Parental cigarette smoking, transforming growth factor-alpha gene variant and the risk of orofacial cleft in Iranian infants

Document Type: Original Article


1 Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Dental Carries Prevention Research Center, Qazvin University of Medical Sciences, Qazvin, Iran

3 Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran

4 Department of Public Health, School of Public Health, Zanjan University of Medical Sciences, Zanjan, Iran

5 Bachelors of Medical Laboratory Sciences, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran


Objective(s):We investigated the influence of genetic variation of the transforming growth-factor alpha (TGFA) locus on the relationship between smoking and oral clefts.
Materials and methods:In this study 105 Iranian infants with non-syndromic cleft lip/palate and 218 controls with non-cleft birth defects were examined to test for associations among maternal exposures, genetic markers, and oral clefts. Maternal and parental smoking histories during pregnancy were obtained through questionnaire. DNA was extracted from newborn screening blood samples, and genotyping of the BamHI polymorphism in the TGFA gene was performed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods.
A number of factors including gender of the newborns, type of oral cleft, consanguinity of the parents, as well as the mother’s age and education were evaluated as potential confounders and effect modifiers.
Results: Maternal smoking, in the absence of paternal smoking, was associated with an increased risk for CL/P (OR = 19.2, 95% CI = [(6.2-59.5)])   and cleft palate only (OR =48.7, 95% CI = [(8-29.3)]). If both parents smoked, risks were generally greater (OR = 55.6, 95% CI = [12-20.25]). Analyses for the risk of clefting from maternal smoking, stratified by the presence or absence of the TGFA/BamH1variant, revealed that the risk of clefting among the infants with the TGFA/BamH1 variant when their mothers smoked cigarettes was much greater than the infants who had non-smoker mothers (P=0.001, OR=10.4,95% CI=[3.2,33.6]).
Conclusion:The results of this study indicate that first-trimester maternal smoking and infant TGFA locus mutations are both associated with nonsyndromic cleft lip and/or palate (CL/P).


1.Ranganathan K, Vercler CJ, Warschausky SA, MacEachern MP, Buchman SR, Waljee JF. Comparative effectiveness studies examining patient-reported outcomes among children with cleft lip and/or palate: a systematic review. Plast Reconstr Surg 2015; 1351:198-211.

2.Crockett DJ, Goudy SL. Cleft lip and palate. Facial Plast Surg Clin North Am 2014; 224:573-586.

3.Aldhorae KA, Böhmer AC, Ludwig KU, Esmail AH, Al-Hebshi NN, Lippke B, et al. Nonsyndromic cleft lip with or without cleft palate in arab populations: genetic analysis of 15 risk loci in a novel case-control sample recruited in Yemen. Birth Defects Res A Clin Mol Teratol 2014; 1004:307-713.

4.Rajabian MH, Sherkat M. An epidemiologic study of oral clefts in Iran: analysis of 1669 cases. Cleft Palate Craniofac J 2000; 372:191-196.

5.Vanderas AP. Incidence of cleft lip, cleft palate, and cleft lip and palate among races: a review. Cleft Palate J 1987; 24:216-25.

6.Czeizel AE, Toth M, Rockenbauer M. Population-based case control study of folic acid supplementation during pregnancy. Teratology 1996; 53:345-51.

7.Itikala PR, Watkins ML, Mulinare J, Moore CA, Liu Y. Maternal multivitamin use and orofacial clefts in offspring. Teratology 2001; 63:79-86.

8.Murray JC: Gene–environment causes of cleft lip and/or palate.Clin Genet 2002; 61:248-256.

9.Bille C, Olsen J, Vach W,  Knudsen VK, Olsen SF, Rasmussen K, et al . Oral clefts and life style factors. A case-cohort study based on prospective Danish data. Eur J Epidemiol 2007; 22: 173-181.

10.VanRooij  IALM, Wegerif MJ, Roelofs HM. Smoking, genetic polymorphisms in biotransformation enzymes, and nonsyndromic oral clefting: a gene–environment interaction. Epidemiology 2001; 12:502 507.

11.Druckery H, Ivankovic S, Preussman R. Teratogenic and carcinogenic effects in the offspring after single injection of ethylnitrosuria to pregnant rats. Nature 1966; 210:1378-1379.

12. Zeng N, Wu J, Zhu WC, Shi B, Jia ZL. Evaluation of the association of polymorphisms in EYA1, environmental factors, and non-syndromic orofacial clefts in Western Han Chinese. J Oral Pathol Med. 2015; 44:864-9

13.Botto LD, Khoury MJ. Facing the challenge  of gene-environment interaction: the two-by-four table and beyond. Am J Epidemiol. 2001;153:1016–1020.

14.Croen LA, Shaw GM, Wasserman CR, Tolarová MM. Racial and ethnic variations in the prevalence of orofacial clefts in California. Am J Med Genet 1998; 79: 42-7.

15.Gundlach KK, Maus C. Epidemiological studies on the frequency of clefts in Europe and world-wide. J CraniomaxillofacSurg 2006; 34 Suppl 2:1-2.

16.Jamilian A, Nayeri F, Babayan A. Incidence of cleft lip and palate in Tehran. J Indian Soc Pedod Prev Dent 2007; 25: 174-176.

17.Taher AA. Cleft lip and palate in Tehran. Cleft Palate Craniofac J 1992; 29: 15-16.

Soltani MK, Mohammadi Z, Nasab AZ, Golfeshan F. The incidence of cleft lip and palate in a Kurd population: a prospective study. Community Dent Health 2014; 31: 50-52.

18.Yaqoob M, Mahmood F, Hanif G, Bugvi SM, Sheikh MA. Etiology and genetic factors in clefts of lip and/or palate reported at children's hospital, Lahore, Pakistan. Indian J Hum Genet 2013; 19:136-143.

19.Croen LA, Shaw GM, Wasserman CR, Tolarová MM. Racial and ethnic variations in the prevalence of orofacial clefts in California, 1983-1992. Am J Med Genet 1998; 79: 42-47.

20.Shaw GM, Wasserman CR, Lammer EJ, O'Malley CD, Murray JC, Basart AM, et al. Orofacial clefts, parental cigarette smoking, and transforming growth factor-alpha gene variants. Am J Hum Genet 1996; 58:551–61.

21.Philipp K, Pateisky N, Endler M. Effects of smoking on uteroplacental blood flow. Gynecol Obstet Invest 1984;17:179–182.

22.Romitti PA, Lidral AC, Munger RG. Candidate genes for nonsyndromic cleft lip and palate and maternal cigarette smoking and alcohol consumption: evaluation of genotype-environment interactions from a population-based case-control study of orofacial clefts. Teratology 1999; 59:39–50.

23.Pollack H, Lantz PM, Frohna JG. Maternal smoking and adverse birth outcomes among singletons and twins. Am J Public Health. 2000;90:395–400.

24.Lieff S, Olshan AF, Werler M, Strauss RP, Smith J, Mitchell A. Maternal cigarette smoking during pregnancy and risk of oral clefts in newborns. Am J Epidemiol 1999;150:683–694.

25.Aldhorae KA, Böhmer AC, Ludwig KU, Esmail AH, AlHebshi NN, Lippke B, et al. Nonsyndromic cleft lip with or without cleft palate in arab populations: genetic analysis of 15 risk loci in a novel case-control sample recruited in  Yemen. Birth Defects Res A Clin Mol Teratol 2014;100:307-313

26.Tricoli, JV, Nakai H, Byers MG. The gene for human transforming growth factor alpha is on the short arm of chromosome 2. Cytogenet Cell Genet 1986;42:94–98.

27.Vieira AR. Association between the Transforming Growth Factor Alpha Gene and Nonsyndromic Oral Clefts: A HuGE Review. American Journal of Epidemiology 2006 ;163: 790-810.

28.Ardinger HH, Buetow KH, Bell GI. Association of genetic variation of the transforming growth factor-alpha gene with cleft lip and palate. Am J Hum Genet 1989;45:348–53.

29.Holder SE, Vintiner GM, Farren B. Confirmation of an association between RFLPs at the transforming growth factor-alpha locus and non-syndromic cleft lip and palate. J Med Genet 1992; 29:390–392.

30.Christensen K, Olsen J, Nørgaard-Pedersen B. Oral clefts, transforming growth factor alpha gene variants, and maternal smoking: a population-based case-control study in Denmark. Am J Epidemiol 1999; 149:248–255.

31.Beaty TH, Maestri NE, HetmanskiJB .Testing for interaction between maternal smoking and TGFA genotype among oral cleft cases born in Maryland. Cleft Palate-Craniof J 1997; 34:447–454.

32.Hwang SJ, Beaty TH, Panny SR. Association study of transforming growth factor alpha (TGFa) TaqI polymorphism and oral clefts: indication of gene-environment interactionina population-based sample of infants with birth defects. Am J Epidemiol 1995; 141:629–636.

33.Wyszynski DF, Duffy DL, Beaty TH. Maternal cigarette smoking and oral clefts: a meta-analysis. Cleft Palate Craniofac J. 1997; 34:206–210.

34.Ebadifar A, Hamedi R, Khorram Khorshid HR, Saliminejad K,Kamali K, Moghadam F, et al. Association of Transforming Growth Factor Alpha polymorphisms with nonsyndromic Cleft Lip and Palate in Iranian Population. Avicenna J Med Biotech 2015; 7:168-172.

35.Kallen K. Maternal smoking and orofacial clefts. Cleft Palate Craniofac J.1997;34:11–16.

36.SaxenI. Cleft lip and palate in Finland: parental histories,course of pregnancy and selected environmental factors.Int J Epidemiol 1974;3:263-270.

37.Evans D, Newcombe R. Campbell H. Maternal smoking habits and congenital malformations: a population study. Br Med J 1979 ; 2:171-173.

38.Hemminki K, Mutanen P. Saloniemi I. Smoking and the occurrence of congenital malformations and spontaneous abortions: multivariate analyses. Am J Obstet Gynecol1983;145:61-66.

39.Shiono PH, Klebanoff MA, Berendes HW. Congenital malformations and maternal smoking during pregnancy. Teratology 1986; 34:65-71.

40.Sabbagh HJ, Hassan MH, Innes NP, Elkodary HM, Little J, Mossey PA. Pssive smoking in the etiology of non-syndromic orofacial clefts: a systematic review and meta-analysis. PLoS One 2015;10:1-21.

41.Givelbar HM, DiPaoloJ A. Teratogenic effects of NEthyl-N-nitrosourea in the Syrian hamster. Cancer Res 1969;29:1151-1155.

42.Lambert GH, Nebert DW .genetically mediated induction of drug-metabolizing enzymes associated with congenital defects in the mouse. Teratology 1977 ;16:147-153.

43.Kallen K. The impact of maternal smoking during pregnancy on delivery outcome. Eur J Public Health. 2001;11:329–333.

44.Mortensen JT, Thulstrup AM, Larsen H, Moller M, Sorensen HT. Smoking, sex of the offspring, and risk of placental abruption, placenta previa, and preeclampsia:a population-based cohort study. Acta Obstet Gynecol Scand. 2001;80:894–898.

45.Millicovsky G, JohnstonM C. Hyperoxia and hypoxia in pregnancy: simple experimental manipulation alters the incidence of cleft lip and palate in CL/Frmice. Proc Natl Acad Sci U S A. 1981; 78:5722–5723.

46.Bronsky PT, Johnston MC, Sulik KK Morphogenesis of hypoxia-induced cleft lip in CUFR mice. J Craniofac Genet Dev Biol 1986; 2:113-128.

47.Witter FR, Blake DA, Baumgardner R, Mellits ED, Niebyl JR (1982) Folate, carotene, and smoking. Am J Obstet Gynecol 144:857.

48.Ebadifar A, KhorramKhorshid HR, Kamali  K, SalehiZeinabadi M, Khoshbakht T, Ameli N. Maternal Supplementary Folate Intake, Methyle netetrahydrofolate Reductase (MTHFR) C677T and A1298C Polymorphisms and the Risk of Orofacial Cleft in Iranian Children. Avicenna J Med Biotechnol. 2015; 7:80-84.

49.Elahi MM, Jackson IT, Elahi O, Khan AH, Mubarak F, Tariq GB, et al. Epidemiology of cleft lip and cleft palate in Pakistan. Plast Reconstr Surg 2004; 113: 1548-1555.

50.Bellis TH, B. Wohlgemuth B. The Incidence of Cleft Lip and Palate Deformities in the South-east of Scotland . Journal of Orthodontics  1999; 26: 121-125.

51.Gregg T,  Boyd D,  Richardson A. The incidence of cleft lip and palate in Northern Ireland from 1980-1990. Orthodontics 1994; 21:387-392.

52.Herkrath AP, Herkrath FJ, Rebelo MA, Vettore MV.  Parental age as a risk factor for non-syndromic oral clefts: a meta-analysis. J Dent 2012; 40:3-14.

53.Jagomagi T, Soots M, Saag M. Epidemiologic factors causing cleft lip and palate and their regularities of occurrence in Estonia. Stomato-logija 2010; 12: 105-108.

54.Abramowicz S, Cooper ME, Bardi K, Weyant RJ, Marazita ML. Demographic and prenatal factors of patients with cleft lip and cleft palate: A pilot study. J Am Dent Assoc 2003 134: 1371-1376.

55.Fathololumi MR, FattahiBafghi A, Nuhi S, NasiriAfshar AA, AghazadehNaieeni A. Prevalence of cleft palate and cleft lip among 20000 Iranian neonates. Pejouhandeh 2007; 12: 31-4.

56.Sadri D, Ahmadi N.The frequency of cleft lip and palate and the related risk factors in a group of neonates in the city of Kerman during 1994-2002. J Mashhad Dental School 2007; 31: 71-76.

57.Reddy SG, Reddy RR, Bronkhorst EM, Prasad R, Ettema AM, Sailer HF, et al. Incidence of cleft lip and palate in the state of Andhra Pradesh, South India. Indian J PlastSurg 2010; 43: 184-189.

58.Lebby KD, Tan F, Brown CP. Maternal factors and disparities associated with oral clefts. Ethn Dis 2010; 20(Suppl 1): 146-149.