急性白血病儿童叶酰聚谷氨酸合成酶基因多态性研究

发布时间：2018-03-15 10:23

本文选题：叶酰多聚谷氨酸合成酶基因　切入点：单核苷酸多态性　出处：《重庆医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的研究叶酰多聚谷氨酸合成酶（FPGS）基因编码区单核苷酸多态性在深圳地区汉族急性白血病（AL）患儿与健康儿童中的等位基因频率和基因型分布特征，为进一步探讨FPGS基因多态性与甲氨喋呤（MTX）药物敏感性可能存在的关系，进而为肿瘤化疗的个体化用药提供可能的理论参考依据。方法采集91例AL患儿的骨髓液和124例单纯上呼吸道感染儿童的外周血，提取总RNA，逆转录为cDNA，利用Premier Primer5.0和Oligo6.0软件设计扩增FPGS全编码区的PCR引物，结合PremierPrimer5.0、Oligo6.0和Winmelt2.0软件设计巢式PCR引物，采用PCR-变性梯度凝胶电泳（DGGE）结合DNA直接测序的方法筛查215例样本的FPGS编码区单核苷酸多态性（cSNPs）情况，实验数据应用SPSS13.0统计学软件进行分析，χ2检验比较cSNPs在AL组和正常对照组之间以及在FPGS两亚型之间的差异性，以p0.05为差异有统计学意义。结果本研究采用PCR-DGGE结合DNA直接测序法对深圳地区215例汉族儿童FPGS全编码区进行筛查，发现了5个突变位点，分别为一个新的错义突变和4个已知的cSNPs。首次在中国汉族儿童中发现了一个国内外尚未见报道的FPGS编码区错义突变502/490TC（L151/101P），此突变在FPGS两种亚型的基因中均有发现，总等位基因频率为线粒体型（L151P）0.47%、胞浆型（L101P）0.70%，在正常对照组中的等位基因频率为1.21%（L101P）、0.81%（L151/P），AL患儿组未检出此突变即等位基因频率为0（L101P和L151/P）。本研究还检测到了4个国外已报道但国内尚未见报道的cSNPs，即908/896CT（L286/236L）、1446/1434CT （R466/416C）、1513/1501GA（S488/438N）和1516/1504CT（A489/439V）。它们的等位基因总频率分别为（线粒体型）0.47%、0.70%、0.47%、0.23%和（胞浆型）0.47%、0、0、0.47%。经χ2检验分析此5个突变位点的等位基因频率在FPGS两个亚型之间与两组之间的差异，P0.05，均无统计学意义，表明这5个位点突变并无亚型之间的差异，与儿童AL亦无易感性关系。此外，本研究人群中并未检测到其它27个NCBI SNP数据库及文献报道中录入的已知cSNPs，包括253/241AC、293281GA、303/291CT、335/323GT、350/338CT、380/368CT、417/405CT、426/414GA、441/429CT、471/459CT、473/461CG、756/744GC、930/918AG、962/950CT、965/953CT、1268/1256CG、1360/1348TA、1451/1439CT、1466/1454GA、1485/1473GA、1535/1523TA、1541/1529CT、1544/1532CT、1633/1621GC、1643/1631AG、1766/1754CT和1799/1787CT。结论首次应用PCR-DGGE结合DNA直接测序法对FPGS cSNPs进行筛查，共检测出5个突变位点，证实DGGE技术是一项可以应用于FPGS基因突变检测的实用技术。首次在中国汉族儿童中发现了一个国内外尚未见报道的编码区错义突变502/490TC（L151/101P），，其等位基因频率为线粒体型（L151P）0.47%、胞浆型（L101P）0.70%，推测其可能对FPGS酶活性及MTX类叶酸拮抗剂敏感性有潜在的影响。首次检测到了4个国外已报道但国内尚未见报道的cSNPs，即908/896CT（L286/236L）、1446/1434CT（R466/416C）、1513/1501GA（S488/438N）和1516/1504CT（A489/439V），它们的等位基因总频率分别为（线粒体型）0.47%、0.70%、0.47%、0.23%和（胞浆型）0.47%、0、0、0.47%。502/490TC、908/896CT、1446/1434CT、1513/1501GA和1516/1504CT在FPGS两种亚型中的分布并无差异，与儿童AL易感性未发现相关性。本研究人群中并未检测到其它27个NCBI cSNPs数据库中录入的已知cSNPs，包括253/241AC、293281GA、303/291CT、335/323GT、350/338CT、380/368CT、417/405CT、426/414GA、441/429CT、471/459CT、473/461CG、756/744GC、930/918AG、962/950CT、965/953CT、1268/1256CG、1360/1348TA，1451/1439CT，1466/1454GA，1485/1473GA，1535/1523TA、1541/1529CT、1544/1532CT、1633/1621GC、1643/1631AG、1766/1754CT和1799/1787CT。
[Abstract]:Objective to study the formyltransferase (FPGS) gene encoding single nucleotide polymorphisms in Shenzhen Han children with acute leukemia (AL) and healthy children in allele frequency and genotype distribution, for the further study of FPGS gene polymorphism with methotrexate (MTX) drug sensitivity might exist. It may provide a theoretical basis for individualized chemotherapy.
Methods bone marrow collected in 91 patients with AL and 124 cases of children with upper respiratory tract infection of peripheral blood, extracted the total RNA, reverse transcription cDNA, PCR primers, amplification of the FPGS encoding region is designed by using the Primer5.0 Premier and Oligo6.0 software combined with PremierPrimer5.0, Oligo6.0 and Winmelt2.0 software design of nested PCR primers, using PCR- denaturing gradient gel electrophoresis (DGGE) combined with DNA direct sequencing method for screening 215 samples of FPGS single nucleotide polymorphism encoding region (cSNPs), the experimental data were analyzed by statistical software SPSS13.0, 2 test comparison of cSNPs between AL group and normal control group and the difference in FPGS between the two subtypes of P0.05 had statistical significance the difference.
The results of this study by PCR-DGGE and DNA sequencing were used to screen 215 cases of children of Han nationality in Shenzhen area FPGS encoding region, found 5 mutations, respectively, a new missense mutation and 4 known cSNPs. for the first time in the Chinese Han children found a home and abroad has not been reported missense FPGS encoding region the mutation of 502/490TC (L151/101P), this mutation was found in the two subtypes of FPGS gene, the total allele frequency of mitochondrial (L151P) 0.47%, cytoplasmic (L101P) 0.70%, in the normal control group, the allele frequency was 1.21% (L101P), 0.81% (L151/P). Children with AL were detected the mutation allele frequency was 0 (L101P and L151/P). This study also detected 4 has been reported abroad but there is no report of cSNPs, namely 908/896CT (L286/236L), 1446/1434CT (R466/416C), 1513/1501GA (S488/438N) and 1516/1504CT (A489/43 9V). The allele frequency of their total respectively (mitochondrial) 0.47%, 0.70%, 0.47%, 0.23% and 0.47% (cytosolic), 0,0,0.47%. by 2 test analysis of the 5 mutant allele frequency between the two subtypes of FPGS and the differences between the two groups, P0.05. There were no statistically significant, indicating that these 5 mutations have no differences between subtypes, nor the susceptibility of children with AL. In addition, the study population did not detect the known cSNPs, input the other 27 NCBI SNP database and reported in the literature including 253/241AC, 293281GA, 303/291CT, 335/323GT, 350/338CT, 380/368CT 417/405CT, 426/414GA, 441/429CT, 471/459CT, 473/461CG, 756/744GC, 930/918AG, 962/950CT, 965/953CT, 1268/1256CG, 1360/1348TA, 1451/1439CT, 1466/1454GA, 1485/1473GA, 1535/1523TA, 1541/1529CT, 1544/1532CT, 1633/1621GC, 1643/1631AG, 1766/1754CT, and 1799/1787CT.'s conclusion Screening of FPGS cSNPs combined application of PCR-DGGE direct sequencing of DNA detected a total of 5 mutations, confirmed that DGGE technology is a practical technology can be applied in the detection of mutations in FPGS gene. For the first time in Chinese Han children were found in the coding region of a missense mutation has not been reported at home and abroad 502/490TC (L151/101P) and the allele frequency of mitochondrial (L151P) 0.47%, cytoplasmic (L101P) 0.70%, speculated that it may have potential effects on FPGS activity and MTX antifolate sensitivity. It was first detected in 4 has been reported abroad but in China has not been reported in the cSNPs, namely 908/896CT (L286/236L), 1446/1434CT (R466/416C), 1513/1501GA (S488/438N) and 1516/1504CT (A489/439V), allele frequency of their total respectively (mitochondrial) 0.47%, 0.70%, 0.47%, 0.23% and 0.47% (cytosolic), 0,0,0.47%.502/490TC, 908/896CT, 1446/1434CT, 1513 There was no difference in the distribution of two subtypes of FPGS in /1501GA and 1516/1504CT, and AL found no correlation. The susceptibility of children in the study population was not detected by the known cSNPs, input the other 27 NCBI cSNPs database including 253/241AC, 293281GA, 303/291CT, 335/323GT, 350/, 338CT, 380/368CT, 417/405CT, 426/414GA, 441/429CT, 471/459CT. 473/461CG, 756/744GC, 930/918AG, 962/950CT, 965/953CT, 1268/1256CG, 1360/1348TA, 1451/1439CT, 1466/1454GA, 1485/1473GA, 1535/1523TA, 1541/1529CT, 1544/1532CT, 1633/1621GC, 1643/1631AG, 1766/1754CT and 1799/1787CT.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R733.7

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