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ORIGINAL ARTICLE
Year : 2020  |  Volume : 33  |  Issue : 3  |  Page : 936-941

Relationship of methylenetetrahydrofolate reductase C677T genetic polymorphism and oxidative changes in Egyptian patients with β-thalassemia major


Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission06-Mar-2019
Date of Decision16-Apr-2019
Date of Acceptance27-Apr-2019
Date of Web Publication30-Sep-2020

Correspondence Address:
Alshimaa R. S. Elkholy
Al Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_87_19

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  Abstract 


Objectives
To evaluate the genetic polymorphism of methylenetetrahydrofolate reductase (MTHFR) C677T among patients with beta-thalassemia major and its related oxidative changes.
Background
Accelerated oxidative damage is one of the hallmarks in beta-thalassemia major. Genetic polymorphism of MTHFR C677T has been shown to cause hyperhomocysteinemia, which acts as a prooxidant.
Material and methods
Genotyping for MTHFR C677T was evaluated by PCR-restriction fragment length polymorphism technique. Complete blood picture, hemoglobin electrophoresis, serum ferritin, and total antioxidant capacity (TAC) were determined in 60 patients with beta-thalassemia major and 20 controls of matched age and sex.
Results
MTHFR 677TT genotype was significantly higher among patients with beta-thalassemia major (23.3%) compared with controls (5%). There was significant decrease in TAC in thalassemic patients as compared with controls. TAC was significantly lower in TT group than both of CC and CT groups. Ferritin level was significantly higher in thalassemic group than controls.
Conclusion
Detection of MTHFR (C6777T) genetic polymorphism among thalassemic patients could be helpful in assessment of oxidative stress among these patients.

Keywords: beta-thalassemia, hemoglobinopathy, homocysteine, oxidative stress, polymorphism


How to cite this article:
El Edel RH, Abdalhalim EF, Alhelbawy MG, Elkholy AR. Relationship of methylenetetrahydrofolate reductase C677T genetic polymorphism and oxidative changes in Egyptian patients with β-thalassemia major. Menoufia Med J 2020;33:936-41

How to cite this URL:
El Edel RH, Abdalhalim EF, Alhelbawy MG, Elkholy AR. Relationship of methylenetetrahydrofolate reductase C677T genetic polymorphism and oxidative changes in Egyptian patients with β-thalassemia major. Menoufia Med J [serial online] 2020 [cited 2020 Oct 29];33:936-41. Available from: http://www.mmj.eg.net/text.asp?2020/33/3/936/296715




  Introduction Top


Beta-thalassemia is an inherited disorder caused by mutations in the beta-globin genes leading to a total lack or reduction in the synthesis of normal beta-globin chains. beta-thalassemia is a major public health problem in Egypt, where the carrier rate was 9–10% [1]. Patients with beta-thalassemia major present clinically with severe transfusion-dependent anemia together with other related complications [2]. Oxidative stress, defined as an imbalance in equilibrium between prooxidant and antioxidant system, is important in the pathology of many diseases [3]. Accelerated oxidative damage is one of the hallmarks in thalassemia major, where the elevation in serum ferritin owing to continuous blood transfusions promotes peroxidative damage in patients with thalassemia [4]. A decreased antioxidant level is found [5]. Methylenetetrahydrofolate reductase (MTHFR) is the most critical enzyme in folate-metabolizing pathway. It plays a key role in the remethylation process of homocysteine and also in de novo thymidine synthesis [6]. A C-to-T missense mutation at nucleotide 677 (C677T SNP) produces a thermolabile form of the enzyme associated with reduction in its activity owing to alanine is converted to valine [7]. This can lead to elevated homocysteine which acts as a prooxidant, generating free radicals by auto-oxidation, inducing lipid peroxidation, decreasing endothelial NO, and causing endothelial cell damage [8]. Several studies have demonstrated a relationship between the MTHFR C677T polymorphism and abnormal levels of oxidative stress markers [9]. The aims of the work were to evaluate the relationship of MTHFR C677T genetic polymorphism and oxidative changes among patients with beta-thalassemia major.


  Materials and Methods Top


This study was done in Clinical Pathology Department, Faculty of Medicine, Menoufia University Hospitals. This study was carried out from November 2017 to December 2018 on 80 patients divided into two groups: group 1 included 60 thalassemic children, and group 2 included 20 age-matched and sex-matched healthy children as controls.

Written informed consents were provided by all participants, and approval for the study was obtained from the ethical committee. All participants underwent complete history taking such as onset of transfusion and frequency of transfusion, complete blood count, hemoglobin (Hb) electrophoresis by high-performance liquid chromatography (HPLC), serum ferritin, and total antioxidant capacity (TAC).

Under complete aseptic conditions, 8 ml of venous blood was collected. Each blood sample was divided as follows: 4 ml was collected into two EDTA containing tubes, one for complete blood picture and Hb electrophoresis and the second for genotyping of MTHFR gene. The remaining 4 ml was collected in plain vacutainer tube for measurement of ferritin level (HITACHI Cobas e 411; High-Technologies Corporation, Tokyo, Japan) and TAC (Biodiagnostic Colorimetric Assay Kits, Giza, Egypt).

DNA analysis

PCR-restriction fragment length polymorphism method was used to determine the distribution of genotype and allele frequencies of MTHFR single nucleotide gene polymorphisms (C677T).

The DNA was extracted using commercially available Spin column technique (Scientifi c Zymo Bead Genomic DNA Kits, Quick-g DNA Mini Prep, Bioscience Bar Hill, Cambridge, USA) for DNA extraction from whole blood, supplied by Zymo Research Corp. (Freiburg, Germany). The eluted genomic DNA was stored at –80°C until amplification PCR (New England Biolabs Inc.: Ipswich, Massachusetts, USA).

The sequences of primers were as follows:

Forward primer (5′- TGAAGGAGAAGGTGTCT GCGGGA-3′).

Reverse primer (5′- AGGACGGTGCGGTGAGA GTG-3′).

Reagents and primers were provided by Thermo Fisher Scientific, USA. PCR reaction with 25 μl final volume was prepared by adding 12.5 μl Master mix, 1 μl Forward primer, 1 μl Reverse primer, 2 μl extracted DNA, and 8.5 μl sterile deionized water into PCR wells.

PCR was done using the following conditions: initial denaturation at 94°C for 5 min, 40 cycles of multiplication of DNA (94°C for 30 s for denaturation, 62°C for 30 s for annealing, and 72°C for 30 s for extension), and final extension for 7 min at 72°C min. The amplified products were digested with Hinf I, restriction enzyme (New England Biolabs Inc.) Thermo Fisher Scientific: (Waltham, Massachusetts, U.S.A).

Overall, 2 μl of amplified product was added to 12 μl of sterile distilled water, followed by addition of 6 μl of restriction enzyme and 5 μl of reaction buffer, which is then incubated at 37°C for 15–20 min.

Electrophoresis was done on the digested products and show the wild genotype (C/C) result in one fragment at 198 bp; homozygous mutant genotype (T/T) result in two fragments at 175 and 23 bp; and the heterozygous mutant genotype (C/T) result in three fragments at 198, 175, and 23 bp [Figure 1].
Figure 1: A representation of MTHFR gene polymorphism by RFLP-PCR. Lanes 1, 3, 6, 8, and 9: wild genotype (C/C) of MTHFR gene, band 198 bp. Lanes 2, 5, and 10: the heterozygous mutant genotype (C/T), bands 198, 175, and 23 bp). Lanes 4 and 7: homozygous mutant genotype (T/T), bands 175 and 23 bp. Band 23 does not appear in the photograph as it is fast migrating. MTHFR, methylenetetrahydrofolate reductase; RFLP, restriction fragment length polymorphism.

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Statistical analysis

Results were collected, tabulated, and statistically analyzed by an International Business Machines (IBM) compatible personal computer with Statistical Package for the Social Sciences (SPSS) version 23 (SPSS Inc. Released 2015, IBM SPSS statistics for Windows, version 23.0; IBM Corp., Armonk, New York, USA).

Two types of statistical analysis were done:

  1. Descriptive statistics, for example, number, percentage, mean, and SD
  2. Analytic statistics, for example, Student's t test, Mann–Whitney's test, Kruskal–Wallis, χ2 test, and analysis of variance (F test).



  Results Top


Genotype analysis of MTHFR C677T using PCR-restriction fragment length polymorphism showed that C allele produced 198-bp band and the T allele produced 175 and 23 bp fragments.

Group 1 are thalassemic patients (34 males and 26 females), and their mean age was 7.57 ± 4.18 years. Group 2 are apparently healthy age-matched and sex-matched individuals as a control group (13 males and seven females), and their mean age was 9.85 ± 4.33 years. There was a statistically significant difference in family history between the studied groups (P < 0.001). Our patients received blood transfusion regularly every 3 weeks in 30% and every month in 70% of cases [Table 1]. There was a statistically significant difference in laboratory findings such as red blood cells count, Hb, mean corpuscular volume, mean corpuscular hemoglobin, platelets counts, and ferritin between the studied groups (P < 0.001). TAC was significantly lower in patients with beta-thalassemia major than in controls (P < 0.01) [Table 2]. Regarding the MTHFR genotyping results, it was found that 38.3% had the C/C genotype (wild type), 38.3% had the C/T genotype, and the remaining 23.3% had T/T genotype in patients with beta-thalassemia major compared with controls (80, 15 and 5%, respectively). The difference was statistically significant (P < 0.05). Regarding C allele frequency, it was significantly higher in control group (87.5%) than in beta-thalassemia group (57.5%) (P < 0.001). The T allele frequency is significantly higher in beta-thalassemia group (42.5%) than in controls (12.5%) (P < 0.001) (allele frequencies of the genotypes examined were in Hardy–Weinberg equilibrium) [Table 3]. TAC was significantly lower in TT group than both of CC and CT groups [Table 4] and [Figure 1].
Table 1: Demographic data, history, and clinical data of the studied groups

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Table 2: Laboratory data of the different studied groups

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Table 3: Distribution of methylenetetrahydrofolate reductase genotype in thalassemic patients and controls

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Table 4: Comparison between methylenetetrahydrofolate reductase polymorphism and different parameters in the case group (n=60)

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  Discussion Top


This study shows the relationship of MTHR C677T genetic polymorphism and oxidative changes among patients with beta-thalassemia major. Patients with beta-thalassemia are mainly exposed to oxidative stress owing to iron overload [10]. Iron has a catalytic role to produce powerful reactive oxidant species and free radicals, which lead to oxidative damage [11]. Therefore, evaluation and maintenance of antioxidant defense can be useful in protecting patients with β-thalassemia from more serious complications of the disease [12]. In the present study, patients with beta-thalassemia major were found in a state of oxidative stress indicated by a significant decrease in TAC as compared with the healthy controls. This agrees with Manafikhi et al. [13] and Tsamesidis et al. [14] who reported that significantly lower levels of TAC were observed in patients when compared with controls. However, Bazvand et al. [15] reported that significant increase of TAC was observed in the patient group, compared with the controls. They explained the enhanced total antioxidant status in their patients owing to either increase in compensatory antioxidant response arising from excessive oxidative stress or owing to chelation therapy by deferoxamine, which has antioxidant properties.

Regarding the MTHFR genotyping results, it was found that 38.3% of patients with beta-thalassemia major had C/C genotype (wild type), 38.3% had the C/T genotype, and remaining 23.3% had T/T genotype compared with controls (80, 15, and 5%, respectively). The difference was statistically significant (P < 0.05). Regarding C allele frequency, it was significantly higher in control group (87.5%) than in beta-thalassemia group (57.5%) (P < 0.001). The T-allele frequency is significantly higher in beta-thalassemia group (42.5%) than in controls (12.5%) (P < 0.001). This was in agreement with Abd-Elmawla et al. [8] who reported that homozygous mutant genotype TT and frequency of T allele was significantly higher in patients with beta-thalassemia major than in controls, revealing a significant association between beta-thalassemia major and MTHFR C677T gene polymorphism. Moreover, Zalloua et al. [16] reported high prevalence of MTHFR C677T gene polymorphism in beta-thalassemia intermedia. Genet et al. [17] reported a higher prevalence of the homozygous mutant genotype TT in Mediterranean countries compared with other countries in Northern Europe. On the contrary, Nagel and Muniz [18] and Sipahi et al. [19] failed to find any association between beta-thalassemia major and such a mutation. This difference may be owing to a marked heterogeneity in the prevalence of the T677 MTHFR allele among the major ethnic groups. In this study, TAC was significantly lower in TT group than both of CC and CT groups (P < 0.001). This was consistent with Abd-Elmawla et al. [8] who reported that ongoing oxidative stress in beta-thalassemia major is exacerbated in patients with TT genotype, and their plasma TAC concentration was severely decreased as compared with those in CC genotype. The mean serum ferritin level was 2735.6 ± 1693.9 ng/ml in thalassemia group, which is significantly higher than the control group. This agrees with Ikram et al. [20], who showed that the serum ferritin levels were more than 2500 ng/ml in 76% of cases. Moreover, Mishra and Tiwari [21] reported that 87.4% of the patients with beta-thalassemia major showed very high ferritin levels. The mean serum ferritin level was found to be 2767.52 ng/ml. The serum ferritin level increases as the frequency of blood transfusion and the age of the patient increase. Hb F % detected by HPLC in patients with beta-thalassemia major was 17.34 ± 16.25. This was consistent withAgouti et al. [22] who reported Hb F % to be 6.4 ± 6.6 in 37 patients with beta-thalassemia major. Moreover, Dadheech et al. [23] reported Hb F % of 18.36 ± 11.75 in 113 patients with beta-thalassemia major. Few thalassemic patients in our study had low Hb F % values owing to previous blood transfusion, which could reduce the Hb F results by HPLC. This was consistent with Wirawan et al. [24], who showed that Hb electrophoresis did not show the typical beta-thalassemia major pattern in patients receiving regular and repeated blood transfusion.


  Conclusion Top


The MTHFR C677T gene polymorphism seems to be prevail in Egyptian patients with beta-thalassemia major. The ongoing oxidative stress in beta-thalassemia major indicated by a significant decrease in TAC is exacerbated in patients with TT genotype. Regular iron chelation and antioxidant therapy should be advised for patients with beta-thalassemia major to improve the antioxidant status.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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