Identifying Novel Sex Determination Genes that protect from B6-YPOS Sex Reversal

描述(由申请人提供)：性发育障碍(DSD)包括广泛的泌尿生殖系统异常，从轻微的尿道下裂到性反转伴外阴不清。令人印象深刻的是，这些疾病发生在大约0.5%-1%的活产儿中。尽管这些疾病普遍存在，但双潜能性腺转变为睾丸或卵巢背后的分子机制尚不完全清楚。对46，XX或46，XY性反转患者的遗传学研究确定，在FISH分析中存在SRY的46，XY性腺发育不良(GD)患者中，SRY或类固醇生成因子-1(SF1)的突变占所有分离的46，XY性腺发育不良(GD)的30%。46，XY GD的其他罕见和孤立原因包括Chromobox-2(CBX2)突变、9p或10q缺失和SOX9重复。然而，这些基因在所有病例中所占比例不到1%。这意味着在哺乳动物性腺发育过程中存在许多未被发现的重要基因。在这项建议中，我们试图通过一个强大的C57BL/6J-YPOS性别逆转小鼠模型来更好地理解哺乳动物性别决定背后的分子机制。在C57BL/6J(B6)遗传背景上存在家鼠Y染色体YPOS，导致60%的B6-YPOS动物发生性别反转，发育成成年雌性(2个卵巢)或两性(1个卵巢，1个睾丸)。B6-YPOS性别逆转发生在胚胎发育的早期，因为所有E15.5动物都发育了一定程度的卵巢。我们已经开发出一种同源菌株，在该菌株中，11号染色体上一个新的129起源的基因位点对B6-YPOS性反转具有显著的保护作用。在这项建议中，我们将把11号染色体上的同源区域的大小缩小到一个可管理的大小，以便进一步研究。到目前为止，我们已经创建了10个亚基因系，每个亚基因系包含原始同源系的一个子集，在成年XY动物中的初步研究已经确定了一个亚同源系，其中1.3Mb的小区域足以提供保护表型。利用生物信息学以及mRNA和蛋白质表达研究，我们将研究1.3Mb区域的一小部分候选基因。性别决定的前三个候选基因的功能将通过性腺器官培养进行分析。第三个目标将集中于一种互补和独立的方法来研究同源区域的功能作用，而不是特定的基因，以了解同源区域在性腺发育早期保护作用背后的机制。我们从性别决定这一新发现的理解中获得的东西，最终将使我们能够更好地诊断和处理性发育障碍患者。 与公共健康相关：在这项建议中，我们研究了决定双潜能性腺成为睾丸或卵巢的分子和细胞机制；这一过程是由Y染色体上男性特有基因Sry的表达触发的。由于只有25%的人类XY性反转病例被遗传学解释，我们建议使用XY性反转的小鼠模型来识别参与这一过程的新基因。这项提议将阐明有关男性和女性如何从子宫内性腺发育开始分化的基本问题，并将改进性发育障碍先天患者的遗传分类和诊断方法。

DESCRIPTION (provided by applicant): Disorders of sex development (DSD) encompass a wide range of urogenital anomalies, ranging from mild hypospadias to sex reversal with genital ambiguity. Impressively, these disorders occur in approximately 0.5-1% of live births. Despite the prevalence of these disorders, the molecular mechanisms behind the transformation of the bipotential gonad into a testis or an ovary are not completely understood. Genetic studies of 46, XX or 46, XY sex reversal patients determined that mutations in either SRY or Steoidogenic Factor-1 (SF1) account for 30% of all isolated 46, XY Gonadal Dysgenesis (GD) where SRY is present by FISH analysis. Other rare and isolated causes of 46, XY GD include mutations in Chromobox-2 (CBX2), deletions in 9p or 10q, and duplications of SOX9. However, these genes account for less than 1% of all cases. This implies that there exist many undiscovered genes that are important in the developing mammalian gonad. In this proposal, we seek to better understand the molecular mechanisms behind mammalian sex determination using a powerful mouse model of C57BL/6J-YPOS sex reversal. The presence of the Mus domesticus poschiavinus Y chromosome, YPOS on the C57BL/6J (B6) genetic background causes 60% of B6- YPOS animals to be sex reversed, developing as adult females (2 ovaries) or hermaphrodites (1 ovary, 1 testis). B6-YPOS sex reversal occurs early in embryonic development, as all E15.5 animals develop some degree of ovotestis. We have developed a congenic strain in which a novel locus of 129-origin on chromosome 11 confers significant protection from B6-YPOS sex reversal. In this proposal, we will narrow the size of the congenic region on chromosome 11 to a manageable size for further study. To date, we have created 10 subcongenic lines, each containing a subset of the original congenic line, and preliminary studies in adult XY animals have identified a subcongenic line in which a small region of 1.3 Mb is sufficient to confer the protection phenotype. Using bioinformatics and mRNA and protein expression studies, we will investigate a small number of candidate genes in our 1.3 Mb region. The function of the top three candidate genes in sex determination will be assayed using gonadal organ culture. The third aim will focus on a complementary and independent approach to investigate the functional role of the congenic region, rather than a specific gene, to understand the mechanism behind the congenic regions' protective effect in early gonadal development. What we gain from this newfound understanding of sex determination will ultimately allow for better diagnosis and management of patients with disorders of sex development. PUBLIC HEALTH RELEVANCE: In this proposal, we investigate the molecular and cellular mechanisms that define the decision of the bipotential gonad to become a testis or an ovary; a process which is triggered by the expression of a male- specific gene, Sry, on the Y-chromosome. As only 25% of human cases of XY-sex reversal are explained genetically, we propose to identify new genes involved in this process using a mouse model of XY-sex reversal. This proposal will elucidate basic questions about how males and females diverge, beginning at gonadal development in utero, and will improve genetic classification and diagnostic methods of patients born with disorders of sex development.