摘要
努南氏症 (Noonan Syndrome, NS) 是一種先天性體染色體突變的顯性遺傳疾病，可能是經由遺傳或是偶發突變造成，其發生率約為 1/1000 ~ 1/2500。努南氏症病患的基因突變，導致轉錄、轉譯出突變的RAS-ERK 訊息傳遞路徑中的蛋白，可能出現的臨床表徵有：發育遲緩、身材矮小、顏面特徵異常、蹼狀頸、先天性心臟方面的疾病以及認知與語言發展遲緩等等。過去的研究顯示，約有 70 ~ 80% 的努南氏症病患是由於帶有 PTPN11、SOS1、 RAF1、 KRAS、 BRAF、 SHOC2 、NRAS、RIT1 基因突變，使轉譯出來的蛋白質產物功能異常，導致 RAS-ERK 訊息傳遞路徑過度活化而致病。然而，仍約有 20% 的病患是由於其他未知的基因突變而致病。於是我們利用外顯子定序法找出24位病患基因的外顯子上帶有的基因突變，排除上述的已知致病基因和 SNP database 中的已知 SNPs後，平均每位病患帶有121個新的基因突變。我們主要將焦點放在 RAS-ERK 訊息傳路徑中的基因，並且在 NF1、RASA和 RIT1 基因上找到了新的突變點。正常的 NF1 和 RASA2 基因的蛋白質產物會刺激 RAS 蛋白內生性的 GTPase 活性，使 GTP 水解成 GDP 進而讓 RAS 蛋白失去活性，我們認為突變的 NF1 和 RASA2 蛋白質沒辦法有效的抑制RAS 蛋白的活性。RIT1 RAS 蛋白具有相似的蛋白質結構，突變的 RIT1 會使 RAF1 持續活化，導致 RAS-ERK 訊息傳遞路徑持續活化。為了確定突變的 NF1、RASA2、RIT1 蛋白質對 RAS-ERK 訊息傳遞路徑的影響，我們分別將基因轉染至 HEK-293T 細胞株中使其表現正常及突變的蛋白，並證實突變的基因蛋白質產物會導致 RAS-ERK 訊息傳遞路徑過度活化。前人研究顯示，帶有不同突變蛋白的 NS 病患可能會有些不同的症狀。因此，找出新的致病基因不僅有助於臨床上的診斷，也利於追蹤病患的疾病進程和可能出現的症狀。

Abstract
Noonan Syndrome (NS), one of the most common developmental diseases, is an autosomal genetic disorder that may occur sporadically or be inherited. NS is caused by germline mutations in genes encoding RAS-ERK signaling pathway. NS, affected about 1 in 1000~2500 newborns, is characterized by developmental delay, short stature, typical facies, webbed neck, distinctive craniofacial dysmorphism, congenital heart diseases, variable cognitive deficits and neurocognitive delay. NS-causing mutations, which may cause hyperactive RAS-ERK signaling pathway, were identified in PTPN11, SOS1, RAF1, KRAS, CBL, SHOC2, NRAS and RIT1 genes in ~80% NS patients. However, the genetic causes in the remaining ~20% of NS patients are still unknown. Hence, whole exome sequencing was performed with genomic DNA from 24 NS patients who carry no mutations in the known NS genes. On average, 121 novel missense mutations were found per patient. Genes involved in RAS-ERK signaling pathway were surveyed and novel mutations in NF1, RASA, and RIT1genes were identified. NF1 and RASA2 genes encode RAS GTPase activating proteins (RASGAP) that negatively regulate RAS activity. RIT1 encodes small GTPase protein, which is a RAS subfamily protein. To validate the effects of mutant proteins, wild type (WT) and mutant NF1, RASA2, or RIT1 genes were expressed in HEK-293 cell lines and in Flp-In TREx 293 cell lines. Loss-of-function mutations of NF1 and RASA2 failed to suppress the RAS activity and gain-of-function mutation in RIT1 lead to ERK hyper-activation. These data suggest that NF1, RASA2, and RIT1 are candidates of NS-causing genes.

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