CLONING OF DISEASE GENES FROM THE HUMAN XP22.3 REGION

提出了一种克隆疾病基因的“反向遗传学”方法 从人类X染色体的末端短臂。 该地区是 特别感兴趣的是，因为它与Y染色体同源， 含有逃避X染色体失活的基因， 基因缺失和易位。 五个致病基因已被指定为 Xp22.3缺失图的特定区间，该缺失图是通过研究 连续基因综合征患者。 这些疾病是：1）X连锁 鱼鳞病，由于类固醇硫酸酯酶缺乏; 2）卡尔曼综合征， 以低促性腺激素性功能减退和嗅觉丧失为特征， 单侧肾发育不全30例; 3）X连锁隐性遗传 点状软骨发育不良，一种以鼻和 趾骨发育不全和骨骺点彩; 4）精神 发育迟缓; 5）身材矮小。 我们计划进行染色体步移 在Xp22.3区域中使用酵母人工染色体（YAC）克隆。 YAC 重叠群已在该区域组装，并将用作起始 点 Xp22.3的缺失和长距离限制性内切酶图谱 将为我们的行走指明方向 将通过两种方法实现重叠群闭合 步移和通过使用衍生自 从Xp 21-pter杂交体和质粒构建粘粒文库 来自保留Xp22.3的辐射杂合体的PCR产物文库 地区 在我们行走的过程中，我们将克隆已经开发出来，并将用于 以达到患者的删除断点。 发现YAC克隆在 将对疾病基因的紧邻进行筛查，以确定是否存在 使用各种传统和新方法表达序列， 包括在λ噬菌体和质粒克隆中亚克隆YAC， 然后与Zoo印迹、北方印迹和cDNA文库杂交， 来自YAC的CpG岛的亚克隆和外显子捕获。 一旦 疾病基因是分离的，表征分子缺陷， 将对患者进行分子诊断测试， 开发 Xp22.3疾病基因在人类和哺乳动物中的表达和功能 还将研究其他物种。 特别是， Kallmann综合征的基因，可能是神经元迁移发育 缺陷，将在小鼠胚胎发育过程中进行研究。 此外，本发明还提供了一种方法， 通过引入缺陷基因， 在胚胎干细胞中。 拟议的研究将导致更好的 了解Xp22.3疾病，更有效地诊断和 可能的治疗 它们还可以使人们了解重要的因素， 参与人类发展的机制。

A "reverse genetics" approach is proposed for the cloning of disease genes from the distal short arm of the human X chromosome. This region is of particular interest since it shares homology with the Y chromosome, it contains genes escaping X inactivation, and it shows a very high frequency of deletions and translocations. Five disease gene have been assigned to specific intervals of an Xp22.3 deletion map which was constructed studying patients with contiguous gene syndromes. These diseases are: 1) X-linked ichthyosis, due to steroid sulfatase deficiency; 2) Kallmann syndrome, characterized by hypogonadotropic hypogonadism and anosmia and in some cases by unilateral renal hypoplasia; 3) X-linked recessive chondrodysplasia punctata, a bone dysplasia characterized by nasal and phalangeal hypoplasia and stippling of the epiphyses; 4) mental retardation; and 5) short stature. We plan to perform chromosome walking in the Xp22.3 region using yeast artificial chromosome (YAC) clones. YAC contigs have been assembled in the region and will be used as starting points. The available deletion and long range restriction maps of Xp22.3 will serve to orient our walks. Contig closure will be achieved by both walking and by the isolation of new YACs using sequences derived from a cosmid library constructed from an Xp21-pter hybrid and from a plasmid library of PCR products from a radiation hybrid retaining the Xp22.3 region. During our walk we will clone has been developed and will be used to reach the patients' deletion breakpoints. YAC clones found to be in the immediate proximity of disease genes will be screened for the presence of expressed sequences using a variety of traditional and new methods, including subcloning of the YACs in lambda phage and plasmid clones, followed by hybridization to Zoo blots, northern blots and cDNA libraries, subcloning of CpG islands from the YACs, and exon trapping. Once the disease genes are isolated, characterization of molecular defects in the patients will be performed and molecular diagnostic tests will be developed. The expression and function of Xp22.3 disease genes in man and other species will also be studied. In particular the expression of the gene for Kallmann syndrome, probably a neuronal migration developmental defect, will be studied during development of mouse embryos. In addition, mouse models of the diseases will be created by introducing defective genes in embryonic stem cells. The proposed studies will lead to a better understanding of Xp22.3 diseases and to more efficient diagnosis and possible therapy. They may also give insights into important factors and mechanisms involved in human development.