房间隔缺损患者GATA4基因新突变的识别

目的 识别房间隔缺损患者新的分子遗传缺陷.方法 收集180例房间隔缺损患者的临床资料和血液标本.以200名健康者作为对照.应用聚合酶链反应扩增GATA4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2例房间隔缺损患者的GATA4基因中各识别出1个新的杂合错义突变,即第21和第87位的密码子分别由GGC和CCG变为GTC和TCG,导致第21和87位的氨基酸分别由甘氨酸和脯氨酸变为缬氨酸和丝氨酸,即G21V和P87S突变.而健康者GATA4基因中未识别出这2个突变.多物种GATA4序列比对显示,突变氨基酸在进化上均高度保守.此外,还识别出1个不改变氨基酸的单核苷酸多态,即c.99 G＞T多态,但是ASD患者与健康者之间基因型和等位基因频率分布差异无统计学意义(GG比GT,x2=0.7556,P=0.3847;G比T,x2=0.7235,P=0.3950).结论 识别出新的GATA4基因杂合错义突变,有助于房间隔缺损的早期防治.     

先天性心脏病相关GATA5基因突变研究

目的:识别先天性心脏病(CHD)相关GATA5基因新突变。方法:收集100例无血缘关系的CHD患者和200名无血缘关系且种族匹配的健康对照者的临床资料和血标本。抽提基因组DNA,通过聚合酶链反应扩增GATA5基因的编码外显子及其剪接位点,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。将所测的序列与GenBank数据库中的GATA5基因序列进行比对以发现GATA5基因突变。应用多序列比对软件ClustalW评估突变氨基酸的保守性,应用致病性预测软件MutationTaster预测突变的致病性。结果:在2例CHD患者各发现1个新的GATA5基因杂合错义突变,突变率为2%。其中1个是GATA5基因编码核苷酸序列第395位的鸟嘌呤(guanine,G)变为胸腺嘧啶(thymine,T),即c.395G>T突变;另一个是GATA5基因编码核苷酸序列第991位的胞嘧啶(cytosine,C)变为腺嘌呤(adenine,A),即c.991C>A突变。多序列比对显示2种突变氨基酸在进化上均高度保守。致病性预测显示2种突变均有致病性。结论:本研究发现了CHD相关GATA5基因新突变,有助于揭示CHD新的分子机制。     

先天性房间隔缺损相关GATA6基因新突变的识别

目的：识别先天性房间隔缺损（ASD）相关GATA6基因新突变。方法：收集110例先天性AsD患者和200名健康对照者的临床资料和血标本,使用DNA纯化试剂盒提取基因组DNA。通过聚合酶链反应扩增GATA6基因的编码外显子和其两侧的部分内含子,应用双脱氧核苷链末端合成终止法进行DNA测序。将所测序列与GenBank数据库中的GATA6基因序列进行比对,识别GATA6基因突变。分别借助在线程序MUSCLE和MutationTaster评估突变氨基酸的保守性和致病性。结果：在1例家族史阴性的先天性ASD患者发现了1种新的GATA6基因杂合错义突变,即P．Q363E突变,突变率约为0．91％。该突变不存在于200名健康对照者,多物种序列比对显示,被改变氨基酸在进化上高度保守,致病性预测表明这种变异为致病性突变。结论：发现ASD相关GATA6基因新突变,有助于揭示ASD新的分子机制。     

特发性房颤相关GATA5基因新突变的识别

目的:识别特发性房颤(AF)相关GATA5基因新突变. 方法:收集120例无血缘关系的特发性AF患者和200名无血缘关系且种族匹配的健康对照者的临床资料和血标本.抽提基因组DNA,通过聚合酶链反应扩增AF候选基因GATA5的全部外显子及其两侧的部分内含子,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.将所测的序列与GenBank数据库中的GATA5基因序列进行比对,以发现GATA5基因突变.应用多序列比对软件ClustalW评估突变氨基酸的保守性,应用致病性预测软件MutationTaster预测突变的致病性. 结果:在2例AF患者各发现1个新的GATA5基因杂合错义突变,突变率约为1.67％.突变分别为GATA5基因编码核苷酸序列第668位的腺嘌呤(adenine,A)变为胸腺嘧啶(thymine,T),即c.668A＞T突变；第863位的A变为胞嘧啶(cytosine,C),即c.863A＞C突变.多序列比对显示2种突变氨基酸在进化上均高度保守,致病性预测表明2种突变均有致病性. 结论:本研究识别出AF相关GATA5基因新突变,有助于揭示AF发生的分子机制.     

先天性心脏病患者GATA4基因突变谱分析

目的:分析先天性心脏病患者GATA4基因的突变谱,揭示先天性心脏病的分子病因。方法:收集110例无血缘关系的先天性心脏病患者的临床资料和血标本,以100名无血缘关系的健康者为对照。应用聚合酶链反应扩增先天性心脏病相关基因GATA4的全部编码外显子和外显子两侧的部分内含子,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。将所测的序列与GenBank数据库中的GATA4基因序列进行比对,识别出GATA4基因突变,并用序列比对在线软件ClustalW2分析突变氨基酸的保守性。结果:在3例无血缘关系的先天性心脏病患者的GATA4基因各识别出1个新的杂合错义突变,即P42T、V48M和S191I突变,这些突变不存在于正常对照者,而且突变氨基酸在哺乳动物进化上有高度的保守性。结论:该研究结果可能揭示了先天性心脏病新的分子病因,有助于先天性心脏病患者的早期防治。     

先天性心脏病相关PITX2c基因新突变研究

目的:研究先天性心脏病(congenital heart disease,CHD)相关PITX2c基因的新突变。方法:收集150例CHD患者和200名正常对照者的外周静脉血标本,使用DNA纯化试剂盒分离基因组DNA。使用DNA聚合酶扩增PITX2c基因的编码区和剪接位点,应用DNA测序试剂盒在DNA分析仪上对扩增片段进行测序。将所测序列与GenBank数据库中的PITX2c基因序列进行比对以发现PITX2c基因突变。使用在线程序MUSCLE分析突变氨基酸的保守性,分别应用MutationTaster和PolyPhen-2分析突变氨基酸的致病性。结果:在1例散发性CHD患者发现了1个新的PITX2c基因杂合错义突变,即p.S101G突变,突变率约为0.67%。该错义突变不存在于200名正常对照者。跨物种PITX2c蛋白之氨基酸序列对比显示第101位的丝氨酸在进化上完全保守,致病性预测显示所发现的PITX2c基因变异是致病性突变。结论:本研究揭示了CHD相关PITX2c基因新突变,对于制定新的CHD防治策略具有潜在的意义。     

孤立性房颤相关SCN4B基因突变谱分析

目的：发现孤立性房颤相关SCN4B基因新突变. 方法：收集160例孤立性房颤患者和200名健康对照者的临床资料和血标本,抽提基因组DNA.通过聚合酶链反应扩增房颤候选基因SCN4B的编码区和剪接位点,采用双脱氧核苷链末端合成终止法测序以发现SCN4B基因突变.应用ClustalW软件评估突变氨基酸的保守性,应用MutationTaster软件预测突变的致病性. 结果：在2例孤立性房颤患者各发现1个新的SCN4B基因杂合错义突变,突变率为1.25％.其中1个是SCN4B基因编码核苷酸序列第409位的腺嘌呤（adenine,A）变为鸟嘌呤（guanine,G）,即c.409A＞G突变；另一个是SCN4B基因编码核苷酸序列第511位的G变为A,即c.511G＞A突变.多序列比对显示2种突变氨基酸在进化上均高度保守.致病性预测表明2种突变均有致病性.结论：本研究发现孤立性房颤相关SCN4B基因新突变,有助于揭示房颤新的分子机制.     

特发性扩张型心肌病相关GATA6基因新突变的识别

目的:识别特发性扩张型心肌病(DCM)相关GATA6基因新突变。方法:入选132例特发性DCM患者和220名健康对照者,收集其临床资料和外周静脉血,使用DNA纯化试剂盒抽提基因组DNA,采用聚合酶链反应试剂扩增GATA6基因的整个编码区,应用循环测序试剂盒对扩增的GATA6基因片段进行测序,将所测序列与Nucleotide数据库中的GATA6基因序列进行对比分析以识别GATA6基因突变。应用MUSCLE和MutationTaste在线系统评估突变氨基酸进化上的保守性和突变的致病性。结果:通过序列分析发现1例散发性DCM患者的GATA6基因新变异c.1165GT,相当于p.E389X突变,在220名健康对照者中没有检测出该无义突变。跨物种GATA6蛋白序列比对分析表明,被改变的氨基酸在物种进化上完全保守,致病性预测显示该GATA6基因突变具有致病性。结论:发现散发性DCM相关GATA6基因新突变,扩大了DCM相关GATA6基因突变谱。     

特发性扩张型心肌病相关LRRC10基因新突变的识别

目的:识别特发性扩张型心肌病(dilated cardiomyopathy,DCM)相关LRRC10基因新突变。方法:收集120例特发性DCM患者和200名健康对照者的血标本,用基因组纯化试剂盒提取基因组DNA。通过聚合酶链式反应扩增LRRC10基因的编码外显子和其两侧的内含子,在DNA测序仪上对扩增片段进行测序。将所测序列与核苷酸数据库中的LRRC10基因序列进行比对,寻找可能的基因突变。借助在线程序MUSCLE分析突变氨基酸的保守性,应用MutationTaster和PolyPhen-2预测突变的致病性。结果:在1例特发性DCM患者中发现了1种新的LRRC10基因杂合错义突变,即p.R157G突变,突变率约为0.83%。在200名健康对照者无此基因突变。多物种LRRC10蛋白的氨基酸序列对比显示,第157位的精氨酸在进化上完全保守,致病性预测表明这种突变具有致病性。结论:本研究发现特发性DCM相关LRRC10基因新突变,揭示了特发性DCM新的分子病因。     

儿童先天性心脏病TBX20基因突变及功能探讨

目的：对先天性心脏病（先心病）患儿进行TBX20基因突变研究,探讨TBX20基因与先心病发生机制之间的关系。方法：对203例中国汉族先心病患儿（病例组）进行TBX20基因编码区测序,同期300例门诊体检健康儿童为对照组,检测可能的先心病相关性基因突变。对新发现的突变应用蛋白质结构预测服务器I-TASSER进行同源建模,对TBX20基因野生型及突变后的蛋白质结构模型进行预测分析。并进一步构建TBX20野生型及突变型表达载体,分别转染到COS7细胞中,通过双荧光素酶报告基因检测它们在NKX2.5、GATA4协同作用下对下游靶基因心钠素（ANF）启动子转录激活作用的影响。结果：（1）病例组1例房间隔缺损患儿发现一个新的TBX20基因错义突变c.187G>A(A63T),DNA序列中第187位鸟嘌呤突变为腺嘌呤,导致氨基酸序列中第63位丙氨酸变为苏氨酸,在对照组中均未发现该变异。（2）对错义突变A63T进一步研究中,TBX20同源建模显示第63位的非极性、疏水性丙氨酸变为极性、亲水性的苏氨酸,与Gly65、Gly68间的氢键断裂,与Asp62、His64形成新的氢键,导致蛋白质构象发生变...     

Analysis of the SLC26A4 gene in patients with Pendred syndrome in Taiwan

Pendred syndrome (PS) is an autosomal recessive disease that is characterized by congenital sensorineural hearing loss, goiter, and a partial iodine organification defect. In this study, we characterized the thyroid status and identified mutations in the SLC26A4 gene in Chinese subjects with PS. We evaluated 7 unrelated Chinese subjects who had PS. Biochemical analysis, formal audiogram, ultrasonography of the thyroid gland, perchlorate discharge test, computerized tomography scan of the vestibular aqueducts, and DNA sequence analysis of SLC26A4 were performed. Levels of thyroid hormones were essentially normal in all patients: 2 patients had goiters and/or elevated serum thyroglobulin levels, whereas 2 other patients had positive thyroid antibodies and a positive perchlorate discharge test. We identified SLC26A4 gene mutations in 6 of 7 probands and their affected relatives. The affected subjects in family I was compound heterozygous for 2 missense mutations: a mutation in exon 9 (1079C>T) that resulted in the replacement of alanine by valine at codon 360 (A360V) and a mutation in exon 19 (2168A>G) that resulted in the replacement of histidine by arginine at codon 723 (H723R). The affected subjects in families II and III all were homozygous for a mutation in intron 7. The probands IV and V were compound heterozygotes for the mutation in intron 7 and in exon 19, and the proband VI was compound heterozygous for the intron 7 mutation and a missense mutation in exon 12 (1343C>T) that resulted in the replacement of serine by leucine at codon 448 (S448L). One novel mutation was identified (A360V). We identified biallelic mutations in the SLC26A4 gene in 6 of 7 probands with PS in Taiwan, including a novel missense mutation. The mild thyroid dysfunction in these patients suggests that PS should be considered in all patients with congenital or early-onset hearing impairment.     

Novel mutation in the gamma-glutamyl carboxylase gene resulting in congenital combined deficiency of all vitamin K-dependent blood coagulation factors

A mutation in the gamma-glutamyl carboxylase gene leading to a combined congenital deficiency of all vitamin K-dependent coagulation factors was identified in a Lebanese boy. He is the first offspring of consanguineous parents and was homozygous for a unique point mutation in exon 11, resulting in the conversion of a tryptophan codon (TGG) to a serine codon (TCG) at amino acid residue 501. Oral vitamin K(1) administration resulted in resolution of the clinical symptoms. Screening of several family members on this mutation with an RFLP technique revealed 10 asymptomatic members who were heterozygous for the mutation, confirming the autosomal recessive pattern of inheritance of this disease. In 50 nonrelated normal subjects, the mutation was not found. This is the second time a missense mutation in the gamma-glutamyl carboxylase gene is described that has serious impact on normal hemostasis.     

Altered Sequences Changing the Operator-Binding Properties of the Lac Repressor: Colinearity of the Repressor Protein with the i-Gene Map

A technique is described for mapping point mutations in the first 59 amino-acid residues of the lac repressor from Escherichia coli, using less than 0.1 mumol (4 mg) of the purified protein. This technique was used to localize five mutations affecting the ability of the i-gene product to repress in vivo. These alterations are located at four different sites in the amino-terminal region of the repressor molecule. Three of these are missense mutations and result in changes from serine to proline (residue 16), threonine to alanine (residue 19), and alanine to valine (residue 53). Each amino-acid substitution alone is sufficient to eliminate repression in vivo, presumably by altering the operator binding activity. The remaining two independently-isolated mutations are identical, and result in a change from a glutamine codon at position 26 to an amber (UAG) codon. Since suppression of this nonsense mutation with amber suppressors that insert leucine, tyrosine, serine, or glutamine restores repressor activity to the molecule, glutamine(26) cannot be crucial for the operator-binding function. A comparison of the position of each altered residue with the genetic map enabled us to estimate the physical distance between several deletion-group endpoints.     

重型遗传性凝血因子Ⅶ缺陷症家系分子遗传学分析

目的 探讨遗传性凝血因子Ⅶ(FⅦ)缺陷症家系基因突变的类型。方法 检测凝血和止血指标以明确诊断;用DNA直接测序法对先证者及其家庭成员FⅦ基因的全部外显子及其侧翼、5’和3’非翻译区进行分析;将含杂合缺失突变外显子克隆入pMD18-TTA克隆载体中,对来自父母的两条染色体的相应序列分别测序。应用限制性内切酶StuI、MspI进行酶切分析。结果 先证者FⅦ的6号、8号外显子分别发现了1个错义杂合突变：9482位G→T,导致Arg(CG-A)152被Leu(CU-G)替代;11348位C→T突变,导致Arg(C-GG)304Trp(U-GG)突变;克隆的pMD18-TTA克隆载体测序结果显示一个杂合缺失：在11487～9位(CCC)缺失一个C,引起框移突变(已证实,后两种杂合突变均来自母亲),使351位His变为Thr,在其后编码与正常氨基酸序列完全不同的6个氨基酸后出现了翻译终止密码。其中,先证者Arg152 Leu杂合突变来自父亲,其祖父具有相同的杂合突变,父亲还含有Arg353Gln杂合多态性;其祖母为Arg304Trp杂合突变且含有Arg353Gln杂合多态性;姑姑和大伯分别为Arg353Gln杂合多态性和Arg304Trp杂合突变。PCR辅助限制性酶切证实了先证者及其家系成员的两种错义突变。结论 在该例家系中发现了Arg304Trp、Arg152Leu、11487～9位(CCC)缺失一个C三种杂合性突变,其中后两种突变为国际首次报道。