使用高分辨率熔解分析检测 CALR 基因中的遗传变异
Summary
高分辨率熔解分析（HRM）是遗传变异检测的灵敏快速解决方案。这取决于导致异质双链体改变熔化曲线形状的序列差异。通过梳理HRM和琼脂糖凝胶电泳，可以鉴定不同类型的遗传变异，如indels。
Abstract
高分辨率熔解分析（HRM）是基因分型和遗传变异扫描的强大方法。大多数HRM应用依赖于检测序列差异的饱和DNA染料，以及改变熔化曲线形状的异质双链体。需要出色的仪器分辨率和特殊的数据分析软件来识别识别变异或基因型的微小熔化曲线差异。在针对特定疾病（尤其是癌症）患者的特异性基因中，以及在费城染色体阴性骨髓增殖性肿瘤患者的 CALR 基因中，可以观察到具有不同频率的不同类型的遗传变异。目标基因中的单核苷酸变化，插入和/或缺失（indels）可以通过HRM分析检测到。不同类型遗传变异的鉴定主要基于qPCR HRM测定中使用的对照。然而，随着产物长度的增加，野生型和杂合子曲线之间的差异变小，遗传变异的类型更难确定。因此，当indels是目标基因中预期的流行遗传变异时，可以使用琼脂糖凝胶电泳等其他方法来澄清HRM结果。在某些情况下，不确定的结果必须通过标准 Sanger 测序重新检查/重新诊断。在这项回顾性研究中，我们将该方法应用于患有MPN的 JAK2 V617F阴性患者。
Introduction
钙质素基因（CALR）的体细胞遗传变异在2013年在骨髓增殖性肿瘤（MPN）患者中得到认可，如原发性血小板增多症和原发性骨髓纤维化1，2。从那时起，在CALR基因中发现了50多个遗传变异，诱导了+1（−1 + 2）帧移位3。两个最常见的CALR遗传变异是52 bp缺失（NM_004343.3（CALR）：c.1099_1150del52，p.（Leu367Thrfs*46）），也称为1型突变，以及5 bp插入（NM_004343.3（CALR）：c.1154_1155insTTGTC，p.（Lys385Asnfs*47）），也称为2型突变。这两个遗传变异代表了所有CALR遗传变异的80%。其他的已被归类为1型或2型相似，使用基于保存接近野生型CALR4的α螺旋的算法。在这里，我们介绍了一种用于CALR遗传变异检测的高灵敏度和快速方法，即高分辨率熔解分析方法（HRM）。该方法能够快速检测1型和2型遗传变异，这些变异代表了大多数CALR突变5。HRM于1997年与实时”聚合酶链反应”（qPCR）相结合引入，作为检测因子V Leiden6突变的工具。与代表黄金标准技术的Sanger测序相比，HRM是一种更灵敏且特异性更低的方法5。HRM方法是一种很好的筛选方法，可以快速分析大量样品，并具有巨大的成本效益5。这是一种在荧光染料存在下进行的简单PCR方法，不需要特定的技能。另一个好处是，该过程本身不会损坏或破坏分析的样品，这使我们能够在HRM程序7之后重复使用样品进行电泳或Sanger测序。唯一的缺点是有时很难解释结果。此外，HRM不能在非1型或2型突变的患者中检测到确切的突变8。在这些患者中，应进行Sanger测序（图1）。
HRM基于在饱和DNA荧光染料存在下对特定DNA区域的扩增，其掺入双链DNA（dsDNA）中。荧光染料在掺入dsDNA时会发光。在温度逐渐升高后，dsDNA分解成单链DNA，可以在熔融曲线上检测到荧光强度的突然降低。熔化曲线的形状取决于用于检测突变的DNA序列。将样品的熔化曲线与已知突变或野生型CALR的熔融曲线进行比较。不同的熔融曲线代表非类型1或类型29的不同突变。
通过HRM、琼脂糖凝胶电泳和测序方法检测 CALR 基因中体细胞遗传变异的算法（图1）在10之前发表的回顾性研究中得到了应用和验证。
Protocol
Representative Results
Discussion
DNA的高分辨率熔化是基因分型和遗传变异扫描的简单解决方案14。它取决于导致异质双面体改变熔化曲线形状的序列差异。在特定癌症患者群体1、2、15、16、10的基因特异性中可以观察到不同频率的不同类型的遗传变异。目的基因中的缺失或插入可以通过HRM?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢卢布尔雅那大学医学中心血液学系血液学系，内科专业血液学实验室的所有学术专家和员工。
Materials
| E-Gel EX 4% Agarose | Invitrogen, Thermo Fischer Scientific | G401004 | |
| Fuorometer 3.0 QUBIT | Invitrogen, Thermo Fischer Scientific | Q33216 | |
| Invitrogen E-Gel iBase and E-Gel Safe Imager Combo Kit | Invitrogen, Thermo Fischer Scientific | G6465EU | |
| MeltDoctor HRM MasterMix 2X | Applied Biosystem, Thermo Fischer Scientific | 4415440 | Components: AmpliTaqGold 360 DNA Polymerase, MeltDoctor trade HRM dye, dNTP blend including dUTP, Magnesium salts and other buffer components, precisely formulated to obtain optimal HRM results |
| MicroAmp Fast 96-well Reaction Plate (0.1 mL) | Applied Biosystems, Thermo Fischer Scientific | 4346907 | |
| MicroAmp Optical adhesive film | Applied Biosystems, Thermo Fischer Scientific | 4311971 | |
| NuGenius | Syngene | NG-1045 | Gel documentation systems |
| Primer CALRex9 Forward | Eurofins Genomics | Sequence: 5'GGCAAGGCCCTGAGGTGT'3 (High-Purity, Salt-Free) | |
| Primer CALRex9 Reverse | Eurofins Genomics | Sequence: 5'GGCCTCAGTCCAGCCCTG'3 (High-Purity, Salt-Free) | |
| QIAamp DNA Mini Kit | QIAGEN | 51306 | DNA isolation kit with the buffer for DNA dilution. |
| Qubit Assay Tubes | Invitrogen, Thermo Fischer Scientific | Q32856 | |
| QUBIT dsDNA HS assay | Invitrogen, Thermo Fischer Scientific | Q32854 | |
| Trackit 100bp DNA Ladder | Invitrogen, Thermo Fischer Scientific | 10488058 | Ladder consists of 13 individual fragments with the reference bands at 2000, 1500, and 600 bp. |
| ViiA7 Real-Time PCR System | Applied Biosystems, Thermo Fischer Scientific | 4453534 | |
| Water nuclease free | VWR, Life Science | 436912C | RNase, DNase and Protease free water |
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