CLONING OF DISEASE GENES FROM THE HUMAN XP22.3 REGION

人们提出了一种“反向遗传学”的方法来克隆疾病基因。 来自人类X染色体的远端短臂。这个地区是 特别有趣，因为它与Y染色体有同源性，它 包含逃脱X失活的基因，它显示出非常高的频率 缺失和易位。五个致病基因已被归类为 研究构建的Xp22.3缺失图谱的特定区间 患有邻近基因综合征的患者。这些疾病是：1)X连锁的 由于类固醇硫酸酯酶缺乏引起的鱼鳞病；2)卡尔曼综合征， 以促性腺激素减退、性腺机能减退和嗅觉障碍为特征 单侧肾发育不全5例；3)X连锁隐性 点状软骨发育不良，是一种以鼻和软骨为特征的骨发育不良 指骨骨发育不全和骨痂点画；4)精神 发育迟缓；5)身材矮小。我们计划进行染色体行走 在Xp22.3区域使用酵母人工染色体(YAC)克隆。YAC 重叠群已在该地区组装完毕，并将用作起点 积分。Xp22.3的有效缺失和长程限制图 将为我们的行走指明方向。重叠群关闭将由两个人实现 行走，并使用源自 Xp21-PTER杂交种和质粒库构建的粘粒文库 保留Xp22.3的辐射杂交种的PCR产物文库 区域。在我们行走的过程中，我们将克隆已经开发出来并将被使用 以达到患者的删除断点。YAC克隆被发现在 将对疾病基因的直接邻近进行筛查，以确定是否存在 使用各种传统和新方法的表达序列， 包括在Lambda噬菌体和质粒克隆中亚克隆YAC， 然后与动物园斑点、Northern斑点和cDNA文库杂交， YAC中CpG岛的亚克隆和外显子捕获。一旦 疾病基因被分离，分子缺陷的特征在 将对患者进行检查，并进行分子诊断测试 发展起来的。Xp22.3致病基因在人类和人类中的表达和功能 其他物种也将被研究。尤其是在表达 Kallmann综合征的基因，可能是神经元迁移发育 缺陷，将在小鼠胚胎发育期间进行研究。此外, 将通过引入有缺陷的基因来建立这种疾病的小鼠模型 在胚胎干细胞中。拟议的研究将导致更好的 了解Xp22.3疾病，以便更有效地诊断和 可能的治疗方法。它们还可以提供对重要因素的见解和 涉及人类发展的机制。

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.