ISOLATING THE GENE FOR CHOROIDEREMIA

无脉络膜症（McKusick#30310）是一种X连锁视网膜营养不良， 导致进行性视力丧失和 光感受器和视网膜色素上皮细胞（RPE）。 通过家系连锁分析，将该位点定位于xq21.2， 染色体缺失和易位。 一名女 在Xq和13 p之间有平衡易位， 显然中断了无脉络膜基因， 正常X染色体失活。 一个匿名探针，pH165.1，分离于 该实验室绘制了易位的近端和<50个酶的图谱， 在这位女性身上。 λ或λ中的重叠基因组序列 分离粘粒载体，直到pH 165.1和165.2之间的整个区域。 已经克隆了断裂点和断裂点远端的序列。 将测试该区域的所有DNA是否包含a的外显子 候选基因基于（i）核酸同源性的保守性， 人和其它哺乳动物，（ii）该序列在视网膜和RPE中的表达， (iii)这些假定的基因突变的证据，缺失或其他， 无脉络膜先证者的外显子序列。 当候选基因 识别， (1)其整个mRNA序列和基因组结构将被表征。 (2)导致疾病的突变将由 测序DNA中基因的外显子和内含子-外显子边界， 患者 (3)将产生针对基因产物的抗体， 表达基因的视网膜组织和表达基因的细胞 和基因产物的亚细胞定位。 (4)负责控制基因启动子和增强子序列 表达将被识别。 (5)将分离同源鼠基因以用于评估 小鼠胚胎中表达的发育时间以及 开始研究通过同源重组建立小鼠模型 胚胎干细胞系统中的突变基因序列。 长期目标是确定人类眼病的基因 通过它的位置，而不是它的功能，然后描述 基因及其表达，以及突变负责 人类疾病。最终目标是了解基因产物是什么 以及基因缺陷如何导致疾病。

Choroideremia (McKusick #30310) is an X-linked retinal dystrophy of unknown pathogenesis that causes progressive vision loss and atrophy of the photoreceptors and retinal pigmented epithelium (RPE) in hemizygous males. The disease locus maps to xq21.2 based on linkage analysis in families and on chromosome deletion and translocation in affected patients. A female with choroideremia has a balanced translocation between Xq and 13p that apparently interrupts the choroideremia gene and causes disease through inactivation of the normal X. An anonymous probe, pH165.1, isolated in this laboratory maps proximal to and <50 kilobases from the translocation breakpoint in this female. Overlapping genomic sequences in lambda or cosmid vectors will be isolated until the entire region between pH165.1 and the breakpoint, and sequences distal to the breakpoint have been cloned. All DNA from this region will be tested for whether it contains exons of a candidate gene based on (i) conservation of nucleic acid homology between man and other mammals, (ii) expression of the sequence in retina and RPE, (iii) demonstration of mutation, deletional or otherwise, of these putative exonic sequences in choroideremia probands. When a candidate gene is identified, (1) its entire mRNA sequence and genomic structure will be characterized. (2) the mutations responsible for the disease will be determining by sequencing the exons and intron-exon boundaries of the gene in the DNA of patients (3) antibodies to the gene product will be raised and used to determine the tissue(s) in the retina in which the gene is expressed and the cellular and subcellular localization of the gene product. (4) the promoter and enhancer sequences responsible for control of gene expression will be identified. (5) the homologous murine gene will be isolated to use for assessing the developmental timing of expression in mouse embryos as well as for beginning studies to develop a mouse model through homologous recombination of mutant gene sequences in the embryonal stem cell system. The long-term objective is to identify the gene for a human eye disease through its location rather than its function and then to characterize the gene and its expression as well as the mutations responsible for the disease in man. The ultimate goal is to understand what the gene product is and how defects in the gene produce disease.