TBX1基因与人类单纯性心脏圆锥动脉干畸形的相关研究
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摘要
前言
心脏圆锥动脉干畸形(Conotruncal Heart Malformation)是一类严重危害婴幼儿健康的先天畸形,占先天性心脏病的1/4～1/3。该疾病属于常见的青紫型先天性心脏病,约占青紫型先天性心脏病的70%,是导致儿童复杂畸形和高死亡率的主要原因。临床表现主要包括法洛四联症(Fallot's Tetralogy,F_4)、肺动脉闭锁(Pulmonary atresia,PA)、大动脉转位(Transposition of the Great Arteries,TGA)、永存动脉干(Persistent Truncus Arteriosus, PTA)、右室双出口(double—outlet right ventricle, DORV)等多种导致紫绀和低氧血症的心脏复杂畸形,其中以法洛四联症最常见。心脏圆锥动脉干畸形有时可伴发于综合征,有时仅表现为单纯心脏畸形而不伴有其他系统异常,后者称之为单纯性心脏圆锥动脉干畸形。
目前认为,心脏圆锥动脉干畸形主要是在胚胎心脏发育的易损期(妊娠3～8周),由于遗传因素和环境因素的共同作用,某些与心脏圆锥动脉干发育有关的基因及其产物出现异常,引起心脏流出道和大动脉的发育异常。国内外学者充分利用模式生物、胚胎学、分子生物学技术等,鉴定了一些与心脏圆锥动脉干发育密切相关的基因,如PAX3、HIRA、dHAND、eHAND、RARs、ET—1、PDGF、NF—1、TBX1、NT—3、connexin43等。
TBX1基因是控制脊椎动物胚胎发育及器官形成的主要基因,其功能改变可直接影响发育。人类TBX1基因首次于1997年在DiGeorge综合征中被克隆。它是T—box基因家族的重要成员,通过其特有的T—box结构域参与多种生物的发育调控。以往研究表明,TBX1基因的突变、缺失或单体型不足是引起一系列伴有心脏圆锥动脉干畸形的综合征的重要原因。然而,也有学者在综合征型心脏圆锥动脉干畸形患者中未发现有意义的TBX1突变,提出TBX1基因突变可能不是引起心脏圆锥动脉干畸形的真正原因。因此,TBX1基因突变是否为心脏圆锥动脉干畸形的致病突变仍
PrefaceConotruncal Heart Malformation is a kind of congenital abnormality that does severe harm to inborn health, accounting for quarter to one-third of congenital heart disease. It belongs to the common congenital heart disease with cyanosis, accounting for 70 percent of congenital heart disease, and it is the main cause of complex malformation and high mortality of child. The main clinical manifestation include Fallot s Tetralogy (F_4) , Pulmonary atresia (PA) , Transposition of the Great Arteries (TGA), Persistent Truncus Arteriosus (PTA), double — outlet right ventricle (DORV) , and other complex heart malformations with cyanosis and hypoxemia. The most common manifestation is TOF. Sometimes conotruncal heart malformation is seen in some syndrome, and sometimes it only display simple heart malformation without other abnormality which was called simple conotruncal heart malformation.It is considered that conotruncal heart malformation results from the abnormal development of the embryonic cardiovascular system. The abnormality of some genes related with the conotruncal heart development might cause the abnormal development of cardiac outflow tract and big arteries due to the interaction between genetic factors and enviromental influence. With the model organism, embryology and molecelar bioligical techniques, some genes related with conotruncal heart development were identified, such as PAX3, HIRA, dHAND, eHAND, RARs, ET-1, PDGF, NF—1, TBX1, NT—3, connexin43, and so on.
TBXl gene is the major gene which controls the vertebrate embryonic development, and the functional changes can influence development directly. Human TBXl gene was cloned from DiGeorge syndrome in 1997. It belongs to T —box gene family and participates in developmental control through T — box domain. Previous researches showed that mutations , deletion and haploinsufficiency of TBXl gene were the major cause of a series of syndrome with conotruncal heart malformation. However, no meaningful mutations were found in patients with syndromic conotruncal heart malformation in some researches, suggesting mutations in TBXl gene might not be the true reseason of conotruncal heart malformation. Therefore, it is controversial whether mutations in TBXl gene are the disease—relative mutations with conotruncal heart malformation or not.Progress in developmental genetics provides a new pathway for the researches on the disease—relative genes. TBXl gene maily expresses at ventricular outflow tract and the structural abnormality of ventricular outflow tract are closely associated with conotruncal heart malformation. Therefore, based on the results of developmental genetics, we introduce a hypothesis that TBXl gene maybe the important candidate gene of simple conotruncal heart malformation. We selected 3 SNPs, those were G2857C, G2963A and A657T, so that we could detect the distribution of 3 SNPs in 130 conotruncal heart malformation patients and 200 normal controls. We also analyzed the association between TBXl gene and conotruncal heart malformation and construct haplotypes by PHASE software. At the same time, we performed mutation screening in the coding region of TBXl gene by PCR—DGGE, and observed the TBXl gene expression py RT—PCR using |3—actin as internal control, in order to reveal the possible pathway or mechanism of TBXl gene to participate in the pathogenesis of conotruncal heart malformation.Methods 1. cSNP association analysis and haplotype analysis in TBXl gene
(1) SNP selection and primer design3 SNPs in TBXl gene, G2857C, G2963A and A6571T, were selected from SNP DataBase. We have obtained the genomic sequences around SNPs and determined whether there are recognizing sites of restriction en-donuclease or not. If there are no recognizing sites of restriction endonu-clease, a mismatched base was introduced to the second reciprocal position at the 3' end of one primer. Primers were designed using primer 5 software.(2) PCR amplicaficationGenomic DNA were extracted using phenol/chloroform and PCR amplification was performed as usual.(3) GenotypeGenotyping were performed by PCR —RFLP. Among the total, Sac II restriction endonuclease were used for G2857C, Age I for G2963A, and Ear I for A6571T.(4) Stastitic analysisHardy—Weinberg Equilibrium was detected between patients and normal controls using Arlequin software. The content of linkage disequ-librium among 3SNPs was estimated by 2LD software. Haplotypes were constructed by PHASE softwar. SPSS10. 0 software was used to analyze the differrence of genotype frequency and allele frequency between patients and normal controls. It is significant when p value is less than 0.05.2. Mutation screening in TBXl gene(1) Primer designPrimers were designed using Primer 3 software. The PCR products must overlap all exons of TBXl gene. 40 bp [GC] clamp was added to the 5' end of the forward primer.(2) PCR-DGGEPCR was performed as usual. The concentration range of denatured gel was determined by vertical DGGE and mutation screening was done by parallel DGGE. Samples with sceptical mutations were done for three
times by PCR—DGGE. PCR products were sent for DNA sequencing after purification in order to determine whether there were mutaions or not. 3. Expression analysis of TBX1 gene(1) PCR designPrimers were designed using Primer 3 software. The PCR products must overlap different introns of TBX1 gene.(2) hemi—qualitative RT—PCRTotal RNA extracted by TRIZOL reagent as usual. The quality of RNA was detected by denatured electrophoresis containing formaldehyde. RNA was digested by DNase I in order to remove the genomic DNA. After the first cDNA strand was synthesized by reverse transcription system KIT, TBX1 gene was amplified by PCR with the internal |3 — actin control. Chemilmager 5500 v2. 03 software was used for hemi—quantative a-nalysis after gel electrophoresis. t—test was performed for statistic analysis. oResults1. cSNP association analysis and haplotype analysis in TBX1 gene(1) Genotype frequency and allele frequency of 3 SNPs in patients and normal controls were consistent with Hardy—Weinberg Equilibrium.(2) The distribution of genotype frequency and allele frequency between patients and normal controls had no significance (P > 0. 05). There were remarkble significance (x2 = 8. 14,P = 0. 0043) with allele G at G2963A locus between patients and normal controls. The distribution of genotype frequency between patients and normal controls was significant (x2==9- 9,P = 0. 0071). The frequency of AA genotype was cut down, and the frequencies of GA and GG genotypes was stepped up. (3) There was strong linkage disequilibrium among 3 SNPs after the analysis using 2LD software.(4) G2857/G2963/A6571 and G2857/G2963/T6571 haplotypes were the common haplotypes in the population. The distribution of 4 haplo-
types between patients and normal controls was significant (x2=22. 39,P = 0. 00005) . G2857/G2963/A6571 and C2857/A2963/T6571 haplotypes were much more common in patients than those in normal controls.2. Mutation screening in TBX1 geneNo mutations were detected in 9 exons of TBX1 gene in 130 simple conotruncal heart malformation patients by PCR—DGGE.3. Expression analysis of TBX1 gene(D18S- and 28S—banding were distinct in the RNA electrophore-sis, and the quantity of 28 S—banding was double that of 18S—banding, which suggested that the quality of total RNA was better.(2) The expression of TBX1 gene mRNA in the right auricle of conotruncal heart malformation patients is lower than that in normal controls (P < 0. 001).Conclusions1. There is an obvious association between G2963 locus in the coding region of TBX1 gene and human simple conotruncal malformation, which was confirmed by cSNP association analysis for the first time. People with allele G have a much higher risk with disease. It suggested that TBX1 gene was associated with conotruncal heart malformation.2. The frequencies of two haplotypes (G2857/G2963/A6571 and C2857/A2963/T6571) in conotruncal heart malformation patients were much higher than those in normal controls. These two haplotype might be linked to the susceptibility gene of human simple conotruncal heart malformation.3. Mutations in the coding region of TBX1 gene might not be the pathological causes of human conotruncal heart malformation.4. Expression abnormalities of TBX1 gene in the transcriptional level might be the potential mechanism participating in the pathogenesis of human conotruncal heart malformation.
心脏圆锥动脉干畸形(Conotruncal Heart Malformation)是一类严重危害婴幼儿健康的先天畸形,占先天性心脏病的1/4～1/3。该疾病属于常见的青紫型先天性心脏病,约占青紫型先天性心脏病的70%,是导致儿童复杂畸形和高死亡率的主要原因。临床表现主要包括法洛四联症(Fallot's Tetralogy,F_4)、肺动脉闭锁(Pulmonary atresia,PA)、大动脉转位(Transposition of the Great Arteries,TGA)、永存动脉干(Persistent Truncus Arteriosus, PTA)、右室双出口(double—outlet right ventricle, DORV)等多种导致紫绀和低氧血症的心脏复杂畸形,其中以法洛四联症最常见。心脏圆锥动脉干畸形有时可伴发于综合征,有时仅表现为单纯心脏畸形而不伴有其他系统异常,后者称之为单纯性心脏圆锥动脉干畸形。
目前认为,心脏圆锥动脉干畸形主要是在胚胎心脏发育的易损期(妊娠3～8周),由于遗传因素和环境因素的共同作用,某些与心脏圆锥动脉干发育有关的基因及其产物出现异常,引起心脏流出道和大动脉的发育异常。国内外学者充分利用模式生物、胚胎学、分子生物学技术等,鉴定了一些与心脏圆锥动脉干发育密切相关的基因,如PAX3、HIRA、dHAND、eHAND、RARs、ET—1、PDGF、NF—1、TBX1、NT—3、connexin43等。
TBX1基因是控制脊椎动物胚胎发育及器官形成的主要基因,其功能改变可直接影响发育。人类TBX1基因首次于1997年在DiGeorge综合征中被克隆。它是T—box基因家族的重要成员,通过其特有的T—box结构域参与多种生物的发育调控。以往研究表明,TBX1基因的突变、缺失或单体型不足是引起一系列伴有心脏圆锥动脉干畸形的综合征的重要原因。然而,也有学者在综合征型心脏圆锥动脉干畸形患者中未发现有意义的TBX1突变,提出TBX1基因突变可能不是引起心脏圆锥动脉干畸形的真正原因。因此,TBX1基因突变是否为心脏圆锥动脉干畸形的致病突变仍
PrefaceConotruncal Heart Malformation is a kind of congenital abnormality that does severe harm to inborn health, accounting for quarter to one-third of congenital heart disease. It belongs to the common congenital heart disease with cyanosis, accounting for 70 percent of congenital heart disease, and it is the main cause of complex malformation and high mortality of child. The main clinical manifestation include Fallot s Tetralogy (F_4) , Pulmonary atresia (PA) , Transposition of the Great Arteries (TGA), Persistent Truncus Arteriosus (PTA), double — outlet right ventricle (DORV) , and other complex heart malformations with cyanosis and hypoxemia. The most common manifestation is TOF. Sometimes conotruncal heart malformation is seen in some syndrome, and sometimes it only display simple heart malformation without other abnormality which was called simple conotruncal heart malformation.It is considered that conotruncal heart malformation results from the abnormal development of the embryonic cardiovascular system. The abnormality of some genes related with the conotruncal heart development might cause the abnormal development of cardiac outflow tract and big arteries due to the interaction between genetic factors and enviromental influence. With the model organism, embryology and molecelar bioligical techniques, some genes related with conotruncal heart development were identified, such as PAX3, HIRA, dHAND, eHAND, RARs, ET-1, PDGF, NF—1, TBX1, NT—3, connexin43, and so on.
TBXl gene is the major gene which controls the vertebrate embryonic development, and the functional changes can influence development directly. Human TBXl gene was cloned from DiGeorge syndrome in 1997. It belongs to T —box gene family and participates in developmental control through T — box domain. Previous researches showed that mutations , deletion and haploinsufficiency of TBXl gene were the major cause of a series of syndrome with conotruncal heart malformation. However, no meaningful mutations were found in patients with syndromic conotruncal heart malformation in some researches, suggesting mutations in TBXl gene might not be the true reseason of conotruncal heart malformation. Therefore, it is controversial whether mutations in TBXl gene are the disease—relative mutations with conotruncal heart malformation or not.Progress in developmental genetics provides a new pathway for the researches on the disease—relative genes. TBXl gene maily expresses at ventricular outflow tract and the structural abnormality of ventricular outflow tract are closely associated with conotruncal heart malformation. Therefore, based on the results of developmental genetics, we introduce a hypothesis that TBXl gene maybe the important candidate gene of simple conotruncal heart malformation. We selected 3 SNPs, those were G2857C, G2963A and A657T, so that we could detect the distribution of 3 SNPs in 130 conotruncal heart malformation patients and 200 normal controls. We also analyzed the association between TBXl gene and conotruncal heart malformation and construct haplotypes by PHASE software. At the same time, we performed mutation screening in the coding region of TBXl gene by PCR—DGGE, and observed the TBXl gene expression py RT—PCR using |3—actin as internal control, in order to reveal the possible pathway or mechanism of TBXl gene to participate in the pathogenesis of conotruncal heart malformation.Methods 1. cSNP association analysis and haplotype analysis in TBXl gene
(1) SNP selection and primer design3 SNPs in TBXl gene, G2857C, G2963A and A6571T, were selected from SNP DataBase. We have obtained the genomic sequences around SNPs and determined whether there are recognizing sites of restriction en-donuclease or not. If there are no recognizing sites of restriction endonu-clease, a mismatched base was introduced to the second reciprocal position at the 3' end of one primer. Primers were designed using primer 5 software.(2) PCR amplicaficationGenomic DNA were extracted using phenol/chloroform and PCR amplification was performed as usual.(3) GenotypeGenotyping were performed by PCR —RFLP. Among the total, Sac II restriction endonuclease were used for G2857C, Age I for G2963A, and Ear I for A6571T.(4) Stastitic analysisHardy—Weinberg Equilibrium was detected between patients and normal controls using Arlequin software. The content of linkage disequ-librium among 3SNPs was estimated by 2LD software. Haplotypes were constructed by PHASE softwar. SPSS10. 0 software was used to analyze the differrence of genotype frequency and allele frequency between patients and normal controls. It is significant when p value is less than 0.05.2. Mutation screening in TBXl gene(1) Primer designPrimers were designed using Primer 3 software. The PCR products must overlap all exons of TBXl gene. 40 bp [GC] clamp was added to the 5' end of the forward primer.(2) PCR-DGGEPCR was performed as usual. The concentration range of denatured gel was determined by vertical DGGE and mutation screening was done by parallel DGGE. Samples with sceptical mutations were done for three
times by PCR—DGGE. PCR products were sent for DNA sequencing after purification in order to determine whether there were mutaions or not. 3. Expression analysis of TBX1 gene(1) PCR designPrimers were designed using Primer 3 software. The PCR products must overlap different introns of TBX1 gene.(2) hemi—qualitative RT—PCRTotal RNA extracted by TRIZOL reagent as usual. The quality of RNA was detected by denatured electrophoresis containing formaldehyde. RNA was digested by DNase I in order to remove the genomic DNA. After the first cDNA strand was synthesized by reverse transcription system KIT, TBX1 gene was amplified by PCR with the internal |3 — actin control. Chemilmager 5500 v2. 03 software was used for hemi—quantative a-nalysis after gel electrophoresis. t—test was performed for statistic analysis. oResults1. cSNP association analysis and haplotype analysis in TBX1 gene(1) Genotype frequency and allele frequency of 3 SNPs in patients and normal controls were consistent with Hardy—Weinberg Equilibrium.(2) The distribution of genotype frequency and allele frequency between patients and normal controls had no significance (P > 0. 05). There were remarkble significance (x2 = 8. 14,P = 0. 0043) with allele G at G2963A locus between patients and normal controls. The distribution of genotype frequency between patients and normal controls was significant (x2==9- 9,P = 0. 0071). The frequency of AA genotype was cut down, and the frequencies of GA and GG genotypes was stepped up. (3) There was strong linkage disequilibrium among 3 SNPs after the analysis using 2LD software.(4) G2857/G2963/A6571 and G2857/G2963/T6571 haplotypes were the common haplotypes in the population. The distribution of 4 haplo-
types between patients and normal controls was significant (x2=22. 39,P = 0. 00005) . G2857/G2963/A6571 and C2857/A2963/T6571 haplotypes were much more common in patients than those in normal controls.2. Mutation screening in TBX1 geneNo mutations were detected in 9 exons of TBX1 gene in 130 simple conotruncal heart malformation patients by PCR—DGGE.3. Expression analysis of TBX1 gene(D18S- and 28S—banding were distinct in the RNA electrophore-sis, and the quantity of 28 S—banding was double that of 18S—banding, which suggested that the quality of total RNA was better.(2) The expression of TBX1 gene mRNA in the right auricle of conotruncal heart malformation patients is lower than that in normal controls (P < 0. 001).Conclusions1. There is an obvious association between G2963 locus in the coding region of TBX1 gene and human simple conotruncal malformation, which was confirmed by cSNP association analysis for the first time. People with allele G have a much higher risk with disease. It suggested that TBX1 gene was associated with conotruncal heart malformation.2. The frequencies of two haplotypes (G2857/G2963/A6571 and C2857/A2963/T6571) in conotruncal heart malformation patients were much higher than those in normal controls. These two haplotype might be linked to the susceptibility gene of human simple conotruncal heart malformation.3. Mutations in the coding region of TBX1 gene might not be the pathological causes of human conotruncal heart malformation.4. Expression abnormalities of TBX1 gene in the transcriptional level might be the potential mechanism participating in the pathogenesis of human conotruncal heart malformation.
引文
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