National Institute of Immunohaematology-ICMR, 13th floor, KEM Hospital Campus, Mumbai, Parel, 400012, India.
Department of Blood Cell Research, Sanquin Blood Supply Organization, Amsterdam, The Netherlands.
J Clin Immunol. 2018 Nov;38(8):898-916. doi: 10.1007/s10875-018-0567-y. Epub 2018 Nov 23.
Chronic granulomatous disease (CGD) is characterized by mutation in any one of the five genes coding NADPH oxidase components that leads to functional abnormality preventing the killing of phagocytosed microbes by affecting the progression of a respiratory burst. CGD patients have an increased susceptibility to infections by opportunistic and pathogenic organisms. Though initial diagnosis of CGD using a nitroblue tetrazolium (NBT) test or dihydrorhodamine (DHR) test is relatively easy, molecular diagnosis is challenging due to involvement of multiple genes, presence of pseudogenes, large deletions, and GC-rich regions, among other factors. The strategies for molecular diagnosis vary depending on the affected gene and the mutation pattern prevalent in the target population. There is a paucity of molecular data related to CGD for Indian population.
This report includes data for a large cohort of CGD patients (n = 90) from India, describing the diagnostic approach, mutation spectrum, and novel mutations identified. We have used mosaicism in mothers and the expression pattern of different NADPH components by flow cytometry as a screening tool to identify the underlying affected gene. The techniques like Sanger sequencing, next-generation sequencing (NGS), and Genescan analysis were used for further molecular analysis.
Of the total molecularly characterized patients (n = 90), 56% of the patients had a mutation in the NCF1 gene, 30% had mutation in the CYBB gene, and 7% each had mutation in the CYBA and NCF2 genes. Among the patients with NCF1 gene mutation, 82% of the patients had 2-bp deletion (DelGT) mutations in the NCF1 gene. In our cohort, 41 different mutations including 9 novel mutations in the CYBB gene and 2 novel mutations each in the NCF2, CYBA, and NCF1 genes were identified.
Substantial number of the patients lack NCF1 gene on both the alleles. This is often missed by advanced molecular techniques like Sanger sequencing and NGS due to the presence of pseudogenes and requires a simple Genescan method for confirmation. Thus, the diagnostic approach may depend on the prevalence of affected genes in respective population. This study identifies potential gene targets with the help of flow cytometric analysis of NADPH oxidase components to design an algorithm for diagnosis of CGD in India. In Indian population, the Genescan method should be preferred as the primary molecular test to rule out NCF1 gene mutations prior to Sanger sequencing and NGS.
慢性肉芽肿病（CGD）的特征是任何一个编码 NADPH 氧化酶组件的五个基因中的突变，导致吞噬微生物后呼吸爆发的进展受阻，从而使吞噬细胞无法杀死微生物，导致功能异常。CGD 患者易受机会性和致病性微生物感染。虽然使用硝基四唑蓝（NBT）试验或二氢罗丹明（DHR）试验进行 CGD 的初步诊断相对容易，但由于涉及多个基因、假基因的存在、大片段缺失和富含 GC 的区域等因素，分子诊断具有挑战性。分子诊断的策略因受影响的基因和目标人群中常见的突变模式而异。印度人群 CGD 的分子数据相对较少。
本报告包括来自印度的 90 例 CGD 患者的大型队列数据，描述了诊断方法、突变谱和鉴定的新突变。我们使用母亲的嵌合体和流式细胞术检测不同 NADPH 成分的表达模式作为筛选工具，以确定潜在的受影响基因。使用 Sanger 测序、下一代测序（NGS）和 Genescan 分析等技术进行进一步的分子分析。
在进行分子特征分析的患者中（n=90），56%的患者 NCF1 基因突变，30%的患者 CYBB 基因突变，7%的患者 CYBA 和 NCF2 基因突变。在 NCF1 基因突变的患者中，82%的患者在 NCF1 基因中有 2 个碱基对缺失（DelGT）突变。在我们的队列中，鉴定了 41 种不同的突变，包括 CYBB 基因中的 9 种新突变，以及 NCF2、CYBA 和 NCF1 基因中的每种 2 种新突变。
大量患者的两个等位基因上都缺乏 NCF1 基因。由于存在假基因，这通常会被高级分子技术（如 Sanger 测序和 NGS）遗漏，需要使用简单的 Genescan 方法进行确认。因此，诊断方法可能取决于各自人群中受影响基因的流行程度。本研究通过 NADPH 氧化酶组件的流式细胞术分析确定了潜在的基因靶点，以设计印度 CGD 诊断的算法。在印度人群中，在进行 Sanger 测序和 NGS 之前，应首选 Genescan 方法作为主要的分子检测，以排除 NCF1 基因突变。
