【作者】 李艺扬；

【导师】 付艳；

【作者基本信息】 吉林大学 ， 妇产科学， 2011， 博士

【摘要】 FOXC2基因在先天发育缺陷中的变异分析在胚胎发育过程中,受到环境、遗传等多种因素的影响,胎儿在形态、组织结构及生理功能上的发育受到影响,常发生胎儿流产、死胎或存在出生缺陷,给家庭、社会带来沉重的经济与心理负担。先天性发育缺陷是流产、死胎、死产和新生儿死亡的主要原因之一。我国近年出生缺陷发生率大约波动在11‰～18‰,是严重影响出生人口健康素质的因素。寻找先天性发育缺陷的确切病因,进一步揭示其发病机理,对于开展产前预防、诊断及干预,提高出生人口素质具有重要的意义。人叉头框-C2基因是具有DNA-结合域的翼状螺旋转录因子家族中的一员,在进化上具有高度保守性。定位于16q22- 16q24,只包含一个单独编码的外显子,没有内含子,cDNA全长为1506bp。其编码的蛋白质共有494个氨基酸残基。已有动物实验证明,Foxc2基因在胚胎发育过程中有着重要的作用,敲除Foxc2基因的小鼠可在心血管、软骨组织、肾脏、主动脉弓及眼睛间质等多部位表现出发育异常。人的FOXC2已经被克隆出来,人类的FOXC2基因和小鼠Foxc2基因有95%以上的同源性。人类FOXC2基因与临床的关联性最早是在家族遗传性疾病淋巴水肿-双睫毛综合征中被确认,LD综合征是一种罕见的常染色体显性遗传病。临床特征是下肢水肿和双重睫毛,还有个体合并静脉曲张、先天性心脏病和上睑下垂等,对LD家族的遗传分析发现FOXC2基因的突变和LD综合征发病密切相关。在动物实验中发现Foxc2与淋巴管的发生、中轴骨骼的发育、心血管系统及泌尿系统的发育有关,然而,在人类胚胎发育过程中,该基因的分子生物学表现及功能状况尚不清楚,故对FOXC2在胚胎发生中的作用及与先天发育缺陷的关系等方面进行研究。第1部分先天发育缺陷胎儿的类型分析目的:分析了长春地区先天性发育缺陷的类型,为重点缺陷的预防提供理论依据。方法:收集长春市多家医院妇产科就诊的流产或引产死胎,伴发育缺陷胎儿73例。对明显的发育畸形进行记录。分析异常器官。结果:在73例先天性发育异常胎儿中,男性41例,女性32例。由于在一个胎儿可以发生多组织器官的畸形或缺陷,共发现了93个器官或组织发育畸形,致宫内死胎共32例。神经管异常47例,占全部发育异常胎儿的64.3%(47/73),占发育畸形的构成比例为50.5%(47/93)。心血管发育异常病例19例,占全部发育异常胎儿的26%(19/73);心血管发育异常死胎15例,心血管发育异常儿胎死宫内发生率为78.9%(15/19),占畸形胎儿死胎总数的46.9%(15/32)。结论:在73例先天性发育缺陷胎儿中,男性比例高于女性。神经管发育缺陷是长春地区最主要的先天发育缺陷。心血管发育异常是胎死宫内最主要原因,也是本地区主要的先天发育缺陷。发现与成人LD综合征相似的淋巴水肿胎儿。第2部分FOXC2是人胚胎多器官发育重要的转录因子目的:研究FOXC2是否参与人胚胎发生发育过程。方法:通过免疫组化方法分析了FOXC2在人胚胎发育过程中的作用。结果:FOXC2在人胚胎最早期的头部间充质发生发育过程中起作用。在肋骨软骨的生发中心肋软骨始基细胞核看到FOXC2蛋白强表达。心脏的大血管、主动脉全层、心室内膜及心室壁外层可见到FOXC2的表达。胚胎肾脏的髓质可见到FOXC2的表达。结论:FOXC2基因是中轴骨骼的发育过程中必不可缺少的转录因子;FOXC2在人胚胎心血管系统发生、发育过程中发挥重要的生物功效;人胚胎期肾脏的发育也需要FOXC2参与。第3部分胎儿先天发育缺陷危险因素及FOXC2基因多态性分析目的:了解先天发育缺陷的危险因素及FOXC2基因的变异与先天发育缺陷发生的关系。方法:对73例先天发育缺陷胎儿的相关危险因素进行调查、登记和分析。提取发育缺陷胎儿基因组DNA,通过PCR反应扩增出包含FOXC2 cDNA的目的片段,送检测序,进行基因多态性分析。结果:父母亲的不良生活习惯、家族发育缺陷遗传史、父母特殊工作史、特殊化学试剂接触史、未行孕前检查及孕早期未补充叶酸、维生素等与胎儿先天性发育缺陷的发生密切相关。对68例发育缺陷胎儿成功测序样本进行分析,共存在20个突变位点,其中引起编码氨基酸改变的突变点共15个。此15个有效突变位点集中在12个病例(包括神经管发育异常、心血管发育异常、淋巴水肿及多囊肾胎儿)中,FOXC2基因突变发生率为17.6%(12/68)。结论:孕期多种因素可以导致先天发育缺陷的发生率增加。基因变异分析发现了胚胎发育期神经管、心血管发育异常、淋巴水肿及多囊肾等先天发育缺陷胎儿存在FOXC2基因多点变异;胚胎期淋巴水肿胎儿显示了与成人LD综合征相似的高FOXC2基因突变率;胚胎期发育缺陷胎儿FOXC2基因的有效突变导致编码氨基酸改变,证明FOXC2基因变化与胎儿先天缺陷的发生密切相关。第4部分先天性心脏病危险因素及FOXC2基因多态性分析目的:了解先心病的危险因素及FOXC2基因的变异与先心病发生的关系。方法:对34例先心病患者的相关危险因素进行调查、登记和分析。提取先心病患者基因组DNA,通过PCR反应扩增出包含FOXC2 cDNA的目的片段,送检测序,进行基因多态性分析。结果:父母亲的吸烟、饮酒等不良生活习惯、父母特殊化学试剂接触史、家族发育缺陷遗传史、孕早期服药、孕早期感冒等均与CHD的发生有关联。成功测序29例先心病患者中,引起编码氨基酸改变的突变位点有9个,集中在7例患者中,有效突变率为24.1%(7/29)。结论:孕期多种不良因素可以导致先心病发病率的增加。CHD患者的FOXC2基因突变引起了编码氨基酸的改变,表明FOXC2的基因突变与CHD的发生密切相关;先心病患者与心脏畸形胎儿共同存在FOXC2基因216bp、255bp和505bp位点的突变,证明这三个突变位点是先心病的突变热点。更多还原

【Abstract】 Mutation analysis of FOXC2 gene in congenital developmental defectIn embryonic development, by environmental, genetic and other factors, the fetus development of the appearance, structure and physiological function were affected, fetal abortion, stillbirth or birth defects often exist,it’s a heavy economic and psychological burden to the family and society. Congenital developmental defects were large proportion mortality in the fetal and neonatal. it’s a serious impact on quality of health diathesis. Find the exact cause of congenital defects, and further reveal its pathogenesis, it’s great significance for prenatal prevention, diagnosis and intervention, and improve the quality of births.Human forkhead box c2 (FOXC2),a member of the winged helix transcription factors family with DNA-binding domains, was evolutionarily highly conserved.The positiong was 16q22-16q24, it only Contained a single coding exon, no intron, the length of cDNA was 1506bp. The protein encoded 494 amino acid residues. Animal experiments have proved, Foxc2 gene in embryonic development played an important role. in gene knockout mice showed abnormalities in cardiovascular, cartilage, kidney, aortic arch and the eyes and other parts of the stroma.Human FOXC2 have been cloned, the human FOXC2 gene and the gene in mice Foxc2 had more than 95% homology.The first relevance of the human FOXC2 gene and clinical disease was confirmed in a hereditary disease-lymphedema -distichiasis syndrome.LD syndrome was a rare autosomal dominant genetic disease. The clinical characteristics were lower limb edema and double eyelashes, and the individual merge varicose veins, congenital heart disease and ptosis.The genetic analysis of LD family found that the FOXC2 gene mutations and LD syndrome were closely related. In animal experiments,it has found that Foxc2 was related with lymphangiogenesis, the development of axial skeleton, cardiovascular system and urinary system.However, in human embryonic development, the gene expression of molecular biology and functional status is not clear, So we did the the research,we want to know the relationship with FOXC2’s role in embryogenesis and congenital developmental defects. PartⅠAutopsy type analysis of congenital developmental defects fetalObjective: Analysis of congenital developmental defects in Changchun, and provide the theoretical basis on the prevention of point defects.Methods: Collected 73 cases of developmental defects fetus that were abortion and induced in Obstetrics and Gynecology department of several hospital in Changchun. We recorded obvious malformations and analysis of abnormal organs. Results: In the 73 cases of congenital abnormalities fetus, male were 41 cases, female were 32 cases. Since a fetus could occur malformations or defects in several tissues and organs, 93 cases of malformations were found in different organ and tissue,they caused 32 cases intrauterine fetal death. The largest number of malformations were neural tube defects, 47 cases. accounting for 64.3%(47/73)of fetal abnormalities,the composition ratio of total malformations was 50.5%(47/93).The number of cardiovascular abnormalities were 19, accounting for 26% (19/73)of fetal abnormalities,the composition ratio of total malformations was 20.4% (19/93). Stillbirth of cardiovascular abnormalities were 15 cases, cardiovascular abnormalities fetal death rate was 78.9% (15/19), accounting for the total number of abnormal stillbirth 46.9% (15/32).Conclusion: In 73 cases of congenital developmental defects fetus, the proportion of male was more than female. Neural tube defects was the most important congenital developmental defects in Changchun area. Cardiovascular abnormalities in this region was also the major congenital developmental defects,it was the main reason caused intrauterine fetal death.We found lymphedema fetus similar to adult LD syndrome.PartⅡFOXC2’s a important transcription factor in the human embryonic development of many organsObjective: To know if FOXC2 was involved in the development of human embryos. Methods:We analyzed FOXC2’s role in the human embryonic development by Immunohistochemical methods.Results: In the human embryo,FOXC2 first occurred in the head mesenchyme. Then In the costal rib cartilage primordial germinal center found strong expression of FOXC2 protein in the nucleus. We could found FOXC2 expression in the the great vessels of heart, whole layer of aortic,the ventricular endocardium and the ventricular outer wall. Embryonic kidney medulla also had FOXC2 expression. Conclusion: FOXC2 gene was the indispensable transcription factor in the process of development of central axial skeleton; FOXC2 played an important biological effect in the process of development of human embryonic cardiovascular system; The development of human embryonic kidney also need FOXC2 participation.PartⅢThe analysis of risk factors for fetal congenital developmental defects and FOXC2 gene polymorphismObjective: To understand the relationship of risk factors with congenital developmental defects ,and FOXC2 gene mutation with congenital developmental defects.Methods: The risk factors were investigated, registed and analysis of 73 cases congenital developmental defects fetus.Extract their genomic DNA, amplified FOXC2 cDNA fragment by PCR,and inspect sequencing,then analysis the genetic polymorphism. Results: The occurrence of congenital defects fetus were closely related with parents’ bad habits, family heredity history of developmental defects,parents’ special work history, history of exposure to specific chemical agents, no progestation examination and without supply folic acid and vitamins in early pregnancy. 68 cases of target DNA fragment were successful sequencing,there were 20 mutations, which caused 15 amino acid changes in sites. The 15 effected mutations concentrated in 12 cases (including the neural tube defects, cardiovascular abnormalities, fetal lymphedema and polycystic kidney fetus), FOXC2 gene mutation rate was 17.6% (12/68). Conclusion: Gene mutation analysis found FOXC2 multiple mutation spots in embryogenesis congenital defects with neural tube defects, cardiovascular dysplasia,lymphedema and polycystic kidney.The embryonic period lymphedema fetus showed similar high FOXC2 mutation rate to adult LD syndrome.The FOXC2 mutations of embryonic developmental defectfetus caused change of encoding amino acids,so FOXC2 gene mutations had close relationship.with birth defects.PartⅣThe analysis of risk factors for congenital heart disease and FOXC2 gene polymorphismObjects: To understand the relationship of risk factors with congenital heart disease,and FOXC2 gene mutation with congenital heart disease.Methods: Collected 34 children’s myocardium with congenital heart disease in cardiac surgery of the First Hospital of Jilin University.The risk factors were investigated, registed and analysis of 34 cases CHD patients.Extract their genomic DNA, amplified FOXC2 cDNA fragment by PCR,and inspect sequencing,then analysis the genetic polymorphism. Results: The occurrence of CHD were closely related with parents’ bad habits(smoking and drinking), history of exposure to specific chemical agents, family heredity history of developmental defects, drug administration and flu in early pregnancy. 29 cases of target DNA fragment were successful sequencing,there were 9 mutations, which caused amino acid changes in sites. The 9 effected mutations concentrated in 7 cases, FOXC2 gene mutation rate was 24.1%(7/29).Conclusion: Variety of factors during pregnancy can cause the increased incidence of CHD.FOXC2 gene mutations had close relationship.with CHD. The FOXC2 mutations of CHD patients caused change of encoding amino acids,so FOXC2 gene mutations had close relationship.with CHD.The site of FOXC2 gene in 216bp, 255bp and 505bp mutation common occurred in CHD patients and cardiac defect fetus,it proved that the three mutation sites were congenital heart disease mutation hot spots.更多还原