Neutrophil Monitoring Laboratory, Applied/Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD.
Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.
Blood Adv. 2019 Jan 22;3(2):136-147. doi: 10.1182/bloodadvances.2018023184.
Mutations in (p47) cause autosomal recessive chronic granulomatous disease (CGD) with abnormal dihydrorhodamine (DHR) assay and absent p47 protein. Genetic identification of mutations is complicated by adjacent highly conserved (>98%) pseudogenes ( and ). has GTGT at the start of exon 2, whereas the pseudogenes each delete 1 GT (ΔGT). In p47 CGD, the most common mutation is ΔGT in (c.75_76delGT; p.Tyr26fsX26). Sequence homology between and its pseudogenes precludes reliable use of standard Sanger sequencing for mutations and for confirming carrier status. We first established by flow cytometry that neutrophils from p47 CGD patients had negligible p47 expression, whereas those from p47 CGD carriers had ∼60% of normal p47 expression, independent of the specific mutation in We developed a droplet digital polymerase chain reaction (ddPCR) with 2 distinct probes, recognizing either the wild-type GTGT sequence or the ΔGT sequence. A second ddPCR established copy number by comparison with the single-copy telomerase reverse transcriptase gene, We showed that 84% of p47 CGD patients were homozygous for ΔGT The ddPCR assay also enabled determination of carrier status of relatives. Furthermore, only 79.2% of normal volunteers had 2 copies of GTGT per 6 total () copies, designated 2/6; 14.7% had 3/6, and 1.6% had 4/6 GTGT copies. In summary, flow cytometry for p47 expression quickly identifies patients and carriers of p47 CGD, and genomic ddPCR identifies patients and carriers of ΔGT , the most common mutation in p47 CGD.
（p47）中的突变导致常染色体隐性慢性肉芽肿病（CGD），二氢罗丹明（DHR）检测异常，p47 蛋白缺失。由于相邻高度保守（>98%）假基因（和）的存在，使得对 突变的基因鉴定变得复杂。在 p47 CGD 中，最常见的突变是 中的ΔGT（c.75_76delGT；p.Tyr26fsX26）。由于 与假基因之间存在序列同源性，因此无法可靠地使用标准 Sanger 测序来检测 突变和确认携带者状态。我们首先通过流式细胞术证实，p47 CGD 患者的中性粒细胞几乎没有 p47 表达，而 p47 CGD 携带者的中性粒细胞则有正常 p47 表达的约 60%，而与 中的特定突变无关。我们开发了一种带有 2 种不同探针的液滴数字聚合酶链反应（ddPCR），可识别野生型 GTGT 序列或 ΔGT 序列。第二种 ddPCR 通过与单拷贝端粒酶逆转录酶基因进行比较来确定拷贝数。我们发现，84%的 p47 CGD 患者均为 ΔGT 纯合子。ddPCR 检测还可确定亲属的携带者状态。此外，只有 79.2%的正常志愿者每 6 个总拷贝（）中都有 2 个 GTGT 拷贝，被指定为 2/6；14.7%有 3/6，1.6%有 4/6 GTGT 拷贝。总之，p47 表达的流式细胞术可快速识别 p47 CGD 的患者和携带者，而基因组 ddPCR 可识别 p47 CGD 中最常见的突变 ΔGT 的患者和携带者。
