ISOLATING THE GENE FOR CHOROIDEREMIA

脉络膜血症是一种X-连锁视网膜营养不良，导致进行性 受影响男性的视力丧失和失明。生物化学和分子生物学 这种疾病的基础尚不清楚。脉络膜血症局限于 与5个限制性片段长度紧密连锁的Xq13-21区域 多态现象。两个不同寻常的视网膜疾病家庭 遗传性耳聋、智力低下和肥胖 对X连锁方式也进行了研究。一个(M)家庭的先证者有一个 Xq21.3间隙缺失，缺失两个紧密连锁的RFLP基因座； 另一个(B)家族的先证者的缺失似乎要小得多 这一点更难通过细胞遗传学或Southern分析来证明。 我们建议分离脉络膜血症的基因，以研究其病因和 发病机制和改善遗传管理。 计划：用二次退火法构建富含B基因缺失DNA的文库 EcoR1的单链正常DNA末端有粘性 使用苯酚的B家族先证者的过量钝端变性DNA 增强的再关联技术(PERT)。 1)用PERT文库序列探测正常、M和B患者的DNA 识别在M或B先证者中缺失的序列。 2)通过筛查符合以下条件的患者来检测B缺失范围内的DNA Southern印迹改变的简单脉络膜血症。查找更多B 48XXXX DNA粘粒文库中的缺失序列。 3)以B缺失的基因组序列作为分离的起点 跨越缺失的更多重叠DNA。测试这个DNA，通过探测 脉络膜血症患者检测视网膜和视网膜中的mRNA 色素上皮细胞用于表达基因。 4)如果1)不成功，则评估交叉点之间的距离 用Southern印迹法检测M个先证者的B基因缺失和标记缺失 脉冲场凝胶分辨100-1000kb限制性片段的分析 电泳法。如果可以在一定距离处定义交汇点，则开始 在标记上分离重叠的粘粒序列，移到B 删除并按照上面的步骤3)继续。

Choroideremia is an X-linked retinal dystrophy that causes progressive visual loss and blindness in affected males. The biochemical and molecular bases for this disorder are unknown. Choroideremia was localized to the region Xq13-21 by tight linkage to five restriction fragment length polymorphisms. Two unusual families in which the retinal disease is being inherited concordantly with deafness, mental retardation and obesity in an X-linked manner have also been studied. Probands in one (M) family have an Xq21.3 interstitial deletion and are missing two tightly linked RFLP loci; probands in the other (B) family appear to have a much smaller deletion that is more difficult to document by cytogenetic or Southern analysis. We propose to isolate the gene for choroideremia to study its etiology and pathogenesis and improve genetic management. PLAN: A library enriched for B deletion DNA was made by reannealling single-stranded normal DNA with EcoR1 "sticky" ends in the presence of excess blunt end denatured DNA from a B family proband using the phenol enhanced reassociation technique (PERT). 1) Probe normal, M and B patients' DNA with PERT library sequences to identify sequences deleted in the M or B probands. 2) Test DNA from within the B deletion by screening patients with uncomplicated choroideremia for Southern blot alterations. Find more B deletion sequences in a cosmid library made from 48XXXX DNA. 3) Use genomic sequences in the B deletion as starting points for isolating more overlapping DNA spanning the deletion. Test this DNA by probing the panel of choroideremia patients and examining mRNA from retina and retinal pigmented epithelium for expressed genes. 4) If 1) is unsuccessful, assess the distance between the junction point of the B deletion and the markers missing in the M probands by Southern blot analysis of 100-1000 kb restriction fragments resolved by pulsed field gel electrophoresis. If the junction point can be defined at a distance, begin at the markers to isolate overlapping cosmid sequences, move into the B deletion and proceed as in step 3) above.