserved in women. The greater growth potential of female myocytes may be critical for the longer lifespan and decreased incidence of heart failure in women.

M. Xian, N. Honbo, J. Zhang, C.-C. Liew, J. S. Karliner and Y.-F. C. Lau. The Green Fluorescent Protein is an Efficient Biological Marker for Cardiac Myocytes. Journal of Molecular and Cellular Cardiology (1999) 31, 2155–2165. There is a need for a non-toxic marker for cardiac myocytes in studies of cardiac development and in experimentally induced pathophysiologic states in adult animals. We investigated the possibility of using the enhanced green fluorescent protein (EGFP) gene as such a biological marker for cardiac myocytes in both whole animal and cell culture systems. Several lines of transgenic mice were constructed harboring an EGFP gene directed by a 2.38-kb promoter fragment of the hamsterβ -myosin heavy chain gene. The transgene was preferentially expressed in the cardiac progenitor cells of embryos at E7.5, a developmental stage that precedes the formation of the cardiomyotube. It was specifically expressed in the cardiomyotube and myotomes along the somites of embryos at E8.5. The EGFP transgene expression continued in the heart throughout gestation and became very intense at birth. When neonatal cardiac cells were fractionated into myocytes and non-myocytes by a differential plating procedure, only myocytes from the transgenic mice showed specific green fluorescence of the transgene product that can be used as a marker for flow cytometry analysis. Although the expression levels were heterogeneous, EGFP expression persisted in the hearts of postnatal animals. In addition to the heart, some skeletal and smooth muscles from transgenic animals also expressed the transgene. The transgenic mice were healthy and had a normal life span, identical to their non-transgenic littermates. These results demonstrate that EGFP is an efficient non-toxic biological marker for cardiac myocytes.

The regulation of extracellular calcium concentration by parathyroid hormone is mediated by a calcium-sensing, G-protein-coupled cell-surface receptor (CASR). Mutations of the CASR gene alter the set-point for extracellular ionised calcium [Ca2+]o and cause familial hypercalcaemia or hypocalcaemia. The CASR missense polymorphism, A986S, is common in the general population and is, therefore, a prime candidate as a genetic determinant of extracellular calcium concentration.We genotyped the CASR A986S variant (S allele frequency of 16.3%) in 163 healthy adult women and tested samples of their serum for total calcium, albumin, total protein, creatinine, phosphate, pH, and parathyroid hormone. A prospectively generated, random subset of 84 of these women provided a whole blood sample for assay of [Ca2+]o.The A986S genotype showed no association with total serum concentration of calcium, until corrected for albumin. In a multivariate regression model, biochemical and genetic variables accounted for 74% of the total variation in calcium. The significant predictors of serum calcium were: albumin (p0.001), phosphate (p=0.02), parathyroid hormone (p=0.007), pH (p=0.001), and A986S genotype (p=0.009). Fasting whole-blood [Ca2+]o also showed an independent positive association with the 986S variant (p=0.013).The CASR A986S variant has a significant effect on extracellular calcium. The CASR A986S polymorphism is a likely candidate locus for genetic predisposition to various bone and mineral disorders in which extracellular calcium concentrations have a prominent part.

Gene activation is often preceded by or accompanied by a perturbation of the chromatin structure. Recently, several co-factors of transcription factors have been identified as histone acetyltransferases. In addition, retinoblastoma protein and some co-factors can form a complex with histone deacetylases. These discoveries provide direct evidence that modification of chromatin structure is crucial for gene activation. However, the role of chromatin structure in cardiac-specific expression has not yet been elucidated. The potential significance of chromatin structure in cardiac-specific gene expression is indicated by: (1) heterogeneous human SWI/SNF chromatin remodelling factors among various tissues; (2) several chromatin remodelling factors shown to be expressed preferentially in the heart; (3) the demonstration of chromatin remodelling of the cardiac beta-myosin heavy chain gene (MyHC) during cardiac development. We therefore propose to study cardiac-specific chromatin remodelling activity in order to elucidate mechanisms controlling the reactivation of the fetal heart genetic program in the hypertrophic heart.

We have isolated and sequenced a human heart cDNA clone encoding a novel LIM-only protein. This full-length cDNA clone has a predicted open reading frame (ORF) encoding 280 amino acids. The ORF of this cDNA codes for a LIM-only protein that possesses four repeats of LIM domain and an extra zinc finger and this putative protein is named four-and-a-half LIM domain protein 1 (FHL1). FHL1 is unique when compared with other LIM-only proteins because it possesses an odd number of zinc fingers. When the FHL1 cDNA probe was used to hybridize with poly-(A) RNA of various human tissues, a very strong signal was detected in skeletal muscle, a moderate one in the heart; only weak signals were associated with the placenta, ovary, prostate, testis, small intestine, colon and spleen, and virtually no signal could be detected in brain, lung, liver, kidney, pancreas, thymus and peripheral blood leukocytes. The FHL1 gene was located to human chromosome at Xq27.2 by somatic cell hybrid mapping, fluorescent in situ hybridization (FISH) and radiation hybrid mapping.

Mutations in the gene for cardiac myosin-binding protein C account for approximately 15 percent of cases of familial hypertrophic cardiomyopathy. The spectrum of disease-causing mutations and the associated clinical features of these gene defects are unknown.DNA sequences encoding cardiac myosin-binding protein C were determined in unrelated patients with familial hypertrophic cardiomyopathy. Mutations were found in 16 probands, who had 574 family members at risk of inheriting these defects. The genotypes of these family members were determined, and the clinical status of 212 family members with mutations in the gene for cardiac myosin-binding protein C was assessed.Twelve novel mutations were identified in probands from 16 families. Four were missense mutations; eight defects (insertions, deletions, and splice mutations) were predicted to truncate cardiac myosin-binding protein C. The clinical expression of either missense or truncation mutations was similar to that observed for other genetic causes of hypertrophic cardiomyopathy, but the age at onset of the disease differed markedly. Only 58 percent of adults under the age of 50 years who had a mutation in the cardiac myosin-binding protein C gene (68 of 117 patients) had cardiac hypertrophy; disease penetrance remained incomplete through the age of 60 years. Survival was generally better than that observed among patients with hypertrophic cardiomyopathy caused by other mutations in the genes for sarcomere proteins. Most deaths due to cardiac causes in these families occurred suddenly.The clinical expression of mutations in the gene for cardiac myosin-binding protein C is often delayed until middle age or old age. Delayed expression of cardiac hypertrophy and a favorable clinical course may hinder recognition of the heritable nature of mutations in the cardiac myosin-binding protein C gene. Clinical screening in adult life may be warranted for members of families characterized by hypertrophic cardiomyopathy.

Recent studies have revealed that the γ-chain of the IL-2 receptor is shared by the receptors for IL-4, IL-7, IL-9, IL-13, and IL-15, and it is therefore also referred to as the common γ-chain (γc). Mutations of γc result in X-linked severe combined immunodeficiency syndrome in humans, indicating that γc is essential for normal development and function of the immune system. We demonstrate that human hematopoietic cells express two γc transcripts differing in their carboxyl terminal coding region. One transcript is the previously reported sequence (γc-long), whereas the newly identified sequence exhibits a deletion of 72 nucleotides close to the 3′-end of the open reading frame (γc-short). This alteration predicts a loss of 24 amino acids including a conserved tyrosine residue which is shared by several members of the cytokine receptor family. The presence of these two distinct forms of γc transcripts was demonstrated by sequencing of reversely transcribed and polymerase chain reaction (RT-PCR) amplified mRNA, restriction digestion of the RT-PCR products, RNAse protection, and Northern blotting from human cell lines and human peripheral blood lymphocytes. Furthermore, the two variants were present in peripheral blood lymphocytes from both female and male donors, which rules out allelic variants since γc is a single copy gene located on the X chromosome. A truncation mutant at a site near the observed changes in γc-short has been reported by others to alter biochemical events activated by cytokines. This combined with the loss of a potential SH2 “docking” site in γc-short suggests that γc-long and γc-short may link to different signaling pathways and may play an important role in determining the cellular response to IL-2, IL-4, IL-7, IL-9, IL-13, IL-15.

The multi-subunit NADH-ubiquinone oxidoreductase (complex I) is the first enzyme complex in the electron transport chain of mitochondria. A small number of NADH-ubiquinone oxidoreductase subunits are the products of mitochondrial genes (subunits 1-7), while the remainder are nuclear encoded and imported from the cytoplasm. We have isolated and sequenced five subunits of the human complex I from a human heart lambda ZAP Express cDNA library. Comparison of the deduced amino acid sequences of the human subunits with the corresponding bovine sequences revealed greater than 80% amino acid identity. The high degree of similarity between human and bovine sequences suggests functional conservation of these subunits in the complex I. In silico Northern analysis revealed that two of the subunits were expressed ubiquitously while the remainder may have more restricted patterns of expression.

Using nuclei isolated from neonatal cardiomyocytes, we have mapped the DNase I hypersensitive sites (DHSs) residing within the 5'-upstream regions of the hamster cardiac myosin heavy-chain (MyHC) gene. Two cardiac-specific DHSs within the 5 kb upstream region of the cardiac MyHC gene were identified. One of the DHSs was mapped to the -2.3 kb (beta-2.3 kb) region and the other to the proximal promoter region. We further localized the beta-2.3 kb site to a range of 250 bp. Multiple, conserved, muscle regulatory motifs were found within the beta-2.3 kb site, consisting of three E-boxes, one AP-2 site, one CArG motif, one CT/ACCC box and one myocyte-specific enhancer factor-2 site. This cluster of regulatory elements is strikingly similar to a cluster found in the enhancer of the mouse muscle creatine kinase gene (-1256 to -1050). The specific interaction of the motifs within the beta-2.3 kb site and the cardiac nuclear proteins was demonstrated using gel mobility-shift assays and footprinting analysis. In addition, transfection analysis revealed a significant increase in chloramphenicol acetyltransferase activity when the beta-2.3 kb site was linked to a heterologous promoter. These results suggest that previously undefined regulatory elements of the beta-MyHC gene may be associated with the beta-2.3 kb site.

A common feature of many tumors is an increase in glucose catabolism during tumor growth. We studied the mechanism of this phenomenon by using Ehrlich ascites tumor bearing mice as the animal model. We found that Ehrlich ascites tumor cells possess only glucose transporter 1 (GLUT1) and GLUT3 but no GLUT2, GLUT4, or GLUT5. The mRNA levels of GLUT1 and GLUT3 increased progressively in the tumour during development; however, there were no changes observable in mRNA levels of glucose transporters of all types in brain, liver, and heart of the host mice. These findings suggest that Ehrlich ascites tumor augments its glucose transport mechanism relative to other tissues in response to its unique growth needs. J. Cell. Biochem. 67:131–135, 1997. © 1997 Wiley-Liss, Inc.

Transgenic analysis has indicated that far upstream regulatory elements of the cardiac α-myosin heavy chain (MyHC) gene are required for appropriate transgene expression [Subramaniam, Gulick, Neumann, Knotts and Robbins (1993) J. Biol. Chem. 268, 4331–4336]. In an attempt to identify these as-yet-undefined regulatory elements, we mapped the DNase I hypersensitive sites (DHSs) in the 4 kb upstream region of the hamster cardiac α-MyHC gene. When using nuclei isolated from late-gestational and adult heart ventricles, a strong DHS was identified in the -1.9 kb region (α-1.9 kb site). It cannot be detected in kidney, liver or cardiofibroblast nuclei. Within this site, we found a conserved GATA-motif that interacts specifically with GATA-binding factors in nuclear extracts of cardiomyocytes at various developmental stages. These data provide further evidence to support the role of GATA factors in the regulation of cardiac α-MyHC gene expression.

We have isolated the full-length human 56 kDa selenium binding protein (hSP56) cDNA clone, which is the human homolog of mouse 56 kDa selenium binding protein. The cDNA is 1,668 bp long and has an open reading frame encoding 472 amino acids. The calculated molecular weight is 52.25 kDa and the estimated isoelectric point is 6.13. Using Northern blot hybridization, we found that this 56 kDa selenium binding protein is expressed in mouse heart with an intermediate level between those found in liver/lung/kidney and intestine. We have also successfully expressed hSP56 in Escherichia coli using the expression vector-pAED4. The hSP56 gene is located at human chromosome 1q21–22. J. Cell. Biochem. 64:217–224. © 1997 Wiley-Liss, Inc.

The role that the Calcium Sensing Receptor (CASR) plays in extracellular calcium regulation had been ascertained through studies of inactivating as well as activating mutations of CASR gene in a number of multiplex families. We have extended these observations to a polymorphism analysis of the intercellular domain of CASR in a cohort of healthy young women. The results demonstrate significant allelic polymorphism as a result of nonconservative changes at two specific sites. Further studies will be required to determine what, if any, relationship this may have to CASR phenotype.

The Zn 2+ -finger DNA-binding domain has been identified in several developmental control proteins, transcription factors and gene products associated with diseases, as well as in several RNA-binding proteins. We applied library screening, expressed sequence tagging (EST sequencing), Zn 2+ -binding assays and Northern blot hybridization, in order to characterize novel cDNA clones of the human cardiovascular system which contain Zn 2+ -finger motifs. An embryonic (8–10 weeks gestation) heart lambda ZAP Express cDNA library was screened with an oligonucleotide probe deduced from a consensus amino acid sequence which is highly conserved for Zn 2+ -finger proteins, and approximately 350 positive clones were isolated from 1×10 4 plaque-forming units (pfu) initially plated. The isolated clones were classified as known and novel following single pass automated DNA sequencing. Analysis of Northern blot hybridization delineated the tissue specificity of these clones, as well as their association with cardiac growth and development. Existence of Zn 2+ -finger motifs in the novel clones was confirmed by Zn 2+ -binding assay. In this report, we present the characterization of eight novel clones, including the complete cDNA sequences of one of these clones (HHZ-123).

Background Colonoscopy is widely regarded to be the gold standard for colorectal cancer (CRC) detection. Recent studies, however, suggest that the effectiveness of colonoscopy is mostly confined to tumors on the left side of the colon (descending, sigmoid, rectum), and that the technology has poor tumor detection for right-sided (cecum, ascending, transverse) lesions. A minimally invasive test that can detect both left-sided and right-sided lesions could increase the effectiveness of screening colonoscopy by revealing the potential presence of neoplasms in the right-sided “blind spot”. Methods We previously reported on a seven-gene, blood-based biomarker panel that effectively stratifies a patient’s risk of having CRC. For the current study, we assessed the effectiveness of the seven-gene panel for the detection of left- and right-sided CRC lesions. Results were evaluated for 314 patients with CRC (left-sided: TNM I, 65; TNM II, 57; TNM III, 60; TNM IV, 17; unknown, 9. right-sided: TNM I, 28; TNM II, 29; TNM III, 38; TNM IV, 12; unknown, 1 and including two samples with both left and right lesions) and 328 control samples. Blood samples were obtained prior to clinical staging and therapy. Most CRC subjects had localized disease (stages I and II, 58%); regional (stage III) and systemic (stage IV) disease represented 32% and 9%, respectively, of the study population. Results The panel detected left-sided (74%, 154/208) and right-sided (85%, 92/108) lesions with an overall sensitivity of 78% (215/316) at a specificity of 66% (215/328). Treatable cancer (stages I to III) was detected with left-sided lesion sensitivity of 76% (138/182) and right-sided sensitivity of 84% (80/95). Conclusion This seven-gene biomarker panel detected right-sided CRC lesions across all cancer stages with a sensitivity that is at least equal to that for left-sided lesions. This study supports the use of this panel as the basis for a patient-friendly, blood-based test that can be easily incorporated into a routine physical examination in advance of colonoscopy to provide a convenient companion diagnostic and a pre-screening alert, ultimately leading to enhanced CRC screening effectiveness.

hnifU, a gene exhibiting similarity to nifU genes of nitrogen fixation gene clusters, was identified in the course of expressed sequence tag (EST) generation from a human fetal heart cDNA library. Northern blot of human tissues and polymerase chain reaction (PCR) using human genomic DNA verified that the hnifU gene represented a human gene rather than a microbial contaminant of the cDNA library. Conceptual translation of the hnifU cDNA yielded a protein product bearing 77% and 70% amino acid identity to NifU-like hypothetical proteins from Haemophilus influenzae and Saccharomyces cerevisiae, respectively, and 40-44% identity to the N-terminal regions of NifU proteins from several diazatrophs (i.e., nitrogen-fixing organisms). Pairwise determination of amino acid identities between the NifU-like proteins of nondiazatrophs showed that these NifU-like proteins exhibited higher sequence identity to each other (63-77%) than to the diazatrophic NifU proteins (40-48%). Further, the NifU-like proteins of non-nitrogen-fixing organisms were similar only to the N-terminal region of diazatrophic NifU proteins and therefore identified a novel modular domain in these NifU proteins. These findings support the hypothesis that NifU is indeed a modular protein. The high degree of sequence similarity between NifU-like proteins from species as divergent as humans and H. influenzae suggests that these proteins perform some basic cellular function and may be among the most highly conserved proteins.

The delineation of the mechanisms that regulate cardiac gene expression is central to our understanding of cardiac growth and development. Much progress has been made toward the identification of factors involved in tissue-restricted gene expression, especially in skeletal muscle cells. However, the mechanisms regulating the expression of cardiac-specific genes remain less well understood. Certain homeodomain proteins have been implicated in commitment to the cardiac phenotype. Among the best characterized are the murine proteins Csx, Nkx-2.5, and Nkx-2.6, related to the protein tinman, which is essential for heart formation in Drosophila . The expression of these genes precedes that of cardiac-specific genes and is therefore believed to play a critical role in the development of the heart. The GATA proteins are a family of zinc finger proteins that are also expressed early in cardiac development and may act separately from, or in concert with, the homeodomain proteins as crucial regulators of heart development. The myosin heavy and light chain genes, the actin genes, the troponin genes, and the atrial natriuretic factor and muscle creatine kinase genes have served as excellent paradigms for the study of cardiac gene expression. Although differences in cis -acting elements and their behavior in binding assays have been observed between different genes, there exist similarities that are noteworthy. In this review, we will discuss the factors involved in the regulation of cardiac-specific gene expression in an attempt to provide a better understanding of the process of cardiogenesis.

We are conducting a cDNA sequencing project using human heart cDNA libraries to study expression of genes in the human heart. From our human heart cDNA libraries, we have accumulated over 10,000 partial cDNA sequences (expressed sequence tags-ESTs) representing both the previously uncharacterized and known transcripts expressed in the human heart (Liew et al., 1994). Currently, we have applied dot blot hybridization as a rapid approach to determine the genes putatively involved in myocardial development. Differential expression patterns of gene transcripts represented by the cDNA clones can be revealed by comparing dot intensities on the autoradiographs, after hybridization with cDNA probes generated from neonatal and adult heart mRNAs, cDNA clones (1505) have been processed by dot blot hybridization, of which 924 and 581 represented novel and known transcripts respectively. Among the screened clones, about 1.4% were found to be differentially expressed during heart development. Further verification was accomplished by Northern blot analysis. By grouping the 581 clones corresponding to known transcripts, a study of the gene expression profile of the heart in the cardiovascular system can be achieved.

An oligonucleotide probe for the consensus sequence of the linker region of zinc finger proteins was used to isolate cDNA clones from a human fetal heart cDNA library. Following DNA sequencing analysis and comparison, genes for the novel clones were mapped by fluorescence in situ hybridization. We report the chromosomal localization of three zinc finger-coding genes to the region of centromere on human chromosome 10p11.1q-11.2, indicating involvement in gene duplication and chromosome rearrangement during primate evolution.