GENE INVOLVED IN SEX REVERSAL MAPS TO CHROMOSOME 9P

涉及性别逆转的基因映射至染色体 9P

• 批准号: 2810732
• 负责人: WENDY L FLEIJTER
• 金额: $ 9.66万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2810732
• 关键词: artificial chromosomes; chemical fingerprinting; chromosome disorders; clinical research; congenital reproductive system disorder; cytogenetics; developmental genetics; fluorescent in situ hybridization; gene deletion mutation; gene expression; genetic library; genetic mapping; human genetic material tag; human subject; linkage mapping; loss of heterozygosity; molecular cloning; nucleic acid sequence; polymerase chain reaction; sex determination; single strand conformation polymorphism; western blottings

DESCRIPTION (Adapted from investigator's abstract): Sex reversal of the 46, XY type can have several causes. Besides mutations in the testis determining gene SRY, autosomal genes have also been implicated. One of the chromosomal regions involved in such disorders is located at the distal end of the short arm of chromosome 9 (9p). Patients with deletions of this region of chromosome 9 present with a number of congenital malformations, including various degrees of sex reversal ranging from ambiguous genitalia to complete sex reversal. The present proposal is to isolate one or more genes responsible for sex reversal from the distal 9p region by positional cloning. Previous work by the PI has identified a small deletion of chromosome 9p which will be combed for expressed sequence. These sequences will then be searched for mutations in patients with sex reversal but no visible abnormalities of chromosome 9. In the first aim, the PI proposes to extend her mapping resources within the 9p critical region that was previously delineated. Previously identified cosmid and YAC probes will be used to screen genomic libraries, initially focusing on cosmid and P1 page libraries and chromosome 9- specific cosmid libraries. If necessary YAC and BAC libraries will also be screened. The probes using interphase FISH followed by determination of overlapping regions using dot blot hybridization, vector/insert function sequencing and fingerprinting. In the second aim, candidate sex determining gene will be searched for within the contig corresponding to the critical region of 9p. Strategies proposed include screening for sequences homologous to two genes, implicated in other forms of sex reversal. SOX9 and SRY, using PCR amplification with HMG hox specific primers. In addition, cDNA libraries will be screened for clones in the region using pools of genomic sequences from the contig as probes. Elimination of false positives and redundant clones will be followed by sequencing and database searches. A third approach is to use magnetic bead capture of relevant cDNAs with multiple cycles of enrichment. In the third aim, a search for mutations in specific genes from the critical region will be conducted in 46, XY females with no apparent deletion of chromosome 9. Patients will be recruited through collaborative efforts and by advertisement in genetic journals. At present, 10 patients have been collected. A combination of approaches, including FISH and LOH analyses will be done to search for deletions. Microsatellite markers will be generated from the new cDNAs isolated to carry out the LOH study. Sequence analysis or SSCP assays on specific genes will be done to look for mutations in the cDNAs isolated in aim 2.

描述（改编自研究者的摘要）：性逆转 46、XY型可能有多种原因。除了睾丸突变之外 决定基因SRY，常染色体基因也受到牵连。之一 与此类疾病有关的染色体区域位于 9 号染色体短臂的远端 (9p)。存在缺失的患者 9 号染色体的这个区域存在许多先天性 畸形，包括不同程度的性逆转 模糊的生殖器来完成性别逆转。目前的建议是 从远端 9p 分离出一个或多个负责性逆转的基因 通过位置克隆确定区域。 PI 之前的工作发现了 9p 号染色体的一个小缺失 将对其进行梳理以获得表达序列。这些序列将被 在性逆转患者中寻找突变，但没有发现明显的突变 9号染色体异常。 在第一个目标中，PI 建议将其制图资源扩展至 先前划定的 9p 关键区域。之前 鉴定出的粘粒和 YAC 探针将用于筛选基因组文库， 最初关注粘粒和 P1 页文库以及 9-号染色体 特定的粘粒库。如果需要，YAC 和 BAC 库也将被 筛选。使用间期 FISH 进行探针检测，然后测定 使用点印迹杂交、载体/插入功能重叠区域 测序和指纹识别。 第二个目标是寻找候选性别决定基因 在对应于 9p 关键区域的重叠群内。策略 建议包括筛选与两个基因同源的序列， 与其他形式的性逆转有关。 SOX9 和 SRY，使用 PCR 使用 HMG hox 特异性引物进行扩增。此外，cDNA 文库 将使用基因组序列池筛选该区域中的克隆 来自重叠群作为探针。消除误报和冗余 克隆之后将进行测序和数据库搜索。第三个 方法是使用磁珠捕获相关 cDNA 富集循环。 第三个目标是寻找特定基因的突变 关键区域将在 46 个 XY 女性中进行，没有明显的 删除 9 号染色体。患者将通过合作招募 努力和遗传期刊上的广告。目前有10名患者 已被收集。多种方法的组合，包括 FISH 和 LOH 将进行分析以搜索删除内容。微卫星标记将 从分离的新 cDNA 中生成以进行 LOH 研究。 对特定基因进行序列分析或 SSCP 分析以了解 针对目标 2 中分离的 cDNA 中的突变。