综合征型先天性心脏病与拷贝数变异遗传学研究

发布时间：2018-06-03 16:37

本文选题：综合征型先天性心脏病 + 拷贝数变异　；参考：《中南大学》2012年博士论文

【摘要】：目的：探讨中国汉族人群中拷贝数变异与综合征型先天性心脏病的关系,发现其新的致病性拷贝数变异。同时尽可能的缩小拷贝数缺失或者重复的致病区间,寻找与先天性心脏病相关的候选致病基因。研究对象：2009年4月到2011年8月就诊于中南大学湘雅二医院小儿心脏外科的140例综合征型先天性心脏病患儿。该140例患儿排除了常见的三体综合征及GATA4,NKX2.5,TBX5和TBXl基因突变。其中,男性患儿75例,女性患儿65例。研究方法：利用美国QIAGEN公司gDNA抽提试剂盒,按照其标准抽提流程对上述140例患儿进行全外周静脉血gDNA抽提。利用美国illumina公司生产的Human660W-Quad及Human Omni1-quad芯片,采用Array-SNP高通量测序技术对这其进行全基因组CNVs分析。我们从中选出四例国内较少报道的综合征型患儿gDNA样本,利用美国Applied Biosystems公司生产的ABI Stepone定量PCR仪实行SYBR(?) Green I嵌合荧光法实时定量PCR,并利用Applied Biosystems的StepOne Software v2.1软件进行数据分析验证芯片结果。结果：在140例综合征型先天性心脏病患儿中共发现21个致病性CNVs,其发生比率约为15%。其中22q11.2微缺失9例,发生比率约为6.7%,在综合征型先天性心脏病中最为常见。同时,发现4例国内研究较少的综合征型先天性心脏病致病性CNVs,包括13q33.1-34,17p11.2缺失各一例及2例11q24-25缺失。对其关键基因进行实施定量PCR,其结果发现这4个致病性CNVs中的关键基因拷贝数约为正常对照组的0.5倍,与芯片结果一致。结论：1)illumina公司生产的Human660W-Quad及Human Omni1-quad芯片可对综合征型先天性心脏病患儿致病性CNVs进行快速而准确的分析,并提供可靠的遗传学信息； 2)13q33.1-34缺失,17p11.2缺失及11q24-25为综合征型先天性心脏病的致病性CNVs区间。背景：利用美国illumina公司的高通量Array-SNP从140例综合征型先天性心脏病筛选出21个致病性CNVs。从这21个致病性CNVs中选出EFNB2, ZFPM2/FOG2为先天性心脏病的候选致病基因。目的：本研究旨在探讨EFNB2,FOG2两种候选基因与先天性心脏病的相关性。研究对象：2009年4月到2011年8月就诊于中南大学湘雅二医院小儿心脏外科的孤立型先天性心脏病患儿。从中选出孤立型房间隔缺损15例,室间隔缺损15例,完全性房室间隔缺损15例,共45例患者进行EFNB2基因突变筛选；右室双出口患儿38例进行ZFPM2/FOG2基因突变筛选,上述83例孤立型先天性心脏病gDNA样本已排除常见的GATA4,NKX2.5,TBX5,TBXl基因突变。研究方法：利用美国QIAGEN公司gDNA抽提试剂盒,按照其标准抽提流程对上述83例患儿进行外周静脉血gDNA抽提。对EFNB2, ZFPM2/FOG2分别设计相应的特异性引物扩增,利用ABI公司BigDye Terminator v1.1测序试剂盒及其3100测序仪对其PCR扩增产物进行测序分析。结果：在38例孤立型右室双出口中共发现5例ZFPM2/FOG2错意突变,其发生比率约为13.2%。其中c.G1015A(p.V339I)、c.G1831A(p.A611T)、c.A2209G(p.K737E)3例为新发的错意突变,而另外两例突变(c.A2107C(p.M703L)及c.C2665G(p.Q889E))常见于先天性膈疝,目前在先天性心脏病中尚无报道。以上5种突变都未收录于1000genomes或者the Single Nucleotide Polymor Polymorphism数据库(dbSNP)中。在对45例先天性间隔型缺损进行EFNB2突变筛查时发现1个同义突变,余样本未发现突变。结论：1)ZFPM/FOG2在先天性右室双出口的发生中起着重要的作用,然而不能确定其突变与某种类型先天性右室双出口相关联。 2)EFNB2不是先天性间隔型缺损的候选致病基因,在13q33.1-34区域中仍存在有其他的候选致病基因。
[Abstract]:Objective : To explore the relationship between copy number variation and syndrome type congenital heart disease in Chinese Han population , and find out its new pathogenic copy number variation .

Object : From April 2009 to August 2011 , 140 children with congenital heart disease were seen from pediatric cardiac surgery in Xiangya Hospital , Central South University . Among these 140 cases , the common three - body syndrome and GATA4 , NKX2.5 , TBX5 and TBXl gene mutations were excluded .

Methods : The whole genome CNVs was extracted from the above 140 cases by using the DNA extraction kit from QIANUS , USA . Using the human 660W - Quad and Human Omni1 - quad chip produced by American Inc . , the whole genome CNVs was analyzed by using the array - SNP high - throughput sequencing technique . Four cases of syndrome - type children with less reported syndrome types were selected from them , SYBR ( ? ) was carried out using ABI Stepone quantitative PCR instrument produced by Applied Biosystems , USA . Real - time quantitative PCR was carried out by Green I chimeric fluorescence method , and the results of the chip were verified by using the software of Applied Biosystems StepOne Software v2.1 .

Results : A total of 21 pathogenic CNVs were found in 140 children with congenital heart disease with a rate of about 15 % . Among them , 22q11.2 was absent in 9 cases , the rate of occurrence was about 6.7 % , the most common in syndrome type congenital heart disease was found .

Conclusion : 1 ) Human660W - Quad and Human Omni1 - quad chip can be used for rapid and accurate analysis of pathogenic CNVs in children with syndrome type congenital heart disease , and provide reliable genetic information .

2 ) The 13q33 . 1 - 34 deletion , 17p11.2 deletion and 11q24 - 25 are the pathogenic CNVs of the syndrome type congenital heart disease .

BACKGROUND : Twenty - one pathogenic CNVs were isolated from 140 syndrome - type congenital heart disease by high - throughput Array - SNP of American company A . EFNB2 and ZFPM2 / FOG2 were selected as candidate pathogenic genes for congenital heart disease .

Objective : To investigate the correlation between EFNB2 and FOG2 candidate genes and congenital heart disease .

Objective : From April 2009 to August 2011 , we visited the isolated congenital heart disease of children in Xiangya Hospital , Central and South University . Fifteen patients with isolated atrial septal defect , 15 ventricular septal defects and 15 complete atrioventricular septal defects were selected , and 45 patients were screened for EFNB2 gene mutation ;
The mutation of the ZFPM2 / FOG2 gene was performed in 38 children with right ventricular double - export , and the above 83 cases of isolated congenital heart disease gDNA were excluded from common GATA4 , NKX2.5 , TBX5 , TBXl gene mutation .

Methods : The gDNA extraction was carried out on the peripheral venous blood of 83 cases of the above - mentioned 83 children by using the gDNA extraction kit of QIAN2 , USA . The corresponding specific primers were designed for EFNB2 and ZFPM2 / FOG2 , and the PCR products were sequenced and analyzed by ABI Company ' s Big Dye Penetration v1.1 sequencing kit and its 3100 sequencer .

Results : Five cases of ZFPM2 / FOG2 missense mutation were found in 38 isolated right ventricular double exits , with a rate of about 13.2 % . Among them , c . G1015A ( p . V339I ) , c . G1831A ( p . A611T ) , c . A2209G ( p . K737E ) were found to be new missense mutations .

Conclusion : 1 ) ZFPM / FOG2 plays an important role in the pathogenesis of congenital right ventricular double exit , however , it cannot be determined that the mutation is associated with some type of congenital right ventricular double - outlet .

2 ) EFNB2 is not a candidate pathogenic gene for congenital septal defect , and other candidate pathogenic genes still exist in the 13q33 . 1 - 34 region .
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R725.4

【共引文献】