蘇華+陳琛+張斌
[摘要] 目的 對(duì)超聲檢查疑為短肢畸形的胎兒進(jìn)行致病基因成纖維細(xì)胞生長(zhǎng)因子受體3(FGFR3)全外顯子突變分析產(chǎn)前基因診斷及遺傳咨詢(xún)。 方法 從UCSC Genome Bioinformatics數(shù)據(jù)庫(kù)中提取包括FGFR3基因全部17個(gè)外顯子的2個(gè)轉(zhuǎn)錄本的相關(guān)序列作為標(biāo)準(zhǔn)序列,設(shè)計(jì)合成FGFR3全基因外顯子擴(kuò)增的共8對(duì)引物。收集2014年1月~2016年12月在河北北方學(xué)院附屬第一醫(yī)院就診的5例疑為短肢畸形的胎兒,對(duì)高危胎兒于B超引導(dǎo)下行羊水穿刺,富集細(xì)胞,對(duì)FGFR3基因外顯子組進(jìn)行PCR擴(kuò)增,應(yīng)用Sanger基因測(cè)序技術(shù)進(jìn)行FGFR3基因全外顯子測(cè)序,采用CodonCode Aligner軟件對(duì)測(cè)序結(jié)果進(jìn)行錯(cuò)義突變位點(diǎn)分析,判斷其是否存在致病突變。 結(jié)果 5例疑為短肢畸形的胎兒,其中1例胎兒FGFR3基因(轉(zhuǎn)錄本NM_000142.4)第7號(hào)外顯子(FGFR3-7-IS1)攜帶錯(cuò)義突變C.1138G>A(P.Arg380Gly),其余4例未發(fā)現(xiàn)FGFR3基因突變。對(duì)明確了致病突變的5例孕早期胎兒家屬進(jìn)行遺傳咨詢(xún)。 結(jié)論 對(duì)疑似短肢畸形胎兒應(yīng)用基因測(cè)序技術(shù)進(jìn)行產(chǎn)前FGFR3基因突變的檢測(cè)可以預(yù)防短肢畸形患兒的出生。
[關(guān)鍵詞] 短肢畸形;軟骨發(fā)育不全;成纖維細(xì)胞生長(zhǎng)因子受體3;突變;產(chǎn)前診斷
[中圖分類(lèi)號(hào)] R714.53 [文獻(xiàn)標(biāo)識(shí)碼] A [文章編號(hào)] 1673-7210(2017)12(b)-0016-04
[Abstract] Objective To identify the fibroblast growth factor receptor 3 (FGFR3) mutation of fetuses with short limbs deformity and carry out genetic counseling by fetuses found in ultrasound screening. Methods The genomic information of FGFR3 full 17 exon amplification from UCSC Genome Bioinformatics, including 2 genome transcripts were extracted as a standard sequence, 8 pairs of primers were designed. 5 fetuses suspected to be with short limb deformities in the First Affiliated Hospital of Hebei North University from January 2014 to December 2016 were selected and the amniotic fluid of the fetuses in high-risk was collected for detection of mutation of FGFR3 gene by polymerase chain reaction and Sanger gene sequencing. The mutation of the sequencing of FGFR3 gene was carried out by CodonCode Aligner software. Results Among the 5 fetuses with short limbs deformity, 1 case was carried mutations C.1138G> A (P.Arg380Gly) on the 7th (FGFR3-7-IS1, transcript NM_000142.4). FGFR3 mutation were not found in the other 4 cases. Genetic counseling was conducted on 5 cases of fetal parents. Conclusion The detection of prenatal FGFR3 gene mutation in suspected patients using gene sequencing could effectively prevent the birth of short limb deformity patients.
[Key words] Short limb deformity; Achondroplasia; Fibroblast growth factor receptor-3; Mutation; Prenatal diagnosis
胎兒四肢骨骼發(fā)育異常是常見(jiàn)的出生缺陷之一,主要的臨床表現(xiàn)以四肢不同程度的肢端短小為主,多見(jiàn)于染色體的異常及各種綜合征。產(chǎn)前的超聲排畸檢查僅能給出提示而不能進(jìn)行診斷,使遺傳咨詢(xún)的進(jìn)一步處理困難重重,因此,對(duì)超聲排畸發(fā)現(xiàn)的短肢畸形胎兒盡可能的明確診斷,可為遺傳咨詢(xún)提供準(zhǔn)確依據(jù)。成纖維細(xì)胞生長(zhǎng)因子受體3(fibroblast growth factor receptor-3,F(xiàn)GFR3,OMIM 134934)基因被認(rèn)為是與肢端發(fā)育異常相關(guān)的基因[1],其突變所致的肢端發(fā)育異常疾病包括軟骨發(fā)育不全(achondroplasia,ACH)、致死性骨發(fā)育不良、成骨發(fā)育不全等。FGFR3基因突變發(fā)生位點(diǎn)的不同,可導(dǎo)致疾病發(fā)生的不同及程度的不同[2]。本研究對(duì)5例疑為短肢畸形的胎兒進(jìn)行致病基因FGFR3全外顯子突變分析產(chǎn)前基因診斷及遺傳咨詢(xún)?,F(xiàn)報(bào)道如下:
1 資料與方法
1.1 一般資料
收集2014年1月~2016年12月在河北北方學(xué)院附屬第一醫(yī)院(以下簡(jiǎn)稱(chēng)“我院”)就診的5例疑為短肢畸形的胎兒,獲得孕婦本人或家屬知情同意的情況下經(jīng)B超引導(dǎo)行羊水穿刺,收集羊水細(xì)胞,進(jìn)行FGFR3基因突變篩查。本研究經(jīng)我院醫(yī)學(xué)倫理委員會(huì)通過(guò)并獲得家屬知情同意。endprint
1.2 一般情況
5例疑為短肢畸形的胎兒,其中4例為24~28周胎兒進(jìn)行B型超聲排畸檢查,疑似短肢畸形的胎兒。1例孕母為臨床確診為ACH的患者,具有ACH典型的臨床表現(xiàn)[4],身材矮小且不成比例,四肢短且粗,軀干細(xì)長(zhǎng),頭大、前額突出、塌鼻梁,肘關(guān)節(jié)不能伸直,雙腿呈“O”型,腰椎前突,手指短小具有典型的“三叉手”,臨床表現(xiàn)和放射學(xué)檢查符合ACH。對(duì)其胎兒行超聲排畸檢查發(fā)現(xiàn)胎兒具有ACH典型臨床特征:巨頭,與胎兒孕齡不符,枕骨大孔小,額部隆起,胎兒近端肢體進(jìn)行性變短,四肢長(zhǎng)骨短小,骨化差,骨后方聲影不明顯,胸腔狹小,肋骨細(xì)短,腹部或有膨隆并伴腹水,椎骨骨化差伴低回聲,孕婦可伴有全身水腫,羊水過(guò)多等,該孕婦于孕25周自愿進(jìn)行FGFR3產(chǎn)前基因檢測(cè)。所有病例均經(jīng)B超引導(dǎo)行羊水穿刺,收集羊水細(xì)胞,常規(guī)試劑盒提取DNA[血液/細(xì)胞/組織基因組DNA提取試劑盒(DP304)購(gòu)自天根生化科技有限公司],-20℃保存待檢。野生型對(duì)照標(biāo)本取自我院體格正常的健康體檢者靜脈抗凝血。
1.3 FGFR3全外顯子基因檢測(cè)方法的建立
從UCSC Genome Bioinformatics數(shù)據(jù)庫(kù)(http://www.genome.UCSC.edu)中提取FGFR3基因全部17個(gè)外顯子的核酸序列,包含全部2個(gè)轉(zhuǎn)錄本(FGFR3IS1:NM_000142.4;IS3:NM_001163213.1)的相關(guān)序列作為標(biāo)準(zhǔn)序列,設(shè)計(jì)并合成FGFR3基因PCR擴(kuò)增引物共8對(duì)(見(jiàn)表1),應(yīng)用Takara PT600梯度PCR儀對(duì)FGFR3基因外顯子組進(jìn)行PCR擴(kuò)增,反應(yīng)總體系為25 μL,GC緩沖液Ⅱ 12.5 μL,1.25 mmol/L dNTP 4 μL,10 μmol/L引物各0.75 μL,LA Taq聚合酶(大連寶生物工程有限公司)1.25 U,基因組DNA模板0.5 μL(核酸濃度均在20~78 ng/μL,微量核酸濃度檢測(cè)儀側(cè)得吸光度A值為1.8~2.0)。PCR反應(yīng)條件:95℃預(yù)變性4 min,93℃ 30 s,62℃ 30 s,72℃ 30 s,35個(gè)循環(huán);72℃延伸5 min。PCR產(chǎn)物經(jīng)0.8%瓊脂糖凝膠電泳檢測(cè),確定產(chǎn)物片段大小是否與預(yù)期一致,并送公司測(cè)序(北京美吉生物技術(shù)有限公司)。
1.4 FGFR3突變篩查
對(duì)5例疑為短肢畸形的胎兒及其父母進(jìn)行致病基因成纖維細(xì)胞生長(zhǎng)因子受體3(FGFR3)全外顯子突變分析,測(cè)序結(jié)果用CodonCode Aligner軟件尋找突變位點(diǎn),并通過(guò)HGMD數(shù)據(jù)庫(kù)及FGFR3基因數(shù)據(jù)庫(kù)對(duì)有意義的突變位點(diǎn)進(jìn)行篩選。
1.5 遺傳咨詢(xún)
根據(jù)FGFR3基因檢測(cè)結(jié)果對(duì)5例受試胎兒家屬進(jìn)行遺傳咨詢(xún),告知胎兒父母胎兒患ACH可能性大小,指導(dǎo)胎兒父母進(jìn)行是否繼續(xù)妊娠。
2 結(jié)果
2.1 FGFR3全外顯子基因檢測(cè)方法的建立
成功設(shè)計(jì)8對(duì)特異性引物,建立了FGFR3全外顯子基因檢測(cè)方法,對(duì)FGFR3基因兩個(gè)轉(zhuǎn)錄本的17個(gè)外顯子基因進(jìn)行PCR擴(kuò)增,片段長(zhǎng)度符合預(yù)期,1%瓊脂糖凝膠電泳結(jié)果見(jiàn)圖1。
2.2 FGFR3 基因突變檢測(cè)
對(duì)5例短肢畸形胎兒臍血和1例正常對(duì)照全血DNA進(jìn)行FGFR3基因全外顯子組測(cè)序,發(fā)現(xiàn)1例孕母診斷為ACH患者的胎兒其FGFR3基因(轉(zhuǎn)錄本NM_000142.4)第7號(hào)外顯子(FGFR3-7-IS1)發(fā)生雜合突變c.1138G>A(p.Gly380Arg),結(jié)果見(jiàn)圖2A;野生型測(cè)序結(jié)果見(jiàn)圖2B。其余4例未檢出FGFR3基因突變。
2.3遺傳咨詢(xún)
得知FGFR3基因測(cè)序結(jié)果后,4例胎兒FGFR3基因無(wú)突變發(fā)生,未攜帶C.1138G>A(P.Arg380Gly)突變,將來(lái)為ACH患者的可能性小,經(jīng)遺傳咨詢(xún)后其孕母及家屬選擇繼續(xù)妊娠。1例先天攜帶ACH致病突變C.1138G>A(P.Arg380Gly)胎兒,其孕母及家屬在得知產(chǎn)前診斷結(jié)果后,通過(guò)遺傳咨詢(xún)最終選擇終止妊娠。
3 討論
FGFR3基因是肢端發(fā)育異常最主要的致病基因,F(xiàn)GFR3基因定位于人類(lèi)染色體4p16.3,包含17個(gè)外顯子,全長(zhǎng)16.5 kb,編碼806個(gè)氨基酸的蛋白質(zhì)分子,分子量為110 000~135 000 D[3]。FGFR3是酪氨酸激酶受體,是具有骨骼調(diào)節(jié)發(fā)育功能的跨膜蛋白,F(xiàn)GFR3受體蛋白包括3個(gè)部分:即胞外區(qū)、跨膜區(qū)和胞內(nèi)區(qū)3部分組成,①胞外區(qū)為3個(gè)免疫球蛋白樣配體結(jié)構(gòu)域組成(分別為IgⅠ、IgⅡ、IgⅢ),是糖基化的配體結(jié)合區(qū),能與成纖維細(xì)胞生長(zhǎng)因子發(fā)生作用,影響細(xì)胞的有絲分裂;②跨膜區(qū)是一個(gè)疏水區(qū),跨膜1次,沒(méi)有可磷酸化的酪氨酸;③胞內(nèi)區(qū)為近膜區(qū)和2酪氨酸激酶區(qū)(TK1和TK2)構(gòu)成。FGFR3與其配體FGFs結(jié)合后,F(xiàn)GFR3發(fā)生二聚化,使酪氨酸激酶激活,胞內(nèi)區(qū)下游含SH2結(jié)構(gòu)域的信號(hào)分子和絲氨酸、蘇氨酸蛋白激酶磷酸化,進(jìn)而激活細(xì)胞核內(nèi)轉(zhuǎn)錄因子,進(jìn)一步調(diào)節(jié)基因的轉(zhuǎn)錄和蛋白的表達(dá)[4-6]。
研究顯示,95%的ACH由FGFR3基因c.1138G>A(Gly380Arg)突變引起(其他罕見(jiàn)突變包括S84L、R200C、N262H、G268C、Y278C、V381E[7-9]),為常染色體顯性遺傳,該突變位于FGFR3蛋白跨膜區(qū)[10-11]。Zhou等[12]發(fā)現(xiàn)FGFR3蛋白跨膜區(qū)的G380R氨基酸發(fā)生突變使蛋白活化過(guò)程中形成二聚體更容易,可持續(xù)活化胞內(nèi)區(qū)的酪氨酸激酶,從而激活胞內(nèi)的信號(hào)傳導(dǎo)途徑,使軟骨細(xì)胞增殖和分化發(fā)生障礙,導(dǎo)致骨骼生長(zhǎng)受限。Di Rocco等[13]通過(guò)小鼠動(dòng)物模型Fgfr3(Y367C/+)觀(guān)察到,F(xiàn)GFR3基因突變導(dǎo)致FGFR3蛋白超活化,使受體過(guò)度降解,軟骨細(xì)胞增殖分化受到抑制,導(dǎo)致骨膜成熟障礙,是ACH發(fā)生的主要原因。有研究發(fā)現(xiàn)[14],ACH的發(fā)生與父親年齡可能相關(guān),隨著父親年齡的增長(zhǎng),精子生成時(shí)影響DNA復(fù)制、修補(bǔ)的因素可使精子細(xì)胞突變的機(jī)會(huì)增加。endprint
直接針對(duì)熱點(diǎn)突變的篩查可以排除95%的ACH,但少數(shù)罕見(jiàn)突變難以被發(fā)現(xiàn),建議對(duì)肢端發(fā)育異?;颊哌M(jìn)行FGFR3全基因組突變篩查。Makrythanasis等[15]報(bào)道了1例嚴(yán)重的骨骼發(fā)育異常患者,臨床表型為脊柱歪斜,突出,“三叉戟”樣手,不能行走,該例患者首次發(fā)現(xiàn)了NM_000142.4:c.1637C>A:p.(Thr546Lys)突變,該突變位于FGFR3蛋白胞內(nèi)激酶區(qū),是季肋發(fā)育不全的突變熱點(diǎn)區(qū)域,因此FGFR3全基因外顯子突變的篩查對(duì)所有肢端發(fā)育異?;颊叩募膊≡\斷都是有重要意義的。
ACH是完全外顯的常染色體顯性遺傳,建議對(duì)B超診斷為肢端短小疑為ACH患者的胎兒已經(jīng)不能僅僅停留在臨床監(jiān)測(cè)的水平,還應(yīng)進(jìn)行FGFR3突變基因的產(chǎn)前基因診斷[16-17]。本文通過(guò)FGFR3全基因外顯子組測(cè)序的方法,實(shí)現(xiàn)高危胎兒產(chǎn)前基因診斷,結(jié)果顯示,5例短肢畸形胎兒,其中1例孕母診斷為ACH患者,其胎兒攜帶FGFR3基因c.1138G>A(p.Gly380Arg突變,父母通過(guò)遺傳咨詢(xún)選擇終止妊娠;其余4例父母均為野生型,胎兒未檢出FGFR3基因突變,經(jīng)遺傳咨詢(xún)后將來(lái)為ACH患者的可能性小,選擇繼續(xù)妊娠。建議父母身材矮小的胎兒(侏儒癥的除外)應(yīng)引起重視,適當(dāng)選擇產(chǎn)前FGFR3突變熱點(diǎn)及全基因外顯子突變篩查[18-19]。產(chǎn)前突變篩查樣本包括胎兒羊水中提取DNA、絨毛膜絨毛細(xì)胞DNA、臍帶血淋巴及單核細(xì)胞DNA等[20]。方法除測(cè)序外也可采用PCR-高分辨率溶解測(cè)定法快速檢測(cè)熱點(diǎn)突變位點(diǎn)[21]。本研究強(qiáng)調(diào)FGFR3基因突變檢測(cè)在肢端發(fā)育異常胎兒產(chǎn)前診斷中的重要性,高危胎兒進(jìn)行FGFR3產(chǎn)前基因突變篩查,可有效預(yù)防患兒的出生,實(shí)現(xiàn)優(yōu)生優(yōu)育。
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(收稿日期:2017-09-01 本文編輯:任 念)endprint