الفهرس 
CHRONIC GRANULOMATOUS DISEASEOnly 14 pages are availabe for public view
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Abstract
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.
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 depending on which NADPH oxidase component is defective. Autosomal recessive forms of CGD are caused by mutations in CYBA gene encoding protein-22 phagocytic oxidase (p22phox). The incidence of autosomal recessive CGD can be higher than X-linked CGD in countries with high rates of consanguineous marriage.
The molecular diagnosis of CGD involves measuring NADPH oxidase activity in phagocytes, measuring protein expression of NADPH oxidase components and mutation analysis of genes encoding these components. In this regard the present study aimed to assess the real time RT-PCR as an aiding tool in the molecular diagnosis of gene expression of CYBA among CGD child patients.
This case-control study was conducted on 15 provisionally diagnosed CGD child patients (group I). They were recruited from different university hospitals in Egypt. The study also included 12 mothers and 8 fathers 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 CYBA gene expression by real time RT-PCR and DHR testing for patients’ group where its results were retrieved from patients’ files.
Results of gene expression of CYBA (fold change), phagocytic index and lytic index among patients’ group showed that median (range) were 2.4 (1.3-3.7), 6.8% (5.6-8.6%) and 0.8% (0.5-1.1%), respectively in comparison to the control group which showed gene expression of CYBA (fold change), phagocytic index and lytic index median (range) to be 0.9 (0.6-1.8), 8.97% (7.9-10.1%) and 1.3% (1-1.4%), respectively.
The comparative statistics between group I and group III as regards fold change of CYBA gene expression as well as the phagocytic index revealed highly statistical significant differences between both groups regarding these two parameters. In addition, a statistically significant difference between both groups as regards lytic index was revealed. However, no significant difference was found between both groups regarding age, TLC or absolute neutrophil count.
The diagnostic characteristics of fold change of CYBA gene expression at the cut off value 0.6 (calculated by the 25th percentile of fold change of the control group) showed sensitivity (6%), specificity (78%), PPV (25%), NPV (44%) and accuracy (41.3%). At this cut-off value 3 subjects (1 patient and 2 mothers) showed under-expression of CYBA gene. The only patient was a 6-years-old male child, presented by pneumonia at the age of 6 months, and the others were 2 mothers; one of them is a mother of a 9-years-old male with abnormal facies. He was presented with chest and skin infections. The other one was a mother of 4-years-old triplet female patients who were presented by pneumonia.
In conclusion, although there are some limitations in our study, such as the small sample size studied due to the disease rarity, some missed data and restriction to studying only one gene (CYBA) among many others controlling the human NADPH oxidase system, which is because of the limited resources. However, our study could establish the diagnosis of 1 out of 15 CGD cases and two mothers with CYBA gene under-expression which encodes p22phox of NADPH oxidase, without the need to use complex and expensive methods such as genomic DNA sequencing.
Future studies on larger sample size are highly recommended to draw definite conclusions about the clinical significance of real time RT-PCR technique as a diagnostic tool for this immunodeficiency disease. Also, the study of other NADPH enzyme subunit encoding genes is recommended to reach the exact genotype of this genotypically heterogeneous disease. We also recommend applying other established diagnostic techniques on our patients in order to test the relevance of our method in the diagnosis of CGD.