法洛四联症患儿NOTCH2基因3'UTR区域突变分析

Analysis of mutation in 3'UTR region of NOTCH2 gene of children with tetralogy of Fallot disease

目的筛查法洛四联症(TOF)患儿中NOTCH2基因3'UTR区域存在的基因突变,探讨该区域突变影响NOTCH2基因表达的可能机制。方法选取152例诊断明确的TOF患儿(TOF组)为研究对象,107名健康儿童为对照组(CON组),PCR扩增NOTCH2基因3'UTR区域序列,所有扩增片段均进行双向测序。将所测NOTCH2基因3'UTR区序列与Gen Bank中的已知序列(NG_008163.1)通过BLAST程序对比,检出可能存在的基因突变。采用Pic Tar和Target Scan在线预测软件预测可能与NOTCH2基因3'UTR区域结合的微小RNA(miRNA),分析NOTCH2基因3'UTR区域突变对miRNA调控NOTCH2基因表达的影响。结果检测出NOTCH2基因3'UTR区域1个新发突变和5个已报道的单核苷酸多态性(SNP),分别为2 672位(157020T>G)、rs368873082(156595C>T)、rs835576(156691A>G)、rs3795664(156937C>T)、rs699779(156967T>C)和rs699780(156836T>C);5个已报道的SNP在TOF组与CON组中的等位基因频率差异均无统计学意义(P>0.05)。预测结果显示34种miRNA能与NOTCH2基因3'UTR区域结合,该突变并不位于34种miRNA与NOTCH2 3'UTR的结合区域。结论 NOTCH2基因3'UTR区域存在新突变157020T>G,该突变有可能通过空间构象影响miRNA与NOTCH2基因3'UTR区的结合效率,进而影响NOTCH2基因的正常表达;而该区域的5个SNP与TOF易感性无明显关联。

Objective To screen mutations in 3＇UTR region of NOTCH2 gene of children with tetralogy of Fallot disease （TOF） and explore possible mechanisms that how mutations in this region affect the expression of NOTCH2 gene. Methods One hundred and fifty-two children confirmed with TOF were selected as subjects （TOF group）, and another 107 healthy children were chosen as controls （GON group）. PCR was employed to amplify 3 ＇UTR region of NOTCH2 gene. Direct forward and reverse sequencing was performed on the PCR products. The sequence of 3＇ UTR region of NOTCH2 gene was compared with the known sequence （ NG_ 008163.1） in GenBank by the BLAST program and possible mutations were screened. Online software PicTar and TargetScan were used to predict miRNA which could bind to 3 ＇UTR region of NOTCH2 gene. The effects of mutations in 3＇UTR region of NOTCH2 gene on the expression of miRNA-regulated NOTCH2 gene were analyzed. Results A new mutation and 5 reported SNPs in 3＇UTR region of NOTCH2 gene, i.e. 2 672 （157020T〉G）, rs368873082（156595C〉T）, rs835576（156691A〉G）, rs3795664（156937C〉T）, rs699779 （ 156967T 〉 C）, and rs699780 （ 156836T 〉 C） were detected. The differences of the frequencies of alleles of5 reported SNPs of the TOF group and CON group were not statistically significant （ P 〉 0.05）. Results of prediction showed that there were 34 miRNAs that might bind to 3＇UTR region of NOTCH2 gene and this mutation did not locate in binding regions of 34 miRNAs and 3 ＇UTR region of NOTCH2 gene. Conclusion A new mutation 157020T 〉G exists in 3＇UTR region of NOTCH2 gene. This mutation may affect the binding efficiency of miRNA and 3 ＇UTR region of NOT^CH2 gene by spatial configuration changes, and further affect the normal expression of NOTCH2 gene. Five SNPs in 3＇UTR region of NOTCH2 gene are not significantly correlated with the susceptibility of TOF.