Baris Hatice Ezgi, Ogulur Ismail, Akcam Bengu, Kiykim Ayca, Karagoz Dilek, Saraymen Berkay, Akgun Gamze, Eltan Sevgi Bilgic, Aydemir Sezin, Akidagi Zeynep, Bentli Esma, Nain Ercan, Kasap Nurhan, Baser Dilek, Altintas Derya Ufuk, Camcioglu Yildiz, Yesil Gözde, Ozen Ahmet, Koker Mustafa Yavuz, Karakoc-Aydiner Elif, Baris Safa
Faculty of Medicine, Department of Pediatrics, Marmara University, Istanbul, Turkey.
Faculty of Medicine, Pediatric Allergy-Immunology, Marmara University, Istanbul, Turkey; Istanbul Jeffrey Modell Diagnostic and Research Center for Primary Immunodeficiencies, Istanbul, Turkey.
J Allergy Clin Immunol Pract. 2020 Nov-Dec;8(10):3525-3534.e1. doi: 10.1016/j.jaip.2020.07.030. Epub 2020 Jul 28.
Chronic granulomatous disease (CGD) is characterized by defective microbial killing due to mutations affecting subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Definitive genetic identification of disease subtypes may be delayed or not readily available.
Sought to investigate the role of intracellular staining of NADPH oxidase enzyme subunits in predicting the respective genetic defects in patients with CGD and carriers.
Thirty-four patients with genetically inherited CGD, including 12 patients with X-linked CGD (gp91 deficiency due to cytochrome b-245, beta polypeptide [CYBB] mutations) and 22 patients with autosomal-recessive CGD (p22, p47, and p67 deficiency due to cytochrome b-245, alpha polypeptide [CYBA], neutrophil cytosolic factor 1 [NCF1] and NCF2 mutations, respectively) were recruited from different immunology centers and followed up prospectively. Dihydrorhodamine testing and NADPH oxidase subunit expression in white blood cells were determined by flow cytometry.
gp91 and p22 defects, which result in simultaneous loss of both proteins due to their complex formation, were differentiated only by comparative analysis of patients' and mothers' intracellular staining. p47 and p67 protein expression was almost undetectable in patients compared with carrier mothers and healthy controls. The expression values of the respective subunits were found to be significantly higher in all controls as compared with carrier mothers, which in turn were higher than those of patients.
Analysis of NADPH oxidase enzyme subunits by flow cytometry in patients and carriers is useful in the rapid prediction of the genetic defect of patients with CGD, thus guiding targeted sequencing and aiding in their early diagnosis.
慢性肉芽肿病（CGD）的特征是由于影响烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶复合物亚基的突变导致微生物杀伤功能缺陷。疾病亚型的确切基因鉴定可能会延迟或难以获得。
旨在研究NADPH氧化酶亚基的细胞内染色在预测CGD患者和携带者各自基因缺陷中的作用。
从不同的免疫学中心招募了34例遗传性CGD患者，包括12例X连锁CGD患者（由于细胞色素b-245β多肽[CYBB]突变导致gp91缺乏）和22例常染色体隐性CGD患者（分别由于细胞色素b-245α多肽[CYBA]、中性粒细胞胞质因子1[NCF1]和NCF2突变导致p22、p47和p67缺乏），并进行前瞻性随访。通过流式细胞术测定白细胞中的二氢罗丹明试验和NADPH氧化酶亚基表达。
gp91和p22缺陷由于其复合物形成导致两种蛋白质同时缺失，仅通过对患者和母亲细胞内染色的比较分析来区分。与携带者母亲和健康对照相比，患者中p47和p67蛋白表达几乎无法检测到。发现所有对照中各亚基的表达值均显著高于携带者母亲，而携带者母亲又高于患者。
通过流式细胞术分析患者和携带者中的NADPH氧化酶亚基有助于快速预测CGD患者的基因缺陷，从而指导靶向测序并有助于早期诊断。
