Common Mutations of the Methylenetetrahydrofolate Reductase (MTHFR) Gene in Non-Syndromic Cleft Lips and Palates Children in North-West of Iran

Document Type : Original


1 Department of Pediatric Otorhinolaryngology, Children’s Hospital, Tabriz University of Medical Sciences, Tabriz, Iran.

2 Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.

3 Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran

4 Tabriz Genetic Analysis Center (TGAC), Tabriz University of Medical Sciences, Tabriz, Iran.

5 Faculty of Medicine, University of Liverpool, Liverpool, United Kingdom.

6 Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.


Cleft lips and cleft palates are common congenital abnormalities in children. Various chromosomal loci have been suggested to be responsible the development of these abnormalities. The present study was carried out to investigate the association between the suspected genes (methylenetetrahydrofolate reductase [MTHFR] A1298C and C677T) that might contribute into the etiology of these disorders through application of molecular methods.
Materials and Methods:
This cross-sectional and explanatory study was carried out on a study population of 65 affected children, 130 respective parents and 50 healthy individuals between 2009 and 2012 at Tabriz University of Medical Sciences, IR Iran. After DNA extraction, amplification refractory mutation system–polymerase chain reaction (ARMS-PCR) and restriction fragment length polymorphism (RFLP)-PCR were used respectively to investigate the C677T and A1298C mutations for the MTHFR gene.
There was a significant difference in the rates of the C677T mutation when affected patients and their fathers were compared with the control group (odds ratio [OR]=0.44) (OR=0.64). However, there was no significant difference observed in the rate of this mutation between the patients’ mothers and the control group (OR=1.35). In addition, the abnormality rate was higher in patients with the A1298C mutation and their parents, when compared with the control group. This abnormality rate was higher for the affected children and their fathers in comparison with their mothers (Fathers, OR=0.26; Mothers, OR=0.65; Children, OR=0.55). No significant difference was seen in the rate of the polymorphism C677T in its CC, when the affected children and their parents were compared with the control group. However, there was a significant difference in the A1298C mutation.
An association was seen between the A1298C mutation and cleft lip and cleft palate abnormalities in Iran. However, there seems to be a stronger relationship between the C67TT mutation and these abnormalities in other countries, which could be explained by racial differences. Moreover, this association was more notable between the affected children and their fathers than their mothers. The findings in this study may be helpful in future studies and screening programs.


Main Subjects

1. Tanabe A, Taketani Sh, Endo-Ichikava Y, Tokunaga R, Ogawa Y, Hiramatos M. Analysis of the candidate genes responsible for non-syndromic cleft lip and palate in Japanese people. Clinical Science 2000; 99(2):105-111.
2. Jones MC. Etiology of facial clefts. Prospective evaluation of 428 patients. Cleft Palate J 1988; 25 (1):16-20.
3. Cummings CW, Haughey BH, Thomas JR, Harker LA, Flint PW. Cummings Otolaryngology: Head and Neck Surgery, 4st ed. Mosby, USA; 2004:2907-95.
4. Johansson B, Ringsberg KC. Parents' experiences of having a child with cleft lip and palate. Journal of Advanced Nursing 2004; 47(2):165-173.
5. Spritz RA. The genetics and epigenetics of orofacial clefts. Curr Opin Pediatr 2001;13(6):556-60.
6. Lowry RB, Trimble BK. Incidence rates for cleft lip and palate in British Columbia 1951-1971 for North American Indian Japanese, Chinese and total populations: Secular trends over twenty years. Teratology 1977;16(3):277-83.
7. Gorlin RY, Cohen MM, Hannekam R. Syndromes of the head and neck, 4st ed. Oxford University Press: Newyork; 2011:413-15.
8. Rajabian MH, Sherkat M. An epidemiologic study of oral clefts in Iran: Analysis of 1669 cases. Cleft Palate Craniofac J 2000; 37(2):191-6.
9. Taher AA. Cleft lip and palate in Tehran. Cleft Palate Craniofac J 1992; 29 (1): 15-6.
10. Wysznsky DF, Beaty TH. Review the role of potential teratogens in the origin of human non-syndromic oral clefts.Teratology 1996;53(5):309-17.
11. Lammer EJ, Chen DT, Hoar RM. Retinoic acid embryopathy. N Engl J Med 1985; 313(14):837-41.
12. Murray JC. Gene/environment causes of cleft lip and/or palate. Clin Genet 2002; 61(4):248-56.
13. Fogh-Andersen P. Inheritance of harelip and cleft palate. Nyt Nordisk Forlag 1942; 10(2):39-45.
14. Marazita ML, Goldstein AM, Smalley SL. Cleft lip with or without cleft palate: reanalyasis of three generation family study in England. Genet Epidemiol 1986; 3(5):335-42.
15. Sozen MA, Tolarova MM, Spritz RA. The common MTHFR C677T and A1298C variants are not associated with the risk of non-syndromic cleft lip/palate in northern Venezuella. J Genet Genomics 2009; 36(5):283-8.
16. Jencks DA, Mathews RG. Allosteric inhibition of Methylenetetrahydrofolate reductase by adenosylme- thionine. Effects of adenosylmethionine and NADPH on the equilibrium between active and inactive forms of the enzyme and on the kinetics of approach to equilibrium. J Biol Chem 1987; 262 (6): 2485-93.
17. Goyette P, Summer JS, Milos R, Duncan AM, Rosenblatt DS, Matthews RG, et al. Human methylenetetra- hydrofolate reductase: Isolation of cDNA, Maping and mutation identification. Nat Genet 1994; 7(2):195-200.
18. 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 methy- lenetetrahydrofolate reductase. Nat Genet 1995; 510(14):111-13.
19. Blanton SH, Kolle BS, Hecht JT, Mulliken JB, Martin ER. No evidence supporting MTHFR as a risk factor in the development of familial NSCLP. Am J Med Genet 2000; 92(5): 370-1.
20. Shaw GM, Rozen R, Finnell RH, Todorof K, Lammer EJ. Infant C677T mutation in MTHFR gene, maternal periconceptional vitamin use, and cleft lip. Am J Med Genet 1998;80(3):196-8.
21. Martinelli M, Scapoli L, Pezzeti F, Carinci F, Carinci P, Baciliero U, et al. Suggestive linkage between markers on chromosome 19q13.2 and nonsyndromic orofacial cleft information. Genomics
1998; 51(2):177-81.
22. Brandalize AP, Bandinelli E, Borba JB, Felix TM, Roisenberg I, Schuler Faccini I. Polymorphisms in genes MTHFR, MTR and MTRR are not risk factors for cleft lip/palate in South Brazil. Braz J Med Biol Res 2007; 40(6):787-91.
23. Van Rooij IA, Vermeij-Keers C, Kluijtmans LA, Ocke 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 (7): 583-91.
24. Verkleij-Hagoort A, Bliek J, Sayed-Tabatabaei F, Ursen N, Steegers E, Steegers-Theunissen R. Hyperhomocysteinemia and MTHFR polymer- phisms in association with orofacial clefts and congenital heart defects: a meta-analysis. Am J Med Genet A 2007; 143A (9):952-60.
25. Talarova MM, Cervenka J. Classification and birth prevalence of orofacial clefts. Amer J Med Genet 1998; 75(2):126-37.
26. Mills JL, Kirke PN, Molloy AM, Burke H, Conley MR, Lee YJ, et al. Methyenetetrahydrofolate reductase thermolabile variant and oral clefts. Am J Med Genet 1999; 86(1):71-4.
27. Pezzetti F, Martinelli M, Scapoli L, Carinci F, Palmieri A, Marchesini J. Maternal MTHFR variant forms increase the risk in offspring of isolated nonsyndromic cleft lip with or without cleft palate. Hum Mutat 2004; 24(1):104-105.
28. ShotelersukV, Ittiwut EJ, Siriwan P, Angspatt A. Maternal 677CT/1298AC genotype of the MTHFR gene as a risk factor for cleft lip. J Med Genet2003; 40(5):e64.
29. Prescott NJ, Winter RM, Malcolm S. Maternal MTHFR genotypecontributes to the risk of non-syndromic cleft lip and palate. J Med Genet 2002; 39(5):368-9.
30. Jugessur A, Wilcox AJ, Lie RT, Murray JC, Taylor JA, Ulvik A, et al. Exploring the effects of methyenetetrahydrofolate reductase gene variants
C677T and A1298C on the risk of orofacial clefts in 261 Norwegian case-parent triads. Am J Epidemiol 2003; 157(12):1083-91.
31. Blanton SH, Patel S, Hecht JT, Mulliken JB. MTHFR is not a risk factor in the development of isolated nonsyndromic cleft lip and palate. Am J Med Genet 2002; 110(4):404-5.
32. Van der put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK, et al. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects?. Am J Med Genet 1998;62(5):1044-51.
33. Friso S, Girelli D, Trabetti E, Stranieri C, Olivieri O, Tinnazi E, et al. A1298C methylenetetrahydrofolate reductase mutation and coronary artery disease: relationships with C677T polymorphism and homocystein/folate metabolism. Clin Exp Med 2002; 2(1):7-12.