KMT2D基因突变所致的Kabuki综合征6例报告并文献复习

吴冰冰 苏雅洁 王慧君 张 萍 李 龙 周文浩,3,5

·论著·

KMT2D基因突变所致的Kabuki综合征6例报告并文献复习

吴冰冰1,3,4苏雅洁2，4王慧君1张 萍1李 龙2，5周文浩1,3,5

目的 探讨KMT2D突变引起的Kabuki综合征(KS)的临床、遗传学特点及其在新生儿期的临床特征。方法 采用全外显子组测序(WES)和临床panel的二代测序技术，结合复旦大学附属儿科医院分子诊断中心建立的数据分析流程，行相关基因测序和数据分析，对6例KMT2D基因突变患儿的临床及分子生物学特征进行总结。计算机检索 PubMed、中国知网、维普、中国生物医学文献和万方数据库，收集KS相关文献，检索时间从2012年4月至 2017年4月，对描述新生儿期临床特征的文献进行提取、归纳和总结。结果 6例KS患儿，男4例，女2例。其中3例在婴儿期均因KS相关临床表现，家属要求行家系WES确诊，1例新生儿经临床panel检测后确诊，2例因家属要求对患儿进行WES测序确诊。6例KS患儿共检测到7个KMT2D基因的杂合突变，分别位于11、39、51和53号外显子，包括1个终止、4个错义和2个移码突变。其中c.12697C>T(p.Q4233X)、c.16498C>T(p.R5500W)、c.16273G>A(p.E5425K)为人类基因突变数据库(HGMD)已收录的致病突变位点。c.12696G>T(p.Q4232H)、c.3495delC (p.Pro1165LeufsTer47)、c.10881delT(p.Leu3627ArgfsTer31)、c.12560G>A(p.G418E)为新发突变位点。经SIFT、Polyphen 2和MutationTaster 软件预测为有害突变。纳入18篇KS新生儿期起病文献加上本文2例(34例)，新生儿期表现为喂养困难(19例)，心脏发育异常(20例)，特殊容貌(17例)，骨骼发育异常(15例)，低血糖(10例)和肌张力低下(9例)等。结论 KS的典型临床表型在新生儿期还未完全呈现，当新生儿有喂养困难、心脏发育异常、特殊容貌等临床特征时需考虑KS，并尽早完善相关基因检测，实现早诊断、早干预。

Kabuki综合征；KMT2D基因； 新生儿临床特征； 遗传学特征

Kabuki综合征(KS，OMIM 147920)，又称“歌舞伎脸谱综合征”，是以生后发育迟缓、肌张力低下和先天性脏器畸形等为主要临床表型的多发畸形综合征[1,2]。其中，Ⅰ型是由赖氨酸特定的甲基转移酶2D(KMT2D)突变导致，呈常染色体显性遗传[3]。目前，已确诊的KS患儿44%～76%由KMT2D突变引起[4～7]。Ⅱ型由位于X染色体的赖氨酸去甲基转移酶6A (KDM6A)突变引起(1%～6%)，呈X连锁显性遗传[1,8]。由于KS的一些典型表型随着年龄的增长才会出现，如睑裂长、上睑下翻等往往在童年期才较明显，故早期临床诊断困难[9]。目前，超过92%的确诊KS的患儿均因临床特征出现后再行基因测序确诊[1]，此时患儿已出现智力发育落后等表现。高通量测序技术可以从基因水平对遗传病进行早期、精确诊断。本文采用全外显子组测序(WES)及临床panel测序技术，结合复旦大学附属儿科医院(我院)分子诊断中心建立的数据分析流程[10]，分析6例KMT2D突变KS患儿的临床表型和分子生物学特征。

1 病例资料

1.1 临床资料 6例KS患儿，男4例，女2例，基本情况和临床特征见表1。

例1～3因“生后生长发育迟缓”在我院就诊，就诊时2 ～10月龄，有典型的眼部特征和大耳(图1A)，例1有乳房早发育(图1B)和胎指垫(图1C)等；例4因“生后反应差、喂养困难”住我院NICU，就诊日龄为21 d，心脏超声提示：全心增大、心功能不全、继发型小房缺和卵圆孔未闭等；例5(3个月)因外院发现“先天性心脏病、呼吸困难”和例6(7 d，弃婴)因“肛门闭锁”考虑遗传性疾病，送血标本至我院诊断，缺乏临床信息描述。5例患儿或无或不清楚家族史。目前例1～4尚在治疗中，例5死亡，例6预后不详。

1.2 测序结果 例1～5在征得父母知情同意后行基因检测，其中例1～3行家系WES测序，例4行新生儿panel检测，例5和6行患儿WES测序，并结合Sanger 测序验证。

图1 KS患儿特征

表1 6例KS患儿基本情况和临床特征

采患儿和/或父母外周静脉血2 mL，抽提基因组DNA(Qiagen公司mini blood 全血试剂盒)。参照SureSelct Human All Exon 试剂盒说明书进行捕获、建库，采用Illumina HiSeq 2000 平台对全基因组编码区外显子进行测序，捕获目标序列50 Mb，总体测序覆盖度达95%。依据文献[10]数据分析流程，结合WuXi Next CODE 分析软件(CSA)进行分析。测序数据通过Burrows-Wheeler Aligner(BWA)与NCBI RefSeq 进行匹配比对，通过ANNOVAR、VEP软件以及注释程序注释变异数据，包括用NCBI RefSeq、SwissPort进行基因注释，HGMD、OMIM、ClinVar进行疾病相关注释，千人基因组计划、EVC6500、ExAC、内部数据库进行突变频率注释以及SIFT、Polyphen 2、MutationTaster 进行突变预测。通过频率及变异类别的筛选以及与疾病的相关关系，筛选出候选突变。

表2显示，6例KS患儿检测到7个KMT2D基因的杂合突变，分别位于11、39、51和53号外显子，突变类型为：1个终止、4个错义和2个移码突变。其中c.12697C>T(p.Q4233X)、c.16498C>T(p.R5500W)、c.16273G>A(p.E5425K)为人类基因突变数据库(HGMD)已收录的致病突变位点。c.12696G>T(p.Q4232H)、c.3495delC(p.Pro1165LeufsTer47)、c.10881delT(p.Leu3627ArgfsTer31)和c.12560G>A(p.G418E)均为新发突变位点。例4～6未进行父母验证，不能明确变异来源，其余3例均经PCR-Sanger测序验证，为患儿的新发突变。

表2 6例KS患儿测序结果

2 文献复习

检索 PubMed、中国期刊全文数据库、中文科技期刊数据库、 中国生物医学文献数据库和万方数据库，检索时间均从2012年4月至2017年4月。以 PubMed 数据库为例，英文检索式为“(Kabuki [Title/Abstract])”，以CBM 为例，中文检索式为“Kabuki OR歌舞伎综合征”。采用主题和自由途径结合方式检索，筛选出新生儿期起病的KS综合征的文献，排除综述类文献中重复报道病例。共纳入18篇文献，均为英文文献。

表3汇总了包括本文2例在内的34例KS患儿在新生儿期的表现，常见的为喂养困难19例，心脏发育异常20例，特殊容貌17例，骨骼发育异常15例，低血糖10例，肌张力低下9例，唇腭裂8例，肾脏发育异常7例，乳房发育异常4例，生殖器异常5例，呼吸困难4例，小头畸形2例。

3 讨论

KMT2D基因位于染色体12q13.12，全长19.4 kbp，包含54个外显子[29]。目前，HGMD已收录的KMT2D基因突变类型共558种，其中错义或无义突变264种、小片段缺失151种、小片段的插入或重复80种，且多为新发突变。本文检测到的7个突变中2个来自同一患儿(例1)，表现为明显的精神发育迟缓，行全基因组拷贝数变异分析(arrayCGH)未发现异常。WES检测到KMT2D基因外显子39的2处连续杂合变异 (c.12697C>C/T, p.Q4233X和c.12696G>G/T， p.Q4232H)。原始测序数据显示这两处的变异来自同一条染色单体，因此这2个变异也可以表示为2个连续的碱基替换(c.12696_12697het_delGCinsAA)。虽该插入缺失位点尚未见报道，但HGMD已收录c.12697C>C/T, p.Q4233X位点为KS的致病位点[30]，因此分析该突变为有害突变。例2和3中检测到的突变均为新发移码突变，导致不能翻译正常蛋白质，患儿表型与KS描述相符，且未检测到其父母亲携带该突变，分析为患儿的新发突变。例4为新生儿，检测到c.16498C>T的突变，该位点是已报道的KS的致病突变[31]，虽然未出现KS典型的面部特征，但表现为喂养困难、反应差、肺部感染、先天性心脏病和隐睾等特征，仍可明确诊断。

KMT2D基因是组蛋白甲基转移酶SET结构域成员之一，参与赖氨酸组蛋白H3K4的三甲基化过程[32]。在胚胎发育中，KMT2D是组织细胞分化的关键调节基因[33,34]。研究表明，KMT2D基因对小鼠脂肪细胞的生成、肌肉的发育、巨噬细胞的活化和淋巴的生成十分必要[35]。文献认为，KS典型的面部特征是KMT2D基因单倍计量的不足造成的[36]。在对斑马鱼进行KMT2D基因的敲除后，出现了明显的颅面异常，包括鳃弓完全缺失、软骨异常、舌骨角畸形和颅盖骨缺失等[37]。本文确诊的6例患儿中，例1～3在婴儿期逐渐出现多个系统的畸形，尤其脸部的典型特征(拱形眉、睑裂长、上睑下垂和宽鼻梁)，例1为女性患儿出现胸部发育和性早熟，符合KS典型的临床特征[38,2]。KS患儿普遍易患感染，如反复的中耳炎、上呼吸道感染及肺炎等，其免疫功能异常与中耳炎的迁延不愈相关[6]。本文确诊的6例KS患儿均在不同时期出现过反复的呼吸道感染病史。多项研究表明，KS容易合并其他出生缺陷，除免疫缺陷、甲状腺炎等合并症外[39]，还会有少数病例合并其他疾病，如毛母细胞瘤[40]、低血糖、语言功能障碍等[41]。

表3 已报道的和本文报告的34 例KS新生儿期的临床表型

注 例8、10为KDM1A和KDM6A突变，余均为KMT2D突变；NA：未报告胎儿B超检查结果

由于KS涉及人体多个系统，且病情复杂，典型的遗传特征在新生儿期难以明确。本文通过二代测序技术明确诊断6例KS患儿，其中2例为新生儿，补充和丰富了该病的基因突变谱。通过对近5年的文献复习，总结了KS在新生儿期的临床表型。当新生儿存在喂养困难、心脏发育异常、特殊容貌等临床特征时需考虑此病，并尽早完善基因二代测序辅助诊断，为临床干预治疗提供依据，为家庭遗传咨询提供线索。

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(本文编辑：张崇凡，孙晋枫)

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

上海市卫生和计划生育委员会基金面上项目：201440628

1 复旦大学附属儿科医院，上海市出生缺陷防治重点实验室，复旦大学儿童发育与疾病转化医学研究中心 上海，201102；2 新疆维吾尔自治区人民医院新生儿科 乌鲁木齐，830001；3 卫生部新生儿疾病重点实验室 上海，201102；4 共同第一作者；5 共同通讯作者

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

10.3969/j.issn.1673-5501.2017.02.011

2017-03-20

2017-04-20)