Molecular studies of Noonan syndrome and related disorders

努南综合征和狮子综合征（NS和LS）是常染色体显性遗传特征，其特征包括 先天性心脏病（CHD）、身材矮小、畸形和智力迟钝; LS还包括 雀斑。我们已经证明PTPN 11错义突变导致近50%的NS，并产生获得性功能丧失。 蛋白酪氨酸磷酸酶SHP-2 PTPN 11突变导致LS。 最近，我们发现KRAS突变导致1%的NS。SHP-2和KRAS在RAS促分裂原中的作用 活化蛋白激酶（MAPK）信号转导。具体目标1将检验未知NS 基因编码RAS-MAPK信号传导中的蛋白质。将以高通量对候选基因进行重新测序 以无PTPN 11或KRAS突变的NS受试者的大队列的方式。生化和细胞培养 方法将用于测试突变对新NS基因的影响。在具体目标2中，我们 假设SHP-2突变体通过对发育的功能获得效应引起豹综合征 尽管它们的磷酸酶活性降低，并且NS相关的KRAS突变改变了更多的信号传导， 比NS PTPN 11缺陷更严重。为了验证这些想法，我们将诱导产生转基因果蝇 表达同源NS和LS突变蛋白。它们的表型和遗传相互作用将是 表征了此外，我们假设与Egfr相关的翅膀表型基因相互作用 从现有的NS果蝇模型将确定新的方面的信号转导以及新的NS疾病 基因.将进行致敏筛选以鉴定抑制或增强该翅膀表型的基因。 特异性AIM 3的假说是Jak-Stat信号通路，它与果蝇中的NS等位基因相互作用， 在NS的心脏发生过程中受到干扰。我们将描述Jak-Stat信号的作用 在野生型和Ptpn 11 D 61 G小鼠的正常和受干扰心脏发生期间，分别使用 免疫学和遗传学方法。总体而言，本申请中提出的研究将 描述了突变时引起NS和LS的基因的范围，并提供了对 在生物化学、细胞和有机体水平上的突变蛋白产物。获得的见解将是 在未来利用，以阐明非综合征性心脏缺陷的遗传原因，以及开发 改善这些表型的新的治疗策略。

Noonan and LEOPARD syndromes (NS and LS) are autosomal dominant traits with features that include congenital heart disease (CHD), short stature, dysmorphism, and mental retardation; LS also includes lentigines. We have shown that PTPN11 missense mutations cause nearly 50% of NS and engender gain-offunction on its protein, the protein tyrosine phosphatase SHP-2. Loss-of-function PTPN11 mutations cause LS. Recently, we found that KRAS mutations cause 1% of NS. SHP-2 and KRAS play roles in RAS-mitogen activated protein kinase (MAPK) signaling. SPECIFIC AIM 1 will test the hypothesis that the unknown NS genes encode proteins in RAS-MAPK signaling. Candidate genes will be resequenced in a high throughput fashion with a large cohort of NS subjects without PTPN11 or KRAS mutation. Biochemical and cell culture approaches will be used to test the effects of mutations on novel NS genes. In SPECIFIC AIM 2, we hypothesize that SHP-2 mutants cause LEOPARD syndrome through gain-of-function effects on development despite their reduced phosphatase activity and that NS-associated KRAS mutations alter signaling more profoundly than do NS PTPN11 defects. To test these ideas, we will generate transgenic fruit flies inducibly expressing homologous NS and LS mutant proteins. Their phenotypes and genetic interactions will be characterized. Further, we hypothesize that genes interacting genetically with the Egfr-related wing phenotype from the existing NS fruit fly model will identify novel aspects of signal transduction as well as new NS disease genes. A sensitized screen will be performed to identify genes that suppress or enhance that wing phenotype. SPECIFIC AIM 3’s hypothesis is that the Jak-Stat signaling pathway, which interacts with NS alleles in fruit fly development, is perturbed during cardiogenesis in NS. We will characterize the roles of Jak-Stat signaling during normal and perturbed cardiogenesis in wild type and Ptpn11D61G mice, respectively, using immunological and genetic approaches. Taken as a whole, the studies proposed in this application will delineate the range of genes that cause NS and LS when mutated as well as provide insights into the effects of their mutant protein products at the biochemical, cellular, and organismal levels. The insights gained will be leveraged in the future to elucidate genetic causes of non-syndromic cardiac defects as well as to develop novel therapeutic strategies to ameliorate these phenotypes.