先天性心脏病胎儿心肌转录因子GATA-4基因突变检测

目的分析先天性心脏病胎儿中GATA-4基因的突变情况，期望为进一步阐明先天性心脏病发病的分子机理提供新的实验依据。方法应用PCR—DNA测序技术在20例先心病胎儿病变部位的心肌组织中筛查GATA-4基因突变。结果20例中，在1例先心病胎儿（ASD、VSD）中检测到一个突变（V267M），位于外显予4；在3例先心病胎儿中发现编码241位氨基酸（半胱氨酸）密码子的第三位碱基均存在杂合同义突变（C→T），位于外显子3，并证实是一个单核苷酸多态性（SNP）位点（rsl062215）。结论GATA-4基因突变（V267M）可能与胎儿先天性心脏病发病有关：GATA-4723C／T基因多态性（rs1062215）与胎儿先天性心脏病的关系有待进一步证实。     

房室间隔畸形患者GATA-4基因一个新的突变功能初步研究

目的 探讨先天性房室间隔畸形患者GATA-4基因的突变以及对所发现的突变点进行功能初步分析.方法 选择的50例汉族先天性房室间隔畸形患者,用聚合酶链反应方法扩增GATA-4基因6个外显子编码区和邻近序列后分别直接测序,并与100名同民族健康者比较;利用生物信息学方法预测点突变对GATA-4蛋白结构和功能影响;以野生型pcDNA3.1-GATA4为模板,用基因定点突变的方法构建突变型GATA-4真核表达载体,在体外和ANF报告质粒共转染Hela细胞,48 h后检测下游报告基因的活性.结果 发现GATA-4基因一个新的杂合子H436Y突变,在NCBI的SNP数据库中未见报道,且在100名对照者中也未发现;GATA-4基因编码第436位组氨酸在生物进化过程中处于高度保守;SIFT和PolyPhen程序预测H436Y突变可能影响GATA-4蛋白质的功能;突变型GATA-4 H436Y质粒激活下游报告基因活性明显下降,与野生型GATA-4质粒相比是(53.8±6.6)%,差异具有统计学意义(P<0.01).结论 GATA-4基因H436Y杂合子突变可能是中国汉族先天性房室间隔畸形致病原因之一.     

山东地区先天性心脏病患者CFC1基因突变研究

目的对山东地区126例先天性心脏病患者进行CFC1基因突变筛查,阐明CFC1基因突变类型和特点,为先天性心脏病患者的基因诊断和基因治疗提供理论依据。方法．从126例先天性心脏病患者外周血白细胞中提取基因组DNA,进行PCR扩增CFC1第2和3外显子,对PCR产物进行直接测序分析。结果在126例先天性心脏病患者患儿的CFC1基因第2、3外显子测序中,均未发现基因突变。结论CFC1基因突变率低,可能不是山东地区先天性心脏病患儿的主要致病基因。     

GATA4 mutations in 486 Chinese patients with congenital heart disease

Recent studies have reported germline mutations in GATA4 gene in some types of congenital heart disease (CHD). However, the prevalence of GATA4 mutations in CHD and the correlation between the GATA4 genotype and CHD phenotype have not been extensively studied. We screened germline mutations in the coding exons and the flanking intron sequences of the GATA4 gene in 486 CHD patients by denaturing high-performance liquid chromatography (DHPLC), and confirmed the mutations by sequencing. Nine distinct mutations including one small deletion mutation (46delS), two small insertion mutations (118–119insA and 125–126insAA), and six non-synonymous mutations (A6V, P163S, E359K, P407Q, S429T and A442V) were identified in 12 of the 486 patients (nine with ventricular septal defect, two with Tetralogy of Fallot, and one with endocardial cushion defect). Of them, two patients carrying E359K mutation were from two generations in one family with ventricular septal defect (VSD). Interestingly, a nucleotide insertion of c.1146 + 25insA in exon 6 was detected in five VSD patients, but not in 486 normal healthy controls. Our findings are useful in understanding the prevalence of GATA4 mutations and the correlation between the GATA4 genotype and the CHD phenotype in Chinese patients.     

HEXIM1在先天性心脏病室间隔缺损中的突变及表达研究

目的通过筛查HEXIM1基因在室间隔缺损(ventricular septal defect,VSD)外周血中的突变和心肌组织中的表达情况,探讨HEXIM1基因与VSD发病机制的关系。方法采用PCR-DNA测序技术对100例单纯性室间隔缺损的患儿外周血进行基因编码序列突变筛查;以β-actin为内对照,用RT-PCR方法检测HEXIM1基因在14例室间隔缺损引产胎儿中mRNA的表达情况。结果所有研究对象的HEXIM1基因测序后同GenBank人类HEXIM1编码序列进行比较,有3例患儿(单纯性室间隔缺损)分别存在单核苷酸的多态性(SNP);与正常心肌组织相比,VSD引产胎儿心肌组织中HEXIM1基因mRNA表达呈下降趋势(P<0.05)。结论本实验收集的病例标本中没有发现HEXIM1基因编码区的突变,基因转录水平异常可能是该基因参与VSD形成的一种潜在机制。     

先天性心脏病术后患儿6分钟步行试验前后心电参数对照研究

目的 研究先天性心脏病（CHD）患儿术后中远期随访6 min步行试验（6MWT）前后心电参数的变化.方法 选取复旦大学附属儿科医院心血管中心经心脏外科手术纠治的年龄≥3岁且随访时间≥6个月的CHD患儿为CHD组;选取健康儿童作为对照组.将CHD组和对照组同时分为3～5岁、～8岁、～12岁和～15岁亚组.CHD组根据CHD类型分为复杂CHD亚组和简单CHD亚组.比较各组6MWT前后P波离散度（Pwd）、QRS时限和QT离散度（QTd）变化.结果 CHD组89例,男51例,女38例,行外科手术时的平均年龄为（3.0±2.3）岁（36 d至11.0岁）,术后平均随访时间为（3.6±3.2）（0.5～14.2）年.3～5岁、～8岁、～12岁和～15岁亚组分别为39、27、11和12例.复杂和简单CHD亚组分别为29和60例.对照组133名（男75名,女58名）,3～5岁、～8岁、～12岁和～15岁亚组分别为26、39、39和29名.①CHD组和对照组各年龄亚组6MWT后Pwd、QRS时限和QTd均较6MWT前减小,但差异无统计学意义（均P〉0.05）.②CHD组3～5岁和～12岁亚组6MWT前QTd均较对照组显著增大,3～5岁亚组：（0.025±0.018） vs （0.012±0.011） s,P＝0.004;～12岁亚组：（0.029±0.014） vs （0.019±0.012） s,P＝0.019.CHD组各年龄亚组6MWT后QTd与对照组比较差异无统计学意义（均P〉0.05）.③CHD组～8岁和～15岁亚组6MWT前后QRS时限均较对照组显著延长（P分别为0.000和0.007）;各年龄亚组6MWT后差异均有统计学意义.④复杂CHD亚组6MWT前后QRS时限均较简单CHD亚组延长,6MWT前：（0.102± 0.025） vs （0.080 ± 0.021） s,P=0.000;6MWT后：（0.103 ± 0.026） vs （0.080 ± 0.021） s,P=0.000,两亚组6MWT前后Pwd和QTd差异均无统计学意义.结论 CHD术后中远期随访患儿心脏电活动仍不稳定,但其6MWT前后心电参数Pwd、QRS时限及QTd的变化趋势与健康儿童相一致,提示亚极量运动有助于增强心脏电活动的稳定性.     

P-selectin gene polymorphism associates with pulmonary hypertension in congenital heart disease

Objective: To investigate the relationship between P-selectin gene polymorphism and congenital heart disease (CHD) with pulmonary hypertension (PAH). Methods: 58 CHD patients with PAH (PAH-CHD), 43 CHD patients without PAH and 205 healthy subjects were included in this study. The concentration of plasma P-selectin was determined by ELISA kits; the direct sequencing of PCR products was used to analyze the P-selectin genotypes. Results: The concentration of plasma P-selectin was markedly higher in PAH-CHD patients than that in CHD subjects and controls, while no difference was observed between CHD group and control. A significant difference of P-selectin genotype -825T/C polymorphism was observed between patients with PAH-CHD and healthy subjects (P<0.05). Logistic analysis showed that the subjects with haplotypes A-G and G-G had lower risk of PAH-CHD compared with the ones with haplotype A-A (OR=0.47, 95% CI=0.24-0.92). In the subjects of PAH-CHD and control, plasma P-selectin concentration was higher in subjects with -825TT genotype than the ones with haplotypes T-C and C-C (P<0.05). Conclusion: P-selectin probably involves in the development of PAH-CHD. The polymorphism of -825T/C is associated with the risk of PAH-CHD, and may be one of its risk factors.