|Year : 2020 | Volume
| Issue : 1 | Page : 15-20
MTHFR C677T Polymorphism and risk of nonsyndromic cleft in craniofacial region in a South Indian Population
Betty Anna Jose1, Varsha Mokhasi1, Subramani Arumugam Subramani2, Mahendrakar Shashirekha1
1 Department of Anatomy, Vydehi Institute of Medical Sciences and Research Centre, Whitefield, Bengaluru, Karnataka, India
2 Department of Plastic Surgery, Vydehi Institute of Medical Sciences and Research Centre, Whitefield, Bengaluru, Karnataka, India
|Date of Submission||26-Aug-2019|
|Date of Acceptance||04-Feb-2020|
|Date of Web Publication||11-Apr-2020|
Dr. Betty Anna Jose
Department of Anatomy, Vydehi Institute of Medical Sciences and Research Centre, Whitefield, Bengaluru - 560 066, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism was found associated with cleft palate (CP) and cleft lip with or without CP in different populations and only very few studies were conducted in the Indian population. The aim of this case–control study is to detect whether there is any association of C677T MTHFR gene polymorphism with clefts in the craniofacial region, especially with Nonsyndromic cleft lip with cleft palate (NSCLP) in the South Indian population. Material and Methods: The study was conducted on 179 nonsyndromic cleft cases and 130 healthy individuals without cleft were included as controls. To detect the polymorphism, polymerase chain reaction–restriction fragment length polymorphism method was carried out, and the result was analyzed using Chi-square test and binary logistic regression model. Results: Among the cleft cases, 120 were NSCLP and it showed 6.67% CT genotype and 18.33% TT genotype, while in controls, it was 18.46% and 1.54%, respectively. The TT genotype increased the risk of NSCLP (odds ratio [OR] =0.079, 95% confidence interval [CI]: 0.02–0.34, P = 0.0007) and CT genotype decreased the risk (OR = 2.59, 95% CI: 1.11–6.06, P = 0.028). However, CT + TT model had no association with NSCLP when compared to CC genotype. No significant difference was found between other 59 clefts and polymorphism. Discussion and Conclusion: The association of MTHFR C677T polymorphism with NSCLP varies from population to population. Our study found an association between the polymorphism and NSCLP. The TT genotype increased the risk of NSCLP and CT genotype reduced the risk.
Keywords: Cleft lip/palate, methylenetetrahydrofolate reductase C677T, nonsyndromic, polymorphism
|How to cite this article:|
Jose BA, Mokhasi V, Subramani SA, Shashirekha M. MTHFR C677T Polymorphism and risk of nonsyndromic cleft in craniofacial region in a South Indian Population. J Anat Soc India 2020;69:15-20
|How to cite this URL:|
Jose BA, Mokhasi V, Subramani SA, Shashirekha M. MTHFR C677T Polymorphism and risk of nonsyndromic cleft in craniofacial region in a South Indian Population. J Anat Soc India [serial online] 2020 [cited 2020 Aug 12];69:15-20. Available from: http://www.jasi.org.in/text.asp?2020/69/1/15/282299
| Introduction|| |
Nonsyndromic cleft in the lip and palate has a multifactorial etiology. Several putative genes were studied and specific genes in single or combined with other genes were identified in syndromic and nonsyndromic clefts in various populations. It was found that maternal folic acid intake during the periconceptional period reduces the risk of clefts and it enlightened that the enzymes and coding genes involved in the folate metabolism might be associated with the congenital clefts. Methylenetetrahydrofolate reductase (MTHFR) is one of the important enzymes involved in the folate metabolism, coded by the MTHFR gene.
Folate and MTHFR enzymes are very much required for DNA synthesis and methylation which, in turn, is essential for normal cell division and gene expression during the embryonic period. In the folate cycle, folic acid is reduced to tetrahydrofolate, its active form. Folate is its natural form and folic acid is the synthetic form. For the conversion of 5,10-methylenetetrahydrofolate to 5-methyl tetrahydrofolate, the MTHFR enzyme is required and the methyl group thus formed is used up for the conversion of homocysteine to methionine. Methionine is an essential amino acid. If this conversion is not occurring, the serum homocysteine level increases which has a teratogenic effect in the embryonic life.
The C677T variant in exon 4 is a common functional mutation identified in the MTHFR gene. In C677T polymorphism, C is substituted by T and it changes the amino acid alanine to valine at 222 position. This change in the amino acid results in the thermolabile MTHFR enzyme. Hypofunctional MTHFR leads to hypomethylation and the thermolabile form causes a reduction in the activity of MTHFR enzyme and increased level of plasma homocysteine.
To identify the association between MTHFR gene polymorphism and nonsyndromic orofacial clefts, many studies were conducted in different populations and showed conflicting results,,,,, and very few studies were conducted in the Indian population. No specific gene is identified as a common causative factor of clefting. In different populations, different polymorphisms were identified as the causative factor. Therefore, we conducted a retrospective study to detect the significance of C677T MTHFR gene polymorphism in cases with nonsyndromic clefts in the craniofacial region, especially with nonsyndromic cleft lip palate (NSCLP) in the South Indian population.
| Materials and Methods|| |
The sample size comprised 309 unrelated subjects which included 157 males and 152 females aged between 9 months and 30 years of the South Indian origin. The study was conducted on 179 nonsyndromic cleft cases from the plastic surgery department and 130 healthy individuals without cleft were included as controls. Among the cases, 8 were cranial clefts, 7 with facial clefts, 22 with cleft lip (CL), 22 with isolated cleft palate (CP), and 120 cases with CL palate. The participants were clinically examined, verified their medical records, and interviewed their parents. Informed written consent was obtained from the participants and from their parents in case of minor. Approval of Institutional Ethics Committee was obtained at the beginning of the study.
The peripheral venous blood samples of 3–5 ml were collected in BD lithium heparinized EDTA vacutainer for the DNA extraction. DNA was extracted by phenol–chloroform extraction method with isopropanol precipitation and stored at −20°C for further procedure. At the time of analysis, samples were thawed and processed for polymerase chain reaction (PCR) amplification. The primers for PCR amplification were designed using Primer 3Plus (Sigma Corporation USA) software and the designed oligonucleotides were synthesized in Sigma Corporation USA. The sequence of primers was 5'GTG AAC TAC TGT GGC CTG GAG3' and 5' ACG TCC TTG ATC TCC TGT GG3'. The PCR conditions were as follows: initial denaturation at 95°C for 5 min, annealing at 52°C for 30 s, extension at 72°C for 1 min, for 30 cycles, and final extension at 72°C for 5 min. PCR yielded a 500 bp product that was digested with restriction endonuclease enzyme Hae III (Merck, India) and loaded in 3% agarose gel electrophoresis and visualized by ethidium bromide staining. The C677T mutation caused Hae III restriction site resulting in the cleavage of 500bp fragment into 350bp and 150bp fragments. The digested products were visualized under UV Transilluminator (Bio Bee, India). The PCR–restriction fragment length polymorphism analysis for the genotyping was according to the method followed previously.
Allele frequency was determined by direct counting of alleles. Goodness-of-fit Chi-squared test was used to assess the allele and genotype frequencies. Based on the genotype frequency, Hardy–Weinberg Equilibrium (HWE) was tested in case and control groups. The association was analyzed using Chi-square test and binary logistic regression model using SPSS statistical software version 21.0 (IBM Corp., Chicago, USA).
| Results|| |
Among 179 cases, the distribution of clefts was as follows: 4.5% craniofacial clefts [Figure 1], 3.9% facial clefts [Figure 2], 67% CL palate [Figure 3], 12.3% CL [Figure 4], and 12.3% isolated CP [Figure 5]. The male preponderance was 54.7% and females were 45.3%. The most common laterality was left sided (44.7%) followed by bilateral (22.4%), right (18.4%), and median (14.5%). The C677T MTHFR, TT genotype, was identified only in CL palate cases and not in other cleft types. Among the 120 cases of NSCLP, 30 cases were identified with C677T polymorphism. Among the 30 NSCLP cases with C677T polymorphism, the left laterality was found in 22 subjects in which 18 with TT genotype and 4 with CT genotype and the bilateral cases were 8 where 4 were TT genotype and other 4 with CT genotype. No right laterality was found and the left laterality was more in males than the females in these 30 NSCLP cases with polymorphism. However, this difference in the laterality and polymorphism did not show any significance.
|Figure 3: Cleft lip palate: Unilateral left side cleft lip with cleft palate|
Click here to view
The remaining 59 clefts belonged to craniofacial clefts, facial clefts, CL, and isolated CP with a genotype of CC (71.2%) and CT (28.8%), and its difference with controls was not statistically significant (odds ratio [OR] = 0.57, 95% confidence interval [CI]: 0.278–1.168, P = 0.12). The 59 cases of other clefts was following the HWE (HWp = 0.196, χ2 = 1.67) and the allele frequency was 0.86 and 0.14 for C and T allele, respectively. The OR of CC versus CT was OR = 0.57 (95% CI: 0.28–1.17, P = 0.12) and CC versus CT + TT was OR = 0.617 (95% CI: 0.30–1.25, P = 0.18).
The distribution of MTHFR gene variant in the control group was in HWE, but the 120 cases of NSCLP deviated from the HWE. Among the NSCLP cases, the minor allele frequency was 0.22 in cases, while it was 0.11 in controls. The CT genotype was found more common in controls, and TT genotype of MTHFR C677T polymorphism was more frequent in NSCLP cases. The presence of TT genotype in NSCLP cases (18.33%) and controls (1.54%) showed a statistically significant difference [Table 1].
|Table 1: Allelic and genotypic frequencies of methylenetetrahydrofolate reductase C677T polymorphism in nonsyndromic cleft lip palate cases and control groups|
Click here to view
| Discussion|| |
The present study revealed the distribution of MTHFR C677T polymorphism in cleft cases and controls of South Indian origin. It also showed that the differences in the allele frequency (P = 0.0009) and MTHFR C677T frequency were statistically significant between the NSCLP cases and controls. The CT genotype was higher in controls (18.46%) than in NSCLP cases (6.67%) and it revealed that T-allele in the heterozygote was associated with NSCLP to lower the risk (OR = 2.59, 95% CI: 1.11–6.06). A similar result was observed in a study of MTHFR polymorphism and gene–environment interaction. The possible mechanism of the protective effect of T-allele and the risk of NSCLP can be explained with the changes in folate metabolism. The reduced enzymatic activity of MTHFR might increase the 5,10-methylenetetrahydrofolate which, in turn, increases the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate (dTMP). The dTMP forms deoxythymidine triphosphate and involves in DNA synthesis. Thus, misincorporation of uracil into DNA is reduced. A study from the Indian subcontinent showed that CT genotype was predominant in cases than the controls and was associated with nonsyndromic CL with or without CP, but the result was different in South Indian population, in which increased CT genotype frequency was found in controls. This is in accordance with our study where CT genotype in controls was three times more than the NSCLP cases. This varied difference among the Indian studies might be due to the heterogeneity in the Indian population. This shows that the association of MTHFR C677T polymorphism with NSCLP varies from population to population.
Furthermore, the present study showed that the frequency of TT genotype was 18.33% in NSCLP cases, while it was very low in controls (1.54%), and this difference (OR = 0.079, 95% CI: 0.02–0.34) revealed that there is an association between MTHFR C677T polymorphism in homozygous TT and NSCLP and it increased the risk of NSCLP. A similar result was found in a Chinese study where the TT genotype caused an increased risk of NSCL/P in Northern China, but no association found in the samples from Southern China. This shows the genetic heterogeneity and influence of MTHFR C677T polymorphism. The T-allele frequency was comparatively lower in Indians when compared to Europeans and Americans and the TT genotype was seen in approximately 1% Indian population. The Southern Chinese population showed that MTHFR C677T polymorphism in CT and TT genotypes increased the risk of NSCL/P and CT genotype contributed to the increased risk of CL only. Our study also found an association between the polymorphism and NSCLP but not with CL. No polymorphism was found in CL cases. In our study, we found that the TT genotype increased the risk of NSCLP and CT genotype reduced the risk. A meta-analysis found that CT and TT genotype of the infant increased the risk of NSCLP among Asians.
In humans, any disruption by genetic or environmental factors during the period of 14th–60th day of postconception may predispose to a cleft in the craniofacial region. The gene expression, cell migration, cell transformation, and apoptosis occur in a sequential series during this period. Increased embryonic homocysteine or hyperhomocysteinemia causes apoptosis due to the oxidative stress which may disturb the normal development of the palate. The function of thermolabile MTHFR enzyme is reduced from 30% to70% in CT and TT genotypes, respectively, at 37°C and further lowered to 65% at 46°C or more. This shows that enzyme activity is 30% in TT genotype and 70% in CT genotype. The TT genotype showed lower serum folate and higher homocysteine concentration than the CC genotype and the CT genotype subjects. The maternal homocysteine enters the fetus through the amniotic fluid and induces apoptosis in palatal mesenchyme and prevents the fusion of palatal shelves. Reduced methionine synthesis due to hyperhomocysteinemia may reduce the cell proliferation as methionine is an essential amino acid required for DNA synthesis and cell growth. The study on murine models showed that the silencing of MTHFR gene expression can prevent growth and induce apoptosis in palatal mesenchyme.
The proper folate supplementation during early pregnancy can compensate the decreased enzymatic activity of MTHFR and mask the effect of polymorphism. In such cases, despite the presence of polymorphism, the cleft does not happen. However, all mothers might not be aware of their pregnancy in the early stage. The upper lip formation or fusion of two maxillary swellings with intermaxillary segment is completed by 40th–48th day, and the fusion of palatal shelves is completed by the 60th day of intrauterine life. Hence, the intake of folic acid in the first 60 days of pregnancy has paramount importance. The conditions such as maternal malnutrition, absence of fortification, and no folate intake neither in natural or synthetic form could not mask the effect of C677T polymorphism and may lead to the risk of NSCLP. Different theories were put forward to explain the pathogenesis of craniofacial clefting. The theory of failure of ectodermal fusion states that at the time of fusion, the epithelial cells must disappear in the area of fusion of processes to facilitate the mesoderm to fuse. The absence of the ossification centers and the developmental arrest can cause the cleft. The failure of mesodermal migration, the arrest of neural crest cell migration, and imbalance in the cell formation and apoptosis are the other theories. The premature involution of embryonic arteries is also pointed out as an embryonic change that causes cleft. The stapedial artery derived from the second pharyngeal arch is present only during 33–40 days of fetal development and its absence results in ischemia which may cause facial clefting.
In our study, the other 59 clefts in the craniofacial region had CC and CT genotypes 71.2% and 28.8%, respectively, and it did not show any significant difference with the CT genotype of the control group (18.46%). The TT genotype was not present among the 59 clefts. MTHFR TT genotype was associated with an increased risk of isolated CP in the Ireland population. However, the present study did not show any association between clefts in the craniofacial region other than NSCLP and MTHFR C677T polymorphism. The fusion of palatal shelves occurs initially in the mid-portion and slowly it is continued towards the anterior or primary palate and posterior directions. When the fusion toward the anterior direction is not occurring completely and if the upper lip is not fused, the condition leads to the CL palate. The event of fusion happens only after disrupting the epithelial seam of palatal shelves which lead to the mesenchymal merging of two shelves. In case of CT genotype, the teratogenic effect may be less and it may cause cleft in a small range like CL or CP. However, in TT genotype, the impact may be more and the cleft is extended from the palate to the upper lip could be the reason for the presence of TT genotype in NSCLP cases and the same was not found in other 59 clefts in our study.
A recent study in the Moroccan population revealed a low association between C677T polymorphism and risk of NSCL/P (OR = 0.24; 95% CI; 0.105–0.536). MTHFR C677T polymorphism showed an increased risk of NSCL/P in the Indian population. However, a study by Murthy et al. could not find any association between NSCL/P with MTHFR C677T polymorphism in the South Indian population. The present study revealed that CT genotype reduced the risk and TT genotype increased the risk of NSCLP. According to Tolarova et al., TT genotype is three times more frequent in CL/P patients than in controls. In our study, this frequency was much higher, 18.33% in patients and only 1.54% in controls. Moreover, the TT genotype showed a significant association with increased risk of NSCLP. In NSCLP cases, the CT genotype was very less than the TT genotype and CT genotype was three times lesser when compared to the controls [Table 1]. However, this difference did not contribute to any significant association. This may be due to the fact that the NSCLP case group was not in HWE. In our study, the CC genotype with CT and TT genotypes did not show a significant difference, but the difference was significant in CT genotype with TT genotype [Table 1]. A recent meta-analysis on 22 studies in the overall population showed that MTHFR C677T polymorphism was significantly associated with nonsyndromic orofacial cleft and contributed to the increased risk of NSCL/P.
In our study among the 30 cases of NSCLP who showed polymorphism, 22 were with left laterality and the remaining 8 with a bilateral cleft in lip and palate. No right laterality was found in cases with polymorphism. The presence of polymorphism in the bilateral clefts might be because of the presence of left laterality in those cases. It hints at the association of polymorphism with left laterality. In our study, we could not find any association of polymorphism with laterality. However, interestingly, all cases with polymorphism had left laterality. Further studies should be conducted in a large sample to find out the association between NSCLP and laterality.
Previous studies were dealing with the cleft in palate or CL with or without CP. The present study included various clefts in the craniofacial region and it is grouped into craniofacial clefts, facial clefts, CL palate, CL, and CP. The NSCLP cases were more in our study and its predominance might have caused more study on its etiology in varied populations. To date, very few studies have correlated the laterality with polymorphism despite there was no significance found in our study.
| Conclusion|| |
The MTHFR C677T polymorphism is associated with the risk of NSCLP but not with other clefts in the craniofacial region. The TT genotype increased the risk of NSCLP and CT genotype showed a decrease in the risk. All the cases with polymorphism had left laterality. Further studies should be conducted to identify whether any association exists between the polymorphism and laterality of cleft.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients' parents have given their consent for their images and other clinical information to be reported in the journal. The patients' parents understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Murray JC, Wehby GL, Ferreira M, Felix T, Costa A, Padovani C. Oral cleft prevention programme. BMC Pediatr 2012;12:184.
Greene ND, Dunlevy LE, Copp AJ. Homocysteine is embryotoxic but does not cause neural tube defects in mouse embryos. Anat Embryol (Berl) 2003;206:185-91.
Rai V. Strong association of C677T polymorphism of methylenetetrahydrofolate reductase gene with nosyndromic cleft lip/palate (nsCL/P). Indian J Clin Biochem 2018;33:5-15.
Kang SS, Wong PW, Susmano A, Sora J, Norusis M, Ruggie N. Thermolabile methylenetetrahydrofolate reductase: An inherited risk factor for coronary artery disease. Am J Hum Genet 1991;48:536-45.
Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Rosenberg IH, et al
. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 1996;93:7-9.
Prescott NJ, Winter RM, Malcolm S. Maternal MTHFR genotype contributes to the risk of non-syndromic cleft lip and palate. J Med Genet 2002;39:368-9.
van Rooij IA, Vermeij-Keers C, Kluijtmans LA, Ocké MC, Zielhuis GA, Goorhuis-Brouwer SM, et al
. Does the interaction between maternal folate intake and the methylenetetrahydrofolate reductase polymorphisms affect the risk of cleft lip with or without cleft palate? Am J Epidemiol 2003;157:583-91.
Pezzetti F, Martinelli M, Scapoli L, Carinci F, Palmieri A, Marchesini J, et al
. Maternal MTHFR variant forms increase the risk in offspring of isolated nonsyndromic cleft lip with or without cleft palate. Hum Mutat 2004;24:104-5.
Ali A, Singh SK, Raman R. MTHFR 677TT alone and IRF6 820GG together with MTHFR 677CT, but not MTHFR A1298C, are risks for nonsyndromic cleft lip with or without cleft palate in an Indian population. Genet Test Mol Biomarkers 2009;13:355-60.
Boyles AL, Wilcox AJ, Taylor JA, Meyer K, Fredriksen A, Ueland PM, et al
. Folate and one-carbon metabolism gene polymorphisms and their associations with oral facial clefts. Am J Med Genet A 2008;146A: 440-9.
Xiao WL, Wu M, Shi B. Folic acid rivals methylenetetrahydrofolate reductase (MTHFR) gene-silencing effect on MEPM cell proliferation and apoptosis. Mol Cell Biochem 2006;292:145-54.
Zappacosta B, Romano L, Persichilli S. Genotype prevalence and allele frequencies of 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms in Italian newborns. Laboratory Med 2009;12:732-6.
Estandia-Ortega B, Velázquez-Aragón JA, Alcántara-Ortigoza MA, Reyna-Fabian ME, Villagómez-Martínez S, González-Del Angel A. 5,10-methylenetetrahydrofolate reductase single nucleotide polymorphisms and gene-environment interaction analysis in non-syndromic cleft lip/palate. Eur J Oral Sci 2014;122:109-13.
Ma J, Stampfer MJ, Giovannucci E, Artigas C, Hunter DJ, Fuchs C, et al
. Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer. Cancer Res 1997;57:1098-102.
Murthy J, Gurramkonda VB, Karthik N, Lakkakula BV. MTHFR C677T and A1298C polymorphisms and risk of nonsyndromic orofacial clefts in a south Indian population. Int J Pediatr Otorhinolaryngol 2014;78:339-42.
Zhu J, Ren A, Hao L, Pei L, Liu J, Zhu H, et al
. Variable contribution of the MTHFR C677T polymorphism to non-syndromic cleft lip and palate risk in China. Am J Med Genet A 2006;140:551-7.
Singh K, Singh SK, Sah R, Singh I, Raman R. Mutation C677T in the methylenetetrahydrofolate reductase gene is associated with male infertility in an Indian population. Int J Androl 2005;28:115-9.
Han Y, Pan Y, Du Y, Tong N, Wang M, Zhang Z, et al
. Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and nonsyndromic orofacial clefts susceptibility in a southern Chinese population. DNA Cell Biol 2011;30:1063-8.
Pan Y, Zhang W, Ma J, Du Y, Li D, Cai Q, et al
. Infants' MTHFR polymorphisms and nonsyndromic orofacial clefts susceptibility: A meta-analysis based on 17 case-control studies. Am J Med Genet A 2012;158A: 2162-9.
Sperber GH. Formation of the primary palate. In: Wyszynski DF, editor. Cleft Lip and Palate: From Origin to Treatment. NY: Oxford University Press; 2002. p. 5-13.
Knott L, Hartridge T, Brown NL, Mansell JP, Sandy JR. Homocysteine oxidation and apoptosis: A potential cause of cleft palate.In vitro
Cell Dev Biol Anim 2003;39:98-105.
Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al
. A candidate genetic risk factor for vascular disease: A common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111-3.
Bhaskar LV, Murthy J, Venkatesh Babu G. Polymorphisms in genes involved in folate metabolism and orofacial clefts. Arch Oral Biol 2011;56:723-37.
Braithwaite F, Watson J. A report on three unusual cleft lips. Br J Plast Surg 1949;2:38-49.
Mills JL, Kirke PN, Molloy AM, Burke H, Conley MR, Lee YJ, et al
. Methylenetetrahydrofolate reductase thermolabile variant and oral clefts. Am J Med Genet 1999;86:71-4.
Rafik A, Rachad L, Kone AS, Nadifi S. MTHFR C677T polymorphism and risk of nonsyndromic cleft lip with or without cleft palate in the Moroccan population. Appl Clin Genet 2019;12:51-4.
Tolarova MM, Van der Pul NMJ, Goldberg AC, Hol F. A common mutation in the MTHFR gene as a risk factor for nonsyndromic cleft lip and or palate anomalies. Am J Hum Genet 1998;63:A27.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]