Biochemical Basis of Cortisone Reductase Deficiency

描述(申请人提供)：表观皮质醇还原酶缺乏症(ACRD)是一种人类疾病，皮质醇的代谢清除增加，导致肾上腺皮质活动过度，肾上腺雄激素分泌增加，表型类似多囊卵巢综合征(PCOS)/代谢综合征/综合征X。我们先前的工作表明，ACRD是一种双基因疾病，需要携带两个基因的突变，HSD11B1(编码11β-羟基类固醇脱氢酶的类型1或肝脏同工酶)和H6PD(编码己糖-6-磷酸脱氢酶)。我们建议阐明这些突变相互作用导致疾病的机制。我们将通过使用定量聚合酶链式反应测量mRNA水平，以及通过免疫印迹和酶活性分析测量蛋白质表达，来确定H6PD在各种小鼠和人类组织中的表达。我们将通过在细菌中表达H6PD和11β-HSD1，并通过分析H6PDH在哺乳动物细胞中表达对11β-HSD1氧化还原酶活性的影响，来研究H6PD在确定11β-HSD1活性设定值中的直接作用。我们将确定H6PD活性变化对原代人类脂肪细胞和肝细胞表型的功能影响。我们将开发一种ACRD的小鼠模型。为此，我们将制造一只具有H6PD靶向失活的小鼠，并将其与一只具有失活HSD11B1等位基因的小鼠杂交。我们将培育双杂合突变小鼠，以产生ACRD的小鼠模型。表型特征将集中在基因和蛋白质的表达，11a-HSD1在肝脏和脂肪组织中的活性和平衡设定点，肾上腺皮质的发育和调节类固醇合成的关键基因的表达水平，肾上腺功能的分析，脂肪组织的发育和肝酶的表达。最后，我们将对患有多囊卵巢综合征的妇女进行基因分型，以确定人类HSD11B1和H6PDH基因的多态在多大程度上是这种疾病发展的危险因素。基因分型数据的子集将作为关联研究、受影响的同胞配对研究和传递不平衡测试进行分析。在多囊卵巢综合征受试者中将寻找这些基因的其他多态。

DESCRIPTION (provided by applicant): Apparent cortisone reductase deficiency (ACRD) is a human disease in which the metabolic clearance of cortisol is increased, leading to over activity of the adrenal cortex, increased secretion of adrenal androgens, and a phenotype similar to polycystic ovary syndrome (PCOS)/metabolic syndrome/syndrome X. Our previous work has shown that ACRD is a digenic disease, requiring carriage of mutations in two genes, HSD11B1 (encoding the type 1 or liver isozyme of 11beta-hydroxysteroid dehydrogenase) and H6PD (encoding hexose-6-phosphate dehydrogenase). We propose to elucidate the mechanisms by which these mutations interact to cause disease. We will define the expression of H6PD in a variety of murine and human tissues by measuring mRNA levels using quantitative PCR, and by measuring protein expression using immunoblotting and assays of enzymatic activity. We will investigate the direct role of H6PD in determining the set point of 11beta-HSD1 activity by expressing H6PD and 11beta-HSD1 in bacteria, and by analyzing the effect of varying H6PDH expression upon 11beta-HSD1 oxo-reductase activity in mammalian cells. We will determine functional consequences of variations in H6PD activity upon phenotype of primary human adipocytes and hepatocytes. We will develop a mouse model of ACRD. To do this, we will produce a mouse with a targeted inactivation of H6PD, and cross this to a mouse with an inactivated HSD11B1 allele. We will breed double heterozygous mutant mice to produce a mouse model of ACRD. Phenotypic characterization will focus on gene and protein expression, 11a-HSD1 activity and equilibrium set-point in liver and adipose tissue, development of the adrenal cortex and levels of expression for key genes regulating steroidogenesis, assays of adrenal function, adipose tissue development, and hepatic enzyme expression. Finally, we will genotype women with polycystic ovary syndrome to determine the degree to which polymorphisms in the human HSD11B1 and H6PDH genes are risk factors for the development of this condition. Subsets of the genotyping data will be analyzed as an association study, as an affected sib pair study, and by transmission disequilibrium testing. Additional polymorphisms in these genes will be sought in PCOS subjects.