ABSTRACT
BACKGROUND: Like glucose-6-phosphate dehydrogenase (G6PD) deficient hemizygous males and homozygous females, heterozygous females could also manifest hemolytic crisis, neonatal hyperbilirubinemia or kernicterus upon exposure to oxidative stress induced by certain foods such as fava beans, drugs or infections. Although hemizygous males and homozygous females are easily detected by conventional G6PD enzyme assay method, the heterozygous state could be missed by the conventional methods as the mosaic population of both normal and deficient RBCs circulates in the blood. Thus the present study aimed to apply high resolution melting (HRM) curve analysis approach to see whether HRM could be used as a supplemental approach to increase the chance of detection of G6PD heterozygosity. RESULTS: Sixty-three clinically suspected females were evaluated for G6PD status using both enzyme assay and HRM analysis. Four out of sixty-three participants came out as G6PD deficient by the enzyme assay method, whereas HRM approach could identify nine participants with G6PD variants, one homozygous and eight heterozygous. Although only three out of eight heterozygous samples had G6PD enzyme deficiency, the HRM-based heterozygous G6PD variants detection for the rest of the samples with normal G6PD enzyme activities could have significance because their newborns might fall victim to serious consequences under certain oxidative stress. CONCLUSIONS: In addition to the G6PD enzyme assay, HRM curve analysis could be useful as a supplemental approach for detection of G6PD heterozygosity.
Subject(s)
DNA Mutational Analysis/methods , Glucosephosphate Dehydrogenase Deficiency/diagnosis , Glucosephosphate Dehydrogenase/genetics , Heterozygote , Mutation , Adolescent , Child , Child, Preschool , Female , Glucosephosphate Dehydrogenase Deficiency/genetics , Humans , Infant , Infant, Newborn , Nucleic Acid DenaturationABSTRACT
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked human enzyme defect of red blood cells (RBCs). Individuals with this gene defect appear normal until exposed to oxidative stress which induces hemolysis. Consumption of certain foods such as fava beans, legumes; infection with bacteria or virus; and use of certain drugs such as primaquine, sulfa drugs etc. may result in lysis of RBCs in G6PD deficient individuals. The genetic defect that causes G6PD deficiency has been identified mostly as single base missense mutations. One hundred and sixty G6PD gene mutations, which lead to amino acid substitutions, have been described worldwide. The purpose of this study was to detect G6PD gene mutations in hospital-based settings in the local population of Dhaka city, Bangladesh. Qualitative fluorescent spot test and quantitative enzyme activity measurement using RANDOX G6PDH kit were performed for analysis of blood specimens and detection of G6PD-deficient participants. For G6PD-deficient samples, PCR was done with six sets of primers specific for G6PD gene. Automated Sanger sequencing of the PCR products was performed to identify the mutations in the gene. Based on fluorescence spot test and quantitative enzyme assay followed by G6PD gene sequencing, 12 specimens (11 males and one female) among 121 clinically suspected patient-specimens were found to be deficient, suggesting a frequency of 9.9% G6PD deficiency. Sequencing of the G6PD-deficient samples revealed c.C131G substitution (exon-3: Ala44Gly) in six samples, c.G487A substitution (exon-6:Gly163Ser) in five samples and c.G949A substitution (exon-9: Glu317Lys) of coding sequence in one sample. These mutations either affect NADP binding or disrupt protein structure. From the study it appears that Ala44Gly and Gly163Ser are the most common G6PD mutations in Dhaka, Bangladesh. This is the first study of G6PD mutations in Bangladesh.
Subject(s)
Exons , Glucosephosphate Dehydrogenase Deficiency/genetics , Glucosephosphate Dehydrogenase/genetics , Mutation, Missense , Adolescent , Amino Acid Substitution , Bangladesh , Child , Child, Preschool , DNA Mutational Analysis , Female , Glucosephosphate Dehydrogenase Deficiency/enzymology , Humans , Infant , Infant, Newborn , MaleABSTRACT
OBJECTIVE: To determine the agreement/variability between colposcopic findings, Pap smear cytology and histopathological diagnosis in gynaecology patients. METHODS: The cross-sectional cohort study was conducted from October 2010 to September 2011 at the Fauji Foundation Hospital, Rawalpindi, and comprised women who presented to the out-patient department with various gynaecological complaints. Colposcopy was performed in all women with unhealthy cervix during gynaecological examination, abnormal Pap smear report, recurrent vaginal discharge and postcoital bleeding. Pap smear was performed before colposcopy if not done earlier. Colposcopic findings were recorded on a specially-designed proforma. Biopsies from abnormal areas were taken and sent for histopathology. Colposcopic findings were compared with histopathology and Pap smear reports The agreement between the methods was evaluated by using Kappa coefficient and chi square test at a significance level of 5%. RESULTS: The mean age of the 143 women was 44 8.5 years (range: 25-72 years). Colposcopic findings were normal in 66(46%) women, while 77(54%) had abnormal findings and among the latter, 62(80.5%) had abnormal histopathology, indicating strong agreement (K=0.65; p<0.001). Pap smear report was abnormal in 48(33.5%) cases and among them histopathology was abnormal in 28(58%). In the remaining 95(66.4%) patients with normal Pap smear, histopathology was abnormal in 44((46%), indicating weak agreement between Pap smear and histopathological diagnosis (K=0.10; p=0.08). CONCLUSIONS: There was a strong agreement between colposcopic findings and histopathological diagnosis. However, agreement between cytological findings and colposcopic findings and cytology and histopathological diagnosis remained weak.
