Adrenal Zonation and Androgen Synthesis

描述(申请人提供)：先天性肾上腺增生症最常见的形式，21-羟基酶缺乏症(21OHD)，也是最常见的遗传性疾病之一。所有210HD患者的一个特征是肾上腺雄激素过多。这些雄激素来源于缺陷酶步骤附近的前体物质的积累，如17-羟孕酮(17OHP4)，在辅因子蛋白细胞色素b5(CyB5A)的刺激下，这些前体通过参与类固醇17-羟基酶/17，20-裂解酶(CYP17A1)的可访问途径转化为睾酮(T)。脱氢表雄酮(DHEA)、脱氢表雄酮硫酸盐(DHEAS)和雄烯二酮(AD)被认为是肾上腺来源的主要雄激素前体。虽然血清17OHP4长期以来一直被用来监测CAH的治疗，但它与DHEA和AD的相关性很差；此外，这些常规测量的雄激素与雄激素过剩的临床证据之间没有很好的相关性。此外，对患有21OHD的成年人进行AD和T的常规测量可能会令人困惑，因为这些类固醇也来自性腺。肾上腺特有的雄激素产生的生物标记物将是210HD治疗的重大进步。我们建议将11b-羟化的19碳类固醇及其11-酮代谢物作为候选生物标记物，因为11b-羟基酶(CYP11B1)只在肾上腺表达，而在性腺中不表达。此外，21OHD患者的其他雄激素和雄激素前体也可能升高。最近的数据显示，210HD患者的代谢物从所谓的“后门途径”上升到双氢睾酮(DHT)。这些中间体通过17OHP4的5�-还原启动的酶促级联反应出现，绕过了脱氢表雄酮、AD或T的中介作用，到达最有效的雄激素dht。此外，我们假设，这些患者体内D4-雄激素的丰富是由于肾上腺雄激素酶和辅因子的表达中断，赋予肾上腺一个性腺样区。这些研究的目的是(1)确定21OHD患者肾上腺皮质内关键雄激素酶和辅因子的地带性分布；(2)详细描述21OHD患者肾上腺雄激素及其中间体的分布，最终目的是确定雄激素过剩的肾上腺特异性生物标志物。在目标1中，我们将从21OHD和ACTH依赖型库欣综合征患者的正常肾上腺组织和肾上腺组织中免疫标记CyB5A和3b-羟基类固醇脱氢酶2(HSD3B2)，这两种酶是雄激素合成所必需的，但在不同的肾上腺区域正常表达。我们假设，210HD和慢性ACTH刺激扰乱了正常的肾上腺酶分布，导致雄激素合成过多。在目标2中，我们将使用来自经典型和非经典型21OHD患者和来自正常对照的血液样本的液相色谱-串联质谱仪分析来表征类固醇的特征。

DESCRIPTION (provided by applicant): The most common form of congenital adrenal hyperplasia, 21-hydroxylase deficiency (21OHD), is also one of the most common genetic diseases. A hallmark characteristic of all patients with 21OHD is adrenal androgen excess. These androgens derive from accumulation of precursors, such as 17-hydroxyprogesterone (17OHP4), proximal to the defective enzymatic step, which are converted to testosterone (T) via accessible pathways involving steroid 17-hydroxylase/17, 20-lyase (CYP17A1) under the stimulation of cofactor protein cytochrome b5 (CYB5A). Dehydroepiandrosterone (DHEA), its sulfate (DHEAS) and androstenedione (AD) are thought to be the major androgen precursors of adrenal origin. Although serum 17OHP4 has long been used to monitor treatment of CAH, it correlates poorly with DHEA and AD; furthermore, no good correlation between these routinely measured androgens and clinical evidence of androgen excess has been demonstrated. In addition, routine measurement of AD and T in adults with 21OHD can be confusing, because these steroids also derive from the gonads. A biomarker of androgen production unique to the adrenal gland would be a major advance in the management of 21OHD. We propose as biomarker candidate's 11b-hydroxylated 19- carbon steroids and their 11-keto metabolites, because 11b-hydroxylase (CYP11B1) is expressed only in the adrenal gland and not in the gonad. In addition, other androgens and precursors might be elevated in patients with 21OHD. Recent data has shown elevation of metabolites from the so-called "backdoor pathway" to dihydrotestosterone (DHT) in patients with 21OHD. These intermediates emerge via an enzymatic cascade initiated by the 5�-reduction of 17OHP4, which bypasses the intermediacy of DHEA, AD, or T to the most potent androgen, DHT. In addition, we hypothesize that the abundance of D4-androgens in these patients is facilitated by disrupted adrenal expression of androgenic enzymes and cofactors, conferring the adrenal a gonadal-like zone. The proposed studies aim to (1) determine the zonal distribution of key androgenic enzymes and cofactors within the adrenal cortex in patients with 21OHD and (2) to characterize in detail the adrenal androgen and intermediates in 21OHD, which derive via traditional and novel pathways, with the ultimate goal of identifying adrenal-specific biomarkers of androgen excess. In Aim 1, we will immunostain normal adrenal tissue and adrenal glands obtained from patients with 21OHD and ACTH-dependent Cushing syndrome for CYB5A and 3b-hydroxysteroid dehydrogenase type 2 (HSD3B2), two enzymes necessary for androgen synthesis, but normally expressed in different adrenal zones. We hypothesize that 21OHD and chronic ACTH stimulation disrupt the normal adrenal enzyme distribution, contributing to excessive androgen synthesis. In Aim 2, we will characterize steroid profiles using liquid chromatography-tandem mass spectrometry analysis of blood samples obtained from patients with classic and non-classic 21OHD and from normal controls.