吳冰冰 蘇雅潔 王慧君 張 萍 李 龍 周文浩,3,5

·論著·

KMT2D基因突變所致的Kabuki綜合征6例報告并文獻復(fù)習

吳冰冰1,3,4蘇雅潔2，4王慧君1張 萍1李 龍2，5周文浩1,3,5

目的 探討KMT2D突變引起的Kabuki綜合征(KS)的臨床、遺傳學特點及其在新生兒期的臨床特征。方法 采用全外顯子組測序(WES)和臨床panel的二代測序技術(shù)，結(jié)合復(fù)旦大學附屬兒科醫(yī)院分子診斷中心建立的數(shù)據(jù)分析流程，行相關(guān)基因測序和數(shù)據(jù)分析，對6例KMT2D基因突變患兒的臨床及分子生物學特征進行總結(jié)。計算機檢索 PubMed、中國知網(wǎng)、維普、中國生物醫(yī)學文獻和萬方數(shù)據(jù)庫，收集KS相關(guān)文獻，檢索時間從2012年4月至 2017年4月，對描述新生兒期臨床特征的文獻進行提取、歸納和總結(jié)。結(jié)果 6例KS患兒，男4例，女2例。其中3例在嬰兒期均因KS相關(guān)臨床表現(xiàn)，家屬要求行家系WES確診，1例新生兒經(jīng)臨床panel檢測后確診，2例因家屬要求對患兒進行WES測序確診。6例KS患兒共檢測到7個KMT2D基因的雜合突變，分別位于11、39、51和53號外顯子，包括1個終止、4個錯義和2個移碼突變。其中c.12697C>T(p.Q4233X)、c.16498C>T(p.R5500W)、c.16273G>A(p.E5425K)為人類基因突變數(shù)據(jù)庫(HGMD)已收錄的致病突變位點。c.12696G>T(p.Q4232H)、c.3495delC (p.Pro1165LeufsTer47)、c.10881delT(p.Leu3627ArgfsTer31)、c.12560G>A(p.G418E)為新發(fā)突變位點。經(jīng)SIFT、Polyphen 2和MutationTaster 軟件預(yù)測為有害突變。納入18篇KS新生兒期起病文獻加上本文2例(34例)，新生兒期表現(xiàn)為喂養(yǎng)困難(19例)，心臟發(fā)育異常(20例)，特殊容貌(17例)，骨骼發(fā)育異常(15例)，低血糖(10例)和肌張力低下(9例)等。結(jié)論 KS的典型臨床表型在新生兒期還未完全呈現(xiàn)，當新生兒有喂養(yǎng)困難、心臟發(fā)育異常、特殊容貌等臨床特征時需考慮KS，并盡早完善相關(guān)基因檢測，實現(xiàn)早診斷、早干預(yù)。

Kabuki綜合征；KMT2D基因； 新生兒臨床特征； 遺傳學特征

Kabuki綜合征(KS，OMIM 147920)，又稱“歌舞伎臉譜綜合征”，是以生后發(fā)育遲緩、肌張力低下和先天性臟器畸形等為主要臨床表型的多發(fā)畸形綜合征[1,2]。其中，Ⅰ型是由賴氨酸特定的甲基轉(zhuǎn)移酶2D(KMT2D)突變導(dǎo)致，呈常染色體顯性遺傳[3]。目前，已確診的KS患兒44%～76%由KMT2D突變引起[4～7]。Ⅱ型由位于X染色體的賴氨酸去甲基轉(zhuǎn)移酶6A (KDM6A)突變引起(1%～6%)，呈X連鎖顯性遺傳[1,8]。由于KS的一些典型表型隨著年齡的增長才會出現(xiàn)，如瞼裂長、上瞼下翻等往往在童年期才較明顯，故早期臨床診斷困難[9]。目前，超過92%的確診KS的患兒均因臨床特征出現(xiàn)后再行基因測序確診[1]，此時患兒已出現(xiàn)智力發(fā)育落后等表現(xiàn)。高通量測序技術(shù)可以從基因水平對遺傳病進行早期、精確診斷。本文采用全外顯子組測序(WES)及臨床panel測序技術(shù)，結(jié)合復(fù)旦大學附屬兒科醫(yī)院(我院)分子診斷中心建立的數(shù)據(jù)分析流程[10]，分析6例KMT2D突變KS患兒的臨床表型和分子生物學特征。

1 病例資料

1.1 臨床資料 6例KS患兒，男4例，女2例，基本情況和臨床特征見表1。

例1～3因“生后生長發(fā)育遲緩”在我院就診，就診時2 ～10月齡，有典型的眼部特征和大耳(圖1A)，例1有乳房早發(fā)育(圖1B)和胎指墊(圖1C)等；例4因“生后反應(yīng)差、喂養(yǎng)困難”住我院NICU，就診日齡為21 d，心臟超聲提示：全心增大、心功能不全、繼發(fā)型小房缺和卵圓孔未閉等；例5(3個月)因外院發(fā)現(xiàn)“先天性心臟病、呼吸困難”和例6(7 d，棄嬰)因“肛門閉鎖”考慮遺傳性疾病，送血標本至我院診斷，缺乏臨床信息描述。5例患兒或無或不清楚家族史。目前例1～4尚在治療中，例5死亡，例6預(yù)后不詳。

1.2 測序結(jié)果 例1～5在征得父母知情同意后行基因檢測，其中例1～3行家系WES測序，例4行新生兒panel檢測，例5和6行患兒WES測序，并結(jié)合Sanger 測序驗證。

圖1 KS患兒特征

表1 6例KS患兒基本情況和臨床特征

采患兒和/或父母外周靜脈血2 mL，抽提基因組DNA(Qiagen公司mini blood 全血試劑盒)。參照SureSelct Human All Exon 試劑盒說明書進行捕獲、建庫，采用Illumina HiSeq 2000 平臺對全基因組編碼區(qū)外顯子進行測序，捕獲目標序列50 Mb，總體測序覆蓋度達95%。依據(jù)文獻[10]數(shù)據(jù)分析流程，結(jié)合WuXi Next CODE 分析軟件(CSA)進行分析。測序數(shù)據(jù)通過Burrows-Wheeler Aligner(BWA)與NCBI RefSeq 進行匹配比對，通過ANNOVAR、VEP軟件以及注釋程序注釋變異數(shù)據(jù)，包括用NCBI RefSeq、SwissPort進行基因注釋，HGMD、OMIM、ClinVar進行疾病相關(guān)注釋，千人基因組計劃、EVC6500、ExAC、內(nèi)部數(shù)據(jù)庫進行突變頻率注釋以及SIFT、Polyphen 2、MutationTaster 進行突變預(yù)測。通過頻率及變異類別的篩選以及與疾病的相關(guān)關(guān)系，篩選出候選突變。

表2顯示，6例KS患兒檢測到7個KMT2D基因的雜合突變，分別位于11、39、51和53號外顯子，突變類型為：1個終止、4個錯義和2個移碼突變。其中c.12697C>T(p.Q4233X)、c.16498C>T(p.R5500W)、c.16273G>A(p.E5425K)為人類基因突變數(shù)據(jù)庫(HGMD)已收錄的致病突變位點。c.12696G>T(p.Q4232H)、c.3495delC(p.Pro1165LeufsTer47)、c.10881delT(p.Leu3627ArgfsTer31)和c.12560G>A(p.G418E)均為新發(fā)突變位點。例4～6未進行父母驗證，不能明確變異來源，其余3例均經(jīng)PCR-Sanger測序驗證，為患兒的新發(fā)突變。

表2 6例KS患兒測序結(jié)果

2 文獻復(fù)習

檢索 PubMed、中國期刊全文數(shù)據(jù)庫、中文科技期刊數(shù)據(jù)庫、 中國生物醫(yī)學文獻數(shù)據(jù)庫和萬方數(shù)據(jù)庫，檢索時間均從2012年4月至2017年4月。以 PubMed 數(shù)據(jù)庫為例，英文檢索式為“(Kabuki [Title/Abstract])”，以CBM 為例，中文檢索式為“Kabuki OR歌舞伎綜合征”。采用主題和自由途徑結(jié)合方式檢索，篩選出新生兒期起病的KS綜合征的文獻，排除綜述類文獻中重復(fù)報道病例。共納入18篇文獻，均為英文文獻。

表3匯總了包括本文2例在內(nèi)的34例KS患兒在新生兒期的表現(xiàn)，常見的為喂養(yǎng)困難19例，心臟發(fā)育異常20例，特殊容貌17例，骨骼發(fā)育異常15例，低血糖10例，肌張力低下9例，唇腭裂8例，腎臟發(fā)育異常7例，乳房發(fā)育異常4例，生殖器異常5例，呼吸困難4例，小頭畸形2例。

3 討論

KMT2D基因位于染色體12q13.12，全長19.4 kbp，包含54個外顯子[29]。目前，HGMD已收錄的KMT2D基因突變類型共558種，其中錯義或無義突變264種、小片段缺失151種、小片段的插入或重復(fù)80種，且多為新發(fā)突變。本文檢測到的7個突變中2個來自同一患兒(例1)，表現(xiàn)為明顯的精神發(fā)育遲緩，行全基因組拷貝數(shù)變異分析(arrayCGH)未發(fā)現(xiàn)異常。WES檢測到KMT2D基因外顯子39的2處連續(xù)雜合變異 (c.12697C>C/T, p.Q4233X和c.12696G>G/T， p.Q4232H)。原始測序數(shù)據(jù)顯示這兩處的變異來自同一條染色單體，因此這2個變異也可以表示為2個連續(xù)的堿基替換(c.12696_12697het_delGCinsAA)。雖該插入缺失位點尚未見報道，但HGMD已收錄c.12697C>C/T, p.Q4233X位點為KS的致病位點[30]，因此分析該突變?yōu)橛泻ν蛔儭＠?和3中檢測到的突變均為新發(fā)移碼突變，導(dǎo)致不能翻譯正常蛋白質(zhì)，患兒表型與KS描述相符，且未檢測到其父母親攜帶該突變，分析為患兒的新發(fā)突變。例4為新生兒，檢測到c.16498C>T的突變，該位點是已報道的KS的致病突變[31]，雖然未出現(xiàn)KS典型的面部特征，但表現(xiàn)為喂養(yǎng)困難、反應(yīng)差、肺部感染、先天性心臟病和隱睪等特征，仍可明確診斷。

KMT2D基因是組蛋白甲基轉(zhuǎn)移酶SET結(jié)構(gòu)域成員之一，參與賴氨酸組蛋白H3K4的三甲基化過程[32]。在胚胎發(fā)育中，KMT2D是組織細胞分化的關(guān)鍵調(diào)節(jié)基因[33,34]。研究表明，KMT2D基因?qū)π∈笾炯毎纳伞⒓∪獾陌l(fā)育、巨噬細胞的活化和淋巴的生成十分必要[35]。文獻認為，KS典型的面部特征是KMT2D基因單倍計量的不足造成的[36]。在對斑馬魚進行KMT2D基因的敲除后，出現(xiàn)了明顯的顱面異常，包括鰓弓完全缺失、軟骨異常、舌骨角畸形和顱蓋骨缺失等[37]。本文確診的6例患兒中，例1～3在嬰兒期逐漸出現(xiàn)多個系統(tǒng)的畸形，尤其臉部的典型特征(拱形眉、瞼裂長、上瞼下垂和寬鼻梁)，例1為女性患兒出現(xiàn)胸部發(fā)育和性早熟，符合KS典型的臨床特征[38,2]。KS患兒普遍易患感染，如反復(fù)的中耳炎、上呼吸道感染及肺炎等，其免疫功能異常與中耳炎的遷延不愈相關(guān)[6]。本文確診的6例KS患兒均在不同時期出現(xiàn)過反復(fù)的呼吸道感染病史。多項研究表明，KS容易合并其他出生缺陷，除免疫缺陷、甲狀腺炎等合并癥外[39]，還會有少數(shù)病例合并其他疾病，如毛母細胞瘤[40]、低血糖、語言功能障礙等[41]。

表3 已報道的和本文報告的34 例KS新生兒期的臨床表型

注 例8、10為KDM1A和KDM6A突變，余均為KMT2D突變；NA：未報告胎兒B超檢查結(jié)果

由于KS涉及人體多個系統(tǒng)，且病情復(fù)雜，典型的遺傳特征在新生兒期難以明確。本文通過二代測序技術(shù)明確診斷6例KS患兒，其中2例為新生兒，補充和豐富了該病的基因突變譜。通過對近5年的文獻復(fù)習，總結(jié)了KS在新生兒期的臨床表型。當新生兒存在喂養(yǎng)困難、心臟發(fā)育異常、特殊容貌等臨床特征時需考慮此病，并盡早完善基因二代測序輔助診斷，為臨床干預(yù)治療提供依據(jù)，為家庭遺傳咨詢提供線索。

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(本文編輯：張崇凡，孫晉楓)

Report of 6 Kabuki syndrome cases caused by KMT2D gene mutation and literature review

WUBing-bing1,3,4,SUYa-jie2,4,WANGHui-jun1,ZHANGPing1,LILong2,5,ZHOUWen-hao1,3,5

(1Children'sHospitalofFudanUniversity,ShanghaiKeyLaboratoryofBirthDefects,TheTranslationalMedicineCenterofChildrenDevelopmentandDiseaseofFudanUniversity,Shanghai201102,China; 2DepartmentofNeonatology,People'sHospitalofXinjiangUygurAutonomousRegion,Urumqi830001,China; 3KeyLaboratoryofNeonatalDiseases,MinistryofHealth,Shanghai201102,China; 4.Co-firstauthor；5.Co-correspondingauthor)

Corresponding Author：ZHOU Wen-hao, E-mail: zhouwenhao@fudan.edu.cn; LI Long, E-mail::lilong65@126.com

ObjectiveTo investigate the clinical and genetic features of Kabuki syndrome caused byKMT2Dmutation and summarize the clinical features in neonate. MethodsUsing Whole-Exome Sequencing (WES) and Clinical panel deep sequencing，combined with data analysis pipeline established by molecular diagnostic center of Children's Hospital of Fudan University, the clinical and molecular features of 6 children withKMT2Dmutations were summarized. Databases including PubMed，CNKI，WanFang Database and VIP were searched to collect literature of KS, which describe the clinical features of neonatal period from April 2012 to April 2017. ResultsFour males and two females were diagnosed as KS. Three cases were diagnosed by WES due to KS related manifestations were present and the families came to order trio-WES. One case was diagnosed by clinical neonatal panel screening. Another two cases were diagnosed by WES. Seven heterozygous mutations were detected in six patients with KS，mutations were located in exon 11, exon 39, exon 51 and exon 53 respectively. The types of mutations were one stop gained, four missenses and two frameshifts. Mutation of c.12697C>T(p.Q4233X)、c.16498C>T(p.R5500W)、c.16273G>A(p.E5425K)were reported as pathogenic mutations and had recorded in Human Gene Mutation Database (HGMD). Mutation of c.12696G>T(p.Q4232H), c.3495delC (p.Pro1165LeufsTer47), c.10881delT (p.Leu3627Argfs Ter31)and c.12560G>A(p.G418E)were novel, which predicted as harmful variants by SIFT, polyphen 2 and MutationTaster software. In a total of 18 literatures，together with the 2 cases in this study, there were 34 neonates were included. The most common clinical features were as follows: feeding problem was in 19cases, cardiac dysplasia in 20 cases, special appearance in 17 cases, skeletal dysplasia in 15 cases, hypoglycemia in 10 cases and hypotonia in 9 cases. ConclusionThe typical clinical features of KS are not shown in the neonatal period. This disease should be considered when the newborn has feeding problem, abnormal cardiac morphololy, special appearance and other clinical features. Genetic test can help to diagnose earlier in clinical. Early diagnosis can provide accurate information to clinic, may help patients to acquire appropriate treatment and family genetic counseling.

Kabuki syndrome;KMT2Dgene; Neonatal clinical features; Genetic features

上海市衛(wèi)生和計劃生育委員會基金面上項目：201440628

1 復(fù)旦大學附屬兒科醫(yī)院，上海市出生缺陷防治重點實驗室，復(fù)旦大學兒童發(fā)育與疾病轉(zhuǎn)化醫(yī)學研究中心 上海，201102；2 新疆維吾爾自治區(qū)人民醫(yī)院新生兒科 烏魯木齊，830001；3 衛(wèi)生部新生兒疾病重點實驗室 上海，201102；4 共同第一作者；5 共同通訊作者

周文浩，E-mail: zhouwenhao@fudan.edu.cn; 李龍，E-mail: lilong65@126.com

10.3969/j.issn.1673-5501.2017.02.011

2017-03-20

2017-04-20)