Molecular Basis of G6PD Deficiency in Babylon : Iraq

Abstract

Objective:
The objective of this study was to investigate the molecular basis of glucose-6-phosphate
dehydrogenase (G6PD) genes in hyperbilirubinemic neonates in Babylon province of Iraq by
using molecular methods (genomic DNA extraction, PCR and RFLP analysis) and then to
investigate the type of G6PD variant predominantly present.
Methods:
The study included a total of 236 full-term male neonates, 183 of them were associated
with severe hyperbilirubinemia which were admitted in Teaching Hospital of Pediatric and
Maternity / Babylon during 1st , Oct., 2007 to 14th , July, 2008 with age ranged between 1 –
28 days, their TSB levels ≥ 15 mg/dl , while another 53 neonates were used as control group.
The blood sample taken from each neonate was divided into two aliquots: the first aliquot
was used for hemoglobin (Hb), total and conjugated bilirubin (TSB and SCB), G6PD
activity. The second aliquot was used for molecular analysis 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
measurements, severe G6PD deficiency was detected in 22 of the total 183
hyperbilirubinemic neonates included and their activity levels was significantly decreased (P
< 0.05) to 0.34 ± 0.17 U/g Hb as compared with control value 10.02 ± 1.17 U/g Hb. The
incidence of severe G6PD deficiency in neonatal hyperbilirubinemic neonates identified was
12.02 %. TSB levels were markedly elevated to (23.01 ± 5.0 mg/dl), whereas the control
value was 0.75 ± 0.23 mg/dl. The mean ± SD values of each of SCB and Hb were
significantly lower than that found in controls (P < 0.05) and reached to 0.063 ± 0.036 mg/dl ;
13.32 ± 0.94 g/dl as compared with that found in control neonates 0.19 ± 0.11 mg/dl ; 15.91 ±
1.62 g/dl respectively. Conjugated bilirubin was undetectable in sera of 10 of 22 neonates
(45.5%) 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 detected by agarose
electrophoresis and then amplified by PCR and finally was subjected to digestion by
endonuclease restriction enzymes to create RFLP to enable the detection of mutation that
caused G6PD deficiency. The overall majority of affected severe G6PD-deficient neonates
with hyperbilirubinemia in Babylon province : Iraq were due to G6PD Med variant (C563T,
Ser 188 Phe) in which 19 out of 22 (86.4%) have this type of gene mutation, and only one
G6PD A- variant (4.55%) (G202A ; A376G mutations) was diagnosed, whereas 2 of 22
(9.1%) remain with unknown G6PD variants.
Conclusion:
The predominant G6PD gene detected in hyperbilirubinemic neonate with severe G6PD
deficiency in Babylon province was G6PD Med.
Keywords : Hyperbilirubinemia, G6PD gene, Polymerase Chain Reaction , RFLP.