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Polymorphisms in maternal selected folate metabolism-related genes in neural tube defect-affected pregnancy
Winner K Dewelle1, Daniel S Melka2, Abenezer T Aklilu3, Mahlet Y Gebremariam4, Markos A Alemayehu5, Dawit H Alemayehu5, Tamrayehu S Woldemichael5, Solomon G Gebre1
1 Department of Biochemistry, School of Medicine, College of Health Sciences, Addis Ababa University, Ethiopia, East Africa
2 Biochemistry Division of Basic Sciences, University of Global Health Equity, Kigali, Rwanda, East Africa
3 Department of Neurosurgery, School of Medicine, College of Health Sciences, Addis Ababa University, Ethiopia, East Africa
4 Department of Obstetrics and Gynecology, School of Medicine, College of Health Sciences, Addis Ababa University, Ethiopia, East Africa
5 Department of Biotechnology and Bioinformatics, Armauer Hansen Research Institute, Addis Ababa, Ethiopia, East Africa
| Date of Submission | 04-Apr-2022 |
| Date of Acceptance | 03-Mar-2023 |
| Date of Web Publication | 30-Jun-2023 |
Correspondence Address:
Dr. Winner K Dewelle
Department of Biochemistry, College of Health Sciences, Addis Ababa University, Addis Ababa
East Africa
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/abr.abr_103_22
Background: Neural tube defects (NTDs) are abnormalities of the brain and spinal cord, which occur as a result of failure in neural tube closure during embryogenesis. Causes of NTDs are complex and multiple, with hereditary, lifestyle, and environmental factors appearing to play a role. In spite of their impact on public health, the role genetics play on NTDs in Ethiopia is lacking. In this study, the role of polymorphisms in MTHFR 677C > T (rs1801133), MTHFR 1298A > C (rs1801131), MTRR 66A > G (rs1801394), RFC1 80A > G (rs1051266), and TCN2 776C > G (rs1801198) on the risk of having NTD-affected pregnancy was investigated.
Materials and Methods: One hundred women with NTD-affected pregnancy and 100 women with normal pregnancy were included in the study. DNA was extracted from saliva and genotyping for five polymorphisms in four genes was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The departure of the genotype's distribution from Hardy–Weinberg equilibrium (HWE) was evaluated using the x2 goodness-of-fit test. Frequencies of genotypes and alleles in case and control mothers were determined and differences between relative frequencies were evaluated by the x2 or the Fisher's exact test.
Results: The statistically significant difference was absent in the genotype and allele frequencies for all the analyzed polymorphisms between cases and controls (P > 0.05).
Conclusion: MTHFR 677C > T, MTHFR 1298A > C, MTRR 66A > G, RFC1 80A > G, and TCN2 776C > G polymorphisms lack association with the risk of having a pregnancy affected by NTD. The role of other genes or environmental factors in NTD etiology needs to be investigated.
Keywords: MTHFR, MTRR, NTDs, RFC-1, TCN2
How to cite this article:
Dewelle WK, Melka DS, Aklilu AT, Gebremariam MY, Alemayehu MA, Alemayehu DH, Woldemichael TS, Gebre SG. Polymorphisms in maternal selected folate metabolism-related genes in neural tube defect-affected pregnancy. Adv Biomed Res 2023;12:160 |
How to cite this URL:
Dewelle WK, Melka DS, Aklilu AT, Gebremariam MY, Alemayehu MA, Alemayehu DH, Woldemichael TS, Gebre SG. Polymorphisms in maternal selected folate metabolism-related genes in neural tube defect-affected pregnancy. Adv Biomed Res [serial online] 2023 [cited 2023 Sep 28];12:160. Available from: https://www.advbiores.net/text.asp?2023/12/1/160/379634 |
| Introduction | |  |
Genes involved in the metabolism of folate and homocysteine contribute to the problem of neonatal abnormalities.[1] It has been demonstrated that polymorphisms in the genes that produce the proteins or enzymes necessary for folate absorption and metabolism affect their expression or catalytic activity, which can significantly increase the chance of developing an abnormality. Despite substantial evidence demonstrating the importance of genetic variables in the etiology of neural tube defects (NTDs), there are currently no single candidate genes for NTDs that influence the management of NTDs in high-risk pregnancies.[2]
The methylenetetrahydrofolate reductase (MTHFR) enzyme, which is encoded by the MTHFR gene, catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the donor of a methyl group for the conversion of homocysteine to methionine.[3] Due to its crucial roles in the one-carbon cycle, which include the metabolism of folate and methionine, the MTHFR enzyme is essential for maintaining cellular homeostasis.[4]
The two frequently investigated single nucleotide polymorphisms (SNPs) in the MTHFR gene are 677C > T and 1298A > C. The 677C > T SNP causes a missense mutation that changes a cytosine into thymine, resulting in an amino acid change from alanine to valine.[4] MTHFR polymorphisms result in reduced enzyme activity and have been linked to increased risks of several pathologies.[5] People with the TT genotype have around 30% of the MTHFR enzyme activity of the wild-type genotype (CC), while individuals with CT genotype have roughly 65% of enzyme activity.[6] Fetal NTDs were observed to be substantially associated with maternal MTHFR 677C > T polymorphism.[1] The 1298A > C SNP leads to the substitution of alanine for glutamate in the MTHFR enzyme and impairs enzyme activity to a lesser degree[7]; however, with 677C > T polymorphisms, it has a stronger impact on the reduction of MTHFR enzyme function. Previous studies have reported an association between 1298A > C and NTD.[8]
The MTRR gene codes methionine synthase reductase (MTRR) enzyme,[9] which is necessary for the regeneration of functional methionine synthase (MS) by reductive methylation in which the methyl donor is S-adenosylmethionine (SAM). The prevalent SNP in the MTRR gene is the substitution of G for A at position 66 which causes isoleucine to methionine substitution. This polymorphism negatively affects enzyme activity.[10] Previous research has suggested additive or synergistic action of MTHFR 677C > T and MTRR 66A > G polymorphisms on levels of plasma homocysteine[11] and an increased risk of NTD with low cobalamin status.[12]
The RFC-1 (SLC19A1) gene encodes a protein involved in folate metabolism, which serves as a bidirectional transporter, receiving folate cofactors while exporting numerous organic anions. In cells and tissues of mammals, the ubiquitously expressed reduced folate carrier (RFC) is the main system for transporting folate cofactors.[13] The 80A > G polymorphism, in the RFC-1 gene was reported to be a risk factor for NTDs.[14]
The TCN2 gene codes for transcobalamin, a protein required for transporting vitamin B12 into cells. The most extensively studied SNP in the TCN2 gene is 776C > G.[15] In the protein, this polymorphism causes the substitution of proline with arginine, which may change the secondary structure of the protein.[16] This SNP has been reported as strongly predictive of NTDs.[17]
| Materials and Methods | | |
Subjects
Pregnant women, 18+ years, with a diagnosis of fetal NTD on prenatal ultrasound, were considered as cases. Controls consisted of pregnant women of the same gestational age whose pregnancy was not affected by NTD. The study participants were chosen from three public hospitals in Addis Ababa, Ethiopia (Zewditu Memorial, Gandhi Memorial, and Tikur Anbessa Specialized Hospitals) between September 2019 and August 2020. The study included 100 cases and 100 controls.
Genetic analysis
Five polymorphisms in four selected folate metabolism-related genes were invetigated; MTHFR 677C > T (rs1801133), MTHFR 1298A > C (rs1801131), MTRR 66A > G (rs1801394), RFC1 80A > G (rs1051266), and TCN2 776C > G (rs1801198) were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).
For DNA extraction, saliva was collected by the study participants themselves using Oragene® DNA self-collection kit (DNA Genotek inc, Ottawa, ON, Canada) after receiving instructions on how to collect saliva. The participants were advised not to eat, drink, or chew gum and requested to rinse their mouths with water before collection. Genomic DNA was extracted by ethanol precipitation using prepIT-L2P reagent according to the manufacturer's protocol (DNA Genotek Inc., Ottawa, ON, Canada). The concentration and purity of the DNA were measured by NanoDrop 2000 spectrophotometer (Thermo Scientific; Thermo Fisher Scientific, Waltham, MA). All of the extracted DNA yielded a 260/280 ratio of greater than 1.8.
PCR was performed in a mixture containing 6.25 μl of HotStarTaq Master Mix (Qiagen Inc., Chatsworth, CA, USA), 0.25 μl of each of forward and reverse primers obtained from previous studies (Inqaba Biotec East Africa Ltd, Nairobi, 00606, Kenya), 4.5 μl of RNase-free water, and 1.25 μl of template DNA. For all the tested polymorphisms, the PCR reactions included an initial step of activation of the HotStarTaq DNA Polymerase at 95°C for 15 min, and termination of the reaction by a final extension at 72°C for 10 min. The PCR conditions, primer sequences, and length of PCR amplification products are presented in [Table 1]. Electrophoresis of PCR amplified products and a DNA ladder of 100 bp were conducted on an agarose gel. The size of PCR products on the gel was estimated by comparing the bands of the sample lane against the DNA ladder. Samples that failed PCR amplification were repeated. | Table 1: PCR conditions, primer sequences, and PCR amplification products
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Restriction enzyme digestion of PCR amplified products
Restriction enzyme digestion of PCR amplified products was carried out in a mixture containing 2 μl of restriction enzyme digestion buffer, 1 μl of specific restriction enzyme (Inqaba Biotec East Africa Ltd, Nairobi, 00606, Kenya), and 7 μl of molecular grade water with 10 μl of PCR amplified product. The reaction was carried out at a digestion temperature of 37°C for 1 h and terminated by heat inactivation at 80°C for MTHFR 677C > T and at 65°C for MTHFR 1298A > C, MTRR 66A > G, RFC1 80A > G, and TCN2 776C > G for 20 min. The restriction enzymes used for each polymorphism and the products obtained after the restriction enzyme digestion are presented in [Table 2]. Electrophoresis of restriction enzyme digestion products and a DNA ladder of 100 bp were carried out on agarose gel. The size of restriction enzyme digestion products on the gel was estimated by comparing the bands of the sample lane against the DNA ladder.
Statistical analysis
Statistical analysis was done using SPSS for windows program version 25.0 (Armonk, NY: IBM Corp.). Discrepancies in variables between cases and controls were evaluated by the x2 test. The departure of the genotype's distribution from Hardy–Weinberg equilibrium (HWE) was measured using x2 goodness-of-fit test. Genotypic and allelic frequencies of cases and control mothers were determined and differences between relative frequencies were analyzed by the x2 or the Fisher's exact test where suitable. Statistical significance was set at P < 0.05.
Ethical approval
Ethical approval was obtained from the National Research Ethics Review Committee at the Ministry of Science and Higher Education-Ethiopia (Ref. No. MoSHE/RD/14.1/465/19, signed by Professor Afework Kassu) on July 17, 2019. The aim of the study was briefly described to the study participants and they were notified that their results will be managed with privacy. Data and samples had been collected after the study participants gave written informed consent.
| Results | | |
The study enrolled 200 pregnant women, of whom 100 were case mothers and 100 were control mothers. Among the 100 pregnancies affected by NTDs, there were 79, 17, and 4 spina bifida, anencephaly, and encephalocele cases, respectively.
Cases had a mean age of 26.8 ± 5.25 while that of controls was 26.5 ± 4 years. Forty-six percent of cases and 45% of controls were in the age group of 20–24 and 25–29 years, respectively.
Five percent of the cases reported they had a sibling with NTD out of which one had a previous child with NTD. None of the controls reported having an NTD sibling or a previous child with NTD. However, this result has no statistical significance (P = 0.059).
HWE matching
To test for the distribution of genotypes for HWE, the Chi-square goodness-of-fit test was used. For MTHFR 677C > T and MTHFR 1298A > C, the alleles were out of HWE in both the cases and controls. For MTRR 66A > G, RFC1 80A > G, and TCN2 776C > G, the alleles were according to HWE in both the cases and controls [Table 3]. | Table 3: Hardy–Weinberg equilibrium matching of case and control mothers
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Genotype and allele distribution between cases and controls
Genotypic and allelic distribution of the selected genes among cases and controls is presented in [Table 4]. | Table 4: Genotype and allele distribution of the selected genes of case and control mothers
Click here to view |
For the MTHFR rs1801133 polymorphism, the CT genotype frequency in cases was 12% while it was 7% in controls, however, this difference was not statistically significant (P = 0.209). Statistical significance was not seen for the TT genotype (P = 0.434) as well as the T allele (P = 0.092).
For MTHFR rs1801131 polymorphism, the AC genotype frequency was higher in case mothers than controls (27% vs. 16%). However, this difference was not statistically significant (P = 0.117). In contrast, the CC genotype frequency was greater in controls than in the cases (35% vs. 27%). However, this difference was not statistically significant (P = 0.55). The distribution of the C allele between cases and controls also lacks statistical significance (P = 0.612).
For MTRR rs1801394 polymorphism, the AG genotype frequency was greater in controls than in cases (31% vs. 21%); however, this difference lacks statistical significance (P = 0.114). A statistically significant difference was not observed in the frequency of the GG genotype (P = 1) and the G allele (P = 0.444) between cases and controls.
For the RFC1 rs1051266 polymorphism, a relatively higher frequency is observed for the AG genotype (44% vs. 38%) but without statistical significance (P = 0.578). Neither the frequency of the GG genotype (P = 0.343) nor did the G allele frequency (P = 0.665) differ between the two groups.
For the TCN2 rs1801198 polymorphism, the frequency of the CG genotype was higher in cases than in controls but without statistical significance (P = 0.089). The GG genotype and the G allele frequencies were not different between the two groups (P = 0.660 and 0.186, respectively).
| Discussion | | |
In this study, the role of polymorphisms in MTHFR 677C > T, MTHFR 1298A > C, MTRR 66A > G, RFC-1 80A > G, and TCN2 776C > G genes on NTD risk in women with NTD-affected pregnancy was explored.
The association between maternal MTHFR 677 gene polymorphism and NTD susceptibility has been debated in many communities throughout the world. Our result indicated that there is no difference in the frequency of the CT (P = 0.209) and TT (P = 0.434) genotypes and the T allele (P = 0.092) between cases and controls implying that neither the genotypes nor the allele had an association with the risk of having NTDs affected pregnancy. Similar to our finding, lack of statistically significant difference in genotype or allele frequencies between the two groups was reported[14],[23] and lack of association with NTD-affected pregnancy[17],[24],[25] was reported. Previous data suggest that it is the TT genotype in the NTD-affected embryo, rather than the maternal TT genotype, which is the primary predictor of NTD.[26]
For the MTHFR 1298A > C, no statistically significant difference was found in the frequency of the AC (P = 0.117) and CC (P = 0.55) genotypes and the C allele (P = 0.612) between cases and controls indicating a lack of association of the genotypes and the allele with risk of having NTD-affected offspring. Our finding agrees with previous reports.[23],[25],[27],[28],[29] Even the 1298A > C polymorphism was found to be protective.[17],[30] However, a direct association between the MTHFR 1298C and NTD was reported.[8] These conflicting findings may be attributable to other factors that may influence the occurrence of NTDs. MTHFR 677C > T and 1298A > C polymorphisms may thus be a maternal risk factor for NTD-affected pregnancies in some populations but not others.
MTRR restores methylated vitamin B12 from the oxidized form and in so doing plays a vital role in sustaining the active state of the enzyme MS which catalyzes the methylation of homocysteine to methionine.[31] It has been indicated that MTRR 66A > G polymorphisms may potentially impair MS action resulting in increased homocysteine concentrations which is a recognized risk factor for NTDs. A meta-analysis indicated that MTRR 66A > G polymorphism may increase the maternal risk for NTDs.[32] It was reported that the G allele frequency was significantly higher in cases than in controls.[33] However, we found no difference in the distribution of the AG (P = 0.114) and GG (P = 1.00) genotypes as well as the G allele (P = 0.444) between cases and controls implying an absence of connection between MTRR 66A > G polymorphism and NTDs. Our result is similar to previous reports.[12],[32] Even the MTRR 66A > G polymorphism was indicated to provide a protective effect in NTD cases.[18] In our study population, it is likely that other factors affected NTD risk.
Our findings indicated that there is no difference in the distribution of the AG genotype (P = 0.578) and the GG genotype (P = 0.343) as well as the G allele (P = 0.665) between cases and controls implying a lack of connection between RFC-1 80A > G polymorphism and NTDs, similar with previous studies.[34],[35] A meta-analysis was unable to provide a positive relationship between RFC1 80A > G polymorphism and vulnerability to NTDs.[36] It appears that our study did not find any positive relationship between RFC1 80A > G polymorphism and the risk of having NTDs affected the fetus.
For the TCN2 776C > G (rs1801198), our finding indicated that the G allele (P = 0.186) and the CG (P = 0.089) and GG (P = 0.660) genotype frequencies did not differ between cases and controls; an indication for absence of a relationship between the polymorphism and NTD risk. Our finding is similar to previous reports.[16],[24],[37] A meta-analysis also indicated that the TCN2 776C > G polymorphism has no significant association with the risks of congenital abnormalities.[38]
| Conclusion | | |
The disagreements about the association of the different genotypes and alleles and NTDs among the various studies could be justified by the multiple causes of NTD. We investigated the association between five polymorphisms in four selected folate metabolism genes and the likelihood of having an NTD-affected pregnancy. A statistically significant difference was absent in the frequencies of all the tested genotypes and alleles between case and control mothers; hence, the MTHFR 677C > T, MTHFR 1298A > C, MTRR 66A > G, RFC1 80A > G, and TCN2 776C > G polymorphisms do not appear to have any role in NTDs in our study population, an observation that highlights the importance of other genes or environmental factors in NTD etiology. Additional studies are needed to get a deeper insight into the etiology of NTDs in Ethiopia.
Acknowledgement
We gratefully acknowledge AAU and AHRI. We are also thankful to Hiwot K Dewele, from UT, Dallas, Tx for covering the cost of manuscript processing fee.
Financial support and sponsorship
Addis Ababa University (AAU) supported expenses related to data and sample collection. Armauer Hansen Research Institute (AHRI), Addis Ababa, Ethiopia supported expenses related to laboratory analysis. AAU and AHRI had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]
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