Orofacial Clefts (OFCs) are the largest group of craniofacial malformations in humans and are a significant public health problem, due to the medical, psychological, social, and economic ramifications.1–3 OFCs include three general categories: those affecting the lip only (CL), those affecting the lip and palate (CLP), and those affecting the palate only (CP).4 However, previous genetic and embryological studies have suggested that CL and CLP, collectively known as CL/P, have distinct etiological mechanisms when compared to CP.5,6 OFCs are characterized by incomplete formation or fusion of structures separating the nasal and oropharyngeal cavities and can affect the upper lip, the hard and/or soft palate, and other parts of the face.7

Most OFCs are not associated with additional malformations or anomalies and are thus classified as Non-Syndromic OFCs (NSOFCs). Despite the important influence of environmental risk factors, such as maternal consumption of alcohol and cigarettes, genetic factors have the biggest etiological contribution to NSOFC. Indeed, the first studies of Fogh-Anderson and then subsequent segregation analysis and twin studies have demonstrated a high rate of family occurrence, with a concordance rate of 40%–60% in monozygotic twins and 3%–5% in dizygotic twins.8,9

To date, there has been considerable statistical and biological support for more than 15 NSOFC susceptibility loci. Of these, 1q32, which includes Interferon Regulatory Factor 6 (IRF6), was initially identified in candidate gene studies,10 and is the most validated locus in Genome-Wide Association Studies (GWAS) of NSOFC.11 Furthermore, structural mutations in IRF6 can also cause Van der Woude and popliteal pterygium syndromes.12 Analysis of animal models indicates that IRF6 plays an important role in facial development, participating in proliferation and differentiation of keratinocytes during the formation of the oral periderm and its spatio-temporal regulation is essential for appropriate palatal adhesion.13,14

Several genetic polymorphisms of IRF6 have been associated with NSOFC risk in many populations, including Non-Syndromic Cleft Lip with or without cleft Palate (NSCL/P) subtype. The SNP, rs2235371, which changes valine to isoleucine at amino acid position 274 (V274I), was one of the first polymorphisms significantly associated with NSCL/P in Asian and Amerindian populations.15,16 However, this result has not been replicated in other studies.17–19 The variant, rs642961, which is located within an enhancer ∼10kb upstream of the IRF6 transcription start, was found to be a causative variant in people of European ancestry, being responsible for 18% of NSCL occurrence.20 The A allele of rs642961 was found to be significantly associated with NSCL/P in some studies,21,22 but this has not been replicated in populations from Brazil, Iran, or Scandinavia.19,23 Another GWAS has suggested evidence of an association between rs1044516 and NSCL/P in Asian populations, but not in other populations.11 This same polymorphism also showed evidence of an interaction with maternal tobacco smoke in Chinese case-parent trios.24

The association between IRF6 variants and NSOFC has been extensively explored in several populations in an effort to determine if IRF6 mutation screening should become a routine procedure, given its crucial role in OFCs. Nonetheless, in genetically heterogeneous populations, such as those in Brazil, the association between NSOFCs and common IRF6 variants has not been confirmed and unrecognized IRF6 variants are poorly investigated. Therefore, the aim of this study was to investigate the association of IRF6 polymorphisms (rs2235371, rs642961, rs2236907, rs861019 and rs1044516) with the development of NSOFC in a population from northeast Brazil. In addition, we evaluated the potential effect of allelic combinations of IRF6 polymorphisms on the development of NSOFC.

Table 1 shows the clinical characteristics of the case and control groups. Among the 186 NSOFC patients, there was a predominance of males, notably with CL and CLP types and an excess of females with the CP type, which is in accordance with previous studies. There were no differences in age distribution between the case and control groups. CLP was the cleft type most reported, followed by CP and CL. Approximately one third of the patients had a family history of clefts.

All studied polymorphisms were in Hardy-Weinberg equilibrium in both groups. Allelic and genotypic distributions of the IRF6 polymorphisms (rs2235371, rs642961, rs2236907, rs861019, and rs1044516) in cases and controls are presented in Table 2. Taking into account embryological differences of NSOFC, the frequencies of IRF6 polymorphisms in the case group were analyzed in CL/P (CL+CLP) and CP subgroups. There was a significant association between rs2235371 and CP, with The T allele (p=0.004) and TT genotype (p=0.016) being significantly more frequent in the CP group than the control group.

None of the polymorphisms studied showed differences in allele or genotype frequencies between the total case group and controls (data not shown). The LD analysis of IRF6 polymorphisms is shown in Fig. 1. One haplotype block was constructed between rs861019 and rs642961; however, it did not show a significant association with NSOFC (p>0.05).

Figure 1.

Pairwise linkage disequilibrium plot of IRF6/rs1044516, rs2235371, rs2236907, rs861019, and rs642961 in the combined sample (cases & controls) showing r2 (×100) values. The block is designed according to the internally developed solid spine of linkage disequilibrium (LD). The value within each diamond represents the pairwise correlation between pairs of Single-nucleotide polymorphisms (SNPs) (measured as 100×r2) defined by the upper left and the upper right sides of the diamond. Below are showed the frequency of each haplotype.

(0.15MB).

NSOFCs have been studied for many years in an attempt to understand their etiology since they constitute the majority of OFC cases. Genetic factors are known to play an important role in the development of OFCs, given the increased occurrence risk in relatives up to the third degree, when compared to the general population.26 In previous studies, variation at IRF6 has been responsible for a considerable percentage of the genetic contribution to NSOFCs. IRF6 variants have also been associated with the most severe sub-phenotypes and symmetrical shape variations in these patients, including a more protrusive forehead, a shorter nose, and a steep mandibular plane.10,22,27–30 However, studies investigating IRF6 polymorphisms in different patient populations have given divergent results.17,31 These findings are especially discordant in studies with mixed populations, such as those in Brazil, where results have varied according to the geographic region and ethnicity studied.16,19,32 Thus, in order to better understand the association of IRF6 with NSOFC, we evaluated the rs2235371, rs642961, rs2236907, rs861019 and rs1044516 polymorphisms in a Brazilian population.

None of the alleles or genotypes at the IRF6 polymorphisms evaluated here were associated with an increased risk of NSOFC in the studied population. Although an association was found between rs2235371 and the CP group, this result should be interpreted with caution, since the small number of individuals in this group affected the reliability of the result, and the association lost significance after correction for multiple tests. A similar result for rs2235371 in CP patients has been reported in an Indian population.33

Several studies have reported associations between rs2235371 and NSOFC risk, especially in Asian populations. A GWAS performed by Sun et al.15 showed that the rs2235371 variant was consistently associated with NSCL/P and it appeared to have a higher importance than the previously identified causal variant, rs642961, in Chinese populations.15 The A allele and AA and AG genotypes at rs2235371 were associated with a decreased risk of NSCL/P in this population when compared to GG genotypes. However, in Caucasians, these genotypes are not significantly associated with NSOFCs.31 The low frequency of the allele coding for isoleucine in populations of European descent is a possible explanation for these divergent results among populations.20 Indeed, the rs2235371 seems not to be causal, but rather in LD with another causal variant in IRF6.27

Among studies performed in Brazilian populations, Letra et al.32 investigated 142 case-parent trios from Sao Paulo and found a positive association between the rs2235371 polymorphism and the specific sub-phenotypes, complete left CLP and CP cases with impaction of permanent teeth. Although IRF6 was not a strong risk factor in that study, the authors suggested that IRF6 variants contributed to the specific sub-phenotype by controlling the affected side of unilateral cleft development. De Souza et al.16 studied 673 individuals from southern, southeastern, and northeastern Brazil and observed a significant association between the G allele at rs2235371 and CL/P and CL, but not CP in subjects with European ancestry. In contrast, Paranaiba et al.19 studied 228 patients from Minas Gerais, Brazil and observed no association between rs2235371 and NSOFCs.

The rs642961 SNP has been shown to be associated with NSCL/P in both European and Asian populations.21,31 The A allele and AA and GA genotypes at rs642961 were found to be significantly associated with NSCL/P, especially in CL individuals.20,31 Do Rego-Borges et al.34 studied 293 unrelated NSCL/P patients from a Brazilian population with a high level of African ancestry (Bahia State) and found a marginal association with rs642961 heterozygotes (GA), which they attributed to the significant European-derived ancestry component in this population.

However, as in the present study, many investigations have failed to replicate this association with rs642961. In the present study, the lack of association may be attributed to the smaller sample size and the effect of clinical heterogeneity among affected individuals (e.g., variation in disease severity).22 In fact, some studies only found an association with this SNP when analyzing specific cleft subtypes or when analyzing haplotypic combinations with other polymorphisms. Sun et al.15 demonstrated that the effect of rs642961 can be weakened by rs2235371, suggesting that the key variants may be located at rs2235371 in related haplotypes.

Brito et al.35 studied 563 NSCL/P patients from five different Brazilian cities and found no significant allelic association with rs642961. However, when stratified by sub-phenotypes, an association was found between rs642961 and CL was observed. De Souza et al.16 did not report an association when rs642961 was analyzed individually, but haplotype analyses showed an association of the A allele of rs642961 and the G allele of rs2235371 with an increased risk of NSCL/P in children and their mothers.

Although it is known that the haplotype information can increase the resolution of genetic data compared with examining SNPs individually, in our study there was no significant association with the constructed haplotype and NSOFC risk.

Although contributions from single genes, such as IRF6, now seem to explain approximately 15% of NSOFC cases, in the present case-control study, there were no significant associations between rs2235371, rs642961, rs2236907, rs861019, or rs1044516 SNPs in IRF6 and NSOFCs. Despite finding an association between rs2235371 and CP risk in our patients, additional studies in similar populations, but with larger sample sizes, are required to further investigate potential associations with different NSOFC sub-phenotypes and to identify the pattern of IRF6 genetic variants in admixed populations, such as those seen in Brazil.

This work was supported by the National Council for Scientific and Technological Development CNPq), Brazil, grant number 477608/2011-6.

The authors declare no conflicts of interest.