论文标题:DPYD基因分型芯片的制备及应用基础研究
Basic Research in Preparation and Application of Oligonucleotide Microarray for DPYD Genotyping
论文作者 王晓云
论文导师 胡小电；王升启,论文学位 硕士,论文专业 肿瘤学
论文单位 中国人民解放军军事医学科学院,点击次数 139,论文页数 70页File Size3049k
2003-05-16论文网 http://www.lw23.com/lunwen_1019237187/ 5-Fu;DPYD;基因芯片;基因分型;基因多态性
5-Fu,DPYD,gene chips,genotyping,gene polymorphism
DPYD基因分型芯片的制备及应用基础研究 5-Fu对于肿瘤病人应用较为广泛，且疗效显著，但有时会出现严重的毒副作用甚至死亡。如果在使用5-Fu之前能够预测病人对它的反应，将减少病人的痛苦和损失，从根本上改变以前的治疗模式，实现个体化医疗。 由于85％以上的5-Fu通过DPD酶代谢而失去活性，故DPD酶活性下降或缺失的病人将遭受严重的毒副作用。目前随着分子生物学的发展，以及对DPYD基因的深入了解，使得从基因水平直接检测5-Fu的反应成为可能。研究发现，DPD酶是由DPYD基因编码的，该基因EXON2T85C(DPYD9)位点的突变的人体内的DPD酶活性完全缺失，故当接受5-Fu治疗时可能发生严重的毒副反应。 本实验以基因芯片为手段通过检测DPYD基因的EXON2T85C(DPYD9)位点突变直接预测5-Fu的反应。同时也检测了DPYD基因中文献报道的突变较高的EXON13A1627G(DPYD5)位点及中国人群中尚未报道的EXON2G62A(DPYD12)，EXON13G1601A(DPYD4)，EXON14deleteC1897(DPYD3)，和EXON14intron14GIA(DPYD2)。目前已完成了DPYD基因分型芯片的制备及条件的优化，同时首次在中国人群中对肿瘤病人、正常人及肾病病人的临床标本进行了基因分型。 第一部分DPYD基因芯片的制备及条件的优化 我们检索了重要SNPs位点及其侧翼序列，并计算机辅助设计引物，探针，本室合成引物和探针。我们将靶DNA分别点在溴片及醛基片上制成芯片备用。同时优化了PCR及杂交条件，也对多重PCR进行了尝试。还以质粒为模板进行了重复性试验，结果显示该芯片杂交结果可靠。另外，我们也考察了紫外交联强度对杂交信号的影响。同时构建了标准模板和建立了分型标准。 第二部分利用DPYD基因芯片对临床样本进行基因分型 实验发现在中国人群中，EXON2T85C(DPYD9)位点的突变在肿瘤病人、正常人及肾病病人中分别为15/112，3/45，9/83，EXON13A1627G(DPYD5)的突变在上述人群中为40/112，14/45，23/83。另外，我们将307医院的肿瘤病人中肺军事医学科学院硕士论文癌与非肺癌及男女病人的上述位点的突变进行了比较，结果:肺癌与非肺癌的EXONZT85C归pYI)9)位点的突变分为6/72，9/40，EXoNl3A1627G(DpYD5)的突变为26/72，14/4o。男女病人中的EXo卜rZT85c(D pYDg)的突变为11/70，4/42，EXON13A1627G(DpYDS)的突变为25/70，25/42。 另外，我们将上述结果进行了统计学处理得出结论:肿瘤病人、正常人及肾病病人在EXONZT85C(DpYDg)和EX0N13A1627侧DpYDS)两个位点的突变均无显著性差异(P>0.05)。肺癌与非肺癌患者在Ex0N2T85C(DPYD9)位点的突变存在显著性差异(P<005)。肺癌与非肺癌患者在EXoNI 3A1627侧DPYDS)位点的突变不存在显著性差异(P>0.05)。男女病人在上述位点的突变不存在显著性差异(P>005)。 本实验还对肿瘤病人，正常人及肾病病人样本中EXONZG62A (DPYD12)，EXON13G160lA(DPYD4)，EXON14del以eC1897(DPYD3)，Ex0N14intronl4GIA(DPYD2)进行了分型，结果未发现突变。 另外，为验证基因芯片的准确性我们采用直接测序法将样本中17例PCR产物直接测序，发现与芯片的分型结果符合的有16例，符合率达到94.12%。证明基因分型芯片具有较高的灵敏性和准确性。 本实验为在中国人群中建立DPYD基因芯片的检测技术平台，发展个体化医疗奠定了基础。同时，对于今后进一步研究DPYD基因多态性与5一氟尿嚓陡药物疗效及毒副作用的关系具有十分重要的意义。
Although the application of 5-Fu is very wide to cancer patients and curative effect is notable, severe side-effects even death will happen sometimes. If we can predict its reaction before using 5-Fu, it will alleviate patients" suffering, change previous treatment pattern radically and realize individual medical treatment.Because more than 85% of 5-Fu is degradated by DPD, patients who have deficiency or descent in DPD activity will suffer severe side effects. At present, along with molecule biology"s development and deeply understanding to DPYD, it is possible to predict reactions of 5-Fu in gene level. It is known the gene that encodes for DPD expression is called DPYD whose polymorphism on EXON2T85C(DPYD9) implies deficiency in DPD activity. So the patient with EXON2T85C(DPYD9) mutation could experience toxicity in response to 5-Fu therapy.The test predicted reaction of 5-Fu by detecting DPYD polymorphism on EXON2T85C(DPYD9) using gene chips directly. At the same time, we detected DPYD polymorphism on EXON13A1627G(DPYD5) for its higher mutation frequency in papers. Also the mutation frequency in Chinese population was detected on EXON2G62A(DPYD12),EXON13G1601A(DPYD4),EXON14deleteC1897(DPYD3) and EXON14intronl4GIA(DPYD2). Now DPYD gene chip has been prepared, and its some influence factors have been optimized, at one time, we have screened a lot of clinic samples by gene chips in Chinese (cancer patients, normal person, nephropathy patients) for the first time. 1 Preparation and optimization of DPYD gene chipWe searched important SNPS and its flanking sequences. Primers and probes were designed aidedly by computer then synthesized. DPYD gene chip was prepared byprinting the target DNA in bromine or aldehyde modified glass. Optimization of PCR and hybridization were excuted. Mjalty PCR was attempted. Repeated trial with plasmid showed hybridization results is credibility. In addition, effect of ultraviolet crosslinking energy was made on its hybridization.We constructed successfully standard template and constituted criteria for classification . 2 DPYD genechips" application in classifying of drug catabolic enzyme geneGenotyping results displayed the allele frequency on EXON2T85C(DPYD9) in Chinese (cancer patients,normal person,nephropathy patients) were 15/112,3/45,9/83, allele frequency on EXON13A1627G(DPYD5) were 40/112,14/45,23/83. At the same time, it were compared on EXON2T85C(DPYD9) and EXON13A1627G(DPYD5) in cancer patients and different gender, the allele frequency of EXON2T85C(DPYD9) in cancer patients was 6/72, it was 9/40 in other patients. Mutation frequency of EXON13A1627G(DPYD5) was 26/72 in cancer patients and was 14/40 in other patients. It was 11/70 in men and 4/42 in women for EXON2T85C(DPYD9). For EXON13A1627G(DPYD5 it were 25/70 and 15/42.On statistics we concluded the allele frequency on EXON2T85C(DPYD9) and EXON13A1627G(DPYD5) were not different obviously in Chinese.the difference was obvious on EXON2T85C(DPYD9) and not obvious on EXON13A1627G(DPYD5) in cancer and other patients.it was not evident on EXON13A1627G(DPYD5).these was not evident difference on EXON2T85C(DPYD9) and EXON13A1627G(DPYD5) in men and women.We also screened these alleles in these people by DPYD genotyping chips, such as EXON2G62A(DPYD12),EXON13G1601A(DPYD4),EXON14deleteC1897(DPYD3) and EXON14intronl4GIA(DPYD2),mutation is not found.17 PCR products were sequenced for proving veracity of genechip, 16 was accordant to its hybridization results, it is about 94.12%. it proved the results" reliability again.These results set the foundation to establish gene chips technique flatporm and develop individuation project in Chinese population. At the same time, it will play animportant role in proving the connection between DPYD polymorphism and clinicalreaction of 5-Fu.
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