Biochemical Basis of Cortisone Reductase Deficiency

描述（由申请人提供）：表观可的松还原酶缺乏症（ACRD）是一种人类疾病，其中皮质醇的代谢清除率增加，导致肾上腺皮质过度活跃，肾上腺雄激素分泌增加，其表型类似于多囊卵巢综合征(PCOS)/代谢综合征/ x综合征。我们之前的工作表明，ACRD是一种遗传性疾病，需要携带两个基因突变。HSD11B1（编码11β -羟基类固醇脱氢酶的1型或肝脏同工酶）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PCR测量mRNA水平，以及使用免疫印迹法和酶活性测定法测量蛋白质表达来定义H6PD在各种小鼠和人体组织中的表达。我们将通过在细菌中表达H6PD和11β - hsd1，以及在哺乳动物细胞中分析不同H6PDH表达对11β - hsd1氧还原酶活性的影响，探讨H6PD在确定11β - hsd1活性设定点中的直接作用。我们将确定H6PD活性变化对原代人脂肪细胞和肝细胞表型的功能影响。我们将建立小鼠ACRD模型。为了做到这一点，我们将产生一个具有H6PD失活的小鼠，并将其与具有HSD11B1失活等位基因的小鼠杂交。我们将培育双杂合突变小鼠，建立ACRD小鼠模型。表型表征将集中于基因和蛋白质表达、肝脏和脂肪组织中的11a-HSD1活性和平衡设定点、肾上腺皮质发育和调节类固醇生成的关键基因的表达水平、肾上腺功能测定、脂肪组织发育和肝酶表达。最后，我们将对患有多囊卵巢综合征的女性进行基因分型，以确定人类HSD11B1和H6PDH基因多态性在多大程度上是多囊卵巢综合征发生的危险因素。基因分型数据的子集将作为关联研究、受影响的兄弟姐妹研究和传播不平衡测试进行分析。这些基因的其他多态性将在多囊卵巢综合征患者中寻找。

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.