Gene-scan method for the recognition of carriers and patients with p47phox-deficient autosomal recessive chronic granulomatous disease
Gene-scan method for the recognition of carriers and patients with p47phox-deficient autosomal recessive chronic granulomatous disease
复制标题
DOI：
10.1016/s0301-472x(01)00731-7
复制
发表时间：
2001-11-01
影响因子：
2.6
通讯作者：
Roos, D
中科院分区：
文献类型：
--
作者：
Dekker, J;de Boer, M;Roos, D
客观的。我们设计了一种方法来识别 p47(phox) 缺陷型慢性肉芽肿病 (A47 CGD) 携带者和患者，这是该疾病最常见的常染色体形式。 CGD 的特点是由于 NADPH 氧化酶系统缺陷，吞噬性白细胞无法杀死微生物。导致 p47(phox) 缺陷 CGD 的主要遗传缺陷是 p47(phox) 基因 NCF1 中外显子 2 开头的 GT 缺失，很可能是由 NCF1 与其假基因之一之间的重组事件引起的。由于 NCF1 与其假基因之间的同源性超过 99%，因此几乎不可能使用标准 PCR/测序技术来研究患者、携带者和正常个体的序列。在我们的基因扫描方法中，NCF1 外显子 2 周围的基因组 DNA 的 198 bp 区域通过非特异性 PCR 使用一种荧光染料标记的引物进行扩增。 NCF1和假基因产物的混合物，其长度相差两个核苷酸，根据大小进行分离。峰高之间的比率表示个体基因组内 NCF1 基因和假基因的相对数量。结果。该方法具有高度重现性 (SD = 4%) 和灵敏性 (r = 0.998)。在16名健康个体中，15名具有2：4的比例（2个基因，4个假基因），10/12的A47 CGD携带者具有1：5的比例，34名患者仅具有假基因。此外，基因扫描包括假基因内含子 2 中 20 bp 的重复，揭示了 NCF1 和假基因之间的交叉事件。结论。我们的方法将具有一种 NCF1 基因（携带者）的个体与对照组和 NCF1 缺陷患者区分开来。 (C) 2001 年国际实验血液学学会。由爱思唯尔科学公司出版
Objective. We devised a method to recognize carriers and patients with p47(phox)-deficient chronic granulomatous disease (A47 CGD), the most common autosomal form of the disease. CGD is characterized by the inability of phagocytic leukocytes to kill microorganisms, due to a defective NADPH oxidase system. The predominant genetic defect leading to p47(phox)-deficient CGD is a GT deletion at the beginning of exon 2 in the p47(phox) gene NCF1, most likely caused by recombination events between the NCF1 and one of its pseudogenes. It is hardly possible to investigate sequences of patients, carriers, and normal individuals using standard PCR/sequencing techniques, due to greater than 99% homology between NCF1 and its pseudogenes.Methods. In our gene-scan method, a 198-bp region of genomic DNA around exon 2 of NCF1 is amplified by nonspecific PCR with one fluorochrome-labeled primer. The mixture of NCF1 and pseudogene product, which differs by two nucleotides in length, is separated according to size. The ratio between the peak heights indicates the relative number of NCF1 genes and pseudogenes within an individual's genome.Results. The method is highly reproducible (SD = 4%) and sensitive (r = 0.998). Of the 16 healthy individuals, 15 had a 2:4 ratio (2 genes, 4 pseudogenes), 10/12 A47 CGD carriers had a 1:5 ratio, and 34 patients had only pseudogenes. In addition, gene-scans including a 20-bp duplication in intron 2 of the pseudogenes revealed insight in the crossing-over events between NCF1 and pseudogenes.Conclusions. Our method distinguishes individuals with one NCF1 gene (carriers) from controls and from NCF1-deficient patients. (C) 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.