Identification of mediterranean mutation in the G 6 PD gene on molecular level in Iraqi population . G 6 PD

The study involved extraction of Deoxyribonucleic acid (DNA) from 71 samples of random G6PD patients and 85 samples of apparently healthy individuals from different Iraqi populations respectively, which was then amplified by polymerase chain reaction (PCR) and later subjected to digestion by restriction enzyme to create restriction fragment length polymorphism (RFLP) to enable the detection of mutation that caused G6PD deficiency namely Mediterranean (Med). The results of the current study showed that Iraqies were affected by G6PD deficiency in a percentage of 7.2% and showed that the majority of affected cases were caused by Med mutation (95.8%). It could be concluded that Med mutation causes a serious impact on pediatric health and its the most prevalent cause of G6PD deficiency. 71 G6PD 85 PCR RFLP . 7.2 % ) ( 95.8 % G6PD . . Introduction Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common hereditary disorder in humans and is found in people of Mediterranean, South-east Asian and African descent. The common clinical manifestations of G6PD deficiency are neonatal jaundice and acute hemolytic anemia triggered by certain drugs, infections or ingestion of Biotechnology Research Center (special edition) Vol. 3No. 22009 91 fava beans. G6PD deficiency is a heterogeneous enzyme abnormality. There are few published population-based studies on G6PD molecular heterogeneity in the Middle East. G6PD is a "housekeeping" enzyme that performs vital functions within all cells of the body. However, within the erythrocyte which lacks a nucleus, mitochondria, and other organelles, there are certain constraints on metabolism and this enzyme has a particularly important role. G6PD catalyzes the first step of the pentose phosphate pathway (hexosemonophosphate pathway) a series of side reactions of the main glycolytic pathway in the erythrocyte and in all cells of the body. The G6PD is one of a cluster of genes on the distal long arm of the X-chromosome (Xq28). Included in this group of genes are those for the fragile X, hemophilia A, color vision, a putative gene for bipolar affective illness, the ABP-280 filamin gene Bornholm eye disease, clasped-thumb, mental retardation (MASA) syndrome, and dyskeratosis congenital, the gene of G-6PD contains 13 exons and is 18 Kb in length . The first exon contains no coding sequence and the intron between exons 2 and 3 is extraordinarily long, extending for 9,857bp [1, 2]. G6PD Mediterranean is a deficient variant with enzyme activity that is less than 10% of the activity of G6PD B; it reaches allele frequencies of between 10 and 25 or higher in many populations in the Mediterranean region and the near and middle east. This variant is a consequence of a cytosine to thymine substitution at nucleotide number 563 resulting in a serine to phenylalanine substitution at amino acid number 188 [3]. The aim of this study was to determine the prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Iraqi population, and describe the Mediterranean (Med) mutation in the population. Materials and methods Blood samples Blood samples were collected from seventy one random G6PD patients from different Iraqi populations were chosen during the period from March to May 2006. Patients were admitted to several Iraqi hospitals which were as follows: Azady Teaching General Hospital in Duhok, AlIskan teaching hospital, pediatrics hospital in Baghdad, AlKadhumia teaching hospital in Baghdad, Al-Zahra a hospital in Diyala. Eighty five of apparently healthy individuals from different Iraqi populations were randomly included in the study. According to the questionnaire form and the study of the patient s files the geographical and demographical distributions of both patient and control groups were recorded. DNA Extraction The genomic DNA was extracted from white blood cells from the G6PD deficient and apparently healthy individuals, the whole fresh blood collected in EDTA anticoagulant tubes using Wizard genomic DNA purification kits. Agrose gel (1%) electrophoresis was adopted to confirm the presence and integrity of the extracted DNA [6]. PCR/RFLP Analysis PCR/RFLP method was adopted to characterize mutation of the DNA samples. According to the common distribution of G6PD mutations in the neighboring countries [4,5]. Biotechnology Research Center (special edition) Vol. 3No. 22009 92 Mediterranean mutation was chosen for molecular diagnosis in Iraq. Table (1) shows the type of mutation and its descriptive characteristics. Table (1): Types of G6PD mutations G6PD variant Origin Base substitution Exon no. Amino acid substitution Mediterranean Mediterranean 563 C A 6 Ser Phe Primer sets which were selected for PCR analysis of mutations are shown in