先天性房间隔缺损相关GATA4基因新突变的筛查

目的 筛查先天性房间隔缺损相关GATA4基因的新突变.方法 收集85例先天性房间隔缺损患者和200名健康者的临床资料及外周静脉血标本.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.以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.采用Clustal W软件分析突变氨基酸的保守性.结果 在其中3例先天性房间隔缺损患者的GATA4基因各识别出1个新的杂合错义突变,即第267、354和407位的密码子分别由GTG、ACC和CCA变为ATG、GCC和CAA,亦即c.799G>A、c.1060A>G和c.1220C>A突变,相应地导致第267、354和407位的氨基酸分别由缬氨酸、苏氨酸和脯氨酸变为蛋氨酸、丙氨酸和谷氨酰胺,也称为p.V267M、p.T354A和p.P407Q突变.200名健康对照者均无上述突变.多物种GATA4序列比对显示,第267位的缬氨酸和第407位的脯氨酸在进化上完全保守.结论 在先天性房间隔缺损患者的GATA4基因识别出3个新的杂合错义突变,揭示了先天性房间隔缺损新的分子病因,有助于先天性房间隔缺损的早期防治.     

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。     

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。     

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。     

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。     

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。     

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。     

新疆地区维吾尔族先天性心脏病患者CITED2基因突变分析

【】 目的：研究新疆维吾尔族先天性心脏病(congenital heart disease,CHD)患者与CITED2基因突变的关系。方法：收集150例散发型维吾尔族CHD患者和150例健康维吾尔族人群血液样本进行DNA提取、目的基因聚合酶链反应及测序,并与GeneBank进行比较以识别基因突变,并分析氨基酸序列。结果：在1例室间隔缺损患者发现1个新的纯合突变c.574A>G,并导致相应氨基酸序列错义突变(p.Ser192Gly),该突变在CITED2基因丝氨酸-甘氨酸富含区,而在健康对照组中未发现此突变。结论：新疆维吾尔族CHD患者中首次发现了CITED2基因纯合突变,Serl92GIy突变所在序列呈现一定保守性,可能与CHD发生有关。     

Identification of three novel mutations in the GATA3 gene responsible for familial hypoparathyroidism and deafness in the Chinese population

BACKGROUND: Familial hypoparathyroidism may be caused by mutations of several genes. The CaSR and GATA3 genes are the two candidates most commonly responsible for this condition. OBJECTIVES: We collected five unrelated families with familial hypoparathyroidism and examined their CaSR and GATA3 genes. METHODS: Blood samples from these five families and 50 ethnically matched unrelated controls were acquired. Biochemistry screening and formal audiogram were performed to evaluate the affected individuals. All the exons and exon-intron boundaries of the GATA3 and CaSR genes were sequenced. RESULTS: We identified three novel mutations in the GATA3 gene responsible for familial hypoparathyroidism and deafness: 1) a frameshift deletion occurring in codon 160 (478delG) was hypothesized to disrupt dual zinc fingers as well as one transactivating domain; 2) a donor splice site mutation at exon 4/intron 4 boundary (IVS4 + 2 T to GCTTACTTCCC) was predicted to lead to truncated GATA3 proteins that lack both N- and C-terminal zinc-containing fingers; and 3) a missense mutation R353S was predicted to disrupt the helical turn and thus changed the angle between the C-terminal zinc finger and the adjacent C-terminal tail. Except for a previously described polymorphism, G990R, we did not find any genetic variants in the CaSR gene. CONCLUSIONS: This is the first article presenting mutations of the GATA3 gene responsible for familial hypoparathyroidism and deafness in the Chinese population. Our results expand the spectrum of mutations and report the first splice donor site mutation of the GATA3 gene. In contrast, we do not find causal sequence variants of the CaSR gene from our collection of familial hypoparathyroidism.     

NKX2—5基因与先天性心脏病的关系

目的:探讨NKX2-5基因与先天性心脏病(congenital heart disease,CHD)的关系. 方法:收集13()例无血缘关系的CHD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CHD候选基因NKX2-5的全部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数据库中公布的NKX2-5基因序列进行比对以识别出NKX2-5基因变异.比较识别出的NKX2-5基因多态在CHD患者和健康对照者间的频率分布差异. 结果:在CHD患者的NKX2-5基因识别出2个单核苷酸多态,一个是NKX2-5基因编码序列第63位的腺嘌呤(A)变为鸟嘌呤(G),即C.63A>G多态;另一个是NKX2-5基因编码序列第606位的G变为胞嘧啶(C),即c.606G>C多态.在NKX2-5基因第606位点的等位基因G、C及其构成的3种基因型GG、GC、CC在CHD患者和健康对照者间的频率分布有显著性差异(等位基因频率比较:X2=4.125,P=0.042;基因型频率比较:X2=4.279,P=0.039).但在NKX2-5基因第63位点的等位基因A、G及其构成的3种基因型AA、AG、GG在CHD患者和健康对照者间的频率分布无显著性差异(等位基因频率比较:X2=0.704,P=0.402;基因型频率比较:X2=0.626,P=0.429). 结论:NKX2-5基因C.606G>C多态可能与CHD有关,等位基因606C携带者可能对CHD的易感性增加.     

Molecular basis of beta-thalassemia in the population of Tunisia

The present study attempts to delineate the spectrum of beta-thalassemia (thal) mutations in Tunisia by studying a large population from different parts of the country. A total of 285 unrelated subjects, 190 of whom had beta-thal major, 72 with Hb S/beta-thal, one with Hb C/beta-thal, one with Hb O-Arab/beta-thal and 21 beta-thal carriers, were studied. The molecular defects were detected in 97.7% of the beta-thalassemic chromosomes (n=475). Nineteen different beta-thalassemic alleles were identified. Two mutations, namely codon 39 (C-->T) and IVS-I-110 (G-->A) accounted for 70.0% of the studied chromosomes, followed by IVS-I-1 (G-->A) (4.5%). Five other mutations, frameshift codon (FSC) 44 (-C), codon 30 (G-->C), IVS-I-2 (T-->G), IVS-II-745 (C-->G), and FSC 6 (-A), are not uncommon in this population, while the remaining 11 mutations, IVS-I-5 (G-->A), -30 (T-->A), codons 25/26 (+T), IVS-I-6 (T-->C), FSC 5 (-CT), IVS-II-848 (C-->A), FSC 8 (-AA), -87 (C-->G), IVS-I-5 (G-->C), IVS-II-1 (G-->A) and IVS-II-849 (A-->C) are quite rare; four of these have not been previously reported in the Tunisian population. Potential origin and spread of these mutations to Tunisia are also discussed.     

Three novel CYP21A2 mutations and their protein modelling in patients with classical 21-hydroxylase deficiency from northeastern Iran

OBJECTIVE: Congenital adrenal hyperplasia (CAH) refers to a group of autosomal recessive disorders frequently caused by mutations in the steroid 21-hydroxylase gene (CYP21A2). We describe three novel CYP21A2 mutations in CAH patients. DESIGN AND METHODS: Sequence analysis of the entire CYP21A2 gene followed by molecular modelling was performed in three unrelated classical CAH patients of northeastern Iranian origin. The active (CYP21A2) and pseudogene (CYP21A1P) alleles were screened for the presence of the new variations in controls. RESULTS: Two novel missense mutations, F404S in exon 9 and T450P in exon 10, were found in homozygous forms in two female patients with a salt-wasting (SW) phenotype. These novel variants were screened by allele-specific polymerase chain reaction (PCR) and excluded in 100 unrelated normal alleles. Prediction of clinical severity, based on molecular modelling and sequence conservation, correlates well with the clinical diagnosis of the patients carrying these mutations. The third novel mutation, a small 10-bp deletion in exon 1, g.19_28del, was found in a female patient with a simple virilizing phenotype in a compound heterozygous form with the common intron 2 splice mutation (IVS2-13A/C>G). This frameshift mutation causes a premature stop codon at amino acid position 48, L48X, resulting in a nonfunctional protein. The CYP21A1P pseudogene alleles were also screened and none of these novel mutations could be detected. CONCLUSIONS: Three novel mutations were found in the CYP21A2 gene and predicted to drastically impair enzyme activity resulting in severe classic CAH. None of these mutations occurs in the CYP21A1P pseudogene.     

Hb Lusaka [α131(H14)Ser→Phe (α1)]: A New Variant Found in a Woman Heterozygous for Hb S [β6(A3)Glu→Val]

The present study attempts to delineate the spectrum of β‐thalassemia (thal) mutations in Tunisia by studying a large population from different parts of the country. A total of 285 unrelated subjects, 190 of whom had β‐thal major, 72 with Hb S/β‐thal, one with Hb C/β‐thal, one with Hb O‐Arab/β‐thal and 21 β‐thal carriers, were studied. The molecular defects were detected in 97.7% of the β‐thalassemic chromosomes (n?=?475). Nineteen different β‐thalassemic alleles were identified. Two mutations, namely codon 39 (C→T) and IVS‐I‐110 (G→A) accounted for 70.0% of the studied chromosomes, followed by IVS‐I‐1 (G→A) (4.5%). Five other mutations, frameshift codon (FSC) 44 (–C), codon 30 (G→C), IVS‐I‐2 (T→G), IVS‐II‐745 (C→G), and FSC 6 (–A), are not uncommon in this population, while the remaining 11 mutations, IVS‐I‐5 (G→A), ??30 (T→A), codons 25/26 (+?T), IVS‐I‐6 (T→C), FSC 5 (–CT), IVS‐II‐848 (C→A), FSC 8 (–AA), –87 (C→G), IVS‐I‐5 (G→C), IVS‐II‐1 (G→A) and IVS‐II‐849 (A→C) are quite rare; four of these have not been previously reported in the Tunisian population. Potential origin and spread of these mutations to Tunisia are also discussed.     

Identification of new and known polymorphisms in glycoprotein IIb and IIIa genes by denaturing gradient gel electrophoresis

Glycoprotein IIb and IIIa contain antigenic determinants involved in the potential production of allo- or autoantibodies directed against platelets, that may result in severe thrombocytopenia. Most of these epitopes appear to be supported by single nucleotide substitutions. We have used denaturing gradient gel electrophoresis (DGGE) to identify sequence variations within the promoter and the coding regions of the glycoprotein IIb and glycoprotein IIIa genes. Using genomic DNA from 60 unrelated normal individuals, we have amplified short domains that encompass the coding sequences and the exon-intron boundaries of both genes that were further separated according to their melting behaviour during the denaturant electrophoretic migration. Only the fragments with an abnormal migration pattern were sequenced. We confirmed the sensitivity of this method by recognizing both previously described Human Platelet Antigen polymorphisms and mutations affecting either the glycoprotein IIb or the glycoprotein IIIa genes in thrombasthenic patients. We also identified four other polymorphisms. Two were located in the glycoprotein IIb gene, involving intron 21 (C G at nucleotide 10480) and first codon of exon 30 (codon GTC GTT coding for residue Val 990), and two in the glycoprotein IIIa gene (exon 6 CCC CCT coding for residue Pro 268; intron 14 C T at position 37126). The screening of the GPIIIa promoter also revealed three different polymorphisms located at position-468 (A/T polymorphism), -425 (A/C polymorphism) and-400 (A/C polymorphism), which could influence the expression of the complex at the cell surface. Denaturing gradient gel electrophoresis appears to be a sensitive and specific technique for identifying polymorphisms and mutations in the GPIIb and GPIIIa genes.