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1、新型亚甲基四氢叶酸还原酶单核苷酸多态性研究方法检测恶性血液病易感性 作者：陈宝安姜妮祭美菊侯鹏 陆祖宏高冲 丁家华,孙耘玉王骏程坚赵刚【摘要】 本研究探讨一种新型亚甲基四氢叶酸还原酶（MTHFR）单核苷酸多态性(SNP)检测方法，并用其检测恶性血液病遗传易感性。根据cDNA芯片原理制作一种目的基因芯片，利用双色荧光探针杂交进行SNP位点检测，测序法对该芯片检测结果的准确性进行验证,并以此对来自中国江苏地区的157例健康对照和127例恶性血液病患者(30例多发性骨髓瘤，28例非霍奇金淋巴瘤，22例急性淋巴细胞白血病，40例急性髓系白血病，7例慢性髓系白血病)的MTHFR基因C677T多态位点进行。
2、检测。结果表明，为野生型、杂合型和突变型的叠加荧光分别显示为绿色、黄色和红色。测序结果与芯片结果吻合。677C和677T在病例和对照组的基因频率分别为58.7%、66.9%、41.3%和33.1%，差异有显著性（2=4.077, P=0.043）。677TT基因型发生MM相对风险明显增加（OR=4.21；95%CI=1.50-11.83；P=0.006）。结论：本芯片检测方法准确、高通量且价格低廉，适用于大规模样本SNP调查；C677T多态改变影响恶性血液病的发病风险。677TT基因型是MM的易感因素。 【关键词】 单核苷酸多态性； 基因芯片； 双色荧光杂交； 亚甲基四氢叶酸还原酶； 恶性血液。
3、病A New Method for 5,10-Methylenetetrahydrofolate Reductase Single Nucleotide Polymorphisms Genotyping Used to Study Susceptibility of Hematological MalignancyAbstractThe aim of this study was to set up a new method for 5,10- Methylenetetrahydrofolate reductase (MTHFR) single nucleotide polymorphisms。
4、 (SNP) genotyping, and to investigate the hereditary susceptibility of hematological malignancy. Prepared an aimed gene microarray based on cDNA microarray theory, dual- color fluorescenece hybri- dization was used to detect SNP loci, and DNA sequencing was performed to confirm the results. The MTHF。
5、R C677T SNP loci of 157 controls and 127 patients with hematological malignancies (30 multiple myeloma, 28 non- Hodgkins lymphoma, 22 acute lymphoblastic leukemia, 40 acute myeloid leukemia, 7 chronic myeloid leukemia) from Jiangsu province were detected. The results showed that after overlapping, h。
6、omozygous wild type, heterozygote type and homozygous mutant type yielded green, yellow and red fluorescence, respectively. DNA sequencing validated these results. The allele frequency of 677C and 677T in patients and controls were 58.7% and 66.9%, 41.3% and 33.1% respectively, showing statistically。
7、 significant difference (2=4.077, P=0.043). 677TT genotype showed a significantly higher risk of MM (OR=4.21; 95%CI=1.50-11.83; P=0.006). It is concluded that this microarray- based method is accurate, high- throughput and inexpensive, suitable for SNP genotyping in a large numbeer of individuals. C。
8、677T polymorphisms influence the risk of hematological malignancies. 677TT genotype is susceptive to MM.Key words single nucleotide polymorphism; gene microarray; dual- color fluorescenece hybridization; 5,10- methylenetetrahydrofolate reductase; hematological malignancy染色体DNA序列的某个位点上存在的单个碱基变化如果在人群中。
9、的发生频率超过1%，即为单核苷酸多态性（SNP）。表达基因平均每1 000 bp即出现一个SNP位点，是人类基因组序列变异的主要形式。在不同的人群中，SNP的频率分布有差异，这些差异可以代表某一种族或人1群间的遗传差异。研究SNP有助于解释个体的表型差异、不同群体和个体对疾病，特别是对复杂疾病的易感性，以及对各种药物的耐受性和对环境因子的反应性。因此，有必要建立一种准确、费用低、适合大样本的SNP检测方法。近年来研究表明，叶酸缺乏和（或）叶酸代谢异常可导致多种肿瘤的发生。亚甲基四氢叶酸还原酶（5，10-methylenetetrahydrofolate reductase，MTHFR）是叶酸代。
10、谢的关键酶，其基因C677T位点多态改变引起酶活性下降，已被证实它与多种肿瘤的易感性有关。为此我们建立一种芯片检测方法，并对恶性血液病患者的C677T位点进行初步分析。材料和方法研究对象127例病例样本取自东南大学附属中大医院、江苏省人民医院和南京鼓楼医院2004年1月至2004年11月收治的血液肿瘤患者。在127例病例中多发性骨髓瘤（MM）30例，非霍奇金淋巴瘤（NHL）28例，急性淋巴细胞白血病（ALL）22例，急性髓系白血病（AML）40例，慢性髓系白血病（CML）7例。所有MM经骨髓细胞形态学、血或尿M蛋白、本- 周氏蛋白检测及扁骨X光片确诊，NHL经组织病理确诊，白血病患者经骨髓细胞。
11、形态学、免疫学、细胞遗传学确诊，所有患者排除已行异基因造血干细胞移植。157例健康对照样本取自同期在东南大学附属中大医院、江苏省人民医院健康查体人群，对照人群排除恶性肿瘤、冠心病。所有标本均为EDTA抗凝的外周血。病例组男76人，女51人，年龄18-79岁，平均年龄47.39岁；对照组男98人，女59人，年龄22-75岁，平均年龄48.55岁。两组性别、年龄分布无显著差异（P值分别为0.657和0.533）。常规酚- 氯仿法提取基因组DNA。目的片段的PCR扩增和纯化采用已报道的引物序列1，C677T位点的PCR反应体系为30 l，其中含10的缓冲液3 l，25 mmol/L的Mg2+溶液2 。
12、l，10 mmol/L的dNTP 0.6 l，10 mol/L的双侧引物各1 l ，1.25 U的Taq DNA聚合酶（5 U/L），提取的基因组DNA 1 l。扩增条件：94预变性5分钟，94变性30秒，62退火30秒，72延伸30秒，共35个循环，72延伸7分钟。扩增在Gene Amp 2400 PCR System上进行。QIAquick PCR产物纯化试剂盒纯化PCR产物。分光光度计对PCR产物进行定量并将产物溶于3SSC缓冲液中，浓度为300 ng/l。目的片段芯片的制备及杂交将PCR产物通过PixSys 5500点样仪（Cartesian Technology Inc产品）点于氨基。
13、玻片上。点样后玻片水化30分钟，100快干2秒，400 mJ紫外交联，80干烤2小时。野生型探针677CC：5- Cy3-CGGGAGCCGATTT- 3，突变型探针677TT：5- Cy3-CGGGAGTCGATTT- 3。将荧光标记探针1 mol/L（野生型和突变型探针各半）与杂交液（Telechem）按体积13混合。在潮湿的杂交盒中37杂交2小时。室温下分别用2SSC- 0.1% SDS 和 0.1SSC -0.1% SDS清洗5分钟，灭菌双蒸水清洗2分钟后吹干玻片。用ScanArray Lite 微阵列分析系统 （BioScience Company产品）进行芯片扫描。统计学处理病例组。
14、和对照组的MTHFR基因型的频率差异以2检验确定，P0.05为差异有显著意义。恶性血液病和MTHFR基因多态性的相关性用比值比（odds ratio，OR）以及95的可信度（95% confidence interval，95% CI）表示，OR值经过年龄、性别校正。所有统计分析均用SPSS 11.5 软件计算。结果样品微阵列的分析双色荧光杂交进行SNP分型原理为经过杂交扫描后，野生型样本能获得一个较强的Cy3荧光（绿色荧光），突变型样本获得一个较强的Cy5荧光（红色荧光），杂合型样本既可获得Cy3荧光又可获得Cy5荧光，经过叠加后能显示一个较强的“黄色”荧光。我们分析了恶性血液病的6个样本的。
15、C677T位点。附图A、B和C中显示了6个样品的荧光信号和附图D中显示了这些样品的荧光信号的强度值。从图中可以看到4个样品（1-4）显示了较强的Cy3荧光信号，并且显示了和背景接近的Cy5荧光值，Cy5/Cy3的比值在0.09和之间，表明这4个样品的DNA仅仅与检测子677CC相匹配，因此判断它们在C677T位点为纯合野生型。样品5显示了“红色”的荧光，Cy5/Cy3的比值是11.59，因此可以判断这个样品在C677T位点为纯合变异型。样品6分别得到了较强的Cy3和Cy5荧光信号，Cy5/Cy3的比值是1.05，因此可以判断这个样品在C677T位点为杂合型。我们对上述6个样品进行了测序验证（附。
16、图E），测序结果和芯片结果相一致。双色荧光杂交微阵列的高通量应用我们利用双色杂交微阵列芯片技术对127例恶性血液病患者和157例健康对照的MTHFR基因的C677T位点进行了分析。两组样本分别点制了4个微阵列，每个微阵列中同一样本重复3个点，病例组点制的微阵列大小为1822，其中第22行仅有1个样本，对照组点制的微阵列大小为2123，第23行有3个样本。所有样本PCR产物的点样浓度控制在200-300 ng/l之间。根据Cy3和Cy5双色荧光扫描结果叠加图所呈现的绿、红、黄3种颜色进行分型。同一样本在同一微阵列中有很好的重复性，在不同微阵列中荧光强度略有差别，但叠加图颜色一致，分型结果一致。两。
17、组基因型频率均符合Hardy- Wenberg平衡，样本具有群体代表性。病例组的C和T基因频率为58.7%和41.3%，对照组的C和T基因频率为66.9%和33.1%，差异有统计学意义（2=4.077, P=0.043）。我们对两组的MTHFR C677T基因型及恶性血液病的易感性进行了单因素分析，677CC、677CT和677TT在患者组和对照组分别为48例（37.8%）、53例(41.7%)、26例(20.5%)和72例（45.9%）、66例(42.0%)、19例(12.1%)。发现677CT和677TT基因型相对677CC基因型发生恶性血液病的风险度有增加趋势，但差异无统计学意义（分别为。
18、OR=1.23,95%CI=0.74-2.05,P=0.434和OR=1.97,95%CI=0.98-3.97,P=0.057）。进而我们将患者组4种疾病（CML因例数过少除外）分别与对照组进行了单因素分析，发现677TT基因型发生MM的风险明显增加（OR=4.21,95%CI=1.50-11.83,P=0.006）。677CT和677TT基因型的NHL易感程度有增加的趋势，且677TT较677CT增加明显，呈等位基因计量- 效应关系（表1，表2）。相反，677TT基因型的ALL易感程度有下降趋势。Table 1. Correlation between MTHFR C667T and ris。
19、k of MM and NHL（略）Table 2. Correlation between MTHFR C667T and risk of ALL and AML（略）讨论近年来SNP芯片检测备受关注。目前芯片的制作主要有两种方法，一种是将探针固定于芯片，将PCR扩增并标记的待检测目的DNA混合物与探针杂交，这种方法可以检测有限样本的多个SNP位点；另一种方法是将PCR扩增后的待检测目的DNA固定于芯片，以标记的探针进行杂交，这种方法可以检测多个样本的有限SNP位点。Flavell等2研制了基于后一种方法的SNP检测芯片，但该方法昂贵的链霉抗生物素蛋白（streptavidin）修饰的玻片只。
20、能在PBS中保存3天。我们介绍的双色荧光杂交微阵列方法在等位基因的区分上过程简单、自动化程度高，大样本检测时成本相对低，能在一个实验中分析上千个样品，因此节省了时间和增强了效率。测序法证明，含有13个碱基的荧光标记检测子在37的杂交温度下能够精确地进行基因分型。而且该芯片能够较长时间地储存，实验中能够使用较多的微阵列，因此它能对结果的分析提供一个较好的统计基础。叶酸缺乏和(或)叶酸代谢异常可导致多种肿瘤的发生，这与叶酸的两种主要作用有关：提供甲基使dUMP转变为dTMP，参与DNA的合成；作为甲基供体维持基因组甲基化的独特模式。MTHFR通过影响叶酸代谢而与肿瘤易感相关。无论何种基因型导致何种。
21、疾病易感，增加叶酸摄入似乎都可以降低发病的危险性。但许多疾病的发生与父母甚至祖父母的叶酸缺乏有关，提高个体叶酸水平从而达到预防疾病的目的似乎也需要长期的过程；且由于基因型的不同，个体对叶酸的需求量有差异。因此鉴别遗传易感个体进行目标明确的预防十分重要。患者组和对照组677C和677T基因频率的显著性差异提示C677T多态改变影响恶性血液病的发病风险。具体疾病分析提示,该位点变异在各种恶性血液病的易感性及易感程度上不完全相同。Gonzalez-Ordonez等3发现，MM患者组的677CC基因型明显少于对照组（分别为19%和46%；P=0.001），其中15例IgG型MM中仅有1例为677CC，。
22、677CC可以使MM发病风险下降75%，Gonzalez-Fraile等4的研究数据也显示MTHFR 677CC在MM患者组和对照组中的分布频率明显不同（分别为34.4%和48.1%），这与我们的研究结果一致。虽然我们的样本数较少，但677TT基因型比例在两组中差异显著，难以完全用抽样误差解释。多数研究认为677CT/TT对ALL有保护作用，但最近一项在意大利人群中的研究显示C677T与ALL发病风险无关，认为这可能是该地区人群的677T基因频率过高以及高叶酸摄入水平造成的5。我们的研究也得出C677T与ALL发病风险无关（P0.05），在我们的对照组中677T基因频率为33.1%，与上海地区。
23、人群的基因频率相似6，明显低于中国北方人群7以及上述意大利人群，这不足以解释该结果。同时我们的数据显示677TT的ALL发病风险有下降趋势（OR=0.38； 95%CI=0.39-4.8），与大多数的研究结论一致，但获得能反映真实情况的结果可能还需要扩大样本含量。C677T与AML发病风险无关，这与目前所有AML的研究结果一致。NHL发病风险与C677T没有明显相关，这符合高加索人种间的研究结果8；但降低的酶活性似乎使患病趋势增加，这与Skibola等9报道的677TT基因型的个体发生滤泡淋巴瘤的相对风险度升高存在一致性。相反，对日本人群的研究结果却发现677T发生NHL的风险下降10。不尽相。
24、同的研究结果说明MTHFR C677T与疾病易感性关系可能因地区人群的基因频率而异，因生活习惯而异，或者还受某些相关基因多态的影响（如MTHFR A1298C；蛋氨酸合酶 A2756G；亚甲基四氢叶酸脱氢酶 G1958A）。因此要取得反应真实情况的研究结果可能需要更大的样本含量，精确的疾病分型，合理的对照，多因素的分析。我们将在MTHFR SNP与恶性血液病遗传易感性的后续研究中加以完善，而双色荧光杂交微阵列方法将为我们的研究提供高通量平台。【参考文献】 1 Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor f。
25、or vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet, 1995; 10:111-1132 Flavell AJ, Bolshakov VN, Booth A, et al. A microarray-based highthroughput molecular marker genotyping methods: the tagged microarray marker (TAM) approach. Nucl Acids Res, 2003; 31:e1153 Gon。
26、zalez-Ordonez AJ, Fernandez-Carreira JM, Fernandez-Alvarez CR, et al. Normal frequencies of the C677T genotypes on the methylenetetrahydrofolate reductase (MTHFR) gene among lymphoproliferative disorders but not in multiple myeloma. Leuk Lymphoma, 2000; 39: 607-612 4 Gonzalez- Fraile MI, Garcia- San。
27、z R, Mateos MV, et al. Methylenetetrahydrofolate reductase genotype does not play a role in multiple myeloma pathogenesis. Br J Haematol, 2002; 117:890-8925 Chiusolo P, Reddiconto G, Cimino G, et al. Methylenetetrahy-drofolate reductase genotypes do not play a role in acute lymhoblastic leukemia pat。
28、hogenesis in the Italian population. Haematologica, 2004; 89:139-1446 张国栋, 项坤三, 翁青等. 亚甲基四氢叶酸还原酶C677T多态性与上海地区2型糖尿病合并大血管并发症的关系. 中华内分泌代谢杂志, 2002; 18:362-3657 Song C, Xing D, Tan W, et al. Methylenetetrahydrofolate reductase polymorphisms increase risk of esophageal squamous cell carcinoma in a Chinese 。
29、population. Cancer Res, 2001; 61:3272-32758 Lincz LF, Scorgie FE, Kerridge I, et al. Methionine synthase genetic polymorphism MS A2756G alters susceptibility to follicular but not diffuse large B-cell non-Hodgkins lymphoma or multiple myeloma. Br J Haematol, 2003; 120:1051-10549 Skibola CF, Forrest 。
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31、phoma. Blood, 2001; 97: 3205-3209 and League branch of manual work, to promote the use of two manuals in the citys organizations at various levels. The county committee, directly under the group (work) is under the jurisdiction of grassroots League and group (total) using branch ratio shall not be l。
32、ess than 70%.Three, to further strengthen the groups leadership and team building(a) to strengthen the leagues leadership at all levels of construction. Continue to do a good job of municipal Party Committee Organization Department on the strengthening of the Communist Youth League cadres management。
33、 opinions implementation. To promote the group counties (autonomous counties, cities) Commission and city directly under the group (workers) appointed to carry out the Five team building activities. Improve the League Association Department working mechanism. Conscientiously fulfill the duties of CO。
34、, combined with the city group directly under (the) Committee of the general election and the adjustment, actively jointly with the unit where the party organization department good group leadership selection with the work. Do a good job in municipal Party committee assistant committee leadership an。
35、d leading cadres of the annual assessment work, adhere to and improve the assistant secretary of the Communist Youth League talk system group To weave individual conversation and collective conversation, to strengthen the committees of all levels, the Standing Committee of the Standing Committee, th。
36、e establishment of a sound system of central group learning, the system of investigation and study, the system of important issues and the reporting system.(two) to strengthen the work of education and training of cadres to conscientiously implement the Central Committee. On the implementation of gr。
37、assroots cadres training project and opinions on carrying out the mission municipal CYL Cadres training project implementation opinions, full implementation of the project of the City Youth League cadres training education. Focus on the county grassroots cadres and workers focused on training, the n。
38、ew school system vocational training League cadres, for the Municipal Organization Department jointly held the third session of the Communist Youth League system training cadres in party. Focusing on the construction of new socialist countryside, strengthening village cadres and rural construction t。
39、eam group member of the young team, the county organizations at various levels throughout the year is expected to train 100 00 rural grassroots cadres, plans to train 1000 village secretary of the village Party branch.(three) actively promote grassroots cadres election work. Under the unified leader。
40、ship of the party organization, to strengthen grassroots organizations direct work, standardize the election work rules and procedures, in the designated candidate, open competition to democratically elected branch secretary at the same time, actively and steadily the development of Township, street。
41、s and other grassroots organizations in the direct election of the pilot work, on the basis of summarizing the experience and gradually expand the scope of the pilot, and gradually formed a set of measures and cadres election system.(four) to strengthen the cadres testing exercise. To study the deve。
42、lopment of XX city from 2006 to 2008 cadres testing exercise planning. To select outstanding cadres to the central mission in Beijing, Shanghai, Guangzhou and other central organs and sending work in developed areas. With the Central Committee the implementation of the western region and ethnic mino。
43、rities cadres training plan . To carry out the eighth batch of municipal organs attachment cadre management, selection, training work. The outstanding cadres to start the National Development Bank XX branch testing exercise.(five) to strengthen the group construction. The main position to play the r。
44、ole of Youth League cadre education and training as the city, in accordance with the requirements of post training, further curriculum scientifically and rationally, adopt arrangement menu course, basic theory includes group work, focus, hot and difficult issues, to enhance the content and practice;。
45、 to strengthen the construction of teaching staff, the integration of teachers, establish a quality teacher training team, establish and implement course evaluation system; take delivery of training to the countryside, on-site training, and increase the rural grassroots cadres training efforts.Four,。
46、 efforts to strengthen the work of young talent(a) to tenth XX young 54 medal in recognition of the publicity, and actively explore the XX youth service. 54 Medal of institutionalization, standardization and socialization mechanism. Give full play to the XX award in recognition of 54 youth medal Ass。
47、ociation, Youth Award winner in the 54 chapter, investigation activities. Who won the Youth Service medal, dedication to the society to create conditions, build the stage. During the 54, named in recognition of the tenth XX young 54 medal. The association of XX editing of young talent , the exchange。
48、 of information and communication platform for the XX of outstanding young talents.(two) the establishment of municipal Party committee of young talent information database. In accordance with the feasible, effective, classification management, implement step by step principle, the establishment of 。
49、municipal Party committee of youth talent database; guiding organizations at various levels generally established the young talent database, construction of city, county (directly under the Communist Youth League), grassroots organizations three young talent information database the relevant informa。
50、tion collection system, and gradually establish the citys young people, the Communist Youth League youth talent information network, provide information platform for young talent resource development.Five, take effective measures to strengthen the mission management(a) to strengthen the mission mana。
51、gement work. Strengthen the dues collection and management, continue to adhere to the dues collection regularly reporting system, the annual comprehensive award in recognition of the tour and the tour one vote veto system audit system. Do statistical work in the group. The group service award in rec。
52、ognition of the advanced units, standardization, promoting group management work institutionalized.(two) to strengthen the organization of research and information exchange efforts. Grassroots carry out research, efforts to form a number of theoretical results. Make full use of the network carrier, 。
53、to explore the establishment of electronic group service system, promote the work of Communist Youth League information.Attachment 4:XX municipal Party Committee Propaganda Department of the Communist Youth League work pointsIn 2006, the Communist Youth League propaganda ideological work XX to Deng 。
54、Xiaoping theory and the important thought of Three Represents as guidance, study and implement the partys sixteen and the spirit of the the fifth Plenary Session of the 16th CPC Central Committee implementation of the eight plenary session of the two committee, the citys cultural work conference, th。
55、e Propaganda Department of the meeting and the the fourth Plenary Session of the 15th CPC Central Committee permanent mission spirit, closely around 11th Five-Year XX the goal of full implementation of Scientific Outlook on Development, emancipating the mind, seeking truth from facts, keep pace with。
56、 the times, close to reality, close to life, close to the young, persevere grasp orientation, grasping deepening, grasping construction innovation, further improve the groups propaganda and ideological work ability, unity To guide the majority of members of the youth to achieve the 11th Five-Year pl。
57、anning tasks, the overall construction of a well-off society, building a socialist harmonious society to make new contributions.One, adhere to the Three Represents important thought and Scientific Outlook on Development education to guide the youth, to further strengthen the ideological and politica。
58、l education of youth(a) armed with youth science theory. Unremittingly with Deng Xiaoping theory and the important thought of Three Represents armed members of youth education, to promote the study and implement to expand the depth and breadth. To study and implement the important thought of Three R。
59、epresents with the establishment and implementation of Scientific Outlook on Development combine with the implementation of the requirements of constructing socialist harmonious society with Scientific Outlook on Development, carry out education as an important part of the theory of armed youth in t。
60、he new period, strengthen the establishment and implementation of Scientific Outlook on Development education, educate and guide the youth to fully understand the great significance of establishment and implementation of Scientific Outlook on Development, in-depth understanding of the Scientific Out。
61、look on Development era back The king, the practice foundation, basic connotation, historical status, spirit and practice requirements, a profound understanding of Scientific Outlook on Development with Marxism Leninism, Mao Zedong thought, inheriting and developing relationship between Deng Xiaopin。
62、g theory and the important thought of Three Represents, to help young people establish and consciously practice Scientific Outlook on Development. Persist in learning, grasping the backbone, grasp the practice, grasp the opportunity. Catch club, effective way to grasp the network, with the group of 。
63、cadres, young students and young backbone fronts as the focus, to promote the work of the armed youth theory to develop in depth, to help the youth continue to firm ideals and beliefs, to build a powerful spiritual pillar. To carry out the study and implementation of the partys sixteen session of the five Eight plenary sessio。
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