台灣神經纖維瘤第一型病人的分子基因學研究

Abstract

神經纖維瘤第一型(NF1; MIM# 162200) 是一常見的自體顯性遺傳疾病，亦是常見的單一基因疾病，發生率約1/3000-4000。且會影響神經功能。目前已知造成第一型神經纖維瘤的基因 (NF1 gene)大約佔genomic DNA 350Kb且位於第17號染色體上，涵蓋60個 coding exons，8.6Kb之mRNA序列，其產物 “神經纖維瘤素” (neurofibromin) 是一個250kDa 之蛋白質，涵蓋2818個胺基酸。Neurofibromin 被認為是一種“腫瘤抑製劑”，許多研究證實neurofibromin與Ras 訊息傳遞有關。 神經纖維瘤第一型的臨床表徵包括咖啡牛奶斑、虹膜斑塊 ，腋下及鼠膝部出現雀斑，神經纖維瘤等，這些症狀大多數的病患皆出現於青春期之後。有一些不常見但其臨床特徵是較嚴重的，例如反覆出現的神經纖維瘤及叢狀神經纖維瘤 、頭大身小、視神經膠質瘤、學習障礙、生長遲緩、脊柱側彎、假關節及可能轉變成惡性腫瘤。 目前已經有五百多個突變巳經被找出，其中包括基因缺失、插入、氨基酸取代、提早轉譯停止、剪切位置突變、大段染色体重組等。本論文研究係利用分子生物相關技術，針對基因單點或小片段的缺失、插入突變等，作一篩選。這些技術包括聚合酶反應(PCR)、液相色層分析 (dHPLC)、直接定序 (Direct Sequence)。至於一些少見的大片段基因的缺失、插入或倒轉所產生的突變，則利用Microsatellite maker 的loss of heterogenicity及同步定量聚合酶反應 (Quantitative Realtime PCR) 的方式來偵測，由此檢測及發現 NF1基因 之 突變，並建立國人NF1基因之突變資料。 本論文係將台大神經科門診及各醫療院所轉介之NF1病患共107位病人，藉由上述的方法，共發現68位NF1病患基因發生突變，其偵測率為64%。其中missense mutation 11位(16.25%)， 20位 (29.4%) nonsense mutation，23位 (33.8%) framshift 及 9位splicing or intron mutation 。其中有5位病患是直接定序發現的，另有5位病人是藉著LOH及同步定量聚合酶反應 (Q-Realtime PCR )的方法，證實這些患者產生了大片段的基因缺失。 根據其臨床表徵資料且具有NF1基因突變的68位病患中，以咖啡牛奶斑(Café-au-lait spots)佔最多(68/68,100%)，其次為表皮或皮下神經纖維瘤(Cutaneous neurofibromas)(54/68,79.4%)，雀斑Freckling (axillary, inguinal)(41/68,60.3%)，叢狀神經纖維瘤(plexiform neurofibromas)(19/68,27.9%)及其他較少見的病徵等等。 本論文發現NF1基因突變與臨床症狀的表現，並無明顯的相關性。這也說明NF1病人臨床症狀的變異性相當高。具基因突變的病患，一定會產生臨床症狀，且家族中罹病成員之臨床症狀不一定一樣。同樣的，具家族史且確診為神經纖維瘤第一型的病患，利用本論文所提供的偵測方法，仍有39位(佔36.4%)病患無法偵測其基因的突變位置。因此，可以嘗試用RNA Level Base，或採用更新的分子診斷方法，以提高其檢測率。

Neurofibromatosis type 1 (NF1; MIM# 162200) is a common autosomal dominant disorder, affecting approximately one is every 4000 individuals. NF1 is also one of the most common single-gene disorders influencing neurological function in humans. Neurofibromatosis type1 (NF1) is caused by the mutation of NF1 gene which encoded a 2818 aminocaid protein, neurofibromin. Neurofibromin, harboring a GTP binding domain, interferes with the signal transduction of RAS-ERK-MAPK pathway. The NF1 gene is located at chromosome 17q11.2 and spans approximately 350 kb genomic DNA. The clinical features of the disease include café-au-lait spots, iris harmatomas (Lisch nodules), and freckling of axillary and inguinal regions. The features are presented in most of the patients after puberty. Some less frequent, nevertheless with more severe manifestations, such as deeply situated and arbitrarily located neurofibromas, plexiform neurofibromas, macrocephaly, optic glioma, short stature, learning difficulties with mild retardation, scoliosis, pseudoarthrosis and even certain malignancies occur among patients with NF1. Up to date, five hundred different mutations have been reported, including deletion, insertion, frameshift mutations, missense mutations, nonsense mutations, splicing mutations. However, not any a report demonstrated the genotype of the NF1 mutation in Chinese patients with NF1. The aim of this study is to use the DHPLC to screen the NF1 gene mutation in Taiwanese patients with NF1. The patients were recruited from the Department of Medical Genetics National Taiwan University Hospital or been transfer from other Hospital. In total, samples from 107 NF1 patients were collected. The DHPLC screening identified 58 heteroduplex with further sequencing confirmed. Five small molecular defects, which were not detected by DHPLC, were identified by direct sequencing. The genotypes for the Taiwanese/Chinese NF1 patients composed of missense mutation 11(16.25%), 20 (29.4%) of nonsense mutation, 23 (33.8%) framshift and 9 splicing or intron mutation. To screen the large deletion which involving the whole gene, two methods, loss-of-heterozygosity and quantitative PCR were employed. Loss of heterozygosity with reduced genomic DNA copy number were identified in 5 patients. Thus, using a serial mutation identification methodology, we found 68 mutations in 107 NF1 patients. The detection rate is 64%. Skin manifestion, café-au-lait spots, occurs in all the NF1 patients (68/68, 100%). The cutaneous neurofibromas were found in 79.4% patients (54/68). Forty-one patients had freckling at axillary and inguinal regions (41/68, 60.3%). Nearly, one-fourths patients had plexiform neurofibroma (19/68, 27.9%). Some less frequent, nevertheless with more severe manifestations. According to this study, no phenotype -genotype correlation is identified. Both the NF1 phenotype and genotype are highly variable. Patients harbor a NF1 gene mutation always show the NF1 features. The mutation detection rate is not high enough. Thus, the more cost-effective and energy-saving methods to improve the genetic diagnosis is important in the future. In addition, studies on the functional implications and pathogenesis for the NF1 mutation would be the cornerstone to shed light on the treatment strategy.