MTHFR rs2274976 Polymorphism Is a Risk Marker for Nonsyndromic Cleft Lip with or without Cleft Palate in the Brazilian Population Sibele Nascimento de Aquino 1 , Ryuichi Hoshi 2 , Elizabete Bagordakis 1 , Maria Giulia Rezende Pucciarelli 1 , Ana Camila Messetti 1 , Helenara Moreira 3 , Andreia Bufalino 1 , Andr  ea Borges 2 , Ana Lucia Rangel 4 , Luciano Abreu Brito 5 , Mario Sergio Oliveira Swerts 6 , Hercilio Martelli-Junior 7 , Sergio R. Line 8 , Edgard Graner 1 , S  ılvia R.A. Reis 2 , Maria Rita Passos-Bueno 5 , and Ricardo D. Coletta* 1 Background: Polymorphisms within the MTHFR (rs2274976) and MTHFD1 (rs2236225) genes were previously associated with maternal susceptibility for having an offspring with nonsyndromic cleft lip with or without cleft palate (NSCL/P) in the Brazilian population. However, as the genotypes of the patients with NSCL/P were not evaluated, it is not clear whether the effects are associated with maternal or offspring genotypes. The aim of this study was to evaluate the association of rs2274976 and rs2236225 in the pathogenesis of NSCL/P. Methods: By using the TaqMan 5 0 -exonuclease allelic discrimination assay, the present study genotyped the rs2274976 and rs2236225 polymorphisms in 147 case–parent trios, 181 isolated samples of NSCL/P and 478 healthy controls of the Brazilian population. Transmission disequilibrium test and structured case–control analysis based on the individual ancestry proportions were performed. Results: The transmission disequilibrium test showed a significant overtransmission of the rs2274976 A allele (p 5 0.004), but no preferential parent-of-origin transmission was detected. The structured case–control analysis supported those findings, revealing that the minor A allele of rs2274976 was significantly more frequent in NSCL/P group compared with control group (p 5 0.001), yielding an odds ratio of 3.46 (95% confidence interval, 2.05–5.85). No association of rs2236225 polymorphism with NSCL/P was observed in both transmission disequilibrium test and case–control analysis. Conclusion: The results of the study revealed that the presence of the rs2274976 A allele is a risk marker for the development of NSCL/P in the Brazilian population. Birth Defects Research (Part A) 100:30–35, 2014. V C 2013 Wiley Periodicals, Inc. Introduction Nonsyndromic cleft lip with or without cleft palate (NSCL/ P) is the most common facial birth defect with lifelong dis- tressing consequences for the patient (Rahimov et al., 2012). The prevalence of oral clefts varies among different populations, and its estimated prevalence is between 0.36 and 1.54 per 1000 live births in Brazil (Martelli-Junior et al., 2007; Rodrigues et al., 2009). The risk factors associated with NSCL/P are not completely understood, but there is a clear interaction between genetic and envi- ronmental factors in the etiology of this complex defect (Dixon et al., 2011). Some studies have shown a signifi- cant association between nutritional deficiency, particu- larly folate deficiency, and elevated risk of oral clefts (Wilcox et al., 2007; Jia et al., 2011; Kelly et al., 2012), whereas other studies have not (Ray et al., 2003; Little et al., 2008). One possible reason for those ambiguous results may reside on genetic variations that influence absorption, transport and metabolism of folate more than levels of maternal intake or availability to the fetus. In support, population-based studies did not show a reduced risk of oral clefts with dietary folate reinforce- ment (Ray et al., 2003; L opez-Camelo et al., 2010; Wehby and Murray, 2010). In a previous study, we demonstrated that specific pol- ymorphic variants in 5,10-methylenetetrahydrofolate reductase (MTHFR) and methylenetetrahydrofolate dehy- drogenase 1 (MTHFD1) genes, which encode enzymes involved in reactions of oxidation and reduction during folate metabolism, increase the maternal risk for having an offspring with NSCL/P (Bufalino et al., 2010). However, one of the limitations of this study was that genotypes of 1 Department of Oral Diagnosis, School of Dentistry, State University of Campi- nas, Piracicaba, S~ ao Paulo, Brazil 2 Escola Bahiana de Medicina e Sa ude P ublica, Dentistry Course, Salvador, Bahia, Brazil 3 Department of Physiotherapy, State University of Western Paran a, Paran a, Brazil 4 Department of Pathology, State University of Western Paran a, Paran a, Brazil 5 Human Genome Research Center, Institute of Biosciences, University of S~ ao Paulo, S~ ao Paulo, Brazil 6 Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of Jos e Ros ario Vellano, Minas, Gerais, Brazil 7 Stomatology Clinic, Dental School, State University of Montes Claros, Montes Claros, Minas Gerais, Brazil 8 Department of Morphology, School of Dentistry, State University of Campinas, Piracicaba, S~ ao Paulo, Brazil *Correspondence to: Ricardo D. Coletta, Department of Oral Diagnosis, School of Dentistry, State University of Campinas, CEP 13414-018, Piracicaba, S~ ao Paulo, Brazil, E-mail: coletta@fop.unicamp.br Published online 19 November 2013 in Wiley Online Library (wileyonlinelibrary. com). Doi: 10.1002/bdra.23199 V C 2013 Wiley Periodicals, Inc.