Clinical Studies Of Abnormal Host Defense

1)慢性肉芽肿病（CGD）是由多组分NADPH氧化酶（吞噬细胞氧化酶，NOX2）复合物突变引起的原发性免疫缺陷。在2014财年，通过与Douglas Kuhns博士（Leidos, Inc.）管理的中性粒细胞监测实验室（NML）合作，我们为至少16例新的CGD患者提供了分子诊断。使用免疫检测NADPH氧化酶成分来鉴定16例CGD患者（筛选出27例）。核酸测序确定了代表所有主要CGD基因型的50名患者及其家庭成员的突变。今年，NML发现了一名p40phox缺乏症患者（世界上已知的两名p40phox缺乏症患者之一）。关于p40phox在刺激特异性激活NOX2中的作用的研究正在进行中。在2014财年，NML在开发区分p47phox编码基因突变与该蛋白的两个已知假基因突变的方法方面取得了重大进展。除了CGD外，NML还在45例先前未确诊的WHIM疾病、白细胞粘附缺陷、中性粒细胞减少症和髓过氧化物酶缺乏症患者中发现了突变。在2014财年，我们还与LHD的遗传免疫治疗部门合作，评估了用于纠正CGD患者干细胞的新型基因治疗方法，并与（Su）合作，评估了一种先前未被识别的调节吞噬细胞先天免疫功能的因子突变的患者。

1) Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the multicomponent NADPH oxidase (phagocyte oxidase, NOX2) complex. During FY14, through collaboration with the Neutrophil Monitoring Laboratory (NML) managed by Douglas Kuhns, PhD ( Leidos, Inc.), we have provided molecular diagnoses of at least 16 new CGD patients. Immunodetection of components of the NADPH oxidase was used to identify 16 subjects with CGD (out of 27 screened). Nucleic acid sequencing determined the mutations in 50 patients and family members representing all major CGD genotypes. This year, a patient with p40phox-deficiency (one of two known patients with this mutation world-wide) was identified by the NML. Ongoing studies of the role of p40phox in the stimulus-specific activation of NOX2 are underway. During FY 2014, the NML made significant progress in developing methods to differentiate mutations in the coding gene for p47phox with those occurring in two known pseudogenes for this protein. In addition to CGD, the NML identified the mutations in 45 previously undiagnosed patients with WHIM disease, Leukocyte adhesion deficiency, neutropenia, and myeloperoxidase deficiency. During FY14, we have also collaborated with the Genetic Immunotherapy Section of the LHD to evaluate novel gene therapy approaches to correct stem cells from CGD patients and with the (Su) to evaluate a patient with a mutation in a previously unrecognized factor that regulates innate immune function in phagocytes. 2) Our group continues its clinical and laboratory studies of the emerging Gram-negative CGD pathogen, Granulibacter bethesdensis. During FY14, we screened 59 CGD samples and 97 normal samples for seropositivity toward G. bethesdensis methanol dehydrogenase, an immunodominant antigen. We continue to monitor seropositivity in culture-confirmed patients to evaluate our hypothesis that this organism can establish persistent, clinically unapparent infections. 3) During FY14, we continued to accrue subjects for NIH Protocol #10-I-0029 Non-invasive Assessment of Atherosclerosis in Patients with CGD and other Disorders of the Immune System (current total = 85 subjects). Atherosclerosis, the major cause of heart disease, is thought to relate to dysregulated inflammation in the cardiac blood vessels and over production of reactive oxygen species (ROS) has been implicated. We hypothesized that CGD patients, who have deficient production of reactive oxygen species by their phagocytes and other cells, may be protected from developing atherosclerosis. The primary endpoint of this study was the assessment of atherosclerotic plaque formation/calcium deposition by CT, MRI and other imaging methodologies, in these and other patients with in-born disorders of immune function. We found significant differences in the incidences of pre-clinical signs of atherosclerosis in CGD patients and a manuscript describing these findings is currently under review. Through collaboration with the National Center for Advancing Translational Sciences (NCATS), we are evaluating chemical inhibitors of the enzyme responsible for ROS production, specifically the NADPH oxidase that is mutated in CGD. 4) Several years ago, two patients with an undiagnosed disease characterized by increased infections and neutropenia were seen at the NIH. These subjects bore similarities to 2 other families of patients seen over a time frame of 30 years by the LHD. Cellular studies indicated an abnormal neutrophil morphology with frequent nuclear herniations, subnormal chemotaxis, and aberrant cytoskeletal structure. We have identified, by biochemical and molecular approaches, the molecular defect in this disease and have submitted this paper for publication. (5) In normal PMN, activation of NOX2 results in a rapid (within seconds to minutes) depletion of NADPH and hypoxia. Given the central roles of oxygen and NAPDH in metabolism, we compared the metabolomes of PMN before and after activation of NOX2 in a pilot study and found unexpected alterations in PMN metabolism. We are repeating these studies in PMN from patients with CGD to determine the exact role of ROS in these processes.