Dutta Debargh, Gunasekera Devi, Ragni Margaret V, Pratt Kathleen P
Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD.
Division Hematology/Oncology, University of Pittsburgh Medical Center, Pittsburgh, PA; and.
Blood Adv. 2016 Dec 14;1(3):231-239. doi: 10.1182/bloodadvances.2016001651. eCollection 2016 Dec 27.
The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 gene inversion, which together cause ∼50% of severe hemophilia A cases. We report a simple and accurate RNA-based assay to detect these mutations in patients and heterozygous carriers. The assays do not require specialized equipment or expensive reagents; therefore, they may provide useful and economic protocols that could be standardized for central laboratory testing. RNA is purified from a blood sample, and reverse transcription nested polymerase chain reaction (RT-NPCR) reactions amplify DNA fragments with the sequence spanning the exon 22 to 23 splice site (intron 22 inversion test) or the exon 1 to 2 splice site (intron 1 inversion test). These sequences will be amplified only from RNA without an intron 22 or intron 1 inversion mutation, respectively. Additional RT-NPCR reactions are then carried out to amplify the inverted sequences extending from exon 19 to the first in-frame stop codon within intron 22 or a chimeric transcript containing exon 1 and the gene. These latter 2 products are produced only by individuals with an intron 22 or intron 1 inversion mutation, respectively. The intron 22 inversion mutations may be further classified (eg, as type 1 or type 2, reflecting the specific homologous recombination sites) by the standard DNA-based "inverse-shifting" PCR assay if desired. Efficient Bcl I and T4 DNA ligase enzymes that cleave and ligate DNA in minutes were used, which is a substantial improvement over previous protocols that required overnight incubations. These protocols can accurately detect inversion mutations via same-day testing of patient samples.
导致甲型血友病的最常见突变是内含子22或内含子1基因倒位，这两种突变共同导致约50%的严重甲型血友病病例。我们报告了一种简单且准确的基于RNA的检测方法，用于检测患者和杂合子携带者中的这些突变。这些检测方法不需要专门的设备或昂贵的试剂；因此，它们可能提供有用且经济的方案，可标准化用于中心实验室检测。从血液样本中纯化RNA，然后通过逆转录巢式聚合酶链反应（RT-NPCR）扩增跨越外显子22至23剪接位点的DNA片段（内含子22倒位检测）或外显子1至2剪接位点的DNA片段（内含子1倒位检测）。这些序列将仅分别从没有内含子22或内含子1倒位突变的RNA中扩增出来。然后进行额外的RT-NPCR反应，以扩增从外显子19延伸至内含子22内第一个读框内终止密码子的倒位序列，或包含外显子1和该基因的嵌合转录本。后两种产物仅分别由具有内含子22或内含子1倒位突变的个体产生。如果需要，内含子22倒位突变可通过基于DNA的标准“反向移位”PCR检测进一步分类（例如，分为1型或2型，反映特定的同源重组位点）。使用了能在数分钟内切割和连接DNA的高效Bcl I和T4 DNA连接酶，这相对于之前需要过夜孵育的方案有了实质性改进。这些方案可通过对患者样本进行当日检测准确检测倒位突变。
