一种基于强健聚合酶链反应的测定法，用于定量易碎 X 智力迟钝-1 基因中的细胞氨酸-鸟-鸟-鸟- 三核苷酸重复
1Central Laboratory, Bao’an Maternity and Child Health Hospital,Jinan University, 2Department of Obstetrics and Gynaecology,The Chinese University of Hong Kong, 3Shenzhen Research Institute,The Chinese University of Hong Kong, 4Department of Genetics and Metabolism,Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, 5PerkinElmer Diagnostics, 6Department of Obstetrics and Gynecology, Bao’an Maternity and Child Health Hospital,Jinan University, 7Maternal-Fetal Medicine Institute, Bao’an Maternity and Child Health Hospital,Jinan University
Summary
精确和强大的聚合酶链反应测定，用于量化易碎 X 智力迟钝-1 基因中细胞氨酸-瓜氨酸-鸟-鸟三核苷酸重复，有助于分子诊断和筛选易碎 X 综合征和易碎 X 相关周转时间较短，设备投资时间缩短。
Abstract
易碎 X 综合征 （FXS） 和相关疾病是由易碎 X 智力迟钝-1（FMR1）基因启动子的 5′ 未翻译区域 （UTR） 中细胞氨酸-瓜氨酸 -鸟-鸟苷 （CGG） 三核苷酸重复的扩展引起的。按照惯例，基因分析仪上的毛细血管电泳片段分析用于FMR1的CGG重复大小，但当重复数高于200时，需要额外的南方印斑分析才能进行精确测量。在这里，我们提出了一种准确和强大的聚合酶链反应（PCR）为基础的方法，用于定量FMR1的CGG重复。本测试的第一步是使用易碎 X PCR 试剂盒对FMR1启动子的 5’UTR 中的重复序列进行 PCR 扩增，然后对 PCR 产品进行纯化和微流体毛细管电泳仪器上的片段大小，以及随后通过使用分析软件引用已知重复项的标准来解释 CGG 重复次数。这种基于PCR的测定是可重复的，能够识别FMR1启动子的全部CGG重复，包括重复数超过200（分类为完全突变）、55至200（预突变）、46至54（中间）和10至45（正常）。它是一种经济高效的方法，有助于对 FXS 和易碎 X 相关疾病进行分类，具有鲁棒性和快速报告时间。
Introduction
易碎 X 综合征 （FXS） 和易碎 X 相关疾病，例如震颤和失流综合征 （FX-TAS），以及原发性卵巢功能不全 （FX-POI） 主要是由 5′ 未翻译的细胞氨酸-瓜氨酸 -瓜氨酸 （CGG） 三核苷酸重复扩张引起的Xq27.31，2. 脆弱 X 智力迟钝 -1（FMR1）基因的区域 （UTR）FMR1编码蛋白（FMRP）是一种多核体相关的RNA结合蛋白，通过调节mRNA的替代拼接、稳定性和树突状传输或调制合成，在神经元发育和突触可塑性方面发挥作用部分贴午后蛋白3，4，5，6，7。
CGG重复大小为>200的动态变异被描述为完全突变，导致FMR1启动子8的异常超甲基化和随后转录沉默。由此产生的FMRP蛋白的缺失或缺乏会破坏正常的神经元发育，并导致FXS9，其特征是各种临床症状，包括中度至重度智力残疾、发育迟缓、过度活跃行为、不良接触和孤独症表现10，11，12。女性FXS患者的表达通常比男性温和。CGG 重复大小从 55 到 200 和 45 到 54 分别归类为预突变和中间状态。由于高度不稳定，CGG重复大小在预突变或中间等位基因大概扩大时，从父母传给后代13，14。因此，具有预突变等位基因的携带者由于重复扩张而使儿童受FXS影响的风险很高，在某些情况下，中间等位基因可以将其重复大小扩展到两代15之间的全部突变范围。 16.此外，预变异的男性也增加患晚发病FX-TAS17、18、19的风险，而预突变女性则倾向于FX-TAS和FX-POI20， 21，22.最近，有报道称，患有发育迟缓和社会行为问题的自闭症谱系障碍在预突变FMR1等位基因23、24的儿童中出现。
确定确切的CGG重复大小对于FXS和易碎X相关疾病的分类和预测具有重要意义25，26。从历史上看，CGG重复区域特异性聚合酶链反应（PCR）具有片段大小加上南方斑点分析，是FMR1 CGG重复27分子分析的黄金标准。然而，传统的特异性PCR对超过100至130次重复的大型预突变不太敏感，并且无法扩增完全突变27，28。此外，用于重复尺寸的传统基因分析仪上的毛细血管电泳无法检测超过 200 CGG 重复的FMR1 PCR 产品。南方印斑分析能够区分更广泛的重复大小，从正常突变到完全突变重复数，并已广泛用于确认完全突变（在男性）和区分异质异位基因与完全突变明显具有正常重复大小的同源性等位子（在女性中）。但是，量化重复的分辨率是有限的。更重要的是，这种分步测试策略是劳动密集型、耗时且成本效益高的。
在这里，我们提出了一种准确和可靠的基于PCR的方法，用于定量FMR1的CGG重复。本测试的第一步是使用易碎 X PCR 试剂盒对FMR1启动器的 5’UTR 中的重复序列进行 PCR 扩增。PCR 产品经过纯化，片段大小在微流体毛细管电泳仪器上执行，随后使用分析软件对 CGG 重复次数进行解释，通过引用基于PCR 片段长度与 CGG 重复次数成正比。PCR系统包括促进高GC富分子三核苷酸重复区域扩增的试剂。这种基于PCR的测定是可重复的，能够识别FMR1启动子的所有CGG重复范围。这是一种经济高效的方法，可在FXS和易碎X相关疾病的分子诊断和筛查中得到广泛应用，周转时间少，设备投资更少，因此可用于更广泛的临床实验室。
Protocol
香港中文大学-新界东集群临床研究伦理委员会获伦理批准（参考编号：2013.055） 1. PCR扩增 开始之前，从-20°C冷冻室中取出PCR缓冲液混合物、样品稀释剂和DNA样品（包括测试和参考DNA）（参见材料表），并将其保持在室温下20-30分钟，以确保所有试剂和DNA完全解冻。涡旋，并在使用前短暂旋转。 使用分光光度计测量DNA样品的浓度（见材料表）?…
Representative Results
预突变母参考样本（NA20240，重复尺寸30和80）和全突变母参考样本（NA20239，重复尺寸为20和200）的大小调整结果分别如图1A和图1B所示。通常，片段大小配置文件中包括两个标记峰（下标记 50 基对 [bp] 和上标记 10，380 bp）。通常有一个底漆复合峰，其大小接近 95 bp。通过参考样本，可以构造一个包含四个点的线性回归标准曲线，如图1C</st…
Discussion
FXS是仅次于三联体21的第二大智力障碍原因，占X相关智力迟钝30的近一半，这可能影响大约四千名男性中的一个和八千名女性中的一个。更重要的是，近1在250-1，000女性携带预突变，这个频率是1在250-1，600男性26，31，32，33。由于 CGG 重复扩张到完全突变的风险在将预突变等位基因传?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究得到了国家自然科学基金应急管理项目（第81741004号赠款）、国家自然科学基金（第81860272号）、广西省科技基金重大研究计划（第81741004号）的资助。授予号AB16380219），中国博士后科学基金资助（批号2018M630993）和广西自然科学基金（批号2018GXNSFAA281067）。
Materials
| Agilent 2100 Bioanalyzer instrument: 0.2 mL PCR tubes | Axygen | PCR-02D-C | |
| Agilent 2100 Bioanalyzer instrument: 1X TE buffer, pH 8.0, Rnase-free | Ambion | AM9849 | |
| Agilent 2100 Bioanalyzer instrument: 2100 Bioanalyzer instrument | Agilent | G2939AA | |
| Agilent 2100 Bioanalyzer instrument: 96-well PCR Plate | Thermo Fisher | AB0800 | |
| Agilent 2100 Bioanalyzer instrument: Electrode cartridge | Agilent | Supplies equipment of the 2100 Bioanayzer instrument | |
| Agilent 2100 Bioanalyzer instrument: IKA vortex mixer | Agilent | Supplies equipment of the 2100 Bioanayzer instrument | |
| Agilent 2100 Bioanalyzer instrument: Sizing software 2100 Expert software | Agilent | Supplies equipment of the 2100 Bioanayzer instrument | |
| Agilent 2100 Bioanalyzer instrument: Test chips | Agilent | Supplies equipment of the 2100 Bioanayzer instrument | |
| Agilent DNA 7500 kit | Agilent | 5067-1506 | For Fragment sizing |
| Agilent DNA 7500 kit: DNA 7500 Ladder (yellow cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: DNA 7500 Markers (green cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: DNA chips | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: DNA Dye Concentrate (blue cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: DNA Gel Matrix Vial (red cap) | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: Electrode Cleaner | Agilent | In kit: Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: Spin Filter | Agilent | Supplies of Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Agilent DNA 7500 kit: Syringe | Agilent | Supplies of Agilent DNA 7500 kit (catalog number: 5067-1506) | |
| Chip priming station | Agilent | 5065-4401 | Supplies equipment of the 2100 Bioanayzer instrument |
| Cubee Mini-centrifuge | GeneReach | aqbd-i | |
| Filter plate vacuum Manifold: MultiScreenHTS Vacuum Manifold | Merck Millipore | MSVMHTS00 | Vacuum instrument for Filter plate vacuum Manifold for PCR product purification |
| Filter plate vacuum Manifold: Silicone stopper | Merck Millipore | XX2004718 | Filter plate vacuum Manifold |
| Filter plate vacuum Manifold: Vacuum pump | Merck Millipore | WP6122050 | Filter plate vacuum Manifold |
| Filter plate vacuum Manifold: Waste collection vessel | Merck Millipore | XX1004705 | Filter plate vacuum Manifold |
| FragilEase Fragile X PCR kit | PerkinElmer | 3101-0010 | For PCR amplification |
| FragilEase Fragile X PCR kit: Sample Diluent | PerkinElmer | In kit: FragilEase Fragile X PCR kit (catalog number: 3101-0010 ) | |
| FragilEase PCR Buffer mix | PerkinElmer | In kit: FragilEase Fragile X PCR kit (catalog number: 3101-0010 ), containing primers. Primer sequences: TCAGGCGCTCAGCTCCGTTTCGGTTTCA (forward) FAM-AAGCGCCATTGGAGCCCCGCACTTCC (reverse) |
|
| FragilEase Polymerase | PerkinElmer | In kit: FragilEase Fragile X PCR kit (catalog number: 3101-0010 ) | |
| FraXsoft analysis software | PerkinElmer | ||
| NanoDrop ND-2000 Spectrophotometer | Thermo Fisher | ||
| Paper towels | |||
| PCR clean up plate: NucleoFast 96 PCR plate | MACHEREY-NAGEL | 743100 | |
| reference DNA sample | Coriell | NA20240 & NA20239 | |
| S1000 96-well Thermal Cycler | Bio-Rad | 1852196 | This can be replaced by other Thermal Cyclers (eg. Veriti™ 96-Well Thermal Cycler, Applied Biosystems, catalog number: 4375786) |
| TriNEST Incubator/Shaker instrument | PerkinElmer | 1296-0050 | |
| UltraPure DNase/RNase-Free Distilled Water | Life Technologies | 10977015 | For 2100 Bioanalyzer electrode cleaning |
| Vortex-Genie 2 | Scientific Industries | SI-0256 (Model G560E) | Conventional vortex mixer |
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Citazione di questo articolo
Wang, H., Zhu, X., Gui, B., Cheung, W. C., Shi, M., Yang, Z., Kwok, K. Y., Lim, R., Pietilä, S., Zhu, Y., Choy, K. W. A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene. J. Vis. Exp. (151), e59963, doi:10.3791/59963 (2019).