Detection of CYBB Gene Expression by Reverse Transcription PCR as a Diagnostic Tool for X-Linked Chronic Granulomatous Disease in Egyptian Children

Background: Chronic granulomatous disease (CGD) is a genetic syndrome characterized by a dysfunction of the res-piratory burst, which is necessary to kill certain phagocytosed pathogens. The fundamental defect in CGD lies in the NADPH oxidase, the enzyme complex responsible for initiating the res-piratory burst. Defects in any of the six subunits of the NADPH oxidase enzyme can manifest as CGD. Thus, CGD patients can be phenotypically similar but genetically heterogeneous de-pending on which NADPH oxidase component is defective. By far the most common form of CGD is the X-linked recessive form. It results from a mutation of the CYBB gene encoding for gp91phox subunit of NADPH oxidase resulting in a greater number of affected males. However, there have been reports of affected females with the diagnosis of X-linked CGD attributed to skewed X chromosome inactivation. Aim of Study: In the present study we aimed to diagnose the X-linked type of CGD in a group of Egyptian children by detection of CYBB gene expression using real time RT-PCR and to investigate if it correlates with the test of phagocytic lytic index as a cheaper diagnostic method for CGD. Patients and Method: This case-control study was con-ducted on 15 provisionally diagnosed CGD 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 (Group II) of the studied patients to detect the genetic mutations in carriers, if any, and 14 apparently healthy children as a control group (Group III). Results: We found that cases with a fold change of CYBB gene expression less than 0.34 (cut-off value calculated by the 25th percentile fold change of the control group), are con-sidered having defective CYBB gene expression. At this cut-off value, 3 males (20% of all cases in group I and 33% of the males in group I) showed under-expression of the CYBB gene, while none of the females in this group showed defective CYBB gene expression. Also, no mother in group (II) showed under-expression of CYBB gene at that cutoff value. The diag-nostic characteristics of fold change of CYBB gene expression by real-time RT-PCR technique at the cut-off 0.34 showed sen-sitivity = 20%, specificity = 86%, PPV = 60%, NPV = 50% and test accuracy = 52%. Conclusion: We could establish the diagnosis of 3 out of 15 CGD cases as X-linked form, derives from defects in the CYBB gene, which encodes gp91phox of the oxidase, without the need to use complex and expensive methodologies such as northern blot, slot blot, or genomic DNA sequencing. Despite the low number of patients included in this study to draw defi-nite conclusions, this molecular analysis provides an insight into the breadth and relative distribution of genetic abnormali-ties responsible for the disease. Overall, we have demonstrated that RT-PCR, a simple and a relatively low cost methodology in comparison to other complex molecular diagnostic methods, may be a suitable tool for diagnosing CGD in laboratories in developing countries.