Alterations in leukocyte transcriptional control pathway activity associated with major depressive disorder and antidepressant treatment.

Major depressive disorder (MDD) is associated with a significantly elevated risk of developing serious medical illnesses such as cardiovascular disease, immune impairments, infection, dementia and premature death. Previous work has demonstrated immune dysregulation in subjects with MDD. Using genome-wide transcriptional profiling and promoter-based bioinformatic strategies, we assessed leukocyte transcription factor (TF) activity in leukocytes from 20 unmedicated MDD subjects versus 20 age-, sex- and ethnicity-matched healthy controls, before initiation of antidepressant therapy, and in 17 of the MDD subjects after 8 weeks of sertraline treatment. In leukocytes from unmedicated MDD subjects, bioinformatic analysis of transcription control pathway activity indicated an increased transcriptional activity of cAMP response element-binding/activating TF (CREB/ATF) and increased activity of TFs associated with cellular responses to oxidative stress (nuclear factor erythroid-derived 2-like 2, NFE2l2 or NRF2). Eight weeks of antidepressant therapy was associated with significant reductions in Hamilton Depression Rating Scale scores and reduced activity of NRF2, but not in CREB/ATF activity. Several other transcriptional regulation pathways, including the glucocorticoid receptor (GR), nuclear factor kappa-B cells (NF-κB), early growth response proteins 1-4 (EGR1-4) and interferon-responsive TFs, showed either no significant differences as a function of disease or treatment, or activities that were opposite to those previously hypothesized to be involved in the etiology of MDD or effective treatment. Our results suggest that CREB/ATF and NRF2 signaling may contribute to MDD by activating immune cell transcriptome dynamics that ultimately influence central nervous system (CNS) motivational and affective processes via circulating mediators.

Adrenal endothelin-1 levels are not associated with aldosterone secretion in primary aldosteronism.

BACKGROUND: Endothelin-1 (ET-1) may function as an aldosterone secretagogue and, in turn, aldosterone can upregulate ET-1 expression. Hence, the existence of a feedforward loop involving ETs and aldosterone has been speculated in primary aldosteronism (PA). In the present study, we sought to examine ET-1 secretion from the adrenal glands in patients with PA. DESIGN: We determined ET-1 levels in blood samples obtained during adrenal venous sampling of patients affected by PA (n=17). Furthermore, we examined the mRNA expression of the ET system in tissue samples from aldosterone-producing adenomas (APAs, n=9) and control normal adrenals (n=3). METHODS: Blood ET-1 levels were determined by RIA. Tissue mRNA expression of the ET system was assayed with Affymetrix microarrays. RESULTS: ET-1 levels did not differ between inferior vena cava and adrenal vein blood in both bilateral adrenal hyperplasia and APA patients. Moreover, cortisol-normalized ET-1 levels did not show lateralized adrenal ET-1 secretion in APAs. Through gene expression profiling with microarray performed in a distinct set of APA individuals (n=9), we confirmed the adrenal expression of a complete ET system, but we did not detect a significant upregulation of ET components within the APA tissue compared with normal adrenals. CONCLUSIONS: The present data argue against the hypothesis of increased ET-1 secretion from APAs and do not support a general role for adrenal ET-1 in the vascular pathophysiology of PA.

Blood-based biomarkers for detecting mild osteoarthritis in the human knee.

OBJECTIVE: This study was designed to test the utility of a blood-based approach to identify mild osteoarthritis (OA) of the knee. METHODS: Blood samples were drawn from 161 subjects, including 85 subjects with arthroscopically diagnosed mild OA of the knee and 76 controls. Following RNA isolation, an in-house custom cDNA microarray was used to screen for differentially expressed genes. A subset of selected genes was then tested using real-time RT-PCR. Logistic regression analysis was used to evaluate linear combinations of the biomarkers and receiver operating characteristic curve analysis was used to assess the discriminatory power of the combinations. RESULTS: Genes differentially expressed (3543 genes) between mild knee OA and control samples were identified through microarray analysis. Subsequent real-time RT-PCR verification identified six genes significantly down-regulated in mild OA: heat shock 90kDa protein 1, alpha; inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein; interleukin 13 receptor, alpha 1; laminin, gamma 1; platelet factor 4 (also known as chemokine (C-X-C motif) ligand 4) and tumor necrosis factor, alpha-induced protein 6. Logistic regression analysis identified linear combinations of nine genes--the above six genes, early growth response 1; alpha glucosidase II alpha subunit; and v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (avian)--as discriminatory between subjects with mild OA and controls, with a sensitivity of 86% and specificity of 83% in a training set of 78 samples. The optimal biomarker combinations were then evaluated using a blind test set (67 subjects) which showed 72% sensitivity and 66% specificity. CONCLUSIONS: Linear combinations of blood RNA biomarkers offer a substantial improvement over currently available diagnostic tools for mild OA. Blood-derived RNA biomarkers may be of significant clinical value for the diagnosis of early, asymptomatic OA of the knee.

Assessing the validity of blood-based gene expression profiles for the classification of schizophrenia and bipolar disorder: a preliminary report.

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.

First comprehensive mapping of cartilage transcripts to the human genome.

We present the first comprehensive transcriptome-to-genome mapping for human cartilage. First, we determined that the cartilage transcriptome represents between 13,200 and 15,800 unique genes. Next, a subset of approximately 10,000 of the best characterized cartilage-expressed transcripts (CETs) was selected and mapped to the human genome. The distribution of CETs across the genome was found to be significantly different compared to the expected distribution. Furthermore, clusters of adjacent coordinately transcribed genes, as well as numerous "hot spots" and "cold spots" for transcription in cartilage, were identified. We propose that transcriptional control in cartilage can be exerted over genomic domains containing as few as four neighboring genes. Our findings, which are consistent with recent "chromatin domain" models of transcription, are further supported by our identification of CETs that putatively encode components of the HDAC- and Swi/SNF-mediated chromatin remodeling pathways. Our study illustrates the value of comprehensive high-resolution scans to detect transcription patterns within the human genome.

Genomics, proteomics and bioinformatics of human heart failure.

Unraveling the molecular complexities of human heart failure, particularly end-stage failure, can be achieved by combining multiple investigative approaches. There are several parts to the problem. Each patient is the product of a complex set of genetic variations, different degrees of influence of diets and lifestyles, and usually heart transplantation patients are treated with multiple drugs. The genomic status of the myocardium of any one transplant patient can be analysed using gene arrays (cDNA- or oligonucleotide-based) each with its own strengths and weaknesses. The proteins expressed by these failing hearts (myocardial proteomics) were first investigated over a decade ago using two-dimensional polyacrylamide gel electrophoresis (2DGE) which promised to resolve several thousand proteins in a single sample of failing heart. However, while 2DGE is very successful for the abundant and moderately expressed proteins, it struggles to identify proteins expressed at low levels. Highly focused first dimension separations combined with recent advances in mass spectrometry now provide new hope for solving this difficulty. Protein arrays are a more recent form of proteomics that hold great promise but, like the above methods, they have their own drawbacks. Our approach to solving the problems inherent in the genomics and proteomics of heart failure is to provide experts in each analytical method with a sample from the same human failing heart. This requires a sufficiently large number of samples from a sufficiently large pool of heart transplant patients as well as a large pool of non-diseased, non-failing human hearts. We have collected more than 200 hearts from patients undergoing heart transplantations and a further 50 non-failing hearts. By combining our expertise we expect to reduce and possibly eliminate the inherent difficulties of each analytical approach. Finally, we recognise the need for bioinformatics to make sense of the large quantities of data that will flow from our laboratories. Thus, we plan to provide meaningful molecular descriptions of a number of different conditions that result in terminal heart failure.

Mutations of the beta myosin heavy chain gene in hypertrophic cardiomyopathy: critical functional sites determine prognosis.

OBJECTIVES: To assess patients with different types of mutations of the beta myosin heavy chain (beta MHC) gene causing hypertrophic cardiomyopathy (HCM) and to determine the prognosis of patients according to the affected functional domain of beta MHC. DESIGN AND SETTING: Cohort study of subjects referred to an HCM clinic at an academic hospital. PATIENTS: 70 probands from the HCM clinic were screened for mutations of the beta MHC gene and 148 family members of the genotype positive probands were further assessed. The control group for the genetic studies consisted of 106 healthy subjects. MAIN OUTCOME MEASURES: Direct DNA sequencing was used to screen 70 probands for mutations of the beta MHC gene. Family members underwent genotypic and detailed clinical, ECG, and echocardiographic assessments. The survival of genotype positive subjects was evaluated according to the type of functional domain affected by the missense mutation and according to phenotypic characteristics. RESULTS: A mutation of the beta MHC gene was detected in 15 of 70 probands (21%). Of 148 family members studied in these 15 families, 74 were identified with a beta MHC defect. Eleven mutations were detected, including four novel mutations: Ala196Thr, Pro211Leu, Val404Leu, and Arg870Cys. Median survival was 66 years (95% confidence interval (CI) 64 to 77 years) in all affected subjects. There was a significant difference in survival between subjects according to the affected functional domain (p = 0.02). Significant independent predictors of decreased survival were the non-conservative (that is, associated with a change in the amino acid charge) missense mutations that affected the actin binding site (hazard ratio 4.4, 95% CI 1.6 to 11.8; p = 0.003) and those that affected the rod portion of beta MHC (hazard ratio 4.8, 95% CI 1.2 to 19.4; p = 0.03). No phenotypic characteristics were associated with decreased survival or cardiovascular morbidity. CONCLUSIONS: The type of beta MHC functional domain affected by the missense mutation is predictive of overall prognosis in HCM.

Profiling genes expressed in human fetal cartilage using 13,155 expressed sequence tags.

OBJECTIVE: To analyze the gene expression profile of human fetal cartilage by expressed sequence tags (ESTs). METHODS: A human fetal cartilage (8-12 weeks) cDNA library was constructed using the lambda ZAP Express vector. ESTs were obtained by partial sequencing of cDNA clones. The basic local alignment search tool algorithm was used to compare all generated ESTs to known sequences. RESULTS: A total of 13,155 ESTs were analyzed, of which 8696 ESTs (66.1%) matched known genes, 53 ESTs (0.4%) were putatively novel (with no match) and the rest matched other ESTs, genomic DNA and repetitive sequences. Importantly, we identified 2448 unique known genes through non-redundancy analysis of the known gene matches, which were then functionally categorized. The tissue specificity of this library was reflected by its EST profile of the extracellular matrix (ECM) proteins. Collagens were the major transcripts, representing 68.5% of the ECM proteins. Proteoglycans were the second most abundant, constituting 9.5%. Collagen type II was the most abundant gene of all. Glypican 3, decorin and aggrecan were the major transcripts of proteoglycans. Many genes involved in cartilage development were identified, such as insulin-like growth factor-II, its receptor and binding proteins, connective tissue growth factor and fibroblast growth factors. Proteases and their regulatory factors were also identified, including matrix metalloprotease 2 and tissue inhibitor of metalloproteinase 1. CONCLUSIONS: The EST approach is an effective way of characterizing the genes expressed in cartilage. These data represent the most extensive molecular information on human fetal cartilage to date. The availability of this information will serve as a basis for further research to identify genes that are essential in cartilage development.

Microarray analysis reveals the involvement of beta-2 microglobulin (B2M) in human osteoarthritis.

OBJECTIVE: To assess whether beta-2 microglobulin (B2M) has effects on articular chondrocytes that would implicate B2M involvement in osteoarthritis (OA) pathogenesis. METHODS: The mRNA levels of B2M in fetal and osteoarthritic chondrocytes were detected by RT-PCR. B2M levels in synovial fluid and tissue cultured media from cartilage explants were tested using B2M ELISA kit. Primary cultured chondrocytes were used for proliferation and microarray experiments. RESULTS: The average B2M level in OA synovial fluid is significantly higher than that found in normal synovial fluid. However, there was no significant difference in B2M synovial fluid levels amongst differing OA stages. The release of B2M by osteoarthritic cartilage was detectable after 24h in culture and continued to increase during the 72 h study period. B2M had an inhibitory effect on chondrocyte growth at 1.0 microg/ml, and became significantly inhibitory at 10.0 microg/ml. Genes regulated by B2M were detected through microarray technology. Twenty genes were found to be up-regulated by B2M, including collagen type III which is known to be up-regulated in OA. Eleven genes were found to be down-regulated at least two-fold by B2M. CONCLUSION: These results indicate that B2M is highly expressed in OA cartilage and synovial fluid compared to normal, and suggest that B2M may have effects on chondrocyte function that could contribute to OA pathogenesis. Published by Elsevier Science Ltd.

Cardiovascular genomics: estimating the total number of genes expressed in the human cardiovascular system.

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.

Reactive oxygen species modulate angiotensin II-induced beta-myosin heavy chain gene expression via Ras/Raf/extracellular signal-regulated kinase pathway in neonatal rat cardiomyocytes.

Angiotensin II (Ang II) causes cardiomyocytes hypertrophy. Cardiac beta-myosin heavy chain (beta-MyHC) gene expression can be altered by Ang II. The molecular mechanisms are not completely known. Reactive oxygen species (ROS) are involved in signal transduction pathways of Ang II. However, the role of ROS on Ang II-induced beta-MyHC gene expression remains unclear. Here we found that Ang II increased beta-MyHC promoter activity and it was blocked by Ang II type 1 receptor antagonist losartan. Ang II dose-dependently increased the intracellular ROS. Cardiomyocytes cotransfected with a dominant negative mutant of Ras (RasN17), Raf-1 (Raf301), or a catalytically inactive mutant of extracellular signal regulated kinase (mERK2) inhibited Ang II-induced beta-MyHC promoter activity, indicating Ras/Raf/ERK pathway was involved. Antioxidants such as catalase or N-acetyl-cysteine decreased Ang II-activated ERK phosphorylation and inhibited Ang II-induced beta-MyHC promoter activity. These data indicate that Ang II increases beta-MyHC gene expression in part via the generation of ROS.

A novel human striated muscle RING zinc finger protein, SMRZ, interacts with SMT3b via its RING domain.

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.

Genomics and the pathophysiology of heart failure.

Heart failure is not a single disease entity, but a syndrome with various causes, including hypertension, ischemic and congenital heart disease, cardiomyopathy, and myocarditis. Because of the multiple etiologies and secondary adaptations contributing to heart failure, the study of the cellular and molecular mechanisms underlying the development and progression of this syndrome has been rather challenging. Much has been learned about the remodeling processes in heart failure, which involve complex interactions among numerous mediators in signaling and regulatory pathways. The Human Genome Project and related projects have provided a preliminary database for a genome-wide analysis of complex polygenic disorders such as heart failure. With the aid of expressed sequence tag technology and microarray applications, both known and previously uncharacterized genes involved in the induction and regression of cardiac hypertrophy and its progression to heart failure can be analyzed simultaneously. Deciphering the complexity of sequence-structure-function relationships in heart failure is a goal for the future, and will require advances in structural biology, proteomics, and computational technology. In this review, we summarize the cellular and molecular aspects of heart failure, and how recent applications of genomic technologies have been successful in achieving a more complete portrait of gene expression in this pathologic state.

Organization of human cardiovascular-expressed genes on chromosomes 21 and 22.

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.

Identification, characterization, and mapping of expressed sequence tags from an embryonic zebrafish heart cDNA library.

The generation of expressed sequence tags (ESTs) has proven to be a rapid and economical approach by which to identify and characterize expressed genes. We generated 5102 ESTs from a 3-d-old embryonic zebrafish heart cDNA library. Of these, 57.6% matched to known genes, 14.2% matched only to other ESTs, and 27.8% showed no match to any ESTs or known genes. Clustering of all ESTs identified 359 unique clusters comprising 1771 ESTs, whereas the remaining 3331 ESTs did not cluster. This estimates the number of unique genes identified in the data set to be approximately 3690. A total of 1242 unique known genes were used to analyze the gene expression patterns in the zebrafish embryonic heart. These were categorized into seven categories on the basis of gene function. The largest class of genes represented those involved in gene/protein expression (25.9% of known transcripts). This class was followed by genes involved in metabolism (18.7%), cell structure/motility (16.4%), cell signaling and communication (9.6%), cell/organism defense (7.1%), and cell division (4.4%). Unclassified genes constituted the remaining 17.91%. Radiation hybrid mapping was performed for 102 ESTs and comparison of map positions between zebrafish and human identified new synteny groups. Continued comparative analysis will be useful in defining the boundaries of conserved chromosome segments between zebrafish and humans, which will facilitate the transfer of genetic information between the two organisms and improve our understanding of vertebrate evolution.

Hydrogen bonding and dipole moment of water at supercritical conditions: a first-principles molecular dynamics study.

We present a first-principles molecular dynamics study of water near and above the critical point ( T = 647 K, rho = 0.32 g/cm(3)). We find that the systems undergo fast dynamics with continuous formation and breaking of H bonds. At low density, the system fragments mostly into trimers, dimers, and single molecules. At a higher density, more complex structures appear and an extended, albeit very dynamical, H-bond network can be identified. These structures have important consequences for the screening properties of the system. This offers a clue to understanding the peculiar chemical behavior of a supercritical system and allows thermodynamical tuning of its solvent properties.

Molecular cloning and characterization of a novel human gene containing ankyrin repeat and double BTB/POZ domain.

A novel human gene containing an ankyrin repeat and BTB/POZ domains (BPOZ) was isolated from a human leukocyte cDNA library. The cDNA sequence contains an open reading frame of 1434 bp that encodes 478 amino acid residues with a predicted molecular mass of 53.9 kDa. Sequence pattern analysis shows that BPOZ contains an N-terminal ankyrin repeat, a bipartite nuclear localization signal and two BTB/POZ domains. Using semiquantitative RT-PCR, the BPOZ transcript was found to be ubiquitously expressed in all fetal tissues examined (heart, brain, liver, and kidney) suggesting that BPOZ is involved in basic cellular function. Low expression of BPOZ in adult tissues (normal and hypertrophic heart) suggests that BPOZ mRNA is developmentally regulated and may play a role in developmental processes. Chromosomal localization by radiation hybrid mapping revealed that this gene is localized between D3S1269 and D3S3606 markers corresponding to the region of chromosome 3q21, a region frequently associated with leukemia. It is thus suggested that BPOZ may be functionally involved in protein-protein interaction, perhaps in forming protein complexes, and may have an important role in normal development and in the development of leukemia.

A cardiovascular EST repertoire: progress and promise for understanding cardiovascular disease.

The application of expressed sequence tag (EST) technology has proven to be an effective tool for gene discovery and the generation of gene expression profiles. The generation of an EST resource for the cardiovascular system has revealed significant insights into the changes in gene expression that guide heart development and disease. Furthermore, an important genetic resource has been developed for cardiovascular biology that is valuable for data mining and disease gene discovery.

Identification of differentially expressed genes in cardiac hypertrophy by analysis of expressed sequence tags.

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.

Role of the adenomatous polyposis coli gene product in human cardiac development and disease.

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.