Characterization of Molecular Defects in Chronic Granulomatous Disease

• Main Authors: Ya-Fang Huang, 黃雅芳
• Other Authors: Chi-Chang Shieh
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/75755153173988702116

碩士 === 國立成功大學 === 分子醫學研究所 === 88 === Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease caused by the inability of the patients' phagocytes to produce sufficient oxygen free radicals in respiratory burst to kill phagocytosed pathogens. CGD patients suffer from recurrent infections, especially by catalase-positive bacteria and fungi. Without appropriate treatment, these patients seldom survive their first decade of life. NADPH oxidase is responsible for the generation of oxygen free radicals in leukocytes. It is a multi-component enzyme complex comprising membrane-bound components (p22phox and gp91phox collectedly known as cytochrome b558) and cytosolic components (p47phox, p67phox and p40phox). Some GTP-binding proteins (rac1/2, rap1A) also involve in this enzyme complex. Gene defects in any one of the four components; gp91phox, p22phox, p47phox and p67phox cause CGD. The mutations encoding gp91phox defects are inherited as X-linked recessive diseases. The other mutations encoding p22phox, p47phox and p67phox defects are located on autosomes and thus are inherited as autosomal recessive diseases. The elucidation of the gene defects in CGD patients is important not only for clinical management of these patients but also for understanding the mechanisms of innate immune response. In this investigation, we first established a convenient and efficient diagnosis system using flow cytometry to detect leukocyte respiratory burst on whole blood. This method is quantitative enough in detecting CGD patients and screening CGD gene carriers. We also found this method could be used to screen for bioactive substances that could prime the granulocytes. We will use this method to study the priming mechanisms in normal condition and in diseases. Secondly, we investigated some X-linked CGD patients, and try characterized their gene defects. We found that patient1 has a missense mutation in codon 1024 C -> T in CYBB gene, which encode 338 His -> Cys mutation in gp91phox. This mutation is located in the potential FAD binding region of gp91phox. This mutation disrupts the binding between FAD and gp91phox and leads to the failure of maturation in cytochrome b558. In patient 2, we found he has a mutation in the intron 7 splicing donor site that leads to exon 7 skipping during mRNA splicing process. This abnormal RNA degrades quickly. We found a 1691G deletion in patient 3. This mutation might lead to protein conformation anomaly and early degradation. Our data suggest that cell quality control play a role in gp91phox biosynthesis of those CGD patients. In summary, our research in flow cytometric analysis of respiratory burst enables us to investigate the leukocyte respiratory burst in physiological and various pathological conditions. Investigating the mutations of these patients not only make it possible for us to do molecular diagnosis and treatment, but also will help us in further understanding the microbial killing mechanisms of leukocyte NADPH oxidase.