先天性心脏病患儿CITED2基因突变及22q11微缺失的研究

发布时间：2018-03-20 18:45

本文选题：先天性心脏病　切入点：CITED2　出处：《新乡医学院》2012年硕士论文　论文类型：学位论文

【摘要】：背景先天性心脏病(congenital heart disease, CHD)是胎儿时期心脏血管发育异常所致的心血管畸形,也是小儿最常见的心脏病。其临床后果极为严重,通常导致流产、死胎、死产、新生儿死亡,以及儿童、青少年和成人残疾。CHD发生率在城市地区一直高居首位,在农村地区仅次于神经管畸形,位于第二位,给家庭和社会造成严重的经济和精神等方面的负担。虽然目前CHD遗传基础的研究已取得了很多进展,但是由于CHD种类繁多、病因复杂,大多数CHD的病因尚未清楚,急需人们多层次、多水平、深入全面综合地探讨。目的分析CHD患儿的CITED2基因编码链突变的情况；检测CHD患儿22q11微缺失的发病及分布情况,探讨多重连接依赖式探针扩增技术(multiplex ligation-dependent probe amplification, MLPA)用于诊断CHD22q11微缺失的可行性。方法以2010～2011年河南省胸科医院及郑州市儿童医院收集的300例散发型CHD患儿为研究对象。300例患儿均为汉族,且均经临床、心脏彩超、手术确诊。50例正常男童及50例正常女童作为本研究的正常对照,均无CHD病史及家族史。使用非试剂盒方法提取外周血基因组DNA,对CITED2基因进行聚合酶链反应并测序,测序结果与GeneBank数据库公布的CITED2基因序列(NM006079.3)进行比较以识别基因突变。用Clustal X软件分析突变氨基酸的保守性。采用MLPA P250试剂盒(共48对探针)对120例CHD患儿进行22q11微缺失检测。所有反应在PCR仪上进行：DNA样品与48对探针杂交16h,然后用连接酶连接半探针,最后用统一的SALSA PCR引物对连接的半探针进行PCR扩增。使用毛细管电泳仪(CEQ8800)和与之配套的软件(CEQ system)来分离和分析PCR产物。将CEQ system分析后的数据转换为Excel格式,最后使用MRC-Holland公司提供的分析软件Coffalyser v9.4来分析数据。10例正常儿童作为分析对照。结果 1.300例CHD患儿中,有5例(1.67%)检测出CITED2基因突变。其余295例(98.33%)没有检测出突变。新发现了两种新的突变(c.481-483delAGC, c.574-579delAGCGGC)。 2.120例CHD患儿中,检出22q11微缺失10例(8.33%),重复1例(0.83%),其余109例(90.84%)没有检测到缺失或重复。10例缺失中,3Mb缺失9例,2Mb缺失1例,包括室间隔缺损3例,法洛四联症5例,肺动脉狭窄1例,右室双出口1例。1例重复CHD类型为肺动脉狭窄。结论 1.在CHD患儿CITED2基因中发现两种突变,两种突变均为国际首次报道。多物种序列对比分析表明CITED2第161、192和193位氨基酸呈现一定的保守性,提示这两种新突变可能通过改变SGJ区蛋白构象导致人类CHD的发生。 2.CHD患儿中存在一定比例的22q11微缺失。MLPA是一种快速、简便且准确的检测22q11微缺失的方法
[Abstract]:Background congenital heart disease (CHD) is a cardiovascular malformation caused by abnormal cardiac vascular development during fetal period, and is also the most common heart disease in children. Its clinical consequences are extremely serious, usually leading to miscarriage, stillbirth, stillbirth and neonatal death. And the incidence of disability in children, adolescents and adults has always been the highest in urban areas, second only to neural tube abnormalities in rural areas, Although much progress has been made in the study of the genetic basis of CHD, because of the wide variety of CHD and the complicated etiology, the etiology of most CHD remains unclear. People in urgent need of multi-level, multi-level, in-depth comprehensive discussion. Objective to analyze the mutation of CITED2 gene coding chain in children with CHD, to detect the occurrence and distribution of 22q11 microdeletion in children with CHD, and to explore the feasibility of multiplex ligation-dependent probe amplification (MLPA) in the diagnosis of CHD22q11 microdeletion. Methods from 2010 to 2011, 300 cases of sporadic CHD were collected from Henan chest Hospital and Zhengzhou Children's Hospital. In this study, 50 normal boys and 50 normal girls had no history of CHD or family history. The genomic DNA of peripheral blood was extracted by non-kit method, and the CITED2 gene was sequenced by polymerase chain reaction (PCR). The results of sequencing were compared with the sequence of CITED2 gene (NM006079.3) published in GeneBank database to identify the mutation. The conserved amino acids of the mutation were analyzed by Clustal X software. MLPA P250 kit (48 pairs of probes) was used to detect 22q11 microdeletion in 120 cases of CHD. Finally, the connected semi-probe was amplified by PCR with a uniform SALSA PCR primer. The PCR product was separated and analyzed by capillary electrophoresis apparatus CEQ8800) and the accompanying software CEQ system. The data analyzed by CEQ system was converted into Excel format. Finally, data were analyzed using Coffalyser v9.4, an analytical software provided by MRC-Holland, to analyze 10 normal children as an analysis control. Results. Of the 1.300 cases of CHD, 5 cases (1.67) were detected CITED2 gene mutation, and the other 295 cases (98.33) were not detected. Two new mutations, c.481-483delAGC, c.574-579delCGCG, were found. 2. Of the 120 cases of CHD, 10 cases with 22q11 microdeletion were found to be 8. 33%, 1 case was repeated with 0. 83% of them, the remaining 10 cases were not detected deletion or repetition. Of 10 cases, 9 cases were missing or duplicated, 9 cases were missing 2 Mb, including 3 cases of ventricular septal defect and 5 cases of tetralogy of Fallot. The results showed that there were 3 cases of ventricular septal defect, 5 cases of tetralogy of Fallot, and 5 cases of Tetralogy of Fallot. Pulmonary artery stenosis in 1 case, double outlet of right ventricle in 1 case, repeat CHD type in 1 case. Conclusion. 1. Two mutations were found in the CITED2 gene of children with CHD, both of which were reported for the first time in the world. The comparative analysis of multiple species sequences showed that the amino acids at position 161,192 and 193 of CITED2 were conserved to some extent. These two new mutations may lead to the occurrence of human CHD by changing the conformation of SGJ domain proteins. 2.There is a certain proportion of 22q11 microdeletions in children with CHD. MLPA is a rapid, simple and accurate method for detecting 22q11 microdeletions.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.4

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