Study On Depth Sequencing And Clinical Phenotype - Genotype Association Analysis Of Pituitary ACTH Adenoma Transcriptome

Context:Pituitary adenomas, accounting for approximately20%of intracranial tumors, can be divided in endocrinologically inactive nonfunctioning pituitary adenomas, associated with mass effects, and functioning pituitary tumors, associated with specific clinical syndromes, due to pituitary hormones (Prolactin, GH, ACTH, and TSH) excess. However, this histopathological classification, based on tumor cell morphology, endocrinology and MRI,could not reveal tumor biological habits, such as nerve-endocrine disorder; Not to mention, accurately guide the clinical diagnosis and treatment. A better understanding of molecular pathology classification of pituitary adenomas may identify new pharmacological targets, with a better chance to control the disease.The objective of the study was to elucidate the signal transduction network of ACTH-secreting pituitary adenoma using the next generation sequencing technology. RNA deep sequencing was used to examine genotypic changes in nine paired female ACTH-secreting pituitary adenomas and adjacent nontumorous pituitary tissues (ANPT). We developed a novel analysis linking disease clinic characteristics and whole transcriptomic changes using Pearson Correlation Coefficient to discover molecular network mechanism. In order to eventually establish a pituitary adenoma new molecular pathology classification, achieve the personalized treatment in patients with pituitary adenomas.Section One:Transsphenoidal approach surgical treatment of ACTH-secreting pituitary adenoma:clinical pheno-phenotype association analysis of118casesObjective:To analyze the Clinical informations of ACTH-secreting pituitary adenoma via transsphenoidal surgery retrospectively, and to explore the clinical phenotypes correlation.Methods:We obtained118pathologically proven fresh ACTH-secreting pituitary adenoma specimens for this study from the Department of Neuro surgery, Peking Union Medical College Hospital (Beijing, China) between May16,2012, and July25,2013. We study106clinical characteristics from118patients, including typical clinical manifestations, endocrine related inspection, blood routine examination, liver and kidney function, electrolyte, sugar&lipid metabolism and operative skills. We developed a novel analysis linking disease clinic characteristics using Pearson Correlation Coefficient.Results:We obtained118pathologically proven fresh ACTH-secreting pituitary adenoma specimens for this study,101cases of women,17cases of men. Ages between14and65years, mean(35.5±12.3). Medical history from2months to19years, mean4years. Endocrinology:blood and24h Urinary free cortisol levels increased, Circadian rhythm disappeared. Dexamethasone suppression test:not be suppressed by low-dose DST (2mg)97%(114/118cases); be suppressed by high-dose DST(8mg)89.5%(105/118cases). Tumor diameter on MRI of the sella with contrast administration mean (7.6±6.4mm). Measure BMD with Dual X-rays absorptiometry (DEXA) at lumbar spine:Approximately28.0%(33/118cases) of patients experience fractures (particularly at the spinal level), consistent with the53.4%(63/118cases) incidence of osteoporosis. Adjacent non-tumorous pituitary tissues resection44.9%(53/118cases). We found that pituitary tumor size was correlated linearly with levels of ACTH, cortisol, LH and E2, as well as clinical characteristics of bone metabolism and mineral density (BMD).Conclusions:We report that osteoporosis is distinguished from the phenotype analysis. Our findings provide new insight into the potential mechanisms of the development of osteoporosis due to ACTH-secreting pituitary adenomas, and may open new potential avenues for osteoporosis intervention and treatment. Section Two:Phenotype-genotype association analysis of ACTH-secreting pituitary adenoma and its molecular Link to patient osteoporosisContext:Adrenocorticotrophin (ACTH)-secreting pituitary adenoma, Cushing disease (CD), is rare and causes metabolic syndrome, cardiovascular disease and osteoporosis due to hypercortisolism. However the molecular pathogenesis of CD is still unclear because of lack of human cell lines and animal models.Objective:The objective of the study was to elucidate the signal transduction network of ACTH-secreting pituitary adenoma by using the next generation sequencing technology.Methods:We obtained118pathologically proven fresh ACTH-secreting pituitary adenoma specimens for this study. Here we study106clinical characteristics and gene expression changes from118patients, the largest cohort of CD in single-center. RNA deep sequencing was used to examine genotypic changes in nine paired female ACTH-secreting pituitary adenomas and adjacent nontumorous pituitary tissues (ANPT). We developed a novel analysis linking disease clinic characteristics and whole transcriptomic changes using Pearson Correlation Coefficient to discover molecular network mechanism.Results:We report that osteoporosis is distinguished from the phenotype and genotype analysis. A cluster of genes involving in osteoporosis is identified using Pearson correlation coefficient analysis. Most of the genes are reported in the bone related literature, confirming the feasibility of phenotype-genotype association analysis, which could be used in analysis of almost all diseases. SPP1, COL1A1, NT5E, HTRA1and ANGPT1and their signalling pathways are shown involving in osteoporosis in CD patients.Conclusions:Our findings provide new insight into the potential mechanisms of the development of osteoporosis due to ACTH-secreting pituitary adenomas, and may open new potential avenues for osteoporosis intervention and treatment. Section Three:Discovery of ACTH-secreting pituitary adenoma related microRNAs using Next generation sequencing technologiesContext:MicroRNAs (miRNAs) are small noncoding RNAs, functioning as antisense regulators ofgene expression by targeting mRNA and contributing to tumor development and progression. More than50%of miRNA genes are located in tumor-associated genomic regions or in fragile sites of the genome.Objective:The aim of the study was to analyze of ACTH-secreting pituitary adenoma related microRNAs using new generation sequencing technologies.Methods:ACTH-secreting pituitary tumor samples and adjacent non-tumorous pituitary tissues (ANPT) were obtained during transphenoidal surgery frompatients with Cushing disease. The relative expression of miRNAs was measured by new generation sequencing technologies.Results:Our next generation sequencing obtained10million reads per sample. We found67microRNAs differentially expressed between paired tumour samples and ANPT. Further studies are needed to explore their functions in the pathogenesis of Cushing disease.Conclusion:Our results support the possibility that altered miRNA expression profile might be involved in corticotrophic tumorigenesis.