Differential expression of the IRF6 gene in the signs of Van der Woude Syndrome: Are distinct genetic modifiers operating?

Objective: The purpose of this study was to report a new variant in the Interferon Regulatory Factor 6 gene (IRF6) and to determine phenotype-genotype correlations in a family segregating Van der Woude syndrome. Methods: A five-generation family of 80 individuals segregating VWS was investigated using a tabulated pedigree but considering that three individuals registered in the pedigree died shortly after birth, the final sample size was 77 individuals. Five individuals had a complete dental clinical examination and molecular analysis performed using direct sequencing of the exon 4 and an adjacent region with 23 base pairs of the IRF6 gene. Results: Features of VWS reported in family history were present in 36.4% (28/77) of all family members; of these 57% (16/28) had pits in the lower lip, 36% (10/28) had both pits and orofacial clefts and 7 % (2/28) had only orofacial clefts. Developmental dental anomalies were observed in three individuals. The sequence analysis of exon 4 of the IRF6 gene carried out for 4 family members revealed the presence of the SNP rs7552506 (c.175-5C> G) located in five base pairs before the start of exon. The analysis of exon 4 of the IRF6 gene also revealed a new variant c.269G>C (p.Ser90Thr) which causes exchange of the Serine amino acid residue for the Threonine residue. Conclusion: The c.269G>C(p.Ser90Thr) can interfere with multimeric interactions and with protein conformation that will be slightly destabilized, because the mutant residue is bigger than the wild-type residue. The phenotypic variations in the cases studied, despite carrying the same genetic mutation, suggest that distinct genetic modifiers operate on the formation of clefts and dental development.


Introduction
Orofacial clefts may occur in association with other birth defects or be isolated. The Van der Woude Syndrome (VWS) (Online Mendelian Inheritance in Man [OMIM]119300) is the most common and well-known syndrome associated with orofacial clefts, being present in approximately 2% of orofacial cleft cases [1,2]. VWS consists of a rare congenital malformation and presents clinical features in addition to orofacial clefts, such as lower lip pits and occasionally, hypodontia. The frequency of VWS in the population varies from 1: 100,000 to 1: 40,000, and there are no reports of differences between males and females [3,4].
The expression of VWS is highly variable, both interfamilial and within the same family. In other words, there is a range of associated phenotypes, from the complete absence of visible abnormalities, only the presence of pits in the lower lip, to the combination of these pits with different types of clefts. These types may include aspects such as laterality (unilateral or bilateral), extension (complete and incomplete) and may involve lip, palate or both [2,[5][6][7].
The etiology of VWS has been attributed to mutations in the IRF6 gene (Interferon Regulatory Factor 6). However, from a molecular point of view, mutations in the IRF6 gene can explain 70% of the cases of VWS. Several studies also point to the possibility of other genes causing VWS, despite the limitations of molecular biology techniques in identifying mutations in many of these genes [8][9][10].
The IRF6 gene is located on the long arm of chromosome 1 (1q32.3 -q41) and contains 10 exons; exons 1, 2 and 10 are not translated. The product encoded by the IRF6 gene is a 467 amino acid polypeptide [8,11,12].
In contrast to the other members of the IRF family (interferon regulating factors), IRF6 does not participate in the innate immune response, however it is essential for embryonic development, can act as a tumor suppressor and participate in the differentiation of ectodermal tissues [13][14][15]. Recently, a new role assigned to the IRF6 ortholog gene is participation in the development of the exocrine gland. This information can be used to investigate the suspicion of disorders in the salivary glands of patients with the two forms of syndrome caused by mutations in the IRF6 gene, the VWS and Popliteal Pterygium Syndrome (SPP), as well as to understand the presence of dental problems, for example, caries, in patients with orofacial clefts [16].
The aim of this study was to report a new variant in the IRF6 gene and to determine phenotype-genotype correlations in a family segregating Van der Woude syndrome.

Genealogical data
The family participating in the study was located in the Service of Assistance to the Patients with Orofacial Clefts at Ophir Loyola Hospital (Belém -Pará, Brazil). At the time, five members of this family were present, a mother and four children (two girls and two boys). All five had congenital pits in the lower lip and only one of them did not present orofacial cleft but instead presented excess of lower lip. The research was carried out in accordance with the basic ethical principles of the guidelines and standards that regulate research in human beings: autonomy, beneficence, non-maleficence and justice. The project was previously approved by the Ethics Committee of the Health Science Center of the Federal University of Pará (CAAE 4879.0.000.073-10, no140 /10).
To obtain genealogical data, the tabulated pedigree method according to Polleta et al. (2014) [17] was used. The graphical construction of the pedigree was performed using an online tool from the Progeny program, available at: www.progenygenetics.com.
Information about other members of this family was obtained during three interviews at different times, resulting in the registration of 80 individuals in the pedigree, belonging to five generations of the index family. It was not possible to specify the gender and the presence of any manifestations of VWS in only three cases, all of whom had died shortly after birth; of these three cases, two were Siamese twin siblings. Of the final sample size of 77 individuals analyzed, 36 (45%) were females and 41 (51%) were males ( Figure 1).

Phenotypic characteristics
Family members with one or more phenotypic characteristics of VWS were considered affected individuals. These characteristics included cleft lip with or without palate, cleft palate, and the presence of pits, whether bilateral or unilateral. The types of orofacial clefts found were grouped into: cleft lip (CL), cleft lip with cleft palate (CLP) and cleft palate (CP). Dental information was gathered through a complete dental clinical examination.

DNA analysis
Four family members underwent molecular analysis of exon 4 of the IRF6 gene. The samples of biological material, 3 to 4 mL of blood, were collected through venipuncture and transferred to test tubes containing EDTA. For the extraction of DNA, the Invirsob spin blood mini kit (Invitek) was used. For the amplification of the sequence of interest in the polymerase chain (Polymerase Chain Reaction, PCR), the primers used were forward -GCTCTGGGCAATGATAGGAC and reverse -ACCTTCTCCCCAGCACCT. The PCR products were subjected to direct sequencing analysis of exon 4 of IRF6 and its exon-intron boundaries based on capillary electrophoresis, using the ABI PRISM BigDye kit, Terminator Cycle Sequencing. The same primers were used for PCR reactions and the ABI PRISM® 3500 Genetic Analyzer Capillary Sequencer by Applied Biosystems.

Data analysis
The family history information obtained from the tabulated pedigree was inserted in the spreadsheet of the  [21] were utilized. The data obtained was analyzed using descriptive statistics.

Results
The family did not present a history of consanguineous marriage and from the second generation on, where a family member has a cleft lip and pits, all subsequent generations have more than one affected relative. It is observed through the pedigree that the affected individuals belong only to the paternal, ascending and descending sides in relation to the index case (indicated with an arrow in the pedigree, Figure 1).
Lip pits were present in 26 of the 28 affected individuals and 12 out of 28 individuals had orofacial clefts, with six of them having the lip and palate affected. Ten individuals had both pits and orofacial clefts ( Table 1).
Out of the five individuals that had a dental examination (Table 2), one had permanent dentition and four had primary dentition. Three out of five had developmental dental anomalies.   According to the SIFT tool this variant is predicted as tolerated with a score of 0.37.
One of the most informative prediction tools was HOPE. The analysis derived from it provided information about the structure of the mutant amino acid residue compared to the wild type of residue, showing that the mutant residue is structurally larger, a factor that can interfere in the multimeric interactions and in the conformation of the protein ( Figure 3).  Differential expression of the IRF6 gene in the signs of Van der Woude Syndrome: Are distinct genetic modifiers operating? Due to the fact that in each generation of the studied family there is at least one affected member and they also had at least one of their parents affected, it's clear that this family follows the expected high penetrance that can vary from 80% to 100% according to previous studies in the literature [2,24].
Variable expression was observed in the studied family, with the main phenotypic manifestation of VWS in the affected individuals being congenital lip pits. According to the literature, this type of pits occurs in around 85% of individuals affected by VWS and in 64% of cases it is the only manifestation of the syndrome [4,7]. Regarding orofacial clefts, the studied family presented mixed types of clefts, with the most prevalent type (50%) being the CLP, a finding similar to other studies [2,7,25].
The presence of mixed types of orofacial clefts in the same family is often observed in families with VWS. In an attempt to explain this occurrence, it has been proposed that individuals with genetic components that cause VWS are subject to some type of multifactorial influence, similar to the non-syndromic form of orofacial clefts [8,[26][27][28]. Furthermore, another possible explanation could be the fact that another gene could be involved in this difference, for example variants in the gene GRHL3 (Grainyhead Like Transcription Factor 3) are more likely to have CP and less likely to have CL or lip pits than individuals with an IRF6 mutations [10] As for the molecular analysis in exon 4, the SNP rs7552506 identified in the investigated family members is a polymorphism already described in the literature in studies with families with non-syndromic orofacial clefts and also in studies with families with VWS [28,29]. In the study by Pegelow et al. (2008) [30] with 17 Swedish families this SNP showed an association with CLP. In addition, in the same study it was found that when there was combined unilateral and bilateral CLP in the same family, there was an association of this polymorphism. This information corroborates the findings in the present study.
According to the analysis performed by the HOPE tool, the c.269G> C variant (p.Ser90Thr) found in the present study results in a mutant amino acid residue that is larger than the wild type residue, which can influence the conformation of the protein that will be slightly destabilized and, in addition, there may be disturbances in the multimeric interactions. The Ser90 position of the polypeptide chain is located in the DNA-binding domain (structural motif of the helix-gyrohelix type), which is important for the binding of other molecules and is in contact with the residue of a regulatory domain. Therefore, this mutation can affect this interaction and disrupt protein regulation. Variants that occur in the IRF6 gene are mostly missense-type and occur mainly in the DNA-binding or proteinbinding domains; variants in these domains are critical to the function of the IRF6 gene [8,29,31]. In addition, the silico analysis to verify the effect on the protein conformation of IRF6, suggests that this mutation is potentially pathogenic.
More specifically, the wild-type residue interacts with the amino acid residue Alanine at position 86 of the polypeptide chain; the size difference observed in the mutant residue prevents it from being in the correct position to perform the hydrogen bonding possible in the wild type of residue. Only two mutations for position 90 of the protein chain encoded by the IRF6 gene are described in the literature, one of which is mutation c. A268G (p.Ser90Gly) as described by Kondo  Among the dental anomalies associated with VWS, the most common among patients is hypodontia [32]. In our study, individuals who presented dental anomalies, as tooth agenesis were in the mandibular arch, which is something already predicted in the literature. Syndromic agenesis generally appears associated with other manifestations such as microdontia and the upper lateral incisor is the most frequently affected tooth in the cleft area [33,34]. Considering the data shown in Table 2, it's possible to see the phenotypic variation of the cases presented despite carrying the same genetic mutation, suggesting that distinct genetic modifiers operate on the formation of clefts and the dental development.
Despite being one of the most studied syndromic forms of orofacial clefts, this study reveals how much remains to be discovered in terms of how genetic factors act underlying the phenotypic expression of VWS. In addition, we emphasize that families with this syndrome still go unnoticed in health systems. Observationally, many families in turn see congenital lower lip pits as a family trait and therefore fail to seek specialized care and appropriate treatment. Thus, it is also expected that this study will contribute in the future to the genetic counseling for these families and also will improve the knowledge and qualifications of health professionals who care for them.