本文選題：葉酰多聚谷氨酸合成酶基因　切入點：單核苷酸多態(tài)性　出處：《重慶醫(yī)科大學》2012年碩士論文　論文類型：學位論文

【摘要】：目的研究葉酰多聚谷氨酸合成酶（FPGS）基因編碼區(qū)單核苷酸多態(tài)性在深圳地區(qū)漢族急性白血�。ˋL）患兒與健康兒童中的等位基因頻率和基因型分布特征，為進一步探討FPGS基因多態(tài)性與甲氨喋呤（MTX）藥物敏感性可能存在的關(guān)系，進而為腫瘤化療的個體化用藥提供可能的理論參考依據(jù)。 方法采集91例AL患兒的骨髓液和124例單純上呼吸道感染兒童的外周血，提取總RNA，逆轉(zhuǎn)錄為cDNA，利用Premier Primer5.0和Oligo6.0軟件設計擴增FPGS全編碼區(qū)的PCR引物，結(jié)合PremierPrimer5.0、Oligo6.0和Winmelt2.0軟件設計巢式PCR引物，采用PCR-變性梯度凝膠電泳（DGGE）結(jié)合DNA直接測序的方法篩查215例樣本的FPGS編碼區(qū)單核苷酸多態(tài)性（cSNPs）情況，實驗數(shù)據(jù)應用SPSS13.0統(tǒng)計學軟件進行分析，χ2檢驗比較cSNPs在AL組和正常對照組之間以及在FPGS兩亞型之間的差異性，以p0.05為差異有統(tǒng)計學意義。 結(jié)果本研究采用PCR-DGGE結(jié)合DNA直接測序法對深圳地區(qū)215例漢族兒童FPGS全編碼區(qū)進行篩查，發(fā)現(xiàn)了5個突變位點，分別為一個新的錯義突變和4個已知的cSNPs。首次在中國漢族兒童中發(fā)現(xiàn)了一個國內(nèi)外尚未見報道的FPGS編碼區(qū)錯義突變502/490TC（L151/101P），此突變在FPGS兩種亞型的基因中均有發(fā)現(xiàn)，總等位基因頻率為線粒體型（L151P）0.47%、胞漿型（L101P）0.70%，在正常對照組中的等位基因頻率為1.21%（L101P）、0.81%（L151/P），AL患兒組未檢出此突變即等位基因頻率為0（L101P和L151/P）。本研究還檢測到了4個國外已報道但國內(nèi)尚未見報道的cSNPs，即908/896CT（L286/236L）、1446/1434CT （R466/416C）、1513/1501GA（S488/438N）和1516/1504CT（A489/439V）。它們的等位基因總頻率分別為（線粒體型）0.47%、0.70%、0.47%、0.23%和（胞漿型）0.47%、0、0、0.47%。經(jīng)χ2檢驗分析此5個突變位點的等位基因頻率在FPGS兩個亞型之間與兩組之間的差異，P0.05，均無統(tǒng)計學意義，表明這5個位點突變并無亞型之間的差異，與兒童AL亦無易感性關(guān)系。此外，本研究人群中并未檢測到其它27個NCBI SNP數(shù)據(jù)庫及文獻報道中錄入的已知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。結(jié)論首次應用PCR-DGGE結(jié)合DNA直接測序法對FPGS cSNPs進行篩查，共檢測出5個突變位點，證實DGGE技術(shù)是一項可以應用于FPGS基因突變檢測的實用技術(shù)。首次在中國漢族兒童中發(fā)現(xiàn)了一個國內(nèi)外尚未見報道的編碼區(qū)錯義突變502/490TC（L151/101P），，其等位基因頻率為線粒體型（L151P）0.47%、胞漿型（L101P）0.70%，推測其可能對FPGS酶活性及MTX類葉酸拮抗劑敏感性有潛在的影響。首次檢測到了4個國外已報道但國內(nèi)尚未見報道的cSNPs，即908/896CT（L286/236L）、1446/1434CT（R466/416C）、1513/1501GA（S488/438N）和1516/1504CT（A489/439V），它們的等位基因總頻率分別為（線粒體型）0.47%、0.70%、0.47%、0.23%和（胞漿型）0.47%、0、0、0.47%。502/490TC、908/896CT、1446/1434CT、1513/1501GA和1516/1504CT在FPGS兩種亞型中的分布并無差異，與兒童AL易感性未發(fā)現(xiàn)相關(guān)性。本研究人群中并未檢測到其它27個NCBI cSNPs數(shù)據(jù)庫中錄入的已知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.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R733.7

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