Alicia Golden

Identification, basic characterization and evolutionary analysis of differentially spliced mRNA isoforms of human YAP1 gene.

The YAP1 gene encodes a potent new oncogene and stem cell factor. However, in some cancers, the YAP1 gene plays a role of tumor suppressor. At present, the gene and its products are intensely studied and its cDNAs are used as transgenes in cellular and animal models. Here, we report 4 new potential mRNA splicing isoforms of the YAP1 gene, bringing the total number of isoforms to 8. We detected all 8 YAP1 isoforms in a panel of human tissues and evaluated the expression of the longest isoform of YAP1 (YAP1-2δ) using Real Time PCR. All YAP1 isoforms are barely detectable in human leukocytes compared to fair levels of expression found in other human tissues. We analyzed the structure of the genomic region that gave rise to alternatively spliced YAP1 transcripts in different metazoans. We found that YAP1 isoforms, which utilize exon 6 emerged in evolution with the appearance of amniotes. Interestingly, 6 YAP1 isoforms, which contain the exon 5 extension, exon 6 or both would have their leucine zipper region disrupted in the predicted protein product, compared to the intact leucine zipper found in two YAP1 (α) isoforms. This observation has direct functional ramifications for YAP1 signaling. We also propose a normalized nomenclature for the mRNA splice variants of the YAP1 gene, which should aid in the characterization of signaling differences among the potential protein products of the YAP1 gene.

Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysms

BackgroundThe infrarenal abdominal aorta exhibits increased disease susceptibility relative to other aortic regions. Allograft studies exchanging thoracic and abdominal segments showed that regional susceptibility is maintained regardless of location, suggesting substantial roles for embryological origin, tissue composition and site-specific gene expression.ResultsWe analyzed gene expression with microarrays in baboon aortas, and found that members of the HOX gene family exhibited spatial expression differences. HOXA4 was chosen for further study, since it had decreased expression in the abdominal compared to the thoracic aorta. Western blot analysis from 24 human aortas demonstrated significantly higher HOXA4 protein levels in thoracic compared to abdominal tissues (P < 0.001). Immunohistochemical staining for HOXA4 showed nuclear and perinuclear staining in endothelial and smooth muscle cells in aorta. The HOXA4 transcript levels were significantly decreased in human abdominal aortic aneurysms (AAAs) compared to age-matched non-aneurysmal controls (P < 0.00004). Cultured human aortic endothelial and smooth muscle cells stimulated with INF-γ (an important inflammatory cytokine in AAA pathogenesis) showed decreased levels of HOXA4 protein (P < 0.0007).ConclusionsOur results demonstrated spatial variation in expression of HOXA4 in human aortas that persisted into adulthood and that downregulation of HOXA4 expression was associated with AAAs, an important aortic disease of the ageing population.

Novel pathways in the pathobiology of human abdominal aortic aneurysms

Objectives: Abdominal aortic aneurysm (AAA), a dilatation of the infrarenal aorta, typically affects males >65 years. The pathobiological mechanisms of human AAA are poorly understood. The goal of this study was to identify novel pathways involved in the development of AAAs. Methods: A custom-designed ‘AAA-chip’ was used to assay 43 of the differentially expressed genes identified in a previously published microarray study between AAA (n = 15) and control (n = 15) infrarenal abdominal aorta. Protein analyses were performed on selected genes. Results: Altogether 38 of the 43 genes on the ‘AAA-chip’ showed significantly different expression. Novel validated genes in AAA pathobiology included ADCY7, ARL4C, BLNK, FOSB, GATM, LYZ, MFGE8, PRUNE2, PTPRC, SMTN, TMODI and TPM2. These genes represent a wide range of biological functions, such as calcium signaling, development and differentiation, as well as cell adhesion not previously implicated in AAA pathobiology. Protein analyses for GATM, CD4, CXCR4, BLNK, PLEK, LYZ, FOSB, DUSP6, ITGA5 and PTPRC confirmed the mRNA findings. Conclusion: The results provide new directions for future research into AAA pathogenesis to study the role of novel genes confirmed here. New treatments and diagnostic tools for AAA could potentially be identified by studying these novel pathways.

Role of Complement Cascade in Abdominal Aortic Aneurysms

Objective—The goal of this study was to investigate the role of complement cascade genes in the pathobiology of human abdominal aortic aneurysms (AAAs). Methods and Results—Results of a genome-wide microarray expression profiling revealed 3274 differentially expressed genes between aneurysmal and control aortic tissue. Interestingly, 13 genes in the complement cascade were significantly differentially expressed between AAA and the controls. In silico analysis of the promoters of the 13 complement cascade genes showed enrichment for transcription factor binding sites for signal transducer and activator of transcription (STAT)5A. Chromatin-immunoprecipitation experiments demonstrated binding of transcription factor STAT5A to the promoters of the majority of the complement cascade genes. Immunohistochemical analysis showed strong staining for C2 in AAA tissues. Conclusion—These results provide strong evidence that the complement cascade plays a role in human AAA. Based on our microarray studies, the pathway is activated in AAA, particularly via the lectin and classical pathways. The overrepresented binding sites of transcription factor STAT5A in the complement cascade gene promoters suggest a role for STAT5A in the coordinated regulation of complement cascade gene expression.

Microarray expression profiling in adhesion and normal peritoneal tissues.

OBJECTIVE To identify molecular markers associated with adhesion and normal peritoneal tissue using microarray expression profiling. DESIGN Comparative study. SETTING University hospital. PATIENT(S) Five premenopausal women. INTERVENTION(S) Adhesion and normal peritoneal tissue samples were obtained from premenopausal women. Ribonucleic acid was extracted using standard protocols and processed for hybridization to Affymetrix Whole Transcript Human Gene Expression Chips. Microarray data were obtained from five different patients, each with adhesion tissue and normal peritoneal samples. Real-time polymerase chain reaction was performed for confirmation using standard protocols. MAIN OUTCOME MEASURE(S) Gene expression in postoperative adhesion and normal peritoneal tissues. RESULT(S) A total of 1,263 genes were differentially expressed between adhesion and normal tissues. One hundred seventy-three genes were found to be up-regulated and 56 genes were down-regulated in the adhesion tissues compared with normal peritoneal tissues. The genes were sorted into functional categories according to Gene Ontology annotations. Twenty-six up-regulated genes and 11 down-regulated genes were identified with functions potentially relevant to the pathophysiology of postoperative adhesions. We evaluated and confirmed expression of 12 of these specific genes via polymerase chain reaction. CONCLUSION(S) The pathogenesis, natural history, and optimal treatment of postoperative adhesive disease remains unanswered. Microarray analysis of adhesions identified specific genes with increased and decreased expression when compared with normal peritoneum. Knowledge of these genes and ontologic pathways with altered expression provide targets for new therapies to treat patients who have or are at risk for postoperative adhesions.

C-B3-03: Association of Epidemiologic and Genetic Factors With Abdominal Aortic Aneurysm (AAA)

Background: Abdominal Aortic Aneurysm (AAA) rupture is the thirteenth leading cause of death in the U.S. and a major cause of death in the elderly. Since most AAAs are asymptomatic, mortality could be greatly reduced by determining which patients are at risk, then screening those patients prior to a rupture. Our aim was to determine which clinical and genetic risk factors are associated with AAA. Methods: This was a case- control study of patients from the Geisinger Vascular Clinic and the Geisinger MyCode Project (a biobanking project of Geisinger Clinic patients who volunteered to participate in genetic research projects). Clinical and environmental risk factor data was obtained from patient electronic medical records (EMR) and analyzed by multivariate logistic regression. In a subset of participants, we also analyzed three promising genetic polymorphisms, two from a prior genome-wide association study in this population (rs12039875, 1q41; rs7635818, 3p12.3) and one from the recent literature (rs10757278, 9p21). The genetic data was combined with the clinical data using multivariate logistic regression. Results: In the clinical analysis the number of Vascular Clinic and MyCode AAA cases totaled 722, with 11,761 controls. Adjusted OR showed a significant AAA risk for age, gender, smoking, intermittent claudication and peripheral artery disease, while body mass index (BMI) and diabetes were surprisingly protective. The genetic analysis consisted of 502 AAA cases and 295 controls from the Vascular Clinic. The GC genotype of rs7635818 showed an increased risk, but the rs12039875 AA genotype showed a significant protective effect when controlling for the clinical variables. This SNP is located in KCNK2, a gene in the potassium channel protein family. Although this gene has not previously been linked with AAA, our laboratory showed that this gene is expressed in vascular tissue. Conclusions: Our study indicates a significant elevated risk of AAA for individuals who are older, male, ever smoked, have peripheral artery disease and a GC genotype of rs7635818. Higher BMI, diabetes and the AA genotype of rs12039875 in the KCNK2 gene significantly lower AAA risk. This is one of the first studies to utilize the MyCode population in a research study and establish this population source as a valuable and convenient asset of Geisinger Health System for clinical research.