Detoxification Genomics in Children with β-Thalassemia Major: Pilot Study of Glutathione S Transferase M1, Pi & Methyltetrahydrofolate Reductase Gene Polymorphisms Combinations in β- Thalassemia Major | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Pediatric Sciences Journal | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Article 7, Volume 1, Issue 2, July 2021, Page 98-106 PDF (842.46 K) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Document Type: Original Research | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
DOI: 10.21608/cupsj.2021.55630.1011 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Authors | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Magd A. Kotb 1; Mona Hamdy1; Khaled Eid1; Mona Aziz2; Mai Abd El Salam1; Hend Abd El Baky 1; Nabil Lotfi3; Niveen Salama 1 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1Department of Pediatrics, Faculty of Medicine, Cairo University, Egypt. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
2Clinical Pathology Department, Faculty of Medicine, Cairo University, Cairo, Egypt | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3Undergraduate Student, Faculty of Medicine, Cairo University, Egypt | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Abstract | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Background: Children with β-thalassemia major differ regarding age at presentation, transfusion requirements and unpredictable timing, rate and severity of hemolytic crisis. The blood transfusions are associated with iron overload. Glutathione S transferase M1 (GSTM1) null mutation was reported to be associated with myocardial iron overload with low body iron. Aim of the Work: To investigate if children with β-thalassemia major have more than a detoxification enzyme defect. Materials and Methods: GSTM1, glutathione S transferase Pi (GSTPi) and methyltetrahydrofolate reductase (MTHFR) polymorphism were studied among 97 children with β-thalassemia major in a cross-sectional study. Results: The studied cohort comprised 24 (24.7%) girls and 73 (75.3%) boys. Mean hemoglobin was 5.9+/- 0.7gm%, serum iron was 145.69 +/- 58.6 mcg% and total iron binding capacity was 222.58 +/-50.66 mcg%. Of them, 68 (70.1%) demonstrated single or multiple polymorphisms (43 had GSTM1, 20 GSTPi and 32 with MTHFR polymorphisms respectively), while 29 (29.2%) did not demonstrate any polymorphism. There was no correlation between type, number of polymorphisms and clinical phenotype. Sample size and cross- sectional nature of our study did not allow genotype-phenotype correlation. Most of studied children express GSTM1, GSTPi and MTHFR gene polymorphism which was not consistent among them. Conclusion: Children with β-thalassemia major may have one or more than a detoxification/ regeneration potential enzyme gene GSTM1, GSTPi and MTHFR polymorphism. Every child with β-thalassemia major has unique genetic detoxification and regeneration abilities. The detected detoxification defects might explain the lack of predictability of occurrence of hemolytic attacks and their severity. More studies are needed to highlight impact of detoxification and regeneration genomics in β-thalassemia. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Glutathione S transferase M1 (GSTM1); glutathione S transferase Pi (GSTPi); me-thyltetrahydrofolate reductase (MTHFR); polymorphism; β-thalassemia | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Introduction The gene hemoglobin beta (HBB) codes for the beta hemoglobin protein. It is located on chromosome 11 short arm 15.5. It is a multigene locus of β-globin; yet, the expression of beta globin is controlled by single locus control region (2). The HBB defects result in beta thalassemia that afflicts millions worldwide (3, 4). It results in reduced or absent production of globin chains of hemoglobin with a clinical spectrum ranging from trait to severe hemoglobinopathy and subsequent development of lifelong chronic hemolytic anemia necessitating lifelong regular blood transfusions. Severe forms of β-thalassemia on regular blood transfusions develop iron overload and are maintained on iron-chelation therapy (5). It is the commonest single gene disorder in the world first noted in the Mediterranean population (3, 6). Yet children with β-thalassemia have individual variations as regards age at presentation, transfusion requirements, number of hemolytic attacks, etc. The different phenotypes were reported to correlate with different β-thalassemia genotype mutations (4). Complications such as iron overload vary as well (7). Detoxification is decreed by interaction of intoxicating material (8) and by host detoxification genomics (9). Host detoxification is decreed by the efficiency of cytochrome p 450, Glutathione S Transferase (GST) glucuronidation, detoxification super families and structural regeneration ability to resolve aftermath (10–14). Detoxification abilities might contribute to pathogenesis and phenotype variation among thalassemia afflicted subjects. Hence, once a child with β- thalassemia is exposed to a chemical that he cannot handle, the child would present by various clinical signs and symptoms related to the chemical, and it would be variable and not consistent with the β- thalassemia genotype. In this work we aimed to investigate glutathione S transferase M1 (GSTM1), glutathione S transferase Pi (GSTPi) and methyltetrahydrofolate reductase (MTHFR) gene mutations in children with β- thalassemia. Subjects and Methods Participants This study was a single center cross sectional pilot study that included 97 consecutive children with documented β-thalassemia major on regular blood transfusion and on iron chelation therapy. The study was approved by the Pediatric Department Committee for Post-Graduate Studies and Research, Faculty of Medicine, Cairo University, Egypt. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. Methods GSTM1, GSTPi and MTHFR polymorphism were studied in 97 children with documented β-thalassemia major. All blood samples were withdrawn on the day of blood transfusion, prior to the transfusion and during the cannulation process. The study was conducted at New Children Hospital, Cairo University, Egypt. Gene testing results were compared to the serum iron and TIBC of the studied children. GSTM1 Polymorphism: Whole peripheral blood was the source for DNA isolation using Qiagen. The polymorphic detection of GSTM1 gene was typed using the multiplex PCR (10, 11). The PCR primers used were as follows: P1: 5’CGCCATCTTGTGCTACATTGCCCG, P2: ‘5ATCTTCTCCTCTTCTGTCTC and P3: ‘5TTCTGGATTGTAGCAGATCA. P1 and P3 amplify a 230 bp product that is specific to GSTM1, whereas P1 and P2 anneal to GSTM1 and GSTM4 genes, yielding a 157 fragment that serves as an internal control. PCR was performed to 20ml containing 20 ng of genomic DNA, 0.5µmol/L of primer, 200 µmol of each dNTPs, 10mmol/L Tris Hcl (pH 8.3), 50 mmol/Kcl, 1.5 mmol/L Mg cl 2 and 0.5 U of amphitaq DNA polymerase (promega). After denaturation for 4 min at 94ºC, the PCR was performed for 35 cycles of 30 seconds at 94ºC, 1min at 58ºC and 1min at 72ºC. The last elongation step was 7 min. The presence of one or both GSTM1 allele identified by a 230 bp, or its complete deletion (null type) was analyzed by electrophoresis using 1.2% agarose gel. The absence of amplifiable GSTM1 (in the presence of the GST4 amplified control) indicated a null genotype. GSTPi Polymorphism: PCR assay adopted the method described by Harries and colleagues. The primers used were as follows: P105F: 5’ACC CCA GGG CTC TAT GGG AA-3’, P105R: 5’- TGA GGG CAC AAG AAG CCCCT-3’. It detected single 176 base pair fragment (homozygous wild type), 91 and 85 base pair fragments (homozygous polymorphic) and 176, 91 and 85 base pair fragments (heterozygote) (12). Genotype Analyses of the MTHFR 677: DNA was extracted from the whole blood (5 ml sample) with a QIAamp DNA blood Mini Kit (Qiagen, Valencia, CA). MTHFR 677 genotyping was performed (13, 14). For the C→T polymorphism, extracted DNA was amplified with the forward primer 5´-TGA AGG AGA AGG TGT CTG CGG GA-3´ and reverse primer 5´- AGG ACG GTG CGG TGA GAG TG -3´. Polymerase chain reaction (PCR) thermal cycling conditions were 2-minute denaturation at 94ºC, then 40 cycles at 94ºC for 30 seconds, 62ºC for 30 seconds and finally 72ºC for 30 seconds. This was followed by 7-minute extension at 72ºC. Amplified 198-bp PCR products were digested with Hinf 1 (Fermentas) and were visualized under electrophoresis on 4% agarose gel with ethidium bromide. The C allele produced 198-bp band, and the T allele produced 175- and 23-bp fragments. Statistical Analysis All the statistical analyses in this study were conducted using Statistical Package for Social Sciences version 15 (SPSS, Chicago, Ill). Simple frequency, cross-tabulation, descriptive analysis, and tests of significance (t test for parametric data and Chi x2for non-parametric data) were used. Data are presented as mean +/- standard deviation (SD). To annul confounding effects of medicines, all children were compliant on iron chelation, and all were on regular blood transfusions. We relied upon historical Egyptian control group for comparison (15–17). Results
Genotyping
GST: glutathione S transferase, MTHFR 677: methyltetrahydrofolate reductase 677.
N= number.
Discussion Conclusion Children with β-thalassemia may have one or more than a detoxification/ regeneration potential enzyme gene GSTM1, GSTPi and MTHFR polymorphism. Every child with β-thalassemia has unique detoxification and regeneration abilities. Sample size and the cross- sectional nature of our pilot study did not allow genotype-phenotype correlation definition. We assume that the unique genomics of each child might lead to phenotypical variation in β-thalassemia symptoms and would be related to time of exposure or to type of chemical, and its quantity if it happens. Hence the unique clinical picture, the individuality of β- thalassemia clinical picture and march among different children afflicted with the disease. Each child has its own genetically determined detoxification-inability combination and has unique DNA regeneration abilities. The children lifestyle differences with regards to nutrition and exposure to specific chemicals provide further innumerable causes of uniqueness and individualization of β-thalassemia phenotype that need to be studied in future research. Acknowledgment We acknowledge Pediatric Hepatology Team and Pediatric Hematology Team, Faculty of Medicine, Cairo University, Cairo, Egypt.
Author Contributions: All authors searched medical literature, databases, conceptualized, conducted the case review andreviewed the final manuscript. All authors have read and agreed to the published version of the manuscript.
FUNDING Authors declare there was no extramural funding provided for this study. CONFLICT OF INTEREST The authors declare no conflict of interest in connection with the reported study. Authors declare veracity of information. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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