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Abstract Primary immunodeficiency disorders are a group of inherited disorders that result in impairment of normal immune development and function. These immunodeficiency diseases are due to genetic disorders that are usually hereditary and present during infancy or childhood. However, some primary immunodeficiency disorders are not recognized until adulthood. The PIDs are broadly classified according to the component of the immune system that is primarily disrupted: innate or adaptive immunity. Chronic granulomatous disease is an inherited immunodeficiency disease caused by defects in the NADPH oxidase complex in neutrophils and macrophages, which cannot effectively produce superoxide anions and their metabolites to kill peroxidase-positive bacteria and fungi. These defects make patients susceptible to recurrent, severe infections and excessive hyper-inflammatory responses at young ages. Defects in any subunit 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 caused by AR mutations in NCF-2 genes encoding P67-phox are considered the rarest and clinically milder than others (found in 5% only of CGD patients). 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 NCF-2 among CGD patients. This case-control study was conducted on 15 provisionally diagnosed CGD patients (group I). They were recruited from different university hospitals in Egypt. The study also included 12 mothers (group IIa) & 8 fathers (group IIb) of the studied patients to detect the genetic mutations in carriers, if any, and 14 apparently healthy children as a control group (group III). Full medical history was taken and clinical examination was done. The selected groups underwent testing for phagocytic and lytic indices, measurement of NCF-2 gene expression by real time RT-PCR. The results of DHR testing for patients group were retrieved from patients’ files. Descriptive statistics of clinical and laboratory data of patients showed that median (25th-75th) of age of onset was 1.5 years (1month- 3 years), median (25th-75th) of TLC was 5.2×103/µL (3.9-8.7×103/µL) and the median (25th-75th) of ANC was 2.4 x103/µL (1.6-5×103/µL). Results showed medians (25th-75th) of gene expression of NCF-2 (fold change), phagocytic index and lytic index among patients’ group were1.55 (0.73-2.10), 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 NCF-2 (fold change), phagocytic index and lytic index median to be 1.01(0.67-1.83), 8.97% (7.9-10.1%) and 1.3% (1-1.4%) respectively. All cases (100%) had a positive history of consanguinity and 60 % of the cases presented by pneumonia, while 26.7% of the cases presented by skin infections and 13.3% presented by mixed infections. About 47% of cases were showing partial or no response to treatment, while 53% of them experienced good response to treatment. Comparative statistics between healthy controls (group III) and CGD patients (group I) showed that the median of both phagocytic and lytic indices were significantly lower in CGD patients than in control group. While there were no statistically significant differences between the 2 groups regarding age, TLC and ANC. Comparative statistics between patients (group I) and mothers (group IIa) regarding fold change of NCF-2 gene expression showed also no significant statistical difference [median (25th-75th) =1.55 (0.73-2.10) and 1.48 (0.77-1.94) respectively](P=0.83). Comparative statistics between patients (group I) and fathers (group IIb) regarding fold change of NCF-2 gene expression showed also no significant statistical difference [median (25th-75th) =1.55 (0.73-2.10) and 2.046(1.02-3.029) respectively] (P=0.20). Cases with a fold change of NCF-2 gene expression less than 0.67 (cut-off value calculated by the 25th percentile fold change of the control group), are considered having defective NCF-2 gene expression. At this cut-off value, 2 children and 1 mother showed under expression of NCF-2 gene. |