CONSEQUENCES OF HUMAN PIT-1 AND GH PROMOTER MUTATIONS

脑下垂体生长激素的产生对正常的出生后是必不可少的 成长。申请人实验室最近的研究表明，突变与 Pit-1基因与一种人类生长障碍有关的缺陷 垂体前叶激素生长激素、催乳素和促甲状腺激素。 在第158位氨基酸中用Pro取代丙氨酸的人类不同 来自Snell矮小鼠，在第261位氨基酸发生突变，因为它们有 垂体前叶大小正常，仅轻度促甲状腺激素缺乏症。这个 观察到Pit-1的POU特异性结构域的变化 消除其已知目标基因生长的转录激活 激素和催乳素，与这些基因的结合损伤最小 而且没有垂体前叶生长的损害不是由 实验室生产的突变型Pit-1蛋白的研究这表明 Pit-1基因及其靶基因启动子的序列变异 可能解释了更广泛的人类生长障碍。这个 拟议的研究通过检测Pit-1基因来解决这个问题 大量GH、PRL和TSH结合家系的序列分析 常染色体隐性遗传性孤立生长激素缺乏，或 常染色体显性遗传性孤立性生长激素缺乏。另一个选择 生长激素调节元件异常的可能性 基因启动子是通过对生长激素进行测序来实现的 常染色体隐性遗传性分离生长激素家系中的启动子 缺乏症。通过聚合酶链检测序列变异 基因组DNA或cDNA的反应扩增和直接定位 DNA测序。Pit-1基因与生长发育的功能关系 通过迁移率变化评估激素启动子序列异常 DNA结合分析、激活剂和报告基因的共转染 HeLa细胞的构建及野生型和突变型的活性测定 腺病毒DNA复制试验中的Pit-1。DNA序列的研究 严重生长障碍儿童的变异伴随有 同一序列在不同受试者之间的多态变异研究 正常生长的极端情况。该项目将定义 Pit-1及其靶基因序列变异在骨肉瘤发病中的作用 垂体前叶激素缺乏症与代际差异 在正常个体中的生长模式。

Pituitary growth hormone production is essential for normal postnatal growth. Recent studies in the applicant's laboratory have linked mutations of the Pit-1 gene to a human growth disorder involving deficiencies of the anterior pituitary hormones growth hormone, prolactin and thyrotropin. Humans with a substitution of proline for alanine at amino acid 158 differ from Snell dwarf mice with a mutation at amino acid 261 in that they have normal anterior pituitary size and only mild thyrotropin deficiency. The observation that an alteration in the POU-specific domain of Pit-1 eliminates transcriptional activation of its known target genes growth hormone and prolactin, with minimal impairment of binding to these genes and no impairment of anterior pituitary growth was not predicted by studies of mutant Pit-1 proteins produced in the laboratory. It suggests that sequence variation in the Pit-1 gene and its target gene promoters may account for a broader spectrum of human growth disorders. The proposed studies address this issue through examination of Pit-1 gene sequence in a large number of families with combined GH, Prl and TSH deficiencies, autosomal recessive isolated growth hormone deficiency, or autosomal dominant isolated growth hormone deficiency. The alternative possibility of an abnormality of regulatory elements in the growth hormone gene promoter is addressed through sequencing of the growth hormone promoter in families with autosomal recessive isolated growth hormone deficiency. Sequence variation is detected through polymerase chain reaction amplification of genomic DNA or cDNA and localized through direct DNA sequencing. The functional consequences of Pit-1 gene and growth hormone promoter sequence abnormalities are assessed by mobility shift DNA-binding assays, co-transfection of activator and reporter gene constructs in HeLa cells, and assays for activity of wild type and mutant Pit-1 in Adenovirus DNA replication assays. Studies of DNA sequence variation in children with severe disorders of growth are accompanied by studies of polymorphic variation in the same sequences among subjects at the extremes for normal growth. This project will define the importance of variations in Pit-1 and its target gene sequences in the pathogenesis of anterior pituitary hormone deficiency and in the generation of differences in patterns of growth among normal individuals.