继发孔房间隔缺损一家系CSX/NKX2.5基因突变研究

目的研究一个继发孔房间隔缺损（ASD）家系同源框转录因子基因CSX／NKX2.5的突变情况。方法收集一个单纯继发孔房间隔缺损家系的临床资料,采用聚合酶链反应及直接测序法对该家系内成员进行CSX／NKX2.5基因突变检测,同时对126名家系外健康对照者的该位点进行单链构象多态性（SSCP）分析。结果该家系中4例ASD患者均存在CSX／NKX2.5基因的3个杂合突变,且3者均为谷氨酸转换为赖氨酸（GAG-AAG）：G270A（Glu32Lys）,G378A（Glu68Lys）和G390A（Glu72Lys）。而在家系内非患者及正常对照者中均未发现该3者突变。结论在中国人一单纯继发孔房间隔缺损家系中发现的CSX／NKX2.5突变,可能是导致此家系房间隔缺损的重要原因。     

应用遗传连锁分析法对伴传导功能障碍的扩张型心肌病家系致病基因的定位

目的: 对一个常染色体显性遗传扩张型心肌病(familial dilated cardiomyopathy,FDCM)家系进行基因定位。方法: 收集FDCM 家系, 对该家系成员进行详细心血管内科检查确诊为扩张型心肌病,且伴发有传导功能障碍;采集外周血3～5 mL,并抽提基因组DNA;选取与该表型相关的已定位区间CMD1A(1q21.2-q21.3),CMD1H(2q14-q22), CMD1E(3p22-p25)和CMD1F(6q22-23)内的共计18个微卫星DNA标记,在该家系中进行排除性定位分析;最后,进行全基因组扫描及连锁分析。结果：①已定位区间的18个微卫星DNA标记位点的LOD值均<-2,证实该家系与已知DCM位点不连锁;②全基因组扫描及两点连锁分析结果显示,该家系致病基因位点与遗传标记D3S1614(3q26)连锁, 在θ= 0时得到最大LOD值2.68。结论: 该家系与已知的4个DCM位点均不连锁,其致病基因位于D3S1614（3q26）附近的一个新位点。     

GATA4 and NKX2.5 gene analysis in Chinese Uygur patients with congenital heart disease

Background Congenital heart disease （CHD） is the most common developmental anomaly in newborns. The germline mutations in GATA4 and NKX2.5 genes have been identified as responsible for CHD. The frequency of GATA4 and NKX2.5 mutations in Chinese Uygur patients with CHD and the correlation between their genotype and CHD phenotype are unknown.
Methods We examined the coding region of GATA4 and NKX2,5genes in 62 Chinese Uygur patients with CHD and 117 Chinese Uygur individuals as the controls by denaturing high pedormance liquid chromatography （DHPLC） and sequencing.
Results Two heterozygous missense mutations of c.1220C〉A and c.1273G〉A in GATA4 gene, which cause the amino acid residue changes of P407Q and D425N in GATA4, were found in a patient with tetralogy of Fallot and a patient with ventricular septal defect, respectively. The two patients did not have atrioventricular conduct defects or non-cardiac abnormalities. The two mutations are expected to affect the protein function. There were no reported NKX2.5 mutations in the patients.
Conclusion Our results provided the primary data on CHD phenotype associated with GATA4 mutation in the Chinese Uygur population.     

A novel candidate locus on chromosome 11p14.1-p11.2 for autosomal dominant hereditary spastic paraplegia

Background Hereditary spastic paraplegia （HSP） is a group of inherited neurodegenerative disorders with the shared characteristics of slowly progressive spasticity and weakness of the lower limbs. Thirteen loci for autosomal dominant HSP have been mapped. Methods A Chinese family with HSP was found in the Shandong province and Inner Mongolia Autonomous Region of China and genomic DNA of all 19 family members was isolated. After exclusion of known autosomal dominant loci, a genome wide scan and linkage analysis were performed. Results The known autosomal dominant loci of SPG3A, SPG4, SPG6, SPG8, SPG9, SPG10, SPG12, SPG13, SPG17, SPG19, SPG29, SPG31 and SPG33 were excluded by linkage analysis. The results of a genome wide scan demonstrated candidate linkage to a locus on chromosome 11 p14.1-p11.2, over an 18.88 cM interval between markers D11 S1324 and D11 S1933. A maximal, two point LOD score of 2.36 for marker D11S935 at a recombination fraction （e） of 0 and a multipoint LOD score of 2.36 for markers D11S1776, D11S1751, D11S1392, D11S4203, D11S935, D11S4083, and D11S4148 at θ=0, suggest linkage to this locus. Conclusion The HSP neuropathy in this family may represent a novel genetic entity, which will facilitate discovery of this causative gene.     

先天性室间隔缺损患者NKX2-5基因新突变的识别

目的 识别先天性室间隔缺损(VSD)患者新的分子遗传缺陷.方法 收集2006年3月至2008年6月在上海儿童医学中心、上海同济医院和上海市儿童医院160例先天性VSD患者的临床资料和血标本,以200名健康者为对照.应用聚合酶链反应扩增NKX2-5基因的全部外显子,采用双脱氧核苷链末端合成终止法对全部扩增片段进行测序.借助BLAST程序将所测序列与GenBank中的已知序列进行比对以识别基因突变,并用Clustal W软件分析突变氨基酸的保守性.结果 在3例VSD患者的NKX2-5基因识别出2个新的杂合错义突变,即2例VSD患者的第179位密码子TCC变为TTC,导致第179位的丝氨酸变为苯丙氨酸,1例VSD患者的第36位密码子CGC变为AGC,导致第36位的精氨酸变为丝氨酸.200名健康对照者均无此突变,多物种NKX2-5序列比对显示突变氨基酸在进化上均高度保守.此外,还发现了2个不改变氨基酸的单核甘酸多态,即常见的c.63A>G多态和少见的c.606G>C多态,但这些多态在VSD患者和健康对照者间的分布差异均无统计学意义(c.63A>G多态:X2=3.403、P=0.0651,c.606G>C多态:X2=3.278、P=0.0702).结论 在先天性VSD患者识别出新的NKX2-5基因突变,揭示了VSD新的分子病因.     

两个房间隔缺损家系的GATA4基因分析

目的探讨GATA4基因变异与房间隔缺损遗传的相关性.方法采集云南省心血管病医院心脏外科2个房间隔缺损（ASD）家系,共4名ASD患者和11名成员,选择GATA4基因编码区内4个可引起氨基酸改变的cSNP和GATA4基因附近的11个STR位点以及5个GATA4基因内STR位点,进行PCR和DNA测序分析.结果 D8S258、D8S552两个位点存在多态性,Q19E（rs1139240）、L39V（rs1139241）、P66A（rs1139244）和G377S（rs3729856）位点未发现多态性及突变.结论 D8S258、D8S552在本文人群中检测到多态但未检测到突变,4个cSNP位点未检测到突变。     

Linkage analysis of five Chinese families with arrhythmogenic right ventricular cardiomyopathy using microsatellite genetic markers

Objective To explore the linkage relationship between specific genetic markers and arrhythmogenic right ventricular cardiomyopathy (ARVC) in Chinese pedigrees. Methods The microsatellite genetic markers D2S152, D14S252, and D10S1664 were studied for their linkages to ARVC in five Chinese ARVC pedigrees and a normal population of 121 Chinese individuals. Genomic DNA of the pedigrees and normal population was amplified using PCR techniques. Denaturing polyacrylamide sequencing gel (4%) electrophoresis was used to detect microsatellite repeat polymorphisms. Gels were silver-stained. A classical linkage analysis program was used assuming models of autosomal dominance and recession. Results The logarithm of the odds (LOD) scores of D2S152 with ARVC in LW, WD, DS, LC and TY pedigrees were 2.174, －0.589, －∞, － （indicating that linkage is not supported in this mode）, and －∞ respectively in autosomal dominant model （recombination fraction=0.000 respectively）and were －∞, －∞, －∞, －∞, and 0.182 respectively in the autosomal recessive model. The LOD scores of D14S252 with ARVC in LW, WD, DS, LC and TY pedigrees were －, －, －∞, －, and 0 respectively in autosomal dominant model, and were －∞, －0.812, －∞, －∞, and 0.087 respectively in autosomal recessive model. The LOD scores of D2S152 with ARVC in LW, WD, DS, LC and TY pedigrees were －, －0.539, －, and 0.602 respectively in autosomal dominant model and were －, －∞, －∞, －∞, and －∞ respectively in autosomal recessive model. Conclusions The LOD score for D2S152 in the LW pedigree was 2.174, indicating that the chance of linkage is about 150∶1. This suggests that there is a possible ARVC-related gene near this marker. There were no clear linkage relationships between ARVC and D10S1664 and D14S252 in this family, and no linkages between ARVC and any of the three genetic markers in the other four families. These results also suggest that there is genetic heterogeneity in LW and in the other pedigrees.     

Association of the GLI gene with ventricular septal defect after the susceptibility gene being narrowed to 3.56 cM in 12q13

Background Our previous research has suggested that genes around D12S1056 in 12q13 may confer susceptibility to ventricular septal defect （VSD） in humans. The present study was to define the chromosome region assignment by transmission disequilibrium test （TDT）, and to identify the important candidate gene by family-based association study and haplotype analysis. Methods Surrounding D12S1056, ten microsatellite markers including D12S329, D12S305, D12S1662, D12S1056, D12S1293, D12S334, D12S102, D12S83, D12S1655 and D12S1691 were chosen, and TDT was performed in 62 nuclear family trios each consisting of an affected child and two healty parents. Subsequently, the GLI gene, a positional candidate gene that maps to the target region, was selected for further analysis. Three single nucleotide polymorphisms （SNPs）, G11888C, G11388A, and G11625T, were selected for family-based association study and haplotype analysis. Results VSD was significantly associated with all selected markers except D12S1691 [72.2 centi morgen （cM）] and D12S1700 （75.76 cM）. VSD was also significantly associated with G11888C （X^2 = 5.918, P = 0.015）, G11388A （X^2 = 8.067, P = 0.005）, and G11625T （X^2 = 11.842, P = 0.001）. Haplotype analysis showed a strong linkage disequilibrium between G11888C and G11388A （D＇=0.999）, but in significant （X^2 = 1.035, df = 2, P 〉 0.05）. Conclusions The susceptibility gene of VSD was mapped to 3.56 cM in 12q13 by TDT, and the GLI gene, an important candidate in the target region, was associated with VSD.     

中国人遗传性并指畸形家系HOXD13基因突变鉴定

目的:研究一个中国人并指(趾)畸形(synpolydactyly,SPD)家系,检测家系成员中是否存在同源盒D13 基因(homeobox D13,HOXD13 )突变.方法:采集SPD 患者及其家系成员的外周血,提取基因组DNA,设计针对HOXD13 基因特异性引物,进行PCR 扩增,产物经2%琼脂糖凝胶电泳检测,并进行测序鉴定.结果:本家系3代共23人, 6人患病,研究发现患者HOXD13 基因第一个外显子中多聚丙氨酸链由15个延长至22个,第二个外显子基因序列正常.结论:首次揭示中国人并指(趾)畸形家系HOXD13 基因存在多聚丙氨酸延展增加7个丙氨酸的突变形式,表明一定数目的丙氨酸延展可能是导致并指(趾)畸形的重要因素.     

Mapping of a gene for severe pediatric gastroesophageal reflux to chromosome 13q14

CONTEXT: Gastroesophageal reflux (GER) has not previously been widely regarded as a hereditary disease. A few reports have suggested, however, that a genetic component may contribute to the incidence of GER, especially in its severe or chronic forms. OBJECTIVE: To identify a genetic locus that cosegregates with a severe pediatric GER phenotype in families with multiple affected members. DESIGN: A genome-wide scan of families affected by severe pediatric GER using polymorphic microsatellite markers spaced at an average of 8 centimorgans (cM), followed by haplotyping and by pairwise and multipoint linkage analyses. SETTING: General US community, with research performed in a university tertiary care hospital. SUBJECTS: Affected and unaffected family members from 5 families having multiple individuals affected by severe pediatric GER, identified through a patient support group. MAIN OUTCOME MEASURES: Determination of inheritance patterns and linkage of a genetic locus with the severe pediatric GER phenotype by logarithm-of-odds (lod) score analysis, considering a lod score of 3 or greater as evidence of linkage. RESULTS: In these families, severe pediatric GER followed an autosomal dominant hereditary pattern with high penetrance. A gene for severe pediatric GER was mapped to a 13-cM region on chromosome 13q between microsatellite markers D13S171 and D13S263. A maximum multifamily 2-point lod score of 5.58 and a maximum multifamily multipoint lod score of 7.15 were obtained for marker D13S1253 at map position 35 cM when presumptively affected persons were modeled as unknown (a maximum multipoint score of 4.88 was obtained when presumptively affected persons were modeled as unaffected). CONCLUSION: These data suggest that a gene for severe pediatric GER maps to chromosome 13q14. JAMA. 2000;284:325-334     

全面性癫痫伴热性惊厥附加症家系致病基因的连锁定位和突变分析

目的 筛查中国全面性癫痫伴热性惊厥附加症(GEFS+)家系的致病基因.方法 采集2个GEFS+家系所有成员的外周静脉血提取基因组DNA,选取GEFS+的候选基因(SCN1B、SCN1A、SCN2A和GABRG2)附近10个微卫星位点用于遗传连锁分析,连锁分析所用的软件为LINKAGE软件包5.1版,根据两点间的LOD值判断连锁关系,以确定两家系致病基因的大致位置.限定性定位后筛选候选致病基因,并对家系所有成员进行候选基因突变分析.结果 标记SCN1A、SCN2A和SCN1B基因的多个微卫星位点在两家系患者中均没有共享等位基因,基本排除两家系与上述3个基因连锁可能.田氏家系在标记GABRG2基因的微卫星位点D5S820、D5S422和D5S1403均有共享等位基因.经两点间连锁分析,在外显率为70%,重组率为0时,D5S820、D5S422和D5S1403处的LOD值分别为0.67,1.00和0.79,提示可能有连锁关系.邸氏家系仅在标记GABRG2基因的微卫星位点D5S1403有共享等位基因.对两家系GABRG2基因9个外显子测序结果 显示,第5号外显子出现一个单核苷酸同义多态位点(c.588C>T),第3号外显子出现一个单核苷酸多态位点(c.604C>T),第7号外显子的非编码区出现一个单核苷酸多态位点,为G/A杂合性改变,未发现GABRG2基因致病突变.结论 我国新发现的两个GEFS+家系的致病基因与目前已知候选基因SCN1B、SCN1A、SCN2A和GABRG2无关.GEFS+家系的常见致病基因仍不清楚.     

CHD病人心肌细胞凋亡及bcl-2家族蛋白和caspase-3的调节作用

目的：观察CHD病人心肌细胞凋亡情况及bcl-2、bcl-xl、bax和bad及caspase-3和心肌氧化性应激对心肌细胞凋亡的调节作用。方法：研究以进行常规CPB手术无肺动脉高压的ASD病人为对照组（ASD组，n=5）、以合并CHD的RHD病人为观察组（RHD组,n=5）。并行循环后留取右心室全层心肌标本。用原位TUNEL技术检测凋亡心肌细胞。用流式细胞仪测定右心室bcl-2、bcl-xl、bax和bad蛋白表达。分别用Z－DEVD－AMC底物降解法和TBARS方法测定右心室caspase-3活性和MDA含量。结果：CHD病人右心室心肌细胞凋亡数、caspase-3活性和MDA含量均明显高于ASD病人（分别P＝0．026，0．013和0．002）；与ASD病人比较，CHD病人右心室促凋亡蛋白bax和bad表达明显上调（P＜0．003和0．001）。而两组抗凋亡蛋白bcl-2和bcl-xl表达之间无显著性差异（均P＞0．01）。结论：心肌细胞凋亡参与CHD的病理生理过程。CHD病人的心肌细胞凋亡与心肌细胞bax和bad蛋白表达上调、caspase-3激活以及心肌氧化性应激增高有关。     

房间隔缺损封堵术后BNP水平变化与左室功能的关系

目的观察房缺修补术后BNP水平变化与左室功能的关系。方法选择我院成功实施经导管ASD封堵治疗术的24例患者,测量其术前、术后3d、术后3、6、12个月的LVEDD、LVESD、LVEDV、LVESV、LVEF及BNP水平。结果房缺修补术后,LEVDD和LVEDV增大,左室前负荷改善;LVSV、LVEF及LVFS增大,左室功能改善,其变化与BNP水平负相关。结论房缺修补术可以纠正血流动力学异常,改良左室结构,对左心功能改善产生长期有利影响。     

中国汉族人群2号染色体上11个STR位点的遗传多态性分析

目的：探讨2号染色体上11个STR位点在中国汉族人群中的遗传多态性。方法：利用PCR结合变性聚丙烯酰胺凝胶电泳方法，对100名无血缘关系的中国汉族个体进行基因型分析。结果：在100名汉族人群中，D2S1780、D2S1360、D2S1788、D2S441、D2S436、D2S1328、D2S1326、D2S1353、D2S1364、D2S1363和D2S427分别观察到6、6、12、8、8、7、9、9、6、6和5个等位基因，杂合度分别为O．65、O．61、O．85、O．77、O．65、O．69、O．86、O．87、O．75、O．70和O．78。结论：本研究所分析的2号染色体上11个STR位点均具有较强的遗传多态性，是进行连锁分析等分析工作的理想位点。     

肝豆状核变性基因连锁图谱的研究

为寻找适于中国人Ｗｉｌｓｏｎ氏病（ＷＤ）基因连锁分析的遗传标记及制定ＷＤ基因所在区域的遗传图谱，我们应用Ｄ１３ｑ１４－２１区域４个ＤＮＡ标记对７５名无亲缘关系个体及９个ＷＤ家系成品进行连锁分析。结果发现４个ＤＮＡ标记中３个标记等位片段与白种人相同而杂合率相异，其中２个ＤＮＡ标记Ｄ１３Ｓ３１，Ｐ１２３Ｍ１．８与ＷＤ基因存在紧密连锁关系。其遗传顺序为：着丝点－Ｒｂ－Ｄ１３Ｓ３１－ＷＮＤ。     

Genetic heterogeneity for familial hypertrophic cardiomyopathy in Chinese: analysis of six Chinese kindreds

OBJECTIVE: Familial hypertrophic cardiomyopathy (FHCM) is a primary myocardial disease characterized by unexplained ventricular hypertrophy. The application of the techniques of reverse genetics has identified at least five chromosomal loci as the major causes for FHCM in diverse ethnic populations, suggesting substantial genetic heterogeneity for FHCM. Recently, the defective gene loci of two Chinese families with FHCM have been mapped to chromosome 11 and 14q1, respectively. For further understanding of the molecular basis of FHCM in Chinese, we analyzed the linkage between four other Chinese kindreds and DNA markers from chromosome 14q1. METHODS: Six unrelated Chinese families with FHCM, including two previously reported, were studied. Totally 90 family members were included for analysis. DNA from 80 individuals was extracted and polymerase chain reactions were performed using the primers designed according to the sequences derived from the alpha and beta myosin heavy chain gene. Totally four polymorphisms were studied, including three polymorphic microsatellite sequences and one single strand conformation polymorphism. Genetic linkage analysis were performed using the Linkage program. RESULTS: In the six studied families, 39 of the 90 family members were found to be affected diagnosed either by echocardiography or by clinical evaluation. The pattern of inheritance in all six studied families was most consistent with an autosomal dominant trait with a high degree of penetrance. Genetic linkage analysis using polymorphisms on the alpha and beta MHC genes showed a combined maximal lod score of 6.2 for trinucleotide repeat polymorphism AMHC-I 15 at theta = 0.00 for three studied families without recombination. Exclusion of linkage to the chromosome 14q1 location was noted in two of three other families with the maximal lod score of -2 or less. CONCLUSIONS: These results provide further evidence that FHCM in Chinese is genetically heterogeneous. Chromosome 14q1 locus, probably the beta myosin heavy chain gene, is important as the molecular basis for FHCM in Chinese.     

A missense mutation S228P in the CRYBB1 gene causes autosomal dominant congenital cataract

Background Congenital cataract is a highly heterogeneous disorder at both the genetic and phenotypic levels. This study was conducted to identify disease locus for autosomal dominant congenital cataracts in a four generation Chinese family. Methods Family history and clinical data were recorded. All the members were genotyped with microsatellite markers which are close to the known genetic loci for autosomal congenital cataracts. Two-point Lod scores were obtained using the MLINK of the LINKAGE program package （vet 5.1）. Candidate genes were amplified by polymerase chain reaction （PCR） and direct cycle sequencing. Results The maximum Lod score of Zmax=2.11 was obtained with three microsatellite markers D22S258, D22S315, and D22S1163 at recombination fraction θ= 0. Haplotype analysis showed that the disease gene was localized to a 18.5 Mbp region on chromosome 22 flanked by markers D22S1174 and D22S270, spanning the β-crystallin gene cluster. A c.752T→C mutation in exon 6 of CRYBB1 gene, which resulted in a heterozygous S228P mutation in predicted protein, was found to cosegregate with cataract in the family. Conclusions This study identified a novel mutation in CRYBB1 gene in a Chinese family with autosomal dominant congenital cataract. These results provide strong evidence that CRYBB1 is a pathogenic gene for congenital cataract.     

Dynamic observation on systolic time intervals in children with ventricular septal defect,atrial septal defect and tetralogy of Fallot before and after Radical Surgery

Summary Dynamic observations on systolic time intervals (STIs) have been undertaken in 24 children with ventricular septal defect (VSD), in 16 with atrial septal defect (ASD) and in 12 with tetralogy of Fallot (TF) before and after radical surgery by simultaneous recording of electrocardiogram, phonocardiogram and carotid pulse tracing. In comparison with the STIs in normal children, the left ventricular ejection time index (LVETI) was significantly shortened, the pre-ejection period index (PEPI) significantly prolonged, and the ratio index of PEP to LVET (PEPI/LVETI) and that of isovolumic contraction time (ICT) to LVET (ICTI/LVETI) were significantly increased in the three groups of congenital heart disease before surgery, indicating definite impairment of the left ventricular performance. The dynamic changes after surgery were: In VSD, the abnormal degree of STIs was even greater at the early postoperative period for about 2–3 weeks, and since then it became improved gradually and nearly normal at the 7th-10th months. In ASD, STIs promptly became entirely normal at the 2nd-3rd weeks after surgery. In TF, STIs became approximately normal at the 2nd-3rd weeks after surgery, and were entirely normal at the 3rd-4th months. The changes of STIs after surgery were chiefly related to the improvement of hemodynamics. The left ventricular performance became normal much more slowly in VSD than in ASD and TF, and the reason was probably that left ventricular hypertrophy was present with impairment of myocardial contractility in the former. The measurements of STIs can sensitively reflect the dynamic changes of the left ventricular performance in children with VSD, ASD and TF before and after radical surgery, and can be used to monitor the state of the left ventricular performance after surgery to provide useful information for the evaluation of the effects of surgery and for supervision of the physical activity of the children during convalescense.