Abstract
Background
Clubfoot is a common congenital foot deformity. Low folate status in mothers has been associated with CTEV. Folate metabolism might be affected by Methylene Tetrahydrofolate Reductase (MTHFR) gene polymorphism. The present study was aimed to investigate MTHFR C677T polymorphism and its association with CTEV.
Methods
This is a Case-mother-Dyad study with 30 pairs of cases and controls. Single Nucleotide Polymorphism (SNP) analysis of the MTHFR gene was done in this hospital-based study by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP).
Results
In this study, we observed less relative risk of CTEV in presence of C allele as compared to T allele in children, with Relative Risk- 0.6281 and likelihood ratio of 0.5714. While analysing the correlation of genotype variation in cases (CC = 8(26.66%) and CT = 22(73.33%)) with there biological mother (CC = 13(43.33%) and CT = 17(56.66%)), no significant correlation (p = 0.3110) was found between cases and their biological mother genotype.
Conclusion
Among the enrolled cases, there was a significant association of increased CTEV risk with 677T variant allele of MTHFR gene. Also, maternal MTHFR genotype was not found to influence CTEV risk of offspring.
Keywords: CTEV, Clubfoot, MTHFR, Etiology of clubfoot, Risk factors for CTEV
1. Introduction
Congenital talipes equino varus (CTEV) is a common developmental disorder of lower limb involving various joints of the foot, which is mostly seen in children. The prevalence of CTEV worldwide is about 1–4 per 1000 births.1 This is a three-dimensional deformity that can be observed immediately at birth. It includes equinus at the ankle, varus and internal rotation of heel, cavus of midfoot, adducted and supinated forefoot, and internal torsion of the tibia.2 There are a few genetic syndromes associated with congenital anomalies that are associated with CTEV.3,4 The cause of isolated CTEV is unclear and requires further investigation. Several studies done in genetically predisposed individuals suggest the role of environmental factors.4, 5, 6 The vitamin folic acid is important in metabolism, including synthesis of DNA, DNA repair, and DNA methylation.7 The risk of congenital malformations in pregnant women has been associated with low folic acid.8,9 Folic acid supplements can reduce the prevalence of CTEV as observed in a study conducted in the USA.10 Similarly, a lower rate of CTEV among mothers taking folic acid supplements was observed in a study conducted in Denmark.11
The folic acid mechanism and its result for pregnancy are not yet known. Folic acid supplementation may facilitate folic acid metabolism and also prevent genetically inherited anomalies.12 The enzyme 5,10-MTHFR is required for the conversion of 5–10 methylene tetrahydrofolate into 5-methyl tetrahydrofolate. Folate metabolism can proceed either towards DNA synthesis and DNA repair, or towards DNA methylation. C677T polymorphism in MTHFR gene leads to change from alanine-to-valine.13 For the T allele variant, enzyme activity is reduced in heterozygotes (CT) as well as homozygotes (TT) in a dose-response fashion.14 MTHFR gene has been investigated in the etiology of various congenital malformations such as orofacial clefts15 and neural tube defects9 but, after Sharp et al.,16 has not been investigated in CTEV.
For methylation of homocysteine to methionine, a methyl donor is required, which comes from methyl tetrahydrofolate which is a product of MTHFR metabolism. A high level of homocysteine harms the developing embryo.17 Some studies have observed an association between raised homocysteine levels in biological mothers and CTEV.18,19 In various studies,20, 21, 22 T allele variants were found to have raised homocysteine levels. However, the relationship between homocysteine and MTHFR has not yet been proved and clear. Some investigators have observed that when folate status is low this relation holds to be true.23, 24, 25, 26, 27, 28 Others investigators reported that relation may be present in some age groups,29,30 and it may vary by gender,26,28 tobacco consumption,26 weight,31 alcohol intake,32 or vitamin riboflavin/folate status.33 However, the two studies with an association between maternal homocysteine levels and CTEV were faulty; because these two studies included relatively fewer numbers of CTEV–affected child (10 in one study) and homocysteine levels were measured of biological mothers sometime after the pregnancies.18,19
The study aimed to investigate MTHFR C677T polymorphism and its association with CTEV using a case-mother-dyad design.
2. Methods
2.1. Study Design
This case control study was conducted at the Department of Paediatric Orthopaedics and Department of Biochemistry in a tertiary care medical and teaching centre. Using a case-mother-dyad design 30 CTEV cases with their biological mothers, and 30 healthy controls with their biological mothers were included.
2.2. Inclusion and exclusion criteria
All patients of either sex of any age group presenting for the management of CTEV along with their biological mothers were included as cases. Previously operated cases, acquired CTEV, cases with associated multiple congenital abnormalities/syndrome and where biological mothers not available for inclusion, were excluded. Age-sex matched children and their biological mothers who were otherwise normal, without any other syndrome or genetic disorder were included as controls.
2.3. Data collection and study protocol
Basic demographic data were collected for all cases, including family history of clubfoot, tobacco consumption history, and folic acid supplement history of biological mothers. All cases were clinically assessed by Pirani scoring and were managed as per Ponseti protocol. Peripheral blood sampling (0.5–1 ml) was done only once in all cases and controls as well as their biological mothers.
2.4. Genomic DNA isolation and genotyping
DNA was isolated from the blood with the QI amp DNA kit (quigen, C A) following the manufacturer’s protocol. The extracted DNA was quantified and then further checked for purity spectrophotometrically (CARY 300 bio –UV/visible spectrophotometry). The integrity of this DNA was analysed by 0.8% agarose gel electrophoresis. Polymerase chain reaction (PCR) followed by enzymatic digestion (restriction fragment length polymorphism) was used for the genotyping of the MTHFR C677T gene Polymorphism. Polymerase chain reaction products were first digested with HinfI and separated by 3% agarose gel electrophoresis. Then they were visualized by ethidium bromide staining and ultraviolet transillumination. A 100bp DNA ladder was used for sizing and approximate quantification of a wide range of ds-DNA fragments on the gel.
2.5. Statistical analysis
Categorical variables were presented in number and percentage (%). Continuous variables were presented as mean ± SD and median. The normality of data was tested by the Kolmogorov-Smirnov test. If normality is rejected then non-parametric tests were used. Odds ratio with 95% Confidence Intervals calculated for selected variables and their significance. Quantitative variables were compared using the Unpaired t-test/Mann-Whitney Test (when the data sets were not normally distributed) between two groups and Anova/Kruskal Wallis test (for non-parametric data) between three groups. Qualitative variables were compared using Chi-Square test/Fisher’s exact test as appropriate. A p-value of <0.05 was considered statistically significant. The data was entered in MS EXCEL spreadsheet and analysis was done using Statistical Package for Social Sciences (SPSS) version 21.0.
3. Results
In this case-mother dyad study, total 30 cases (19 male and 11 females) with CTEV and 30 controls (17 male and 13 females) were enrolled as per inclusion/exclusion criteria. The mean age of CTEV cases and their biological mothers was 2.53 ± 1.02 years and 26.75 ± 3.54 years respectively, whereas the mean age of the controls and their biological mothers was 2.07 ± 0.96 years and 23.28 ± 2.87 years respectively. Based on site of deformity, most of the CTEV cases were bilateral (n = 27; 90%) followed by right side (n = 2; 6.66%) and left side (n = 1; 3.33%).
Most of the cases (n = 27; 90%) had no family history of clubfoot. The difference in numbers was statistically insignificant. There was no control with the history of clubfoot in his/her family. As per the division of biological mothers based on their tobacco consumption, most of the mothers of cases (n = 26; 86.66%) and all mothers of controls (n = 30; 100%) were not consuming tobacco. Most of the biological mothers of cases (n = 22; 73.3%) and controls (n = 19; 63.33%) consumed folic acid during the first trimester of the pregnancy. The difference in numbers was statistically insignificant.
In cases, the allele frequency distribution (genotypic variation) were CC = 8 (26.66%) and CT = 22 (73.33%) whereas in control the allele frequency distribution (genotypic variation) were CC = 14 (46.66%) and CT = 16(53.33%). We observed less relative risk of CTEV in presence of C allele as compared to T allele in children with RR 0.6281 and likelihood ratio of 0.5714 (Table-1). While analysing the correlation of genotype variation in cases (CC = 8(26.66%) and CT = 22(73.33%)) with their biological mother (CC = 13(43.33%) and CT = 17(56.66%)), no significant correlation (p = 0.3110) was found between cases and their biological mother genotype (Table-2).
Table 1.
Genotype variation in cases and controls.
| Genotype | Cases | Controls | Relative Risk | Likelihood Ratio | P-Value |
|---|---|---|---|---|---|
| CC | 8(26.66%) | 14(46.66%) | 0.6281 | 0.5714 | 0.1799 |
| CT | 22(73.33%) | 16(53.33%) |
Table 2.
Correlation of genotype variation in cases’ with their biological mothers.
| Genotypes | Cases | Cases Mothers | 95% Confidence Interval | P-Value |
|---|---|---|---|---|
| CC | 8(26.66%) | 13(43.33%) | −0.1922 to 0.5242 | r = 0.1914 p = 0.3110 |
| CT | 22(73.33%) | 17(56.66%) |
4. Discussion
MTHFR gene has been investigated for several congenital malformations (Botto et al., 20009; Jugessur et al., 200315; Hobbs et al., 200034). To our best knowledge, the present study will be the next in line after Sharp et al., 2006,16 to study polymorphism in the MTHFR gene, genetic variation in folate metabolism and risk of isolated CTEV. As per our objective, by proving the MTHFR genetic polymorphism as a risk factor for CTEV, we may establish a potential biomarker (diagnostic/prognostic) for the early prediction of CTEV by screening of pregnant women and may cure the infant from CTEV before birth via gene therapy. Hence, the study outcomes will be beneficial for general public health too as, with the help of this study we will be able to establish a new approach in CTEV management and also predict the risk of CTEV during anti-natal-care.
Children with the C variant allele (CC) were associated with significantly reduced risk of isolated CTEV, as compared to T variant allele heterozygotes (CT). Whether the mother used folate supplementation during conception period or not this association was not affected. Also, there is no statistically significant difference with the interaction between maternal genotype, folic acid supplement use, and risk of CTEV in the offspring.
The main strength of our study was the ability to investigate the combination of a child’s as well as biological mother’s genotype and maternal use of folate supplement. Also, it avoids any possibility of population stratification.35 It was not clear if the child’s or the biological mother’s genotype was aetiologically relevant. Confounding might occur if the genotype of child is measured when the biological mother’s genotype was relevant and vice versa (Umbach et al., 200036). To avoid this problem, in the present study we analysed both the mother and child genotype with each other as well as with the risk of CTEV.
The observation of an increased risk of CTEV in children with T allele is fascinating since variant T allele is associated with decreased activity of MTHFR enzyme (Rozen et al., 199714). The major methyl donor in the methylation of homocysteine to methionine is 5-methyl tetrahydrofolate which is a product of MTHFR enzyme reaction. In high concentrations, homocysteine and its derivatives may produce toxic effects on developing embryo and tissue (Rosenquist et al., 200117), and 2 studies have reported association between raised maternal homocysteine level and CTEV (Vollset et al., 200018; Karakurt et al., 200319). Not in all but in several studies (Ma et al., 199720; Ma et al., 199921; Casas et al. 200522), carriers of the variant T allele have been found to have increased homocysteine levels.
Several investigators in their studies have observed that this relation holds only with low folate status (Jacques et al., 199623; Girelli et al., 199824; Kim et al., 200425; Brown et al., 200426; Papoutsakis et al., 200527; Papoutsakis et al., 200628). Others have suggested that this relation might be present in some age groups but not others (Spotila et al., 200329; Van et al., 200530) and/or might vary by gender (Brown et al., 200426; Papoutsakis et al., 200527; Papoutsakis et al., 200628), smoking status (Brown et al., 200426), alcohol intake (Chiuve et al., 200532), or riboflavin status (McNulty et al., 200233).
Sharp et al. (2006)16 studied the genotype association of MTHFR C677T polymorphism with CTEV. In their study, although a possible interaction with folate supplement use was found but, the overall maternal MTHFR genotype did not influence CTEV risk for the offspring. They observed that children who carry the T variant of the MTHFR gene are at a decreased risk of CTEV. This is in contrast to our finding that T variant is associated with an increased risk of developing CTEV.
In conclusion, we found a significant association of increased CTEV risk with 677T variant allele of MTHFR gene. Also, maternal MTHFR genotype was not found to influence CTEV risk of offspring. However, further multicentre studies are warranted for the replication of these genetic associations with a larger sample size to validate the generalizability.
Conflicts of interest
The authors declare that they have no competing interests in this article.
Ethical approval
This study was permitted by the ethical committee of our institute (94th ECM II B-Thesis/P 18), and informed consent was taken from each participant guardian.
Funding
The study was funded by the KGMU-DHR [KGMUID:DHRMRU 012].
CRediT authorship contribution statement
Vaishnavi Pandey: Formal analysis, Data curation, Investigation. Pradeep Chaturvedi: Writing - original draft, Data curation. Harshit Gehlot: Methodology, Writing - review & editing, Visualization, Validation. Abbas Ali Mahdi: Investigation, Resources, Project administration. Ajai Singh: Conceptualization, Supervision, Project administration, Funding acquisition. Mayank Mahendra: Methodology, Validation, Writing - review & editing.
Acknowledgements
This study was supported by Department of Paediatric Orthopaedics in collaboration with Department of Biochemistry, and Department of Orthopaedic Surgery King George’s Medical University, Lucknow, Uttar Pradesh, India.
Contributor Information
Ajai Singh, Email: ajaipaedortho62@gmail.com.
Mayank Mahendra, Email: mahendra_mayank@rediffmail.com.
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