Dekker J, de Boer M, Roos D
Central Laboratory of the Netherlands Blood Transfusion Service (CLB), Academic Medical Center, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
Exp Hematol. 2001 Nov;29(11):1319-25. doi: 10.1016/s0301-472x(01)00731-7.
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.
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.
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.
Our method distinguishes individuals with one NCF1 gene (carriers) from controls and from NCF1-deficient patients.
我们设计了一种方法来识别携带p47（phox）缺陷型慢性肉芽肿病（A47 CGD）的携带者和患者，该病是该疾病最常见的常染色体形式。慢性肉芽肿病的特征是吞噬性白细胞由于NADPH氧化酶系统缺陷而无法杀死微生物。导致p47（phox）缺陷型慢性肉芽肿病的主要遗传缺陷是p47（phox）基因NCF1外显子2起始处的GT缺失，很可能是由NCF1与其一个假基因之间的重组事件引起的。由于NCF1与其假基因之间的同源性大于99%，因此使用标准PCR/测序技术几乎不可能研究患者、携带者和正常个体的序列。
在我们的基因扫描方法中，使用一条荧光染料标记的引物通过非特异性PCR扩增NCF1外显子2周围198 bp的基因组DNA区域。长度相差两个核苷酸的NCF1和假基因产物的混合物按大小分离。峰高之间的比率表明个体基因组中NCF1基因和假基因的相对数量。
该方法具有高度可重复性（标准差=4%）和敏感性（r=0.998）。在16名健康个体中，15名的比例为2:4（2个基因，4个假基因），12名A47 CGD携带者中有10名的比例为1:5，34名患者只有假基因。此外，包括假基因内含子2中20 bp重复的基因扫描揭示了NCF1与假基因之间的交叉事件。
我们的方法能够区分携带一个NCF1基因的个体（携带者）与对照以及NCF1缺陷患者。
