Detection of NCF-1 Gene Expression as a Diagnostic Tool for Autosomal Recessive Chronic Granulomatous Disease among Egyptian Children with Phagocytic Dysfunction

Abstract

Abstract Background Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder caused by functional impairment of the nicotinamide dinucleotide phosphate (NADPH) oxidase complex in neutrophilic granulocytes and monocytes and characterized by recurrent and severe infections, dysregulated inflammation and autoimmunity. Aim of the Work To diagnose the autosomal recessive (AR) type of CGD among a group of Egyptian pediatric patients with phagocytic dysfunction by detection of Neutrophil Cytosolic Factor-1 (NCF-1) gene expression using reverse transcription polymerase chain reaction (RT-PCR) and to correlate it with other diagnostic tests. Subjects and Methods A case-control study was conducted on 15 provisionally diagnosed CGD pediatric patients (Group I) by the use of DHR test, with the stimulation index using PMA < 30%. They were recruited from different university hospitals in Egypt. The study also included 12 mothers of the studied patients to detect the genetic mutations in carriers, if any, (Group II) and 14 apparently healthy children as a control group (Group III). Full medical history was taken and thorough clinical examination was done. The selected groups underwent testing for phagocytic and lytic indices, measurement of NCF-1 gene expression by real time RT-PCR. Results The comparative statistics between group I and group III as regards the phagocytic index (PI) revealed a high statistical significant difference between both groups. In addition, a statistically significant difference between both groups as regards lytic index (LI) was also revealed. Correlation studies were done between fold change of NCF-1 gene expression and each of PI, LI and DHR and showed no significant statistical correlation between these parameters in the 3 studied groups. Cases with a fold change of NCF-1 gene expression less than 0.48 (cut-off value calculated by the 25th percentile fold change of the control group), are considered having defective NCF-1 gene expression. At this cut-off value 2 out of 15 CGD cases and 3 mothers out of 12 showed NCF-1 gene under-expression. Conclusion Assessment of NCF-1 gene expression which encodes p47phox by real time RT- PCR in the present study could detect defective gene expression only among two CGD children and their mother as well as two other mothers with no correlation to other studied parameters. Since CGD is a genotypically heterogeneous disease; the findings of the present study highlight the importance of molecular testing for diagnosis after screening for NADPH oxidase protein deficiency by flow cytometry.