張林妹　周淵峰　柴毅明　王　佶　吳冰冰　王　藝　周水珍

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·論著·

結(jié)節(jié)性硬化105例臨床特征和基因型分析

張林妹周淵峰柴毅明王佶吳冰冰王藝周水珍

結(jié)節(jié)性硬化；診斷標(biāo)準(zhǔn)；臨床特征；TSC基因

AbstractObjectiveTo analyse the clinical phenotype and genotype of tuberous sclerosis complex (TSC) and improve the diagnostic level of TSC.MethodsPatients were retrospectively collected from Aug. 2013 to Sept. 2015 hospitalized in Children′s Hospital of Fudan University, who were diagnosed with 2012 revised diagnostic criteria for TSC. Data of clinical phenotype included brain, skin, heart, kidney and eyes were intercepted. Patients were divided into the age of <1 year, -3 years, -6 years, -13 years and -18 years groups, statistics was made to get detection rate and the correlation was analysed between genetic mutation and clinical phenotype.ResultsA total of 105 cases diagnosed as TSC were recruited into the study including 54 males and 51 females. Age of visit was ranged from 2 months to 13 years. ① Reason for the first visit: 83 cases (79.0%) for seizure, 8 cases (7.6%) for abnormal of skin and 5 cases (4.8%) for cardiac tumor. ②39 cases (37.1%) were diagnosed only with clinical phenotype; 47(44.8%) with clinical phenotype and genetic testing; 2 cases (1.9%) with clinical suspicious but gene positive. ③Clinical characteristics detection rate: Subependymal nodules were in 91/99 cases(91.9%), cortical dysplasias were in 81/99 cases(81.8%), which was similar with other different age groups. Hypomelanotic macules were in 97/105 cases(92.4%), angiofibromas were in 55 cases(52.4%), shagreen patch in 46 cases(43.8%), the detection rate of angiofibromas and shagreen patch rised with age. Cardiac rhabdomyomas were in 25/75 cases(33.3%)，the detection rate descended with age; renal lesions were in 14/71 cases(19.7%); ocular lesions were in 5/37 cases(13.5%). ④TSC1 mutation was detected in 15/66 cases (22.7%)，TSC2 mutation was detected in 34/66 cases (51.5%)；spasm was more common in patients withTSC2 mutation (29.4%vs13.3%).ConclusionTSC is an extremely variable disease that can affect multiple important organs. The detection rates of brain lesion were similar among different age and cardiac rhabdomyomas decreased with age. Gene test conduced to the diagnosis of clinical suspect cases.

結(jié)節(jié)性硬化 (TSC) 是一種罕見的常染色體顯性遺傳疾病[1]。TSC皮膚病變影響美觀，且可累及腦、心、腎、肺、肝和眼等重要器官。Gomez于1979年首先提出了TSC診斷標(biāo)準(zhǔn)，1998年國(guó)際TSC臨床共識(shí)會(huì)議對(duì)該標(biāo)準(zhǔn)進(jìn)行了修訂[2,3]，近年來(lái)隨著影像學(xué)和基因檢測(cè)技術(shù)的進(jìn)展， 2012年修訂了新的TSC診斷標(biāo)準(zhǔn)[1]，其中臨床特征仍是TSC診斷的主要方法，補(bǔ)充基因檢測(cè)結(jié)果作為獨(dú)立的診斷標(biāo)準(zhǔn)，診斷的級(jí)別從3個(gè)(可疑、可能、確診)降至2個(gè)(可能、確診)。目前國(guó)內(nèi)尚缺乏應(yīng)用此標(biāo)準(zhǔn)診斷TSC的大宗病例報(bào)道，也缺乏TSC基因型與臨床特征的相關(guān)性分析。本文回顧性分析復(fù)旦大學(xué)附屬兒科醫(yī)院(我院)以2012年修訂標(biāo)準(zhǔn)診斷的TSC病例的臨床特征和基因型，以期提高臨床醫(yī)生對(duì)本病的認(rèn)識(shí)。

1　方法

1.2病例納入標(biāo)準(zhǔn)2013年8月至2015年9月在我院神經(jīng)科TSC專病門診就診的TSC病例，需要說(shuō)明的是我院2013年8月后執(zhí)行2012年修訂的TSC診斷標(biāo)準(zhǔn)。

1.3TSC基因檢測(cè)方法采集外周抗凝血2 mL，使用QIAamp DNA Mini Kit抽提基因組DNA，NanoDrop紫外分光光度儀檢測(cè)DNA濃度和A260/A280比值。參考國(guó)內(nèi)外文獻(xiàn)并使用Primer 3在線軟件設(shè)計(jì)TSC1和TSC2基因PCR引物，TaKaRa LA Taq酶進(jìn)行擴(kuò)增，2%瓊脂糖電泳以判斷產(chǎn)物的擴(kuò)增特異性。參照Ion文庫(kù)構(gòu)建相關(guān)試劑盒使用說(shuō)明書制備測(cè)序文庫(kù)，步驟簡(jiǎn)述如下：將單個(gè)樣本的PCR產(chǎn)物標(biāo)準(zhǔn)化后全部混合，經(jīng)酶切打斷，接頭連接，片段選擇，產(chǎn)物定量，乳液PCR后制備模板，磁珠富集純化。應(yīng)用Ion Personal Genome Machine (PGMTM)測(cè)序系統(tǒng)和Ion 316TMChip v2測(cè)序。

采用Ion torrent PGMTM服務(wù)器自帶的分析軟件，進(jìn)行測(cè)序結(jié)果的初步分析，得到整體數(shù)據(jù)量、平均測(cè)序深度、參考序列匹配程度等數(shù)據(jù)。其后生成FASTA文件，采用NextGENe軟件進(jìn)行后續(xù)的序列數(shù)據(jù)結(jié)果分析。參考千人基因數(shù)據(jù)庫(kù)(http://www.1000genomes.org)，dbSNP數(shù)據(jù)庫(kù)(http://www.ncbi.nlm.nih.gov/projects/SNP)和HGMD(http://www.biobase-international.com/product/hgmd)數(shù)據(jù)庫(kù)對(duì)檢測(cè)到變異的臨床意義進(jìn)行解釋，Mutation taster、SIFT和Polyphen軟件對(duì)檢測(cè)到的變異的功能進(jìn)行預(yù)測(cè)。陽(yáng)性突變采用Sanger測(cè)序法進(jìn)行驗(yàn)證。

1.4資料截取和匯總從病史中截取以下資料，①一般情況:確診年齡、就診年齡、就診原因；②腦部病變：頭顱MR或CT描述或胎兒期B超描述，分為管膜下結(jié)節(jié)、腦皮質(zhì)結(jié)構(gòu)異常和室管膜下巨細(xì)胞星形細(xì)胞瘤；③皮膚病變：包括色素脫失斑、面部纖維腺瘤和鯊革斑，以患兒父母提供的病史或臨床醫(yī)生體檢發(fā)現(xiàn)皮膚病變的時(shí)間為準(zhǔn)；④心臟病變：心臟超聲診斷的心臟橫紋肌瘤；⑤腎臟病變：腹部B超診斷的血管肌脂瘤和多發(fā)性囊腫等；⑥眼部病變：眼底檢查發(fā)現(xiàn)的多發(fā)視網(wǎng)膜結(jié)節(jié)狀錯(cuò)構(gòu)瘤、視網(wǎng)膜脫色斑；⑦肺淋巴管肌瘤?。虎郥SC基因檢查結(jié)果。

分為<1歲、～3歲、～6歲、～13歲和～18歲，統(tǒng)計(jì)TSC臨床特征檢出率，其中皮膚病變以不同年齡段實(shí)際檢出計(jì)，腦部病變、心臟橫紋肌瘤和腎臟病變以不同年齡段新增檢出計(jì)。

2　結(jié)果

2.1一般情況105例TSC病例進(jìn)入分析，男54例，女51例，男∶女為1.06∶1。就診時(shí)中位年齡3歲(2月齡至17歲)，<1歲17例(16.2%)，～3歲44例(41.9%)、～6歲25例(23.8%)、～13歲17例(16.2%)、～18歲2例(1.9%)。

2.3診斷結(jié)果39例(37.1%)依據(jù)臨床特征確診，未行TSC基因檢測(cè)；66例行TSC基因檢測(cè)病例中，符合臨床特征確診且TSC基因檢測(cè)陽(yáng)性47例(44.8%)，符合臨床特征確診但TSC基因檢測(cè)陰性17例(16.2%)，可能符合臨床特征但TSC基因檢測(cè)陽(yáng)性2例(1.9%)。

2.4臨床特征6例(5.7%)有家族史，4例父親或母親患病，2例同胞姐或弟患病。

表1顯示TSC病例各臨床特征在不同年齡段首次檢出率。室管膜下結(jié)節(jié)檢出91.9%，1例為胎兒期檢查時(shí)發(fā)現(xiàn)；腦皮質(zhì)結(jié)構(gòu)異常占81.8%，1例為胎兒期發(fā)現(xiàn)；室管膜下結(jié)節(jié)和腦皮質(zhì)結(jié)構(gòu)異常的檢出率在4個(gè)年齡段相近；4例診斷為室管膜下巨細(xì)胞星形細(xì)胞瘤,其中2例行腫瘤切除術(shù)。

皮膚病變：色素脫失斑占92.4%，面部纖維瘤占52.4%，鯊革斑占43.8%；色素脫失斑檢出率在4個(gè)年齡段相近(均>90%)，面部纖維瘤和鯊革斑檢出率隨年齡的增長(zhǎng)呈增高趨勢(shì)。

心臟橫紋肌瘤占33.3%，其中2例于胎兒期超聲心動(dòng)圖發(fā)現(xiàn)，檢出率隨年齡增長(zhǎng)呈下降趨勢(shì)；腎臟病變占19.7%，包括腎臟血管肌脂瘤9例，多發(fā)腎囊腫6例，檢出率隨年齡的增長(zhǎng)呈增高趨勢(shì)。37例眼底檢查多發(fā)視網(wǎng)膜結(jié)節(jié)狀錯(cuò)構(gòu)瘤5例(13.5%)。1例女性患兒在隨訪至14歲時(shí)發(fā)現(xiàn)肺淋巴管肌瘤病。

表1　TSC病例各臨床特征在不同年齡段檢出率[n/n(%)]

注皮膚病變以不同年齡段實(shí)際檢出計(jì)，腦部病變、心臟橫紋肌瘤和腎臟病變以不同年齡段新增檢出計(jì)；1)含胎兒期發(fā)現(xiàn)1例；2)含胎兒期發(fā)現(xiàn)2例

2.5TSC基因檢測(cè)結(jié)果及其與臨床特征相關(guān)性66例行TSC基因檢測(cè)，49例(74.2%)檢出TSC基因突變。TSC1基因突變15例(30.6%)，其中無(wú)義突變6例，移碼突變4例，同義和錯(cuò)義突變各2例，剪切突變1例；TSC2基因突變34例(69.4%)，其中移碼突變12例，錯(cuò)義突變10例，剪切突變5例，無(wú)義突變4例，同義突變3例。共檢測(cè)到9個(gè)未見報(bào)道的TSC1/TSC2基因新發(fā)突變，包括TSC1：C.482_485dupTGTC(移碼突變)；C.1726T>TC，p.L576L(同義突變)；C.1756het-delT,p.C586V*43(移碼突變)；TSC2： C.136A>AT,p.R46RX(錯(cuò)義突變)；C.267dupG(移碼突變)；C.822C>CA,p.Y274YX(無(wú)義突變)；C.899G>GT,p.G300GV(錯(cuò)義突變)； C.932-936delCTCTC,p.S311X(移碼突變)；C.3607het-delA,p.T1203QfsX7(移碼突變),均通過(guò)父母樣本驗(yàn)證(另文發(fā)表)。

表2顯示，TSC1/TSC2基因突變型與臨床特征相關(guān)性分析顯示，TSC1和TSC2基因突變的各臨床特征差異無(wú)統(tǒng)計(jì)學(xué)意義，但TSC2基因突變者痙攣發(fā)作發(fā)生率較TSC1基因突變有增高趨勢(shì)。

3　討論

Bourneville于1880年首次描述了TSC的神經(jīng)系統(tǒng)特征[4]，但該病直到20世紀(jì)80年代才被真正認(rèn)識(shí)[1]。2/3的TSC病例為散發(fā)性，1/3病例具有家族史[5]。TSC目前已確定腫瘤抑制基因?yàn)槠渲虏』?，分?種類型：定位于9q34的TSC1基因，其產(chǎn)物為錯(cuò)構(gòu)瘤蛋白[6]；定位于16p13.3 的TSC2基因，其產(chǎn)物為馬鈴薯蛋白[7]。TSC基因發(fā)生突變激活哺乳動(dòng)物雷帕霉素靶蛋白(mTOR)，mTOR信號(hào)轉(zhuǎn)導(dǎo)通路異常激活后導(dǎo)致TSC患者各臟器增生異常而產(chǎn)生錯(cuò)構(gòu)瘤。2012年更新的TSC診斷標(biāo)準(zhǔn)中臨床特征仍

表2　TSC1/TSC2 突變基因型的臨床特征比較[n(%)]

注1)χ2檢驗(yàn)；2) Fisher′s精確概率法

是診斷的主要方法， 重要的變化是將TSC基因檢測(cè)作為獨(dú)立的診斷標(biāo)準(zhǔn)。本文103例通過(guò)臨床特征確診，2例不符合臨床特征確診標(biāo)準(zhǔn)，通過(guò)TSC基因檢測(cè)陽(yáng)性確診。TSC基因突變可作為診斷的補(bǔ)充或在缺乏臨床表現(xiàn)的情況下作出診斷[1]。

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(本文編輯：丁俊杰)

The analysis of clinical phenotype and genotype of tuberous sclerosis complex in 105 cases

ZHANGLin-mei,ZHOUYuan-feng,CHAIYi-ming,WANGJi,WUBing-bing,WANGYi,ZHOUShui-zhen

(DepartmentofNeurology,Children′sHospitalofFudanUniversity,Shanghai201102,China)

ZHOU Shui-zhen，E-mail:szzhou@shmu.edu.com

Tuberous sclerosis complex；Diagnostic criteria；Clinical characteristics；TSCgene

復(fù)旦大學(xué)附屬兒科醫(yī)院神經(jīng)內(nèi)科上海，201102

周水珍，E-mail:szzhou@shmu.edu.com

10.3969/j.issn.1673-5501.2016.03.012

2015-12-30

2016-05-17)