Mohammad Abdul Mannan

Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations in Bangladeshi Individuals

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.

High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh

BackgroundBangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh.ResultsOur HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result.ConclusionsTargeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and electrophortic indices in order to avoid false positive and false negative results.

Molecular Diagnostic Approach Prevails Superior Over Conventional GelElectrophoresis Method in Detecting Sickle Cell Anemia

Sickle cell anemia is defined as homozygosity caused by the mutation of the glutamic acid residue to valine in the β-globin gene. Sickle cell disease is an increasing global health burden with the estimated number of patients increasing in a concerning manner. Here we report a very interesting and clinically insightful case of hemoglobinopathy which was initially suspected to be Hb S/D Punjab- a rare type of hemoglobinopathy as diagnosed by the hemoglobinelectrophoresis technique. Despite the diagnosis report indicating the rare Hb S/D Punjab, the uncharacteristic clinical presentation of the patient which was not coherent with the classical symptoms of Hb S/D Punjab, forced the clinicians to turn their attention to molecular diagnosis. To clarify the etiology of the clinical case, a sequencing-based molecular diagnosis approach was adopted that revealed the mutational signature of sickle cell anemia (SCA). This case can be regarded as a prominent example where the molecular techniques lead to the correct diagnosis matching with the clinical symptoms while the conventional diagnostic approach failed.

High resolution melting curve analysis enables rapid and reliable detection of G6PD variants in heterozygous females

BackgroundLike 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.ResultsSixty-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.ConclusionsIn addition to the G6PD enzyme assay, HRM curve analysis could be useful as a supplemental approach for detection of G6PD heterozygosity.

Death audit in the neonatal ICU of a tertiary care hospital in Bangladesh: a retrospective chart review

Abstract Background: Expectant reduction of neonatal mortality and formulation of preventive strategies can only be achieved by analysis of risk factors in a particular setting. This study aimed to document incidence of neonatal death and to analyze the risk factors associated with neonatal death. Methods: This retrospective study was carried out in department of Neonatology, Bangabandhu Sheikh Mujib Medical University (BSMMU) over a 12-month period from January to December 2015. The newborns that died within 28 d of life were defined as “Cases” and “Control” were the surviving newborn discharged to home as healthy. Two birth weight and gestational age matched controls were taken for each case. Maternal, obstetric, and newborn characteristics were analyzed between both the groups. Data analysis was performed using SPSS version 20.0 (SPSS Inc., Chicago, IL). A probability of < .05 was considered statistically significant. The strength of association was determined by calculating odds ratio and their 95% confidence intervals (CIs). Results: During the study period, the proportion of death was 9.6% (64/612). Both in Chi-square analysis and in logistic regression analysis, less than four antenatal visits (odds ratio (OR) 2.78; 95% CI: 1.23–6.28, p = .014) and sepsis (OR 2.37; 95% CI: 1.07–5.26, p = .034) were found to be independent risk factors for deaths, whereas LUCS found to be protective for deaths (OR 0.40; 95% CI: 0.19–0.83, p = .015). Conclusion: In conclusion, less than four antenatal visits and presence of sepsis were found to be independent risk factors whereas LUCS protective of newborn death.