【作者】 罗成；

【导师】 尹邦良；

【作者基本信息】 中南大学 ， 外科学， 2012， 博士

【摘要】 第一部分先天性心脏病伴肢体畸形相关基因突变分析背景：先天性心脏病是最常见的出生缺陷；肢体畸形是第二位的出生缺陷,即最常见的心外畸形。在涉及先天性心脏病的综合征中,肢体畸形经常与心脏缺陷相伴发生,尤其以上肢畸形多见。己报道的包括心脏和上肢畸形的综合征超过100种。Holt-Oram综合征（HOS）是最常见的心-手综合征,其中约85%的HOS为新发基因突变所致。己知NKX2-5（RefSeq: NM₀04387.3）和GATA4（RefSeq: NM₀02052.3）是相关先天性心脏病的致病基因；已知TBX5（RefSeq: NM₀00192.3）和SALL4（RefSeq: NM₀20436.3）与先天性心脏病伴肢体畸形综合征有关。目的：筛查先天性心脏病伴肢体畸形中TBX5, NKX2-5, GATA4和SALL4四种候选基因突变及突变基因功能分析,以期发现已知突变基因的新的突变形式,并进一步深化对先天性心脏病相关基因突变的致病机制的理解。方法：选取2009年9月至2012年3月中南大学湘雅二医院心脏外科收治的先天性心脏病伴肢体畸形患者23名,包括：先心病伴上肢畸形16例,先心病伴下肢畸形4例,先心病伴上、下肢畸形3例。对所有患者进行详细的临床检查包括经胸壁心脏彩超和心电图检查,对部分患者行缺陷肢体X-线摄片检查和腹部超声检查。采集所有患者的和部分患者父母的外周血提取gDNA,对TBX5, NKX2-5, GATA4和SALL4基因各外显子进行PCR扩增,然后对扩增的目的片段进行测序,并对基因突变进行功能预测分析。结果：通过对患者缺陷肢体的X-线摄片,明确其缺陷肢体的骨骼畸形。通过基因突变分析,在1名部分型房室间隔缺损合并多指（趾）畸形（24指）的患者中发现一个新的NKX2-5错义突变c.257T>C （p.F86S）;在其表型正常的父亲发现一个新的NKX2-5同义突变c.186T>A （p.A62A）,并携带与女儿相同的错义突变；母亲无NKX2-5突变。结论：NKX2-5错义突变c.257T>C （p.F86S）可以导致先天性心脏病,部分型房室间隔缺损。第二部分先天性心脏病伴肢体畸形基因组拷贝数变异研究背景：已有研究证明拷贝数变异（CNv）可以导致先天性心脏病,而且研究结果一致显示在伴有心外畸形的先天性心脏病,即综合征型先天性心脏病中的致病性CNV发生率更高。但是,目前尚无专门关于先天性心脏病伴肢体畸形相关综合征的CNV研究。目的：1.探讨致病性CNV与先天性心脏病伴肢体畸形的关系及发生率。2.发现其新的致病性CNV,定位新的与先天性心脏病伴肢体畸形相关的候选基因。方法：选取2009年9月至2012年3月中南大学湘雅二医院心脏外科收治的先天性心脏病伴肢体畸形患者19名,包括：先心病伴上肢畸形12例,先心病伴下肢畸形4例,先心病伴上、下肢畸形3例。通过Array-SNP芯片技术,对所有患者和部分患者父母行全基因组CNVs分析,寻找可能与心脏-肢体畸形相关的CNV区域。结果：在3例心脏-肢体畸形患者中发现3个致病性CNV,分别为染色体3p25.2微重复,16p13.3微重复和22q11.21微缺失。其中包含RAF1基因的3p25.2微重复,属于首次被发现和Noonan综合征有关。结论：1.包括RAF1的染色体3p25.2微重复可以导致Noonan综合征。2.扩展了16p13.3微重复综合征的表型谱,增添了膈膨升和小便失禁。更多还原

【Abstract】 SECTION1:Congenital Heart Defects with Limb Anomalies Related Genes Mutation Detection and Functional AnalysisBackground:Congenital heart defects are the most common birth defect. Limb deformities are the most common extracardiac malformation which is considered as the second most common birth defect. Among all the syndromes of cardiac malformations, congenital heart defects and limb deformities often occur in combination, especially upper limb deformities. More than100syndromes including the heart and upper limb malformations have been reported. The known genes associated with heart and/or limb deformities include NKX2-5（RefSeq:NM₀04387.3）, GATA4（RefSeq:NM₀02052.3）, TBX5（RefSeq:NM₀00192.3） and SALL4（RefSeq:NM₀20436.3）Objective:To investigate the mutation frequencies of four candidate genes TBX5, NKX2-5, GATA4, and SALL4and functional analysis.Methods:Twenty-three congenital heart defect patients with limb anomalies were recruited in the Department of Cardiothoracic Surgery of Second Xiangya Hospital from Sep2009to Mar2012, which included sixteen congenital heart defect patients with upper limb anomalies, four congenital heart defect patients with lower limb anomalies and three congenital heart defect patients with both upper and lower limb anomalies. All patients underwent detailed clinical examination including transthoracic echocardiography and ECG, and a part of patients underwent limb X-ray examination and abdominal ultrasound examination. DNA of all patients and some patients’parents was extracted from the peripheral blood for PCR amplification, then TBX5, NKX2-5, GATA4, and SALL4gene sequencing and functional prediction analysis.Results:By X-ray of the defective limbs, defects in skeletal deformities of the limbs were identified. By mutation analysis, a novel missense mutation of NKX2-5c.257T>C （p.F86S） was detected in a patient diagnosed as partial atrioventricular septal defect associated with polydactyly （24digits）. Her father who carried a missense mutation c.257T>C （p.F86S） and synonymous mutation c.186T>A （p.A62A） was phenotypically normal. And her mother bears no NKX2-5mutation.Conclusions:Missense mutation of NKX2-5c.257T>C （p.F86S） could lead to congenital heart defect, partial atrioventricular septum defects. SECTION2:Investigation of CNVs Associated with Congenital Heart Defects with Limb AnomaliesBcakgroud:Copy number variations （CNVs） is known to be associated with congenital heart defects. And it is indicated that pathogenic CNVs more frequently associated with congenital heart defect with extracardiac defects. But now there is no investigation of CNV with congenital heart defects with limb anomalies.Objective:To investigate the relationship between the pathogenic CNVs and congenital heart defects with limb deformities, discover the novel pathogenic CNVs, and ascertain the new candidate genes associated with congenital heart defects with limb deformities.Methods:Nineteen out of twenty-three congenital heart defect patients with limb anomalies were selected in the Department of Cardiac Surgery of Second Xiangya Hospital from Sep2009to Mar2012, which included twelve congenital heart defect patients with upper limb deformities, four congenital heart defect patients with lower limb deformities, and three congenital heart defect patients with both upper and lower limb deformities. In order to seek CNVs regions related to the heart-limb deformities, whole genome CNVs were analyzed in all patients and some patients’parents via Array-SNP chip technology. Results:Three pathogenic CNVs were found in the three congenital heart disease patients with limb anomalies, which were chromosome3p25.2microduplication,16p13.3microduplication and22q11.21microdeletion respectively.Conclusions:1. Microduplication of3p25.2encompassing RAF1associated with congenital heart disease suggestive of Noonan syndrome.2. Our study extends the phenotypic spectrum of the16p13.3microduplication syndrome and increases the phenotype of diaphragmatic eventration and urine incontinence.更多还原