Molecular Basis of G6PD Deficiency in Hyperbilirubinemic Neonates in Middle Euphrates Province : Iraq

M. Frankool, William; Jawad Al-Tu, Fadhil

	Molecular Basis of G6PD Deficiency in Hyperbilirubinemic Neonates in Middle Euphrates Province : Iraq
Kerbala Journal of Medicine
Article 2, Volume 3, Issue 7, 2010, Pages 867-881 PDF (0 K)
Authors
William M. Frankool; Fadhil Jawad Al-Tu
Abstract
Background: Neonates G6PD deficiency screening has been recognized as an essential component of public health care in most developed and some Mediterranean countries. However, such screening is yet to be widely embraced in Iraq. More than 442 variants of G6PD have been identified by various molecular methods. The aim of the present study was to determine the normal values of G6PD and deficiency prevalence of this enzyme in male neonates and then determination of the type molecular variant of G6PD prevalence in Middle Euphrates Province of Iraq. Objective: The objective of this study was to investigate the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency in hyperbilirubinemic neonates in Middle Euphrates province of Iraq. Molecular methods (genomic DNA extraction, polymerase chain reaction and restriction fragment length polymorphism analysis) and then investigate the type of G6PD variant predominantly present have been performed. Methods: The study included a total of 917 full-term male neonates which were divided into two groups: The first group which include 704 neonates (76.8%) associated with severe hyperbilirubinemia were admitted in Middle Euphrates Province Teaching Hospitals of Maternity and Pediatrics during 1st Oct., 2007 to 12th July, 2008 with age ranged between 1 – 28 days, their total serum protein , TSB levels ≥ 15 mg/dl. The second group which include 213 neonates (23.2%) with the same age ranged were used as control group, their TSB levels ˂ 1 mg/dl. The blood sample taken from each neonate was divided into two aliquots: the first aliquot was used for the determination of total and serum conjugated bilirubin (TSB and SCB), and G6PD activity. The second aliquot was used for molecular analyses including genomic DNA extraction and then application of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) protocols. Results and Discussion: Severe hyperbilirubinemic neonates were screened for erythrocyte G6PD enzyme activity, severe G6PD deficiency was detected in 75 of 704 hyperbilirubinemic neonates included and their activity levels was significantly decreased (P < 0.05) to less than 10% of that found in control group. Therefore, the incidence of severe G6PD deficiency identified in Middle Euphrates Province of Iraq was 10.65%. TSB levels were markedly elevated to ( ≥ 15 mg/dl), whereas the mean ± SD values of SCB were significantly lower than that found in controls (P < 0.05) , SCB was undetectable in 32 of 75 (42.67%) of hyperbilirubinemic neonates with severe G6PD deficiency which imply a partial defect of bilirubin conjugation. The molecular part of the study involved the extraction of genomic DNA from hyperbilirubinemic neonates with severe G6PD deficiency which was detected by agarose gel electrophoresis and then amplified by PCR and finally was subjected to digestion by endonuclease restriction enzymes to create RFLP and to enable the detection of mutation that caused G6PD deficiency. The majority of affected severe G6PD deficient neonates with hyperbilirubinemia in Middle Euphrates province – Iraq, were due to G6PD Med variant (C563T, Ser 188 Phe) , of such 67 of 75 neonates (89.3%) have this type of mutation, and 5 of 75 (6.67%) have G6PD A- variant (G202A ; A376G mutations), whereas only 3 of 75 (5.3%) remain unknown G6PD variants which require future molecular studies.Conclusion: The predominant G6PD gene detected in hyperbilirubinemic neonate with severe G6PD deficiency in Middle Euphrates province was G6PD Med.variant. Keywords : Hyperbilirubinemia, G6PD gene, Polymerase Chain Reaction , RFLP.
Keywords
Hyperbilirubinemia; PD gene; Polymerase Chain Reaction; RFLP

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