Detection of 563C-t and 1311C-t as the most common G6PD gene in Pakistani population using RFLP based PCR

Detection Of 563C-T And 1311C-T as the most common G6PD Gene in Pakistani Population Using RFLP based PCR.

 Author: Bushra Moiz, Amna Nasir, Zulfiqar Naqvi, Tariq Moatter, and M Khurshid
Department of Pathology and Microbiology
The Aga Khan University, Karachi.

 Objectives: To detect the most frequent G6PD gene in Pakistani population through RFLP-PCR and evaluate its competence as a diagnostic marker for the enzymopathy

 Subjects and Methods: The study was conducted at Aga Khan University Hospital during April 2006 to present after approval from institutional ethical review committee. It is supported by seed money grant #SM050905.

The patients were selected irrespective of age and gender that were referred from within and outside hospital for G6PD deficiency and were identified by Sigma® screening test (kit no 403). 100 patients were enrolled after informed consent. Total genomic DNA was isolated by QIAmp® DNA mini kit. PCR primers for amplification of fragments which include sites of 563 C-T, and 1311 C-T mutations were designed. The detection was carried out in 97 patients using PCR/RFLP technique and products were digested with restriction enzymes: MboII and BclI and analyzed by gel electrophoresis. PCR products were sent to Macrogen® ((Seoul, Korea) for DNA sequencing.

 Results: Of 97 subjects identified as G6PD deficient,  76 (78.0%) were found carrying 563C-T and  44 (45%) 1311C mutation. Except for 9 patients, all were males. The DNA sequencing data supported the results obtained by RFLP-PCR.

 Conclusions: Thus we conclude that the 563C-T and silent mutation 1311C are the commonest G6PD mutations in Pakistani population and RFLP-PCR is a rapid, inexpensive and accurate method for detection of these mutations.

 

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