Differential hippocampal gene expression and pathway analysis in an etiology-based mouse model of major depressive disorder.

We have recently reported the creation and initial characterization of an etiology-based recombinant mouse model of a severe and inherited form of Major Depressive Disorder (MDD). This was achieved by replacing the corresponding mouse DNA sequence with a 6-base DNA sequence from the human CREB1 promoter that is associated with MDD in individuals from families with recurrent, early-onset MDD (RE-MDD). In the current study, we explored the effect of the pathogenic Creb1 allele on gene expression in the mouse hippocampus, a brain region that is altered in structure and function in MDD. Mouse whole-genome profiling was performed using the Illumina MouseWG-6 v2.0 Expression BeadChip microarray. Univariate analysis identified 269 differentially-expressed genes in the hippocampus of the mutant mouse. Pathway analyses highlighted 11 KEGG pathways: the phosphatidylinositol signaling system, which has been widely implicated in MDD, Bipolar Disorder, and the action of mood stabilizers; gap junction and long-term potentiation, which mediate cognition and memory functions often impaired in MDD; cardiac muscle contraction, insulin signaling pathway, and three neurodegenerative brain disorders (Alzheimer's, Parkinson's, and Huntington's Diseases) that are associated with MDD; ribosome and proteasome pathways affecting protein synthesis/degradation; and the oxidative phosphorylation pathway that is key to energy production. These findings illustrate the merit of this congenic C57BL/6 recombinant mouse as a model of RE-MDD, and demonstrate its potential for highlighting molecular and cellular pathways that contribute to the biology of MDD. The results also inform our understanding of the mechanisms that underlie the comorbidity of MDD with other disorders.

Lung tissues in patients with systemic sclerosis have gene expression patterns unique to pulmonary fibrosis and pulmonary hypertension.

OBJECTIVE: Pulmonary complications, including pulmonary fibrosis (PF) and pulmonary arterial hypertension (PAH), are the leading cause of mortality in patients with systemic sclerosis (SSc). The aim of this study was to compare the molecular fingerprint of lung tissue and matching primary fibroblasts from patients with SSc with that of lung tissue and fibroblasts from normal donors, patients with idiopathic pulmonary fibrosis (IPF), and patients with idiopathic pulmonary arterial hypertension (IPAH). METHODS: Lung tissue samples were obtained from 33 patients with SSc who underwent lung transplantation. Tissues and cells from a subgroup of SSc patients with predominantly PF or PAH were compared to those from normal donors, patients with IPF, and patients with IPAH. Microarray data were analyzed using efficiency analysis for determination of the optimal data-processing methods. Real-time polymerase chain reaction and immunohistochemistry were used to confirm differential levels of messenger RNA and protein, respectively. RESULTS: Consensus efficiency analysis identified 242 and 335 genes that were differentially expressed in lungs and primary fibroblasts, respectively. SSc-PF and IPF lungs shared enriched functional groups in genes implicated in fibrosis, insulin-like growth factor signaling, and caveolin-mediated endocytosis. Gene functional groups shared by SSc-PAH and IPAH lungs included those involved in antigen presentation, chemokine activity, and interleukin-17 signaling. CONCLUSION: Using microarray analysis on carefully phenotyped SSc and comparator lung tissues, we demonstrated distinct molecular profiles in tissues and fibroblasts from patients with SSc-associated lung disease compared to idiopathic forms of lung disease. Unique molecular signatures were generated that are disease specific (SSc) and phenotype specific (PF versus PAH). These signatures provide new insights into the pathogenesis and potential therapeutic targets of SSc-related lung disease.

Performance of whole-genome amplified DNA isolated from serum and plasma on high-density single nucleotide polymorphism arrays.

Defining genetic variation associated with complex human diseases requires standards based on high-quality DNA from well-characterized patients. With the development of robust technologies for whole-genome amplification, sample repositories such as serum banks now represent a potentially valuable source of DNA for both genomic studies and clinical diagnostics. We assessed the performance of whole-genome amplified DNA (wgaDNA) derived from stored serum/plasma on high-density single nucleotide polymorphism arrays. Neither storage time nor usage history affected either DNA extraction or whole-genome amplification yields; however, samples that were thawed and refrozen showed significantly lower call rates (73.9 +/- 7.8%) than samples that were never thawed (92.0 +/- 3.3%) (P < 0.001). Genotype call rates did not differ significantly (P = 0.13) between wgaDNA from never-thawed serum/plasma (92.9 +/- 2.6%) and genomic DNA (97.5 +/- 0.3%) isolated from whole blood. Approximately 400,000 genotypes were consistent between wgaDNA and genomic DNA, but the overall discordance rate of 4.4 +/- 3.8% reflected an average of 11,110 +/- 9502 genotyping errors per sample. No distinct patterns of chromosomal clustering were observed for single nucleotide polymorphisms showing discordant genotypes or homozygote conversion. Because the effects of genotyping errors on whole-genome studies are not well defined, we recommend caution when applying wgaDNA from serum/plasma to high-density single nucleotide polymorphism arrays in addition to the use of stringent quality control requirements for the resulting genotype data.

The genomic heritage of lymph node metastases: implications for clinical management of patients with breast cancer.

BACKGROUND: Metastatic breast cancer is an aggressive disease associated with recurrence and decreased survival. To improve outcomes and develop more effective treatment strategies for patients with breast cancer, it is important to understand the molecular mechanisms underlying metastasis. METHODS: We used allelic imbalance (AI) to determine the molecular heritage of primary breast tumors and corresponding metastases to the axillary lymph nodes. Paraffin-embedded samples from primary breast tumors and matched metastases (n = 146) were collected from 26 patients with node-positive breast cancer involving multiple axillary nodes. Hierarchical clustering was used to assess overall differences in the patterns of AI, and phylogenetic analysis inferred the molecular heritage of axillary lymph node metastases. RESULTS: Overall frequencies of AI were significantly higher (P < 0.01) in primary breast tumors (23%) than in lymph node metastases (15%), and there was a high degree of discordance in patterns of AI between primary breast carcinomas and the metastases. Metastatic tumors in the axillary nodes showed different patterns of chromosomal changes, suggesting that multiple molecular mechanisms may govern the process of metastasis in individual patients. Some metastases progressed with few genomic alterations, while others harbored many chromosomal alterations present in the primary tumor. CONCLUSIONS: The extent of genomic heterogeneity in axillary lymph node metastases differs markedly among individual patients. Genomic diversity may be associated with response to adjuvant therapy, recurrence, and survival, and thus may be important in improving clinical management of breast cancer patients.

Functional identity of genes detectable in expression profiling assays following globin mRNA reduction of peripheral blood samples.

OBJECTIVES: Whole-blood RNA for microarray analysis is easily accessible but contains a large proportion of globin mRNA that interferes with the accurate assessment of other genes. This study investigated the biological significance of genes whose expression was unmasked by globin mRNA reduction in peripheral blood. DESIGN AND METHODS: Samples were collected from healthy subjects using the PAXgene Blood RNA System, and globin mRNA was depleted using GLOBINclear. Genes exhibiting consistent changes in expression on Affymetrix HU133A 2.0 arrays were characterized in three main areas of gene ontology--molecular function, biological process, and cellular component. RESULTS: Globin reduction permitted detection of 2652+/-395 additional genes per assay. Genes unmasked by globin reduction include low abundance transcripts that function primarily as molecular binding proteins and catalytic enzymes in biological processes including transcription, replication, and intracellular transport and signalling. Protein products of these genes are preferentially associated with membranes and the nucleus. CONCLUSIONS: Additional genes detectable only after globin reduction in whole-blood RNA function in a variety of biological processes that may be important to diverse fields of study.

Co-occurrence analysis for discovery of novel breast cancer pathology patterns.

To discover novel patterns in pathology co-occurrence, we have developed algorithms to analyze and visualize pathology co-occurrence. With access to a database of pathology reports, collected under a single protocol and reviewed by a single pathologist, we can conduct an analysis greater in its scope than previous studies looking at breast pathology co-occurrence. Because this data set is unique, specialized methods for pathology co-occurrence analysis and visualization are developed. Primary analysis is through a co-occurrence score based on the Jaccard coefficient. Density maps are used to visualize global co-occurrence. When our co-occurrence analysis is applied to a population stratified by menopausal status, we can successfully identify statistically significant differences in pathology co-occurrence patterns between premenopausal and postmenopausal women. Genomic and proteomic experiments are planned to discover biological mechanisms that may underpin differences seen in pathology patterns between populations.

Circulating MMP2 and MMP9 in breast cancer -- potential role in classification of patients into low risk, high risk, benign disease and breast cancer categories.

Matrix metalloproteinase (MMP) 2 and 9 are involved in cancer invasion and metastasis, and increased levels occur in serum and plasma of breast cancer (BC) patients. It is, however, unclear whether changes in serum levels can be exploited for early detection or classification of patients into different risk/disease categories. In our study, we measured concentration and activity of MMP2/9 in sera of 345 donors classified as low risk (Gail score <1.7), high risk (HR) (Gail score > or =1.7), benign disease or BC. Kruskal-Wallis and Mann-Whitney nonparametric tests showed that total-MMP2 concentration is higher in HR compared to control (p = 0.012), benign (p = 0.001) and cancer (p = 0.007). Active MMP2 (aMMP2) concentration is higher in control than benign and cancer (p < 0.001, respectively). Total and aMMP9 concentrations are higher in cancer than benign (p < 0.001, p = 0.002, respectively). Total-MMP2 and total-MMP9 activities are lower in control than benign (p < 0.001, p = 0.002, respectively) and cancer (p < 0.001, respectively). Total-MMP2 and MMP9 activities are also higher in cancer than benign (p = 0.004, p < 0.001) and HR (p = 0.008, p = 0.007, respectively). These results were not affected by age or inclusion/exclusion of donors with noninvasive cancer or atypical hyperplasia. Linear discriminant analysis revealed that HR donors are characterized by lower total-MMP2 and higher aMMP2. Overall group classification accuracy was 64.5%. Independent validation based on the leave-one-out cross validation approach gave an overall classification of 63%. Our study provides evidence supporting the potential role of serum MMP2/9 as biomarkers for breast disease classification.