來自上海交通大學(xué)、復(fù)旦大學(xué)和第二軍醫(yī)大學(xué)的研究人員，采用外顯子組測(cè)序方法揭示出了肝母細(xì)胞瘤（hepatoblastoma）的一些新突變和癌基因，并證實(shí)它們與Wnt信號(hào)通路以及泛素連接酶復(fù)合體相關(guān)。這一研究發(fā)現(xiàn)已在國際著名肝臟疾病雜志Hepatology（最新影響因子12.003）在線發(fā)表。
上海交通大學(xué)的何祥火(Xianghuo He)教授、復(fù)旦大學(xué)的董巋然（Kuiran Dong）教授以及第二軍醫(yī)大學(xué)的王紅陽（Hongyang Wang）院士是這篇論文的共同通訊作者。
為了鑒別出肝母細(xì)胞瘤的新突變，研究人員對(duì)6對(duì)肝母細(xì)胞瘤及匹配淋巴細(xì)胞進(jìn)行了全外顯子組測(cè)序。由此鑒別出了21個(gè)基因上的24種體細(xì)胞非同義突變，其中大多數(shù)的為新發(fā)突變，包括Wnt信號(hào)通路中CTNNB1 (G512V)和CAPRIN2 (R968H/S969C)基因的三個(gè)新突變。還發(fā)現(xiàn)了從前證實(shí)與泛素連接酶復(fù)合體相關(guān)的一些基因：SPOP、KLHL22、TRPC4AP和RNF169。
研究人員證實(shí)，在功能上CTNNB1 (G512V)和CAPRIN2 (R968H/S969C)均為功能獲得性突變，在肝母細(xì)胞瘤中CAPRIN2 (R968H/S969C)激活了Wnt信號(hào)通路。這些研究結(jié)果表明在肝母細(xì)胞瘤中存在Wnt信號(hào)通路激活，通過在42個(gè)肝母細(xì)胞瘤腫瘤中對(duì)β-catenin進(jìn)行免疫組化染色進(jìn)一步證實(shí)了這一點(diǎn)。
隨后，他們利用shRNA介導(dǎo)RNA干擾，評(píng)估了21種突變基因?qū)τ诟文讣?xì)胞瘤細(xì)胞生存的影響。研究結(jié)果表明1種新的癌基因CAPRIN2和3種抑癌基因SPOP、OR5I1及CDC20B影響了肝母細(xì)胞瘤的細(xì)胞生長(zhǎng)。并且，研究人員發(fā)現(xiàn)在肝母細(xì)胞瘤細(xì)胞中SPOP S119N是一種功能喪失性突變。他們最終證實(shí)SPOP可通過調(diào)控CDKN2B表達(dá)抑制肝母細(xì)胞瘤的細(xì)胞增殖。這些研究結(jié)果擴(kuò)展了肝母細(xì)胞瘤的遺傳變異圖譜，闡明了Wnt和泛素信號(hào)通路調(diào)控異常在肝母細(xì)胞瘤的腫瘤形成中起著重要的作用。
外顯子組測(cè)序 （Exome sequencing）是近年發(fā)展起來的一種利用序列捕獲技術(shù)將全基因組外顯子區(qū)域DNA捕捉并富集后進(jìn)行高通量測(cè)序的基因組分析方法。是一種選擇基因組的編碼序列的高效策略，相對(duì)于基因組測(cè)序其成本較低。目前，外顯子組測(cè)序已經(jīng)在米勒綜合癥、歌舞伎綜合癥、重型顱腦畸形等孟德爾疾病的研究中得到成功應(yīng)用。還有其它一些癌癥和復(fù)雜性疾病也應(yīng)用外顯子組測(cè)序觀察到高度相關(guān)的突變。
文章中外顯子測(cè)序、SNP芯片以及后續(xù)的大部分?jǐn)?shù)據(jù)分析均由晶能生物完成。
Abstract：
Hepatoblastoma (HB) is the most common primary liver tumor in children. Mutations in the β-catenin gene that leads to constitutive activation of Wnt pathway have been detected in a large proportion of HB tumors. To identify novel mutations in HB, we performed whole-exome sequencing of 6 paired HB tumors and their corresponding lymphocytes. This identified 24 somatic non-synonymous mutations in 21 genes, many of which were novel, including three novel mutations targeting the CTNNB1 (G512V) and CAPRIN2 (R968H/S969C) genes in the Wnt pathway, and genes previously shown to be involved in the ubiquitin ligase complex (SPOP,KLHL22, TRPC4AP and RNF169). Functionally, both the CTNNB1 (G512V) and CAPRIN2 (R968H/S969C) were observed to be gain-of-functional mutations, and theCAPRIN2 (R968H/S969C) was also shown to activate the Wnt pathway in HB cells. These findings suggested the activation of the Wnt pathway in HB, which was confirmed by immunohistochemical staining of the β-catenin in 42 HB tumors. We further used shRNA-mediated interference to assess the effect of 21 mutated genes on HB cell survival. The results suggested that 1 novel oncogene (CAPRIN2) and 3 tumor suppressors (SPOP, OR5I1 and CDC20B) influence HB cell growth. Moreover, we found that SPOP S119N is a loss-of-function mutation in HB cells. We finally demonstrated that one of the mechanisms by which SPOP inhibits HB cell proliferation is through regulating CDKN2B expression. Conclusion: these results extend the landscape of genetic alterations in HB and highlight the dysregulation of Wnt and ubiquitin pathways in HB tumorigenesis. (Hepatology 2014;)