Mutations in Van Der Woude Families From Ethiopia

Abstract

Background:

Van der Woude syndrome (VWS) is the most common syndromic orofacial cleft which accounts for approximately 2% of all cleft lip (CL) and/or palate cases. It is characterized by the presence of lower lip pits, in addition to CL, CL with or without cleft palate, cleft palate only, and hypodontia. It is inherited as an autosomal-dominant trait with almost complete penetrance but variable expressivity, and different variants in IRF6 gene have been reported in different populations around the world including African populations (Ethiopian, Ghanaian, and Nigerian).

Methods and Findings:

The authors investigated the role of IRF6 in Ethiopian families with VWS. The DNA of 7 families with VWS from Ethiopia were screened by Sanger sequencing. The authors screened all 9 exons of IRF6 and found a novel missense variant in exon 4 (p. Gly65Glu). This variant was predicted to be deleterious/probably damaging by Sift and PolyPhen, respectively. The IRF6 variant (p. Gly65Glu) segregates in the family since it was identified in the father and a sibling.

Conclusion:

Several of the individuals with lower lip pits in this study did not seek treatment. This is due to lack of awareness about the significance of this minor looking deformity and its consequences, and availability of treatment for birth defects. Therefore, it is important to educate families. Finally, screening for novel variants in known genes has a role in counseling and prenatal diagnosis for high-risk families.

Orofacial clefts are the commonest craniofacial birth defects and one of the most common congenital abnormalities in humans with a prevalence of 1 in 500 to 1 in 2500 births, depending on geographic origin, race, and socioeconomic background.^1–4 Orofacial clefts can occur as isolated in 70% of the cases and the rest 30% can be described as syndromic, where the phenotypes include other developmental or morphological abnormalities.⁵ Van der Woude syndrome (VWS) (OMIM:119300) is the commonest form of syndromic oral cleft that affects 2% of all cases. It is characterized by congenital lower lip pits, cleft lip (CL), CL with or without cleft palate, cleft palate only, and sometimes, hypodontia.⁶ It is inherited as an autosomal-dominant trait with high penetrance (96.7%) with variable expression.⁷

It was first reported by Demarquay in 1845. However, Van der Woude described this syndrome in detail in 1954.⁸ The main features of VWS are the occurrence of CL and palate and cleft palate only within the same family. The typical lower lip pits commonly occur on either side of the midline, they can also be unilateral. Single median or paramedian lip pits are considered an incomplete expression of the trait.⁹

We previously reported studies of Ethiopian and Nigerian families with VWS and Popliteal Pterygium Syndrome (OMIM:119500).¹⁰ In the previous study, we found and reported a novel nonsense variant in exon 4 (p.Lys66X), a novel splice-site variant in exon 4 (p.Pro126Pro), a novel missense variant in exon 4 (p.Phe230Leu), and a known missense variant in exon 7 (p.Leu251Pro). In the Popliteal Pterygium Syndrome family we found a known missense variant in exon 4 (p.Arg84His). The current study was designed to identify additional variants in VWS families from Ethiopia.

METHODS

This study is part of the larger Ethiopian genetics study entitled “Investigating the role of Genetics and environmental factors in the occurrence of orofacial clefts in the Ethiopian population” and the saliva samples collected for this study were used for the current study. Ethical approval was obtained from the Institutional Review Board of the College of Health Sciences Addis Ababa University and has been renewed every year. In addition, we obtained written informed consent from the recruited families and collected saliva samples from both affected probands and parents. Saliva samples from the parents and children were collected using Oragene collection kit (www.dnagenotek.com). For the children, we used saliva sponges to collect saliva sublingually and in the buccal sulcus and these sponges were then placed into the Oragene collection kits. After obtaining permission we shipped the saliva samples to the Butali Laboratory in Iowa, for DNA processing and sequencing. The protocol for DNA processing is available on the Murray

Lab website (http://genetics.uiowa.edu/protocols.php). The DNA samples concentration was measured using a Qubit 2.0 Fluorometer (http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Qubit.html) and a 4 ng/ lL was used for Sanger sequencing.

SEQUENCING

The primer sets that were designed by De Lima et al in 2009 were used to sequence all the exons in the IRF6 gene (NM_006147.2). This sequencing method was reported by Butali et al in 2013.

The samples from the mother and father were sequenced to confirm variants that are de novo or segregating in the family. We then compared the variants found in this study to variants that are present in the African population and other populations in the 1000 genome database (the genomes of 1000 volunteers from multiple populations around the world). Variants not found in the control databases or reported in literatures are referred to as novel. All coding mutations were submitted into the locus-specific database (http://www.centralmutations.org/LsdbAdd.php). The functional effects of these variants on the protein were predicted using bioinformatics tools such as polyphen (http://genetics.bwh.harvar-d.edu/pph2/) (Adzhubei et al 2010), SIFT (http://sift.jcvi.org/) (Kumar et al 2009), and HOPE (http://www.cmbi.ru.nl/hope) (Ven-selaar et al 2010).

RESULTS

In this present study, we identified a novel missense variant in exon 4, which resulted in the substitution of glycine for glutamate at position 65 (p. Gly65Glu) c.194G>A.

Supplemental Table 1, http://links.lww.com/SCS/D219 shows variants found in IRF6 and predicted functional effect on the protein. Figure 1 shows the pedigree of the family with all individuals with lower lip pits. Figure 2 shows the chromatogram with nucleotide change from G to A in the proband (IV-1), his siblings (IV-2 and IV-3), and both parent (III-9 and III-10).

FIGURE 1.

Pedigree of the family with Van der Woude syndrome. All the individuals with shaded boxes have lower lip pits and the star shows that the individual has features of Popliteal Pterygium Syndrome (PPS). Signifies the individuals whose DNA were collected for sequencing in the family.

FIGURE 2.

The chromatogram of the proband (IV-1), his siblings (IV-2 and IV-3), and both parents (III-9 and III-10). The chromatogram of IV-1, IV-2, IV-3, and III-9 showed a nucleotide change from G to A leading to amino acid change from glycine for glutamate at position 65, while the chromatogram of the mother (III-10) showed no change.

The torsion angles for this residue are unusual. Only glycine is flexible enough to make these torsion angles, variant into another residue will force the local backbone into an incorrect conformation and will disturb the local structure (Fig. 3). Thus, a change to another amino acid might abolish this flexibility necessary for the protein functions.

FIGURE 3.

The protein is in gray color, the side chains of both the wild-type and the mutant residue are shown and colored green and red, respectively.

Glycine is highly conserved in this location and found in all almost all the species except for Xenopus-tropicalis that has a serine residue. This observation underscores the fact that the variant can be damaging to the protein.

The proband’s great grandmother (I.2) had lower lip pits. She has 5 sons and 5 daughters (II.1, 2,3,5,6). Four of her sons have lower lip pits (II-1, II-2, II-3, and II-6) and 1 of her daughters has lower lip pits (II-7). One of those who has lower lip pits is the grandfather (II-3) of the proband (IV-1). He has 9 sons and 6 daughters. Seven of his sons (III-1, III-2, III-3, III-5, III-7, and 8) and 2 of his daughters (III-13 and III-15) had lower lip pits. One of those who has lower lip pits is the probands father (III-9). The probands father has 3 sons (IV-1, IV-2, and IV-3) and they all have lower lip pits and 2 of them (IV-2, IV-3) have isolated cleft palate and one of these 2 (IV-2) is a case of popliteal pterygium syndrome (has lower lip pits, webbed knees, and isolated cleft palate).

The penetrance for the lower lip pits is complete whereas for cleft palate it is incomplete. The lower lip pits in some of the victims are discharging saliva but none of them sought treatment for their lower lip pits.

The other variant identified in the other individuals with VWS was a known variant (Supplemental Table 1, http://links.lww.com/SCS/D219). All variants identified in this study are openly available in the locus-specific database at http://www.centralmutations.org/LsdbAdd.php.

DISCUSSION

In the current Ethiopian study, we screened for novel variants in IRF6 in families with VWS from Ethiopia and found a novel missense variant p. Gly65Glu. This variant was found in exon 4 and was predicted to be probably damaging/damaging by Sift and PolyPhen. Currently amino acid substitutions account for approximately half of the known gene lesions responsible for human inherited diseases.¹¹

Hope analysis reveals that the variant residue is bigger than the wild-type residue, and the wild-type residue (glycine) charge is neutral and the variant residue charge (glutamate) is negative. The wild-type residue is more hydrophobic than the variant residue and charge introduced by the variant at this position could cause repulsion between the variant residue and neighboring residues. The residue is located on the surface of the protein and variant of this residue can disturb interactions with other molecules or other parts of the protein. This variant was predicted to be probably damaging/damaging by Sift and Polyphen.

Several studies have reported that variants in IRF6 are non-randomly distributed and mainly in the DNA-binding domain (Exon 3 and 4) and protein-binding domain (Exon 5–9), therefore emphasizing the importance of these domains.^12–15

The phenotype in most families consists of lower lip pits only. Lower lip pits can be surgically excised for cosmetic reasons or to alleviate discomfort. Surgical excision of the lower lip pits will improve the lower lip pits appearance and reduce the mucous discharge.¹⁶ In this study none of the victims with only lower lip pits sought treatment even though in some of them the pits were discharging and causing discomfort. This is likely due to lack of awareness.

Even though genetic counseling and examination is highly suggested for families with a history of van der Woude syndrome no one of the families in this study received genetic counseling. Genetic counselling is not part of the health care system in many developing countries. It should be part of the health care system of all countries. A through collection of family history is very important prior to provision of genetic counselling.¹⁷

CONCLUSION

This study, just like our previous studies, confirms the need for screening of novel variants in known genes in different populations of the world as it has a role in counseling and prenatal diagnosis for high-risk families. We also found out that people in many parts of the developing world still lack basic information on the treatability of birth defects. Therefore, we recommend strengthening public education in developing world. Establishing birth defect registry also will help in creating understanding among the community and health care workers.