The Cleft Palate–Craniofacial Journal 00(00) pp. 000–000 Month 2016 Ó Copyright 2016 American Cleft Palate–Craniofacial Association
ORIGINAL ARTICLE Association Between Genes Involved in Craniofacial Development and Nonsyndromic Cleft Lip and/or Palate in the Brazilian Population Renato Assis Machado, D.D.S., M.S., Ana Camila Messetti, D.D.S., M.S., Sibele Nascimento de Aquino, D.D.S., ´ ´ M.S., PhD., Herc´ılio Martelli-Junior, D.D.S., M.S., Ph.D., Ma´rio Sergio Oliveira Swerts, D.D.S., M.S., Ph.D., Silvia Regina de Almeida Reis, D.D.S., M.S., Ph.D., Helenara Salvati Bertolossi Moreira, Ph.D., Darlene Camati Persuhn, B.S., M.S., Ph.D., Ricardo D. Coletta, D.D.S., M.S., Ph.D. Objective: To determine the association of single-nucleotide polymorphisms (SNPs) in genes related to craniofacial development, which were previously identified as susceptibility signals for nonsyndromic oral clefts, in Brazilians with nonsyndromic cleft lip and/or palate (NSCL/P). Design: The SNPs rs748044 (TNP1), rs1106514 (MSX1), rs28372960, rs15251 and rs2569062 (TCOF1), rs7829058 (FGFR1), rs1793949 (COL2A1), rs11653738 (WNT3), and rs242082 (TIMP3) were assessed in a family-based transmission disequilibrium test (TDT) and a structured casecontrol analysis based on the individual ancestry proportions. Setting: The SNPs were initially analyzed by TDT, and polymorphisms showing a trend toward excess transmission were subsequently studied in an independent case-control sample. Participants: The study sample consisted of 189 case-parent trios of nonsyndromic cleft lip with or without cleft palate (NSCL6P), 107 case-parent trios of nonsyndromic cleft palate (NSCP), 318 isolated samples of NSCL6P, 189 isolated samples of NSCP, and 599 healthy controls. Main Outcome Measure: Association of alleles with NSCL/P pathogenesis. Results: Preferential transmission of SNPs rs28372960 and rs7829058 in NSCL6P trios and rs11653738 in NSCP trios (P ¼ .04) were observed, although the structured case-control analysis did not confirm these associations. The haplotype T-C-C formed by TCOF1 SNPs rs28372960, rs15251, and rs2569062 was more frequently transmitted from healthy parents to NSCL6P offspring, but the P value (P ¼ .01) did not withstand Bonferroni correction for multiple tests. Conclusions: With the modest associations, our results do not support the hypothesis that TNP1, MSX1, TCOF1, FGFR1, COL2A1, WNT3, and TIMP3 variants are risk factors for nonsyndromic oral clefts in the Brazilian population. KEY WORDS:
COL2A1, craniofacial development, FGFR1, MSX1, nonsyndromic cleft lip and/or palate, TCOF1, TIMP3, TNP1, WNT3
Nonsyndromic cleft lip and/or palate (NSCL/P) is the most common craniofacial birth defect in humans and shows a prevalence of approximately 1:700 live births worldwide, but considerable geographic and ethnic variation is reported (Dixon et al., 2011). In Brazil, prevalence ranges from 0.36 to 1.54 per 1000 live births (Martelli-Junior et al., 2007; Rodrigues et al., 2009). The etiology of NSCL/P, which involves both genetic and environmental factors, is highly complex, and the molecular basis remains largely unknown (Wehby and Murray, 2010). Although genetic studies have identified a number of candidate genes and chromosomal regions associated with NSCL/P (Zucchero et al., 2004; Birnbaum et al., 2009; Grant et al., 2009; Beaty et al.,
Mr. Machado and Mrs. Messetti are Ph.D. students, Department of Oral Diagnosis, School of Dentistry, University of Campinas, Piracicaba, Sao ˜ Paulo, Brazil. Dr. de Aquino is Assistant Professor, School of Dentistry, Federal University of Juiz de Fora, Governador Valadares, Minas Gerais, Brazil. Dr. Martelli-Junior ´ is Professor, Stomatology Clinic, Dental School, State University of Montes Claros, Montes Claros, Minas Gerais, Brazil, and Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of Jose´ Rosario Vellano, Alfenas, Minas Gerais, Brazil. Dr. Swerts is Assistant Professor, Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of Jose´ Rosario Vellano, Alfenas, Minas Gerais, Brazil. Dr. Reis is Professor, Department of Basic Science, Bahiana School of Medicine and Public Health, Salvador, Bahia, Brazil. Dr. Moreira is Assistant Professor, Department of Physiotherapy, State University of Western Parana´, Parana´, Brazil. Dr. Persuhn is Assistant Professor, Molecular Biology Department, Federal University of Para ´ıba, Joao ˜ Pessoa, Para ´ıba, Brazil. Dr. Coletta is Associate Professor, Department of Oral Diagnosis, School of Dentistry, University of Campinas, Piracicaba, Sao ˜ Paulo, Brazil. This work was supported by grants from Procad/Casadinho-CNPq/ CAPES, Bras ´ılia, Brazil; the State of Sao ˜ Paulo Research FoundationFAPESP, Sao ˜ Paulo, Brazil; and the State of Minas Gerais Research Foundation-FAPEMIG, Minas Gerais, Brazil. Submitted March 2015; Revised July 2015; Accepted July 2015.
Address correspondence to: Mr. Renato Assis Machado, Department of Oral Diagnosis, School of Dentistry, University of Campinas, CEP 13414-018, Piracicaba, Sao ˜ Paulo, Brazil. E-mail renatoassismachado@ yahoo.com.br. DOI: 10.1597/15-107 0
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2010; Mangold et al., 2010; Ludwig et al., 2012), few of them have been replicated in different populations. Previous studies with those potential susceptibility markers in the Brazilian population have confirmed some associations and failed to confirm others, most likely indicative of the varied genomic ethnic background in the population of Brazil (Parana ´ıba et al., 2010; Brito, Bassi, et al., 2012; Brito, Paranaiba, et al., 2012; Bagordakis et al., 2013; De Aquino et al., 2013; De Aquino et al., 2014). One strategy that has helped in the identification of genetic alterations associated with NSCL/P is the evaluation of single-nucleotide polymorphisms (SNPs) in genes related to craniofacial development. The craniofacial development involves a series of highly coordinated events, including proliferation, migration, epithelial-mesenchymal transition, and apoptosis, and polymorphic variations in genes that control these events may affect the normal development of the lips and palate, resulting in NSCL/P (Butali et al., 2011). Previous studies have shown statistical evidences of association between specific genes, including transition nuclear protein 1 (TNP1; Beaty et al., 2006), muscle segment homeobox 1 (MSX1; Beaty et al., 2002; Vieira et al., 2003; Suazo et al., 2004; Jagomagi et al., 2010; Nikopensius et al., 2010; Butali et al., 2011; Cardoso et al., 2013), Treacher Collins-Franceschetti syndrome 1 (TCOF1; Masotti et al., 2005; Sull et al., 2008), fibroblast growth factor receptor 1 (FGFR1; Menezes et al., 2008; Nikopensius et al., 2010; Lace et al., 2011), collagen type II, alpha 1 (COL2A1; Nikopensius et al., 2010), wingless-type MMTV integration site family, member 3 (WNT3; Chiquet et al., 2008; Nikopensius et al., 2010; Lace et al., 2011; Mostowska et al., 2012), tissue inhibitor of metallopeptidase 3 (TIMP3; Nikopensius et al., 2010), and nonsyndromic oral clefts across different populations. However, the involvement of these genes in the Brazilian population remains uncertain. The Brazilian population shows highly variable genomic ancestry (Pena et al., 2011), due to five centuries of genetic admixture between Amerindians, Europeans, and sub-Saharan Africans, which affects specific genetic associations of NSCL/P loci identified in other populations (Bagordakis et al., 2013; De Aquino et al., 2014). The aim of the present study was to investigate potential associations of markers that were previously identified using candidate-gene approaches in the Brazilian population. These NSCL/P risk markers were represented by the following SNPs: rs748044 in TNP1 (Beaty et al., 2006); rs1106514 in MSX1 (Nikopensius et al., 2010); rs28372960, rs15251, and rs2569062 in TCOF1 (Masotti et al., 2005; Sull et al., 2008); rs7829058 in FGFR1; rs1793949 in COL2A1; rs11653738 in WNT3; and rs242082 in TIMP3 (Nikopensius et al., 2010). The SNP rs28372960 in TCOF1 was not previously investigated in NSCL/P, but its functional effects, decreasing transcriptional activity of the gene (Masotti et al., 2005), suggest a causal role in NSCL/P.
MATERIALS
AND
METHODS
Sample This study included patients with nonsyndromic oral clefts and healthy controls from four Brazilian states: Minas Gerais state (Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of Jose´ Rosa´rio Vellano, Alfenas-MG), located in the southeastern region of Brazil; Parana´ state (Association of Carrier of Lip and Palate Cleft–APOFILAB, Cascavel-PR), in the southern region; and Para ´ıba state (University Hospital of Lauro Wanderley–HULW, Joao ˜ Pessoa-PB) and Bahia state (Santo Antonio Hospital, Salvador-BA), which are located in the northeast region. All patients were diagnosed independently and screened for the presence of associated anomalies or syndromes by the team of specialists from each center, and only patients with the nonsyndromic form of oral clefts were included. The nonsyndromic oral clefts were classified with the incisive foramen as reference. For transmission disequilibrium test (TDT) analysis, 296 trios composed of one affected offspring and two healthy parents (189 with nonsyndromic cleft lip with or without cleft palate [NSCL6P] and 107 with nonsyndromic cleft palate only [NSCP]) were included. In the TDT analysis, we searched for the unbalanced transfer of a specific allele from a nonaffected healthy parent to the affected child, and the polymorphic markers that showed a preferential transmission with a P value ,.05 were selected for the case-control study with correction for ancestry differences. For the casecontrol analysis, 507 patients affected with NSCL/P (318 NSCL6P and 189 NSCP) and 599 healthy individuals without physical or psychiatric diseases, history of congenital malformations, or familial history of orofacial clefts consented to participate in this study. The control group consisted of samples matched for gender and geographical location, and no statistically significant differences between groups were observed. There was no overlap between the samples used in the TDT and the case-control study. The study was approved by the ethics review board of each of the centers or hospitals affiliated with the collaborative study. Written informed consent was obtained from the parents or guardians and/or the participants. Genotyping and Estimation of the Genomic Ancestry The genomic DNA was isolated from buccal mucosa cells obtained by mouthwash with a 3% sucrose solution, using a salting-out protocol previously described (Aidar and Line, 2007). Polymerase chain reaction (PCR)–based genotyping of rs748044, rs1106514, rs28372960, rs15251, rs2569062, rs7829058,
Machado et al., GENES RELATED TO DEVELOPMENT IN NONSYNDROMIC ORAL CLEFTS
TABLE 1
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Characteristics of the Single-Nucleotide Polymorphisms (SNPs)*
SNP
Gene
Chromosome
Position
Alleles
Original Study
rs748044 rs1106514 rs28372960 rs15251 rs2569062 rs7829058 rs1793949 rs11653738 rs242082
TNP1 MSX1 TCOF1 TCOF1 TCOF1 FGFR1 COL2A1 WNT3 TIMP3
2 4 5 5 5 8 12 17 22
217,289,319 4,874,199 150,357,401 150,396,669 150,398,801 38,474,577 47,977,812 46,809,587 32,828,453
C/T G/C C/T C/T C/G G/C C/T T/C G/A
Beaty et al., 2006 Nikopensius et al., 2010 Masotti et al., 2005 Sull et al., 2008 Sull et al., 2008 Nikopensius et al., 2010 Nikopensius et al., 2010 Nikopensius et al., 2010 Nikopensius et al., 2010
* Risk alleles appear in bold.
rs1793949, rs11653738, and rs242082 (Table 1) was performed on the StepOnePlus Real-Time PCR platform (Applied Biosystems, Foster City, CA) using TaqMan 5 0 -exonuclease allelic discrimination assays (assays-on-demand, Applied Biosystems). For quality control purposes, reactions were randomly repeated in 10% of the samples for each SNP, and the concordance rate was 100%. All samples were successfully genotyped, with a genotype call rate of 100%. To determine the genomic ancestry of each individual, samples were genotyped for a set of 40 biallelic short insertion/deletion polymorphisms previously validated as ancestry informative markers of the Brazilian population (Bastos-Rodrigues et al., 2006). Statistical Analysis
Collection) were incorporated to assist the software in the ancestry estimation. Following ancestry assessment, STRAT was used to test the association based on the individual ancestry proportions (Pritchard, Stephens, Rosenberg, et al., 2000). Deviation from Hardy-Weinberg equilibrium was assessed by a chi-square test. The genotypes were analyzed under unrestricted, dominant, and recessive genetic models. The odds ratio associated with the 95% confidence interval was also calculated. A Bonferroni-adjusted P value threshold of a .0056 (.05/9) was considered statistically significant. Power for detecting a P value ,.05 for SNPs analyzed in the casecontrol approach was calculated using the Genetic Power Calculator (Purcell et al., 2003), assuming a prevalence of NSCL/P in Brazil of 0.00146 (MartelliJunior et al., 2007) and using the most conservative odds ratios reported in the original studies.
The TDT was performed with the aid of the Family Based Association Test (FBAT) software (Laird et al., 2000). The haplotype-based analysis was conducted using the HBAT function of the FBAT software. To determine the genomic ancestry of each individual in the case-control study, Structure software version 2.3.4 (Pritchard, Stephens, and Donnelly, 2000) was used in a model assuming K¼3 parental populations based on the tri-hybrid origin of the Brazilian population. Samples with a prespecified population of origin (Amerindian, European, and sub-Saharan African reference populations from the Marshfield Clinic
In the initial screening, we identified a preferential transmission of the rs28372960 T allele (P ¼ .04) and of the rs7829058 C allele (P ¼ .04) from healthy parents to NSCL6P offspring (Table 2). Similarly, the rs11653738 C allele showed association with NSCP offspring (P ¼ .04; Table 3). Although in a low frequency, the haplotype T-CC of TCOF1, composed of the T allele of rs28372960, C allele of rs15251, and C allele of rs2569062, was found to be more frequently transmitted to NSCL6P offspring (P ¼
TABLE 2 Transmission Disequilibrium Test of the SingleNucleotide Polymorphisms (SNPs) in Nonsyndromic Cleft Lip With or Without Cleft Palate Trios*
TABLE 3 Transmission Disequilibrium Test of the SingleNucleotide Polymorphisms (SNPs) in Trios With Nonsyndromic Cleft Palate*
RESULTS
SNP
MAF
Number of Families
T/NT
Z Score
P Value
SNP
MAF
Number of Families
T/NT
Z Score
P Value
rs748044 rs1106514 rs28372960 rs15251 rs2569062 rs7829058 rs1793949 rs11653738 rs242082
0.275 0.447 0.054 0.263 0.461 0.146 0.388 0.271 0.353
116 133 30 122 129 83 134 111 135
77/39 90/43 20/10 72/50 83/46 42/41 111/23 64/47 79/56
0.00 1.49 1.97 0.16 1.14 2.00 0.53 1.71 0.82
1.00 .13 .04 .87 .25 .04 .59 .08 .41
rs748044 rs1106514 rs28372960 rs15251 rs2569062 rs7829058 rs1793949 rs11653738 rs242082
0.254 0.469 0.045 0.250 0.445 0.123 0.385 0.232 0.349
63 82 15 62 79 41 80 64 83
35/28 57/25 6/9 36/26 56/23 19/22 55/25 34/30 52/31
1.42 0.28 0.50 0.87 0.38 0.30 0.19 2.04 0.28
0.15 0.77 0.61 0.38 0.69 0.76 0.84 0.04 0.77
* MAF ¼ minor allele frequency; T/NT ¼ transmission/nontransmission counts. P value .0056 is required after applying multiple testing corrections.
* MAF ¼ minor allele frequency; T/NT ¼ transmission/nontransmission counts. P value .0056 is required after applying multiple testing corrections.
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TABLE 4 Haplotype Family-Based Association Test for TCOF1 rs28372960, rs15251, and rs2569062 Performed Using HBAT
Haplotype* NSCL6P C-C-G C-C-C C-T-C T-C-C C-T-G NSCP C-C-G C-C-C C-T-C T-C-C C-T-G
Frequency
0.451 0.249 0.238 0.037 0.015 0.429 0.272 0.245 0.045 0.008
Number of Families
117 114 105 24 23 72 66 59 14 14
Z Score
0.34 0.46 0.29 2.41 1.42 0.38 0.78 1.09 0.73 0.60
TABLE 5 Average Proportions of the European, African, and Amerindian Ancestry of the Patients in Control and Case Groups* European
African
Amerindian
85.6% 83.3% 78.9%
12.4% 15.1% 18.7%
2.0% 1.6% 2.4%
P Value
.73 .64 .77 .01 .15 .70 .43 .27 .46 .54
* Sequence: rs28372960, rs15251, and rs2569062; NSCL6P ¼ nonsyndromic cleft lip with or without cleft palate; NSCP ¼ nonsyndromic cleft palate. P value .0056 is required after applying multiple testing corrections.
.01; Table 4). However, no association retained statistical significance after adjusting for multiple comparisons. Based on the results that revealed a possible association between NSCL6P and rs28372960 and rs7829058 and between NSCP and rs11653738, we performed the casecontrol analysis structured by genomic ancestry to confirm the possible associations. The European ancestry contribution was the most predominant in the three groups, followed by African and Amerindian (Table 5). There were no statistically significant differences in the ancestry proportions between groups. Frequencies of the alleles and genotypes of rs28372960, rs7829058, and rs11653738 structured by genomic ancestry are presented in Table 6. The genotype frequencies observed for all studied polymorphisms in controls did not reveal statistically significant differences compared with those expected under HardyWeinberg equilibrium. None of the polymorphisms tested showed association with NSCL6P or NSCP in this Brazilian case-control cohort. Further analyses in the dominant and recessive genetic models also revealed no differences in the distribution (data not shown). Power analysis showed good statistical power to detect any association with the current case-control sample size for the polymorphisms rs7829058 (92%) and rs11653738 (86%), indicating that there may be no association between NSCL/P and these SNPs in this population. DISCUSSION The results of our study did not confirm the presumption that the examined SNPs in TNP1, MSX1, TCOF1, FGFR1, COL2A1, WNT3, and TIMP3 are associated with an increased risk of NSCL/P. Although we observed a preferential transmission of the rs28372960 T allele and of the rs7829058 C allele from parents to NSCL6P patients, as well as the rs116 53738 C allele to NSCP offspring, the results of the case-control study with a more robust sample
Control NSCL6P NSCP
* NSCL6P ¼ nonsyndromic cleft lip with or without cleft palate; NSCP ¼ nonsyndromic cleft palate.
did not confirm the association of these SNPs with NSCL/P in the Brazilian population. TCOF1 is a putative candidate gene for nonsyndromic clefts because of its essential participation in neural crest cell formation and proliferation (Dixon et al., 2006). TCOF1 encodes the Treacle protein, which is involved in ribosomal biosynthesis by controlling rDNA transcription and preprocessing of the ribosomal-RNA transcripts (Kadakia et al., 2014). Thus, Treacle is a unique spatiotemporal regulator of ribosome biogenesis, and alterations in its normal function disrupt neural crest cell function, causing among other craniofacial anomalies cleft palate (Dixon et al., 2006). The association of variants in TCOF1 with nonsyndromic clefts was first reported by Sull et al. (2008) in a study showing a statistically significant overtransmission of rs15251 and rs2569062 risk alleles to offspring with NSCP in case-parent trios from Maryland, United States; Taiwan; and Singapore. The rs28372960 polymorphism in TCOF1 replaces a thymidine for a cytosine at position346 of the promoter (346C.T), decreasing the transcriptional activity of the gene by ~40% (Masotti et al., 2005). We observed a tendency toward association between NSCL6P and TCOF1, as revealed by the association of rs28372960 as a single SNP and as a haplotype together with rs15251 and rs2569062. Our findings show a tendency toward association between TCOF1 and nonsyndromic oral clefts, which should encourage further studies with larger samples and higher gene coverage to determine the influence of TCOF1 on NSCL/P. The pleiotropic effects of fibroblast growth factors (FGFs), which are essential for formation of the lip and the palate (Snyder-Warwick and Perlyn, 2012; Stanier and Pauws, 2012) occur after binding to their specific transmembrane receptors (FGFR1-4) with tyrosine kinase activity (Belov and Mohammadi, 2013). The FGF/FGFR signaling network regulates the migration and epithelialmesenchymal interactions involved in the development of the palate and lip (Stanier and Pauws, 2012), and animal models support the involvement of the FGF/FGFR pathway in oral cleft pathogenesis (Huang et al., 2008; Wang et al., 2013). Considering the crucial roles of the FGF/FGFR pathway in craniofacial development, polymorphic variants that disrupt this pathway may contribute to NSCL/P susceptibility. Indeed, several studies have shown statistical evidence of an association between FGF and FGFR SNPs and NSCL/P (Riley et al., 2007; Menezes et al., 2008; Nikopensius et al., 2010; Lace et al., 2011).
Machado et al., GENES RELATED TO DEVELOPMENT IN NONSYNDROMIC ORAL CLEFTS
TABLE 6
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Frequency of Polymorphisms rs28372960, rs7829058, and rs11653738 in the Case-Control Model† HWE (P Value)*
NSCL6P rs28372960 (TCOF1) Allele (C/T) Genotype (CC/CT/TT) rs7829058 (FGFR1) Allele (G/C) Genotype (GG/GC/CC) NSCP rs11653738 (WNT3) Allele (T/C) Genotype (TT/TC/CC)
Control (%)
Case (%)
ORAlelle (95% CI) P Value
ORHet (95% CI) P Value
ORHom (95% CI) P Value
93.4/6.6 87.4/11.9/0.7
94/6 88.7/10.7/0.6
0.89 (0.60–1.33) .62
0.88 (0.57–1.36) .57
0.92 (0.17–5.07) 1.00
84.9/15.1 71.2/27.5/1.3
86.9/13.1 75.8/22.3/1.9
0.84 (0.63–1.2) .26
0.76 (0.55–1.05) .09
1.32 (0.45–3.86) .59
72.4/27.6 52.9/38.9/8.2
71.4/28.6 52.2/38.5/9.3
1.05 (0.81–1.36) .73
1.00 (0.71–1.43) .98
1.15 (0.63–2.09) .64
0.36
0.07
0.49
† HWE ¼ Hardy-Weinberg equilibrium; NSCL6P ¼ nonsyndromic cleft lip with or without cleft palate; NSCP ¼ nonsyndromic cleft palate; OR ¼ odds ratio; CI ¼ confidence interval. * P value .0056 is required after applying multiple testing corrections.
Riley et al. (2007) suggested that impaired FGF signaling contributes to 3% to 5% of all cases of nonsyndromic oral clefts. Significant associations between SNP rs7829058 in FGFR1 and NSCL6P (Lace et al., 2011) and NSCP (Nikopensius et al., 2010) were observed in European populations. Although the sample of the current study was enriched with European-derived ancestry, only a modest association was detected in NSCL6P trios. The failure to replicate previous findings could be explained by the modest sample size, but population differences should also be considered. Several studies have shown evidence of an association between WNT3 and NSCL/P in different populations (Chiquet et al., 2008; Menezes et al., 2010; Nikopensius et al., 2010; Lace et al., 2011; Nikopensius et al., 2011; Mostowska et al., 2012), but the results are inconsistent because both increased and reduced risk of NSCL/P had been reported. Moreover, WNT3 was not found to be significantly associated with NSCL6P in a population from Brazil (Fontoura et al., 2015), but the power to detect effects was limited in this study. Our family-based study showed that the WNT3 rs11653738 C allele was preferentially transmitted to NSCP in the study trios, but the casecontrol approach failed to confirm this association. Combining family-based and case-control studies is an approach based on the premise that if the same effect of a disease-marker association can be obtained from TDT and case control, the magnitude of information is strong and true. Thus, the involvement of WNT3 in NSCL/P in the Brazilian population is unclear, and further studies are required. This study has some strengths and limitations. High geographic coverage by including four different Centers in four Brazilian states and the assessment of ancestry contribution of each patient, correcting for specific effects that the population stratification may have, are among the most important strengths. Where limitations are concerned, the first thing to acknowledge is the modest sample size, especially for the NSCP group. Therefore, modest associ-
ations of polymorphisms and the oral cleft risk may have been missed. However, power calculations indicated that the sample size provided 95% and 86% statistical power to detect an association with rs7829058 and rs11653738, respectively, in the case-control analysis. Patients with NSCL/P were recruited in four reference centers for orofacial cleft treatment, whereas healthy controls were obtained from patients admitted into the associated dental schools for regular dental treatment, so selection bias may not have been avoided. In conclusion, in the present study, by combining familybased and case-control models, we did not find consistent associations between nonsyndromic orofacial clefts and SNPs of the TNP1, MSX1, TCOF1, FGFR1, COL2A1, WNT3, and TIMP3 genes in the Brazilian population. REFERENCES Aidar M, Line SR. A simple and cost-effective protocol for DNA isolation from buccal epithelial cells. Braz Dent J. 2007;18:148– 152. Bagordakis E, Paranaiba LM, Brito LA, Aquino SN, Messetti AC, Martelli-Junior H, Swerts MS, Graner E, Passos-Bueno MR, Coletta RD. Polymorphisms at regions 1p22.1 (rs560426) and 8q24 (rs1530300) are risk markers for nonsyndromic cleft lip and/or palate in the Brazilian population. Am J Med Genet A. 2013;161a:1177–1180. Bastos-Rodrigues L, Pimenta JR, Pena SD. The genetic structure of human populations studied through short insertion-deletion polymorphisms. Ann Hum Genet. 2006;70:658–665. Beaty TH, Hetmanski JB, Fallin MD, Park JW, Sull JW, McIntosh I, Liang KY, Vanderkolk CA, Redett RJ, Boyadjiev SA, et al. Analysis of candidate genes on chromosome 2 in oral cleft caseparent trios from three populations. Hum Genet. 2006;120:501– 518. Beaty TH, Hetmanski JB, Zeiger JS, Fan YT, Liang KY, VanderKolk CA, McIntosh I. Testing candidate genes for non-syndromic oral clefts using a case-parent trio design. Genet Epidemiol. 2002;22:1– 11. Beaty TH, Murray JC, Marazita ML, Munger RG, Ruczinski I, Hetmanski JB, Liang KY, Wu T, Murray T, Fallin MD, et al. A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4. Nat Genet. 2010;42:525–529.
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Belov AA, Mohammadi M. Molecular mechanisms of fibroblast growth factor signaling in physiology and pathology. Cold Spring Harb Perspect Biol. 2013;5:1–28. Birnbaum S, Ludwig KU, Reutter H, Herms S, Steffens M, Rubini M, Baluardo C, Ferrian M, Almeida de Assis N, Alblas MA, et al. Key susceptibility locus for nonsyndromic cleft lip with or without cleft palate on chromosome 8q24. Nat Genet. 2009;41:473–477. Brito LA, Bassi CF, Masotti C, Malcher C, Rocha KM, Schlesinger D, Bueno DF, Cruz LA, Barbara LK, Bertola DR, et al. IRF6 is a risk factor for nonsyndromic cleft lip in the Brazilian population. Am J Med Genet A. 2012;158A:2170–2175. Brito LA, Paranaiba LM, Bassi CF, Masotti C, Malcher C, Schlesinger D, Rocha KM, Cruz LA, Ba´rbara LK, Alonso N, et al. Region 8q24 is a susceptibility locus for nonsyndromic oral clefting in Brazil. Birth Defects Res A Clin Mol Teratol. 2012;94:464–468. Butali A, Mossey PA, Adeyemo WL, Jezewski PA, Onwuamah CK, Ogunlewe MO, Ugboko VI, Adejuyigbe O, Adigun AI, AbdurRahman LO, et al. Genetic studies in the Nigerian population implicate an MSX1 mutation in complex oral facial clefting disorders. Cleft Palate Craniofac J. 2011;48:646–653. Cardoso ML, Bezerra JF, Oliveira GH, Soares CD, Oliveira SR, de Souza KS, da Silva HP, Silbiger VN, Luchessi AD, Fajardo CM, et al. MSX1 gene polymorphisms in non-syndromic cleft lip and/or palate. Oral Dis. 2013;19:507–512. Chiquet BT, Blanton SH, Burt A, Ma D, Stal S, Mulliken JB, Hecht JT. Variation in WNT genes is associated with non-syndromic cleft lip with or without cleft palate. Hum Mol Genet. 2008;17:2212– 2218. De Aquino SN, Messetti AC, Bagordakis E, Martelli-Junior H, Swerts MS, Graner E, Coletta RD. Polymorphisms in FGF12, VCL, CX43 and VAX1 in Brazilian patients with nonsyndromic cleft lip with or without cleft palate. BMC Med Genet. 2013;14:53. De Aquino SN, Messetti AC, Hoshi R, Borges A, Viena CS, Reis SR, Swerts MSO, Graner E, Martelli-Junior H, Coletta RD. Analysis of susceptibility polymorphisms for nonsyndromic cleft lip with or without cleft palate in the Brazilian population. Birth Defects Res A Clin Mol Teratol. 2014;100:36–42. Dixon J, Jones NC, Sandell LL, Jayasinghe SM, Crane J, Rey JP, Dixon MJ, Trainor PA. Tcof1/Treacle is required for neural crest cell formation and proliferation deficiencies that cause craniofacial abnormalities. Proc Natl Acad Sci U S A. 2006;103:13403–13408. Dixon MJ, Marazita ML, Beaty TH, Murray JC. Cleft lip and palate: understanding genetic and environmental influences. Nat Rev Genet. 2011;12:167–178. Fontoura C, Silva RM, Granjeiro JM, Letra A. Association of WNT9B gene polymorphisms with nonsyndromic cleft lip with or without cleft palate in Brazilian nuclear families. Cleft Palate Craniofac J. 2015;52:44–48. Grant SF, Wang K, Zhang H, Glaberson W, Annaiah K, Kim CE, Bradfield JP, Glessner JT, Thomas KA, Garris M, et al. A genomewide association study identifies a locus for nonsyndromic cleft lip with or without cleft palate on 8q24. J Pediatr. 2009;155:909–913. Huang X, Goudy SL, Ketova T, Litingtung Y, Chiang C. Gli3deficient mice exhibit cleft palate associated with abnormal tongue development. Dev Dyn. 2008;237:3079–3087. Jagomagi T, Nikopensius T, Krjutskov K, Tammekivi V, Viltrop T, Saag M, Metspalu A. MTHFR and MSX1 contribute to the risk of nonsyndromic cleft lip/palate. Eur J Oral Sci. 2010;118:213– 220. Kadakia S, Helman SN, Badhey AK, Saman M, Ducic Y. Treacher Collins syndrome: the genetics of a craniofacial disease. Int J Pediatr Otorhinolaryngol. 2014;78:893–898. Lace B, Kempa I, Piekuse L, Grinfelde I, Klovins J, Pliss L, Krumina A, Vieira AR. Association studies of candidate genes and cleft lip and palate taking into consideration geographical origin. Eur J Oral Sci. 2011;119:413–417.
Laird NM, Horvath S, Xu X. Implementing a unified approach to family-based tests of association. Genet Epidemiol. 2000;19:S36– S42. Ludwig KU, Mangold E, Herms S, Nowak S, Reutter H, Paul A, Becker J, Herberz R, AlChawa T, Nasser E, et al. Genome-wide meta-analyses of nonsyndromic cleft lip with or without cleft palate identify six new risk loci. Nat Genet. 2012;44:968–971. Mangold E, Ludwig KU, Birnbaum S, Baluardo C, Ferrian M, Herms S, Reutter H, de Assis NA, Chawa TA, Mattheisen M, et al. Genome-wide association study identifies two susceptibility loci for nonsyndromic cleft lip with or without cleft palate. Nat Genet. 2010;42:24–26. Martelli-Junior H, Porto LV, Martelli DR, Bonan PR, Freitas AB, Della Coletta R. Prevalence of nonsyndromic oral clefts in a reference hospital in the state of Minas Gerais, Brazil, between 2000-2005. Braz Oral Res. 2007;21:314–317. Masotti C, Armelin-Correa LM, Splendore A, Lin CJ, Barbosa A, Sogayar MC, Passos-Bueno MR. A functional SNP in the promoter region of TCOF1 is associated with reduced gene expression and YY1 DNA-protein interaction. Gene. 2005;359:44– 52. Menezes R, Letra A, Kim AH, Kuchler EC, Day A, Tannure PN, ¨ Gomes da Motta L, Paiva KB, Granjeiro JM, Vieira AR. Studies with Wnt genes and nonsyndromic cleft lip and palate. Birth Defects Res A Clin Mol Teratol. 2010;88:995–1000. Menezes R, Letra A, Ruff J, Granjeiro JM, Vieira AR. Studies of genes in the FGF signaling pathway and oral clefts with or without dental anomalies. Am J Med Genet A. 2008;146a:1614–1617. Mostowska A, Hozyasz KK, Biedziak B, Wojcicki P, Lianeri M, Jagodzinski PP. Genotype and haplotype analysis of WNT genes in non-syndromic cleft lip with or without cleft palate. Eur J Oral Sci. 2012;120:1–8. Nikopensius T, Jagomagi T, Krjutskov K, Tammekivi V, Saag M, Prane I, Piekuse L, Akota I, Barkane B, Krumina A, et al. Genetic variants in COL2A1, COL11A2, and IRF6 contribute risk to nonsyndromic cleft palate. Birth Defects Res A Clin Mol Teratol. 2010;88:748–756. Nikopensius T, Kempa I, Ambrozaityte L, Jagomagi T, Saag M, Matuleviciene A, Utkus A, Krjutskov K, Tammekivi V, Piekuse L, et al. Variation in FGF1, FOXE1, and TIMP2 genes is associated with nonsyndromic cleft lip with or without cleft palate. Birth Defects Res A Clin Mol Teratol. 2011;91:218–225. Paranaiba LM, Bufalino A, Martelli-Junior H, de Barros LM, Graner E, Coletta RD. Lack of association between IRF6 polymorphisms (rs2235371 and rs642961) and non-syndromic cleft lip and/or palate in a Brazilian population. Oral Dis. 2010;16:193–197. Pena SD, Di Pietro G, Fuchshuber-Moraes M, Genro JP, Hutz MH, Kehdy FS, Kohlrausch F, Magno LA, Montenegro RC, Moraes MO, et al. The genomic ancestry of individuals from different geographical regions of Brazil is more uniform than expected. PLoS One. 2011;6:e17063. Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000;155:945– 959. Pritchard JK, Stephens M, Rosenberg NA, Donnelly P. Association mapping in structured populations. Am J Hum Genet. 2000;67:170– 181. Purcell S, Cherny SS, Sham PC. Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics. 2003;19:149–150. Riley BM, Mansilla MA, Ma J, Daack-Hirsch S, Maher BS, Raffensperger LM, Russo ET, Vieira AR, Dode C, Mohammadi M, et al. Impaired FGF signaling contributes to cleft lip and palate. Proc Natl Acad Sci U S A. 2007;104:4512–4517. Rodrigues K, Sena MF, Roncalli AG, Ferreira MA. Prevalence of orofacial clefts and social factors in Brazil. Braz Oral Res. 2009;23:38–42.
Machado et al., GENES RELATED TO DEVELOPMENT IN NONSYNDROMIC ORAL CLEFTS
Snyder-Warwick AK, Perlyn CA. Coordinated events: FGF signaling and other related pathways in palatogenesis. J Craniofac Surg. 2012;23:397–400. Stanier P, Pauws E. Development of the lip and palate: FGF signalling. Front Oral Biol. 2012;16:71–80. Suazo J, Santos JL, Carreno H, Jara L, Blanco R. Linkage disequilibrium between MSX1 and non-syndromic cleft lip/palate in the Chilean population. J Dent Res. 2004;83:782–785. Sull JW, Liang KY, Hetmanski JB, Fallin MD, Ingersoll RG, Park JW, Wu-Chou YH, Chen PK, Chong SS, Cheah F, et al. Excess maternal transmission of markers in TCOF1 among cleft palate case-parent trios from three populations. Am J Med Genet A. 2008;146a:2327–2331.
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Vieira AR, Orioli IM, Castilla EE, Cooper ME, Marazita ML, Murray JC. MSX1 and TGFB3 contribute to clefting in South America. J Dent Res. 2003;82:289–292. Wang C, Chang JY, Yang C, Huang Y, Liu J, You P, McKeehan WL, Wang F, Li X. Type 1 fibroblast growth factor receptor in cranial neural crest cell-derived mesenchyme is required for palatogenesis. J Biol Chem. 2013;288:22174–22183. Wehby GL, Murray JC. Folic acid and orofacial clefts: a review of the evidence. Oral Dis. 2010;16:11–19. Zucchero TM, Cooper ME, Maher BS, Daack-Hirsch S, Nepomuceno B, Ribeiro L, Caprau D, Christensen K, Suzuki Y, Machida J, et al. Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate. N Engl J Med. 2004;351:769– 780.
ORIGINAL ARTICLE Association Between Genes Involved in Craniofacial Development and Nonsyndromic Cleft Lip and/or Palate in the Brazilian Population Renato Assis Machado, D.D.S., M.S., Ana Camila Messetti, D.D.S., M.S., Sibele Nascimento de Aquino, D.D.S., ´ ´ M.S., PhD., Herc´ılio Martelli-Junior, D.D.S., M.S., Ph.D., Ma´rio Sergio Oliveira Swerts, D.D.S., M.S., Ph.D., Silvia Regina de Almeida Reis, D.D.S., M.S., Ph.D., Helenara Salvati Bertolossi Moreira, Ph.D., Darlene Camati Persuhn, B.S., M.S., Ph.D., Ricardo D. Coletta, D.D.S., M.S., Ph.D. Objective: To determine the association of single-nucleotide polymorphisms (SNPs) in genes related to craniofacial development, which were previously identified as susceptibility signals for nonsyndromic oral clefts, in Brazilians with nonsyndromic cleft lip and/or palate (NSCL/P). Design: The SNPs rs748044 (TNP1), rs1106514 (MSX1), rs28372960, rs15251 and rs2569062 (TCOF1), rs7829058 (FGFR1), rs1793949 (COL2A1), rs11653738 (WNT3), and rs242082 (TIMP3) were assessed in a family-based transmission disequilibrium test (TDT) and a structured casecontrol analysis based on the individual ancestry proportions. Setting: The SNPs were initially analyzed by TDT, and polymorphisms showing a trend toward excess transmission were subsequently studied in an independent case-control sample. Participants: The study sample consisted of 189 case-parent trios of nonsyndromic cleft lip with or without cleft palate (NSCL6P), 107 case-parent trios of nonsyndromic cleft palate (NSCP), 318 isolated samples of NSCL6P, 189 isolated samples of NSCP, and 599 healthy controls. Main Outcome Measure: Association of alleles with NSCL/P pathogenesis. Results: Preferential transmission of SNPs rs28372960 and rs7829058 in NSCL6P trios and rs11653738 in NSCP trios (P ¼ .04) were observed, although the structured case-control analysis did not confirm these associations. The haplotype T-C-C formed by TCOF1 SNPs rs28372960, rs15251, and rs2569062 was more frequently transmitted from healthy parents to NSCL6P offspring, but the P value (P ¼ .01) did not withstand Bonferroni correction for multiple tests. Conclusions: With the modest associations, our results do not support the hypothesis that TNP1, MSX1, TCOF1, FGFR1, COL2A1, WNT3, and TIMP3 variants are risk factors for nonsyndromic oral clefts in the Brazilian population. KEY WORDS:
COL2A1, craniofacial development, FGFR1, MSX1, nonsyndromic cleft lip and/or palate, TCOF1, TIMP3, TNP1, WNT3
Nonsyndromic cleft lip and/or palate (NSCL/P) is the most common craniofacial birth defect in humans and shows a prevalence of approximately 1:700 live births worldwide, but considerable geographic and ethnic variation is reported (Dixon et al., 2011). In Brazil, prevalence ranges from 0.36 to 1.54 per 1000 live births (Martelli-Junior et al., 2007; Rodrigues et al., 2009). The etiology of NSCL/P, which involves both genetic and environmental factors, is highly complex, and the molecular basis remains largely unknown (Wehby and Murray, 2010). Although genetic studies have identified a number of candidate genes and chromosomal regions associated with NSCL/P (Zucchero et al., 2004; Birnbaum et al., 2009; Grant et al., 2009; Beaty et al.,
Mr. Machado and Mrs. Messetti are Ph.D. students, Department of Oral Diagnosis, School of Dentistry, University of Campinas, Piracicaba, Sao ˜ Paulo, Brazil. Dr. de Aquino is Assistant Professor, School of Dentistry, Federal University of Juiz de Fora, Governador Valadares, Minas Gerais, Brazil. Dr. Martelli-Junior ´ is Professor, Stomatology Clinic, Dental School, State University of Montes Claros, Montes Claros, Minas Gerais, Brazil, and Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of Jose´ Rosario Vellano, Alfenas, Minas Gerais, Brazil. Dr. Swerts is Assistant Professor, Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of Jose´ Rosario Vellano, Alfenas, Minas Gerais, Brazil. Dr. Reis is Professor, Department of Basic Science, Bahiana School of Medicine and Public Health, Salvador, Bahia, Brazil. Dr. Moreira is Assistant Professor, Department of Physiotherapy, State University of Western Parana´, Parana´, Brazil. Dr. Persuhn is Assistant Professor, Molecular Biology Department, Federal University of Para ´ıba, Joao ˜ Pessoa, Para ´ıba, Brazil. Dr. Coletta is Associate Professor, Department of Oral Diagnosis, School of Dentistry, University of Campinas, Piracicaba, Sao ˜ Paulo, Brazil. This work was supported by grants from Procad/Casadinho-CNPq/ CAPES, Bras ´ılia, Brazil; the State of Sao ˜ Paulo Research FoundationFAPESP, Sao ˜ Paulo, Brazil; and the State of Minas Gerais Research Foundation-FAPEMIG, Minas Gerais, Brazil. Submitted March 2015; Revised July 2015; Accepted July 2015.
Address correspondence to: Mr. Renato Assis Machado, Department of Oral Diagnosis, School of Dentistry, University of Campinas, CEP 13414-018, Piracicaba, Sao ˜ Paulo, Brazil. E-mail renatoassismachado@ yahoo.com.br. DOI: 10.1597/15-107 0
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2010; Mangold et al., 2010; Ludwig et al., 2012), few of them have been replicated in different populations. Previous studies with those potential susceptibility markers in the Brazilian population have confirmed some associations and failed to confirm others, most likely indicative of the varied genomic ethnic background in the population of Brazil (Parana ´ıba et al., 2010; Brito, Bassi, et al., 2012; Brito, Paranaiba, et al., 2012; Bagordakis et al., 2013; De Aquino et al., 2013; De Aquino et al., 2014). One strategy that has helped in the identification of genetic alterations associated with NSCL/P is the evaluation of single-nucleotide polymorphisms (SNPs) in genes related to craniofacial development. The craniofacial development involves a series of highly coordinated events, including proliferation, migration, epithelial-mesenchymal transition, and apoptosis, and polymorphic variations in genes that control these events may affect the normal development of the lips and palate, resulting in NSCL/P (Butali et al., 2011). Previous studies have shown statistical evidences of association between specific genes, including transition nuclear protein 1 (TNP1; Beaty et al., 2006), muscle segment homeobox 1 (MSX1; Beaty et al., 2002; Vieira et al., 2003; Suazo et al., 2004; Jagomagi et al., 2010; Nikopensius et al., 2010; Butali et al., 2011; Cardoso et al., 2013), Treacher Collins-Franceschetti syndrome 1 (TCOF1; Masotti et al., 2005; Sull et al., 2008), fibroblast growth factor receptor 1 (FGFR1; Menezes et al., 2008; Nikopensius et al., 2010; Lace et al., 2011), collagen type II, alpha 1 (COL2A1; Nikopensius et al., 2010), wingless-type MMTV integration site family, member 3 (WNT3; Chiquet et al., 2008; Nikopensius et al., 2010; Lace et al., 2011; Mostowska et al., 2012), tissue inhibitor of metallopeptidase 3 (TIMP3; Nikopensius et al., 2010), and nonsyndromic oral clefts across different populations. However, the involvement of these genes in the Brazilian population remains uncertain. The Brazilian population shows highly variable genomic ancestry (Pena et al., 2011), due to five centuries of genetic admixture between Amerindians, Europeans, and sub-Saharan Africans, which affects specific genetic associations of NSCL/P loci identified in other populations (Bagordakis et al., 2013; De Aquino et al., 2014). The aim of the present study was to investigate potential associations of markers that were previously identified using candidate-gene approaches in the Brazilian population. These NSCL/P risk markers were represented by the following SNPs: rs748044 in TNP1 (Beaty et al., 2006); rs1106514 in MSX1 (Nikopensius et al., 2010); rs28372960, rs15251, and rs2569062 in TCOF1 (Masotti et al., 2005; Sull et al., 2008); rs7829058 in FGFR1; rs1793949 in COL2A1; rs11653738 in WNT3; and rs242082 in TIMP3 (Nikopensius et al., 2010). The SNP rs28372960 in TCOF1 was not previously investigated in NSCL/P, but its functional effects, decreasing transcriptional activity of the gene (Masotti et al., 2005), suggest a causal role in NSCL/P.
MATERIALS
AND
METHODS
Sample This study included patients with nonsyndromic oral clefts and healthy controls from four Brazilian states: Minas Gerais state (Center for Rehabilitation of Craniofacial Anomalies, Dental School, University of Jose´ Rosa´rio Vellano, Alfenas-MG), located in the southeastern region of Brazil; Parana´ state (Association of Carrier of Lip and Palate Cleft–APOFILAB, Cascavel-PR), in the southern region; and Para ´ıba state (University Hospital of Lauro Wanderley–HULW, Joao ˜ Pessoa-PB) and Bahia state (Santo Antonio Hospital, Salvador-BA), which are located in the northeast region. All patients were diagnosed independently and screened for the presence of associated anomalies or syndromes by the team of specialists from each center, and only patients with the nonsyndromic form of oral clefts were included. The nonsyndromic oral clefts were classified with the incisive foramen as reference. For transmission disequilibrium test (TDT) analysis, 296 trios composed of one affected offspring and two healthy parents (189 with nonsyndromic cleft lip with or without cleft palate [NSCL6P] and 107 with nonsyndromic cleft palate only [NSCP]) were included. In the TDT analysis, we searched for the unbalanced transfer of a specific allele from a nonaffected healthy parent to the affected child, and the polymorphic markers that showed a preferential transmission with a P value ,.05 were selected for the case-control study with correction for ancestry differences. For the casecontrol analysis, 507 patients affected with NSCL/P (318 NSCL6P and 189 NSCP) and 599 healthy individuals without physical or psychiatric diseases, history of congenital malformations, or familial history of orofacial clefts consented to participate in this study. The control group consisted of samples matched for gender and geographical location, and no statistically significant differences between groups were observed. There was no overlap between the samples used in the TDT and the case-control study. The study was approved by the ethics review board of each of the centers or hospitals affiliated with the collaborative study. Written informed consent was obtained from the parents or guardians and/or the participants. Genotyping and Estimation of the Genomic Ancestry The genomic DNA was isolated from buccal mucosa cells obtained by mouthwash with a 3% sucrose solution, using a salting-out protocol previously described (Aidar and Line, 2007). Polymerase chain reaction (PCR)–based genotyping of rs748044, rs1106514, rs28372960, rs15251, rs2569062, rs7829058,
Machado et al., GENES RELATED TO DEVELOPMENT IN NONSYNDROMIC ORAL CLEFTS
TABLE 1
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Characteristics of the Single-Nucleotide Polymorphisms (SNPs)*
SNP
Gene
Chromosome
Position
Alleles
Original Study
rs748044 rs1106514 rs28372960 rs15251 rs2569062 rs7829058 rs1793949 rs11653738 rs242082
TNP1 MSX1 TCOF1 TCOF1 TCOF1 FGFR1 COL2A1 WNT3 TIMP3
2 4 5 5 5 8 12 17 22
217,289,319 4,874,199 150,357,401 150,396,669 150,398,801 38,474,577 47,977,812 46,809,587 32,828,453
C/T G/C C/T C/T C/G G/C C/T T/C G/A
Beaty et al., 2006 Nikopensius et al., 2010 Masotti et al., 2005 Sull et al., 2008 Sull et al., 2008 Nikopensius et al., 2010 Nikopensius et al., 2010 Nikopensius et al., 2010 Nikopensius et al., 2010
* Risk alleles appear in bold.
rs1793949, rs11653738, and rs242082 (Table 1) was performed on the StepOnePlus Real-Time PCR platform (Applied Biosystems, Foster City, CA) using TaqMan 5 0 -exonuclease allelic discrimination assays (assays-on-demand, Applied Biosystems). For quality control purposes, reactions were randomly repeated in 10% of the samples for each SNP, and the concordance rate was 100%. All samples were successfully genotyped, with a genotype call rate of 100%. To determine the genomic ancestry of each individual, samples were genotyped for a set of 40 biallelic short insertion/deletion polymorphisms previously validated as ancestry informative markers of the Brazilian population (Bastos-Rodrigues et al., 2006). Statistical Analysis
Collection) were incorporated to assist the software in the ancestry estimation. Following ancestry assessment, STRAT was used to test the association based on the individual ancestry proportions (Pritchard, Stephens, Rosenberg, et al., 2000). Deviation from Hardy-Weinberg equilibrium was assessed by a chi-square test. The genotypes were analyzed under unrestricted, dominant, and recessive genetic models. The odds ratio associated with the 95% confidence interval was also calculated. A Bonferroni-adjusted P value threshold of a .0056 (.05/9) was considered statistically significant. Power for detecting a P value ,.05 for SNPs analyzed in the casecontrol approach was calculated using the Genetic Power Calculator (Purcell et al., 2003), assuming a prevalence of NSCL/P in Brazil of 0.00146 (MartelliJunior et al., 2007) and using the most conservative odds ratios reported in the original studies.
The TDT was performed with the aid of the Family Based Association Test (FBAT) software (Laird et al., 2000). The haplotype-based analysis was conducted using the HBAT function of the FBAT software. To determine the genomic ancestry of each individual in the case-control study, Structure software version 2.3.4 (Pritchard, Stephens, and Donnelly, 2000) was used in a model assuming K¼3 parental populations based on the tri-hybrid origin of the Brazilian population. Samples with a prespecified population of origin (Amerindian, European, and sub-Saharan African reference populations from the Marshfield Clinic
In the initial screening, we identified a preferential transmission of the rs28372960 T allele (P ¼ .04) and of the rs7829058 C allele (P ¼ .04) from healthy parents to NSCL6P offspring (Table 2). Similarly, the rs11653738 C allele showed association with NSCP offspring (P ¼ .04; Table 3). Although in a low frequency, the haplotype T-CC of TCOF1, composed of the T allele of rs28372960, C allele of rs15251, and C allele of rs2569062, was found to be more frequently transmitted to NSCL6P offspring (P ¼
TABLE 2 Transmission Disequilibrium Test of the SingleNucleotide Polymorphisms (SNPs) in Nonsyndromic Cleft Lip With or Without Cleft Palate Trios*
TABLE 3 Transmission Disequilibrium Test of the SingleNucleotide Polymorphisms (SNPs) in Trios With Nonsyndromic Cleft Palate*
RESULTS
SNP
MAF
Number of Families
T/NT
Z Score
P Value
SNP
MAF
Number of Families
T/NT
Z Score
P Value
rs748044 rs1106514 rs28372960 rs15251 rs2569062 rs7829058 rs1793949 rs11653738 rs242082
0.275 0.447 0.054 0.263 0.461 0.146 0.388 0.271 0.353
116 133 30 122 129 83 134 111 135
77/39 90/43 20/10 72/50 83/46 42/41 111/23 64/47 79/56
0.00 1.49 1.97 0.16 1.14 2.00 0.53 1.71 0.82
1.00 .13 .04 .87 .25 .04 .59 .08 .41
rs748044 rs1106514 rs28372960 rs15251 rs2569062 rs7829058 rs1793949 rs11653738 rs242082
0.254 0.469 0.045 0.250 0.445 0.123 0.385 0.232 0.349
63 82 15 62 79 41 80 64 83
35/28 57/25 6/9 36/26 56/23 19/22 55/25 34/30 52/31
1.42 0.28 0.50 0.87 0.38 0.30 0.19 2.04 0.28
0.15 0.77 0.61 0.38 0.69 0.76 0.84 0.04 0.77
* MAF ¼ minor allele frequency; T/NT ¼ transmission/nontransmission counts. P value .0056 is required after applying multiple testing corrections.
* MAF ¼ minor allele frequency; T/NT ¼ transmission/nontransmission counts. P value .0056 is required after applying multiple testing corrections.
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TABLE 4 Haplotype Family-Based Association Test for TCOF1 rs28372960, rs15251, and rs2569062 Performed Using HBAT
Haplotype* NSCL6P C-C-G C-C-C C-T-C T-C-C C-T-G NSCP C-C-G C-C-C C-T-C T-C-C C-T-G
Frequency
0.451 0.249 0.238 0.037 0.015 0.429 0.272 0.245 0.045 0.008
Number of Families
117 114 105 24 23 72 66 59 14 14
Z Score
0.34 0.46 0.29 2.41 1.42 0.38 0.78 1.09 0.73 0.60
TABLE 5 Average Proportions of the European, African, and Amerindian Ancestry of the Patients in Control and Case Groups* European
African
Amerindian
85.6% 83.3% 78.9%
12.4% 15.1% 18.7%
2.0% 1.6% 2.4%
P Value
.73 .64 .77 .01 .15 .70 .43 .27 .46 .54
* Sequence: rs28372960, rs15251, and rs2569062; NSCL6P ¼ nonsyndromic cleft lip with or without cleft palate; NSCP ¼ nonsyndromic cleft palate. P value .0056 is required after applying multiple testing corrections.
.01; Table 4). However, no association retained statistical significance after adjusting for multiple comparisons. Based on the results that revealed a possible association between NSCL6P and rs28372960 and rs7829058 and between NSCP and rs11653738, we performed the casecontrol analysis structured by genomic ancestry to confirm the possible associations. The European ancestry contribution was the most predominant in the three groups, followed by African and Amerindian (Table 5). There were no statistically significant differences in the ancestry proportions between groups. Frequencies of the alleles and genotypes of rs28372960, rs7829058, and rs11653738 structured by genomic ancestry are presented in Table 6. The genotype frequencies observed for all studied polymorphisms in controls did not reveal statistically significant differences compared with those expected under HardyWeinberg equilibrium. None of the polymorphisms tested showed association with NSCL6P or NSCP in this Brazilian case-control cohort. Further analyses in the dominant and recessive genetic models also revealed no differences in the distribution (data not shown). Power analysis showed good statistical power to detect any association with the current case-control sample size for the polymorphisms rs7829058 (92%) and rs11653738 (86%), indicating that there may be no association between NSCL/P and these SNPs in this population. DISCUSSION The results of our study did not confirm the presumption that the examined SNPs in TNP1, MSX1, TCOF1, FGFR1, COL2A1, WNT3, and TIMP3 are associated with an increased risk of NSCL/P. Although we observed a preferential transmission of the rs28372960 T allele and of the rs7829058 C allele from parents to NSCL6P patients, as well as the rs116 53738 C allele to NSCP offspring, the results of the case-control study with a more robust sample
Control NSCL6P NSCP
* NSCL6P ¼ nonsyndromic cleft lip with or without cleft palate; NSCP ¼ nonsyndromic cleft palate.
did not confirm the association of these SNPs with NSCL/P in the Brazilian population. TCOF1 is a putative candidate gene for nonsyndromic clefts because of its essential participation in neural crest cell formation and proliferation (Dixon et al., 2006). TCOF1 encodes the Treacle protein, which is involved in ribosomal biosynthesis by controlling rDNA transcription and preprocessing of the ribosomal-RNA transcripts (Kadakia et al., 2014). Thus, Treacle is a unique spatiotemporal regulator of ribosome biogenesis, and alterations in its normal function disrupt neural crest cell function, causing among other craniofacial anomalies cleft palate (Dixon et al., 2006). The association of variants in TCOF1 with nonsyndromic clefts was first reported by Sull et al. (2008) in a study showing a statistically significant overtransmission of rs15251 and rs2569062 risk alleles to offspring with NSCP in case-parent trios from Maryland, United States; Taiwan; and Singapore. The rs28372960 polymorphism in TCOF1 replaces a thymidine for a cytosine at position346 of the promoter (346C.T), decreasing the transcriptional activity of the gene by ~40% (Masotti et al., 2005). We observed a tendency toward association between NSCL6P and TCOF1, as revealed by the association of rs28372960 as a single SNP and as a haplotype together with rs15251 and rs2569062. Our findings show a tendency toward association between TCOF1 and nonsyndromic oral clefts, which should encourage further studies with larger samples and higher gene coverage to determine the influence of TCOF1 on NSCL/P. The pleiotropic effects of fibroblast growth factors (FGFs), which are essential for formation of the lip and the palate (Snyder-Warwick and Perlyn, 2012; Stanier and Pauws, 2012) occur after binding to their specific transmembrane receptors (FGFR1-4) with tyrosine kinase activity (Belov and Mohammadi, 2013). The FGF/FGFR signaling network regulates the migration and epithelialmesenchymal interactions involved in the development of the palate and lip (Stanier and Pauws, 2012), and animal models support the involvement of the FGF/FGFR pathway in oral cleft pathogenesis (Huang et al., 2008; Wang et al., 2013). Considering the crucial roles of the FGF/FGFR pathway in craniofacial development, polymorphic variants that disrupt this pathway may contribute to NSCL/P susceptibility. Indeed, several studies have shown statistical evidence of an association between FGF and FGFR SNPs and NSCL/P (Riley et al., 2007; Menezes et al., 2008; Nikopensius et al., 2010; Lace et al., 2011).
Machado et al., GENES RELATED TO DEVELOPMENT IN NONSYNDROMIC ORAL CLEFTS
TABLE 6
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Frequency of Polymorphisms rs28372960, rs7829058, and rs11653738 in the Case-Control Model† HWE (P Value)*
NSCL6P rs28372960 (TCOF1) Allele (C/T) Genotype (CC/CT/TT) rs7829058 (FGFR1) Allele (G/C) Genotype (GG/GC/CC) NSCP rs11653738 (WNT3) Allele (T/C) Genotype (TT/TC/CC)
Control (%)
Case (%)
ORAlelle (95% CI) P Value
ORHet (95% CI) P Value
ORHom (95% CI) P Value
93.4/6.6 87.4/11.9/0.7
94/6 88.7/10.7/0.6
0.89 (0.60–1.33) .62
0.88 (0.57–1.36) .57
0.92 (0.17–5.07) 1.00
84.9/15.1 71.2/27.5/1.3
86.9/13.1 75.8/22.3/1.9
0.84 (0.63–1.2) .26
0.76 (0.55–1.05) .09
1.32 (0.45–3.86) .59
72.4/27.6 52.9/38.9/8.2
71.4/28.6 52.2/38.5/9.3
1.05 (0.81–1.36) .73
1.00 (0.71–1.43) .98
1.15 (0.63–2.09) .64
0.36
0.07
0.49
† HWE ¼ Hardy-Weinberg equilibrium; NSCL6P ¼ nonsyndromic cleft lip with or without cleft palate; NSCP ¼ nonsyndromic cleft palate; OR ¼ odds ratio; CI ¼ confidence interval. * P value .0056 is required after applying multiple testing corrections.
Riley et al. (2007) suggested that impaired FGF signaling contributes to 3% to 5% of all cases of nonsyndromic oral clefts. Significant associations between SNP rs7829058 in FGFR1 and NSCL6P (Lace et al., 2011) and NSCP (Nikopensius et al., 2010) were observed in European populations. Although the sample of the current study was enriched with European-derived ancestry, only a modest association was detected in NSCL6P trios. The failure to replicate previous findings could be explained by the modest sample size, but population differences should also be considered. Several studies have shown evidence of an association between WNT3 and NSCL/P in different populations (Chiquet et al., 2008; Menezes et al., 2010; Nikopensius et al., 2010; Lace et al., 2011; Nikopensius et al., 2011; Mostowska et al., 2012), but the results are inconsistent because both increased and reduced risk of NSCL/P had been reported. Moreover, WNT3 was not found to be significantly associated with NSCL6P in a population from Brazil (Fontoura et al., 2015), but the power to detect effects was limited in this study. Our family-based study showed that the WNT3 rs11653738 C allele was preferentially transmitted to NSCP in the study trios, but the casecontrol approach failed to confirm this association. Combining family-based and case-control studies is an approach based on the premise that if the same effect of a disease-marker association can be obtained from TDT and case control, the magnitude of information is strong and true. Thus, the involvement of WNT3 in NSCL/P in the Brazilian population is unclear, and further studies are required. This study has some strengths and limitations. High geographic coverage by including four different Centers in four Brazilian states and the assessment of ancestry contribution of each patient, correcting for specific effects that the population stratification may have, are among the most important strengths. Where limitations are concerned, the first thing to acknowledge is the modest sample size, especially for the NSCP group. Therefore, modest associ-
ations of polymorphisms and the oral cleft risk may have been missed. However, power calculations indicated that the sample size provided 95% and 86% statistical power to detect an association with rs7829058 and rs11653738, respectively, in the case-control analysis. Patients with NSCL/P were recruited in four reference centers for orofacial cleft treatment, whereas healthy controls were obtained from patients admitted into the associated dental schools for regular dental treatment, so selection bias may not have been avoided. In conclusion, in the present study, by combining familybased and case-control models, we did not find consistent associations between nonsyndromic orofacial clefts and SNPs of the TNP1, MSX1, TCOF1, FGFR1, COL2A1, WNT3, and TIMP3 genes in the Brazilian population. REFERENCES Aidar M, Line SR. A simple and cost-effective protocol for DNA isolation from buccal epithelial cells. Braz Dent J. 2007;18:148– 152. Bagordakis E, Paranaiba LM, Brito LA, Aquino SN, Messetti AC, Martelli-Junior H, Swerts MS, Graner E, Passos-Bueno MR, Coletta RD. Polymorphisms at regions 1p22.1 (rs560426) and 8q24 (rs1530300) are risk markers for nonsyndromic cleft lip and/or palate in the Brazilian population. Am J Med Genet A. 2013;161a:1177–1180. Bastos-Rodrigues L, Pimenta JR, Pena SD. The genetic structure of human populations studied through short insertion-deletion polymorphisms. Ann Hum Genet. 2006;70:658–665. Beaty TH, Hetmanski JB, Fallin MD, Park JW, Sull JW, McIntosh I, Liang KY, Vanderkolk CA, Redett RJ, Boyadjiev SA, et al. Analysis of candidate genes on chromosome 2 in oral cleft caseparent trios from three populations. Hum Genet. 2006;120:501– 518. Beaty TH, Hetmanski JB, Zeiger JS, Fan YT, Liang KY, VanderKolk CA, McIntosh I. Testing candidate genes for non-syndromic oral clefts using a case-parent trio design. Genet Epidemiol. 2002;22:1– 11. Beaty TH, Murray JC, Marazita ML, Munger RG, Ruczinski I, Hetmanski JB, Liang KY, Wu T, Murray T, Fallin MD, et al. A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4. Nat Genet. 2010;42:525–529.
0
Cleft Palate–Craniofacial Journal, May 2016, Vol. 53 No. 3
Belov AA, Mohammadi M. Molecular mechanisms of fibroblast growth factor signaling in physiology and pathology. Cold Spring Harb Perspect Biol. 2013;5:1–28. Birnbaum S, Ludwig KU, Reutter H, Herms S, Steffens M, Rubini M, Baluardo C, Ferrian M, Almeida de Assis N, Alblas MA, et al. Key susceptibility locus for nonsyndromic cleft lip with or without cleft palate on chromosome 8q24. Nat Genet. 2009;41:473–477. Brito LA, Bassi CF, Masotti C, Malcher C, Rocha KM, Schlesinger D, Bueno DF, Cruz LA, Barbara LK, Bertola DR, et al. IRF6 is a risk factor for nonsyndromic cleft lip in the Brazilian population. Am J Med Genet A. 2012;158A:2170–2175. Brito LA, Paranaiba LM, Bassi CF, Masotti C, Malcher C, Schlesinger D, Rocha KM, Cruz LA, Ba´rbara LK, Alonso N, et al. Region 8q24 is a susceptibility locus for nonsyndromic oral clefting in Brazil. Birth Defects Res A Clin Mol Teratol. 2012;94:464–468. Butali A, Mossey PA, Adeyemo WL, Jezewski PA, Onwuamah CK, Ogunlewe MO, Ugboko VI, Adejuyigbe O, Adigun AI, AbdurRahman LO, et al. Genetic studies in the Nigerian population implicate an MSX1 mutation in complex oral facial clefting disorders. Cleft Palate Craniofac J. 2011;48:646–653. Cardoso ML, Bezerra JF, Oliveira GH, Soares CD, Oliveira SR, de Souza KS, da Silva HP, Silbiger VN, Luchessi AD, Fajardo CM, et al. MSX1 gene polymorphisms in non-syndromic cleft lip and/or palate. Oral Dis. 2013;19:507–512. Chiquet BT, Blanton SH, Burt A, Ma D, Stal S, Mulliken JB, Hecht JT. Variation in WNT genes is associated with non-syndromic cleft lip with or without cleft palate. Hum Mol Genet. 2008;17:2212– 2218. De Aquino SN, Messetti AC, Bagordakis E, Martelli-Junior H, Swerts MS, Graner E, Coletta RD. Polymorphisms in FGF12, VCL, CX43 and VAX1 in Brazilian patients with nonsyndromic cleft lip with or without cleft palate. BMC Med Genet. 2013;14:53. De Aquino SN, Messetti AC, Hoshi R, Borges A, Viena CS, Reis SR, Swerts MSO, Graner E, Martelli-Junior H, Coletta RD. Analysis of susceptibility polymorphisms for nonsyndromic cleft lip with or without cleft palate in the Brazilian population. Birth Defects Res A Clin Mol Teratol. 2014;100:36–42. Dixon J, Jones NC, Sandell LL, Jayasinghe SM, Crane J, Rey JP, Dixon MJ, Trainor PA. Tcof1/Treacle is required for neural crest cell formation and proliferation deficiencies that cause craniofacial abnormalities. Proc Natl Acad Sci U S A. 2006;103:13403–13408. Dixon MJ, Marazita ML, Beaty TH, Murray JC. Cleft lip and palate: understanding genetic and environmental influences. Nat Rev Genet. 2011;12:167–178. Fontoura C, Silva RM, Granjeiro JM, Letra A. Association of WNT9B gene polymorphisms with nonsyndromic cleft lip with or without cleft palate in Brazilian nuclear families. Cleft Palate Craniofac J. 2015;52:44–48. Grant SF, Wang K, Zhang H, Glaberson W, Annaiah K, Kim CE, Bradfield JP, Glessner JT, Thomas KA, Garris M, et al. A genomewide association study identifies a locus for nonsyndromic cleft lip with or without cleft palate on 8q24. J Pediatr. 2009;155:909–913. Huang X, Goudy SL, Ketova T, Litingtung Y, Chiang C. Gli3deficient mice exhibit cleft palate associated with abnormal tongue development. Dev Dyn. 2008;237:3079–3087. Jagomagi T, Nikopensius T, Krjutskov K, Tammekivi V, Viltrop T, Saag M, Metspalu A. MTHFR and MSX1 contribute to the risk of nonsyndromic cleft lip/palate. Eur J Oral Sci. 2010;118:213– 220. Kadakia S, Helman SN, Badhey AK, Saman M, Ducic Y. Treacher Collins syndrome: the genetics of a craniofacial disease. Int J Pediatr Otorhinolaryngol. 2014;78:893–898. Lace B, Kempa I, Piekuse L, Grinfelde I, Klovins J, Pliss L, Krumina A, Vieira AR. Association studies of candidate genes and cleft lip and palate taking into consideration geographical origin. Eur J Oral Sci. 2011;119:413–417.
Laird NM, Horvath S, Xu X. Implementing a unified approach to family-based tests of association. Genet Epidemiol. 2000;19:S36– S42. Ludwig KU, Mangold E, Herms S, Nowak S, Reutter H, Paul A, Becker J, Herberz R, AlChawa T, Nasser E, et al. Genome-wide meta-analyses of nonsyndromic cleft lip with or without cleft palate identify six new risk loci. Nat Genet. 2012;44:968–971. Mangold E, Ludwig KU, Birnbaum S, Baluardo C, Ferrian M, Herms S, Reutter H, de Assis NA, Chawa TA, Mattheisen M, et al. Genome-wide association study identifies two susceptibility loci for nonsyndromic cleft lip with or without cleft palate. Nat Genet. 2010;42:24–26. Martelli-Junior H, Porto LV, Martelli DR, Bonan PR, Freitas AB, Della Coletta R. Prevalence of nonsyndromic oral clefts in a reference hospital in the state of Minas Gerais, Brazil, between 2000-2005. Braz Oral Res. 2007;21:314–317. Masotti C, Armelin-Correa LM, Splendore A, Lin CJ, Barbosa A, Sogayar MC, Passos-Bueno MR. A functional SNP in the promoter region of TCOF1 is associated with reduced gene expression and YY1 DNA-protein interaction. Gene. 2005;359:44– 52. Menezes R, Letra A, Kim AH, Kuchler EC, Day A, Tannure PN, ¨ Gomes da Motta L, Paiva KB, Granjeiro JM, Vieira AR. Studies with Wnt genes and nonsyndromic cleft lip and palate. Birth Defects Res A Clin Mol Teratol. 2010;88:995–1000. Menezes R, Letra A, Ruff J, Granjeiro JM, Vieira AR. Studies of genes in the FGF signaling pathway and oral clefts with or without dental anomalies. Am J Med Genet A. 2008;146a:1614–1617. Mostowska A, Hozyasz KK, Biedziak B, Wojcicki P, Lianeri M, Jagodzinski PP. Genotype and haplotype analysis of WNT genes in non-syndromic cleft lip with or without cleft palate. Eur J Oral Sci. 2012;120:1–8. Nikopensius T, Jagomagi T, Krjutskov K, Tammekivi V, Saag M, Prane I, Piekuse L, Akota I, Barkane B, Krumina A, et al. Genetic variants in COL2A1, COL11A2, and IRF6 contribute risk to nonsyndromic cleft palate. Birth Defects Res A Clin Mol Teratol. 2010;88:748–756. Nikopensius T, Kempa I, Ambrozaityte L, Jagomagi T, Saag M, Matuleviciene A, Utkus A, Krjutskov K, Tammekivi V, Piekuse L, et al. Variation in FGF1, FOXE1, and TIMP2 genes is associated with nonsyndromic cleft lip with or without cleft palate. Birth Defects Res A Clin Mol Teratol. 2011;91:218–225. Paranaiba LM, Bufalino A, Martelli-Junior H, de Barros LM, Graner E, Coletta RD. Lack of association between IRF6 polymorphisms (rs2235371 and rs642961) and non-syndromic cleft lip and/or palate in a Brazilian population. Oral Dis. 2010;16:193–197. Pena SD, Di Pietro G, Fuchshuber-Moraes M, Genro JP, Hutz MH, Kehdy FS, Kohlrausch F, Magno LA, Montenegro RC, Moraes MO, et al. The genomic ancestry of individuals from different geographical regions of Brazil is more uniform than expected. PLoS One. 2011;6:e17063. Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000;155:945– 959. Pritchard JK, Stephens M, Rosenberg NA, Donnelly P. Association mapping in structured populations. Am J Hum Genet. 2000;67:170– 181. Purcell S, Cherny SS, Sham PC. Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics. 2003;19:149–150. Riley BM, Mansilla MA, Ma J, Daack-Hirsch S, Maher BS, Raffensperger LM, Russo ET, Vieira AR, Dode C, Mohammadi M, et al. Impaired FGF signaling contributes to cleft lip and palate. Proc Natl Acad Sci U S A. 2007;104:4512–4517. Rodrigues K, Sena MF, Roncalli AG, Ferreira MA. Prevalence of orofacial clefts and social factors in Brazil. Braz Oral Res. 2009;23:38–42.
Machado et al., GENES RELATED TO DEVELOPMENT IN NONSYNDROMIC ORAL CLEFTS
Snyder-Warwick AK, Perlyn CA. Coordinated events: FGF signaling and other related pathways in palatogenesis. J Craniofac Surg. 2012;23:397–400. Stanier P, Pauws E. Development of the lip and palate: FGF signalling. Front Oral Biol. 2012;16:71–80. Suazo J, Santos JL, Carreno H, Jara L, Blanco R. Linkage disequilibrium between MSX1 and non-syndromic cleft lip/palate in the Chilean population. J Dent Res. 2004;83:782–785. Sull JW, Liang KY, Hetmanski JB, Fallin MD, Ingersoll RG, Park JW, Wu-Chou YH, Chen PK, Chong SS, Cheah F, et al. Excess maternal transmission of markers in TCOF1 among cleft palate case-parent trios from three populations. Am J Med Genet A. 2008;146a:2327–2331.
0
Vieira AR, Orioli IM, Castilla EE, Cooper ME, Marazita ML, Murray JC. MSX1 and TGFB3 contribute to clefting in South America. J Dent Res. 2003;82:289–292. Wang C, Chang JY, Yang C, Huang Y, Liu J, You P, McKeehan WL, Wang F, Li X. Type 1 fibroblast growth factor receptor in cranial neural crest cell-derived mesenchyme is required for palatogenesis. J Biol Chem. 2013;288:22174–22183. Wehby GL, Murray JC. Folic acid and orofacial clefts: a review of the evidence. Oral Dis. 2010;16:11–19. Zucchero TM, Cooper ME, Maher BS, Daack-Hirsch S, Nepomuceno B, Ribeiro L, Caprau D, Christensen K, Suzuki Y, Machida J, et al. Interferon regulatory factor 6 (IRF6) gene variants and the risk of isolated cleft lip or palate. N Engl J Med. 2004;351:769– 780.
