|Year : 2020 | Volume
| Issue : 3 | Page : 176-179
Novel mutations with clinical variability and surgical experience in van der woude syndrome
Ahmet Cevdet Ceylan1, Figen Ozgur2, Ibrahim Vargel2
1 Department of Medical Genetics, Ankara City Hospital, Ankara Yıldırım Beyazıt University, Ankara, Turkey
2 Department of Plastic, Reconstructive, and Aesthetic Surgery, Faculty of Medicine, Hacetepe University, Ankara, Turkey
|Date of Submission||08-Jan-2020|
|Date of Acceptance||25-Mar-2020|
|Date of Web Publication||26-May-2020|
Prof. Ibrahim Vargel
Department of Plastic, Reconstructive, and Aesthetic Surgery, Faculty of Medicine, Hacetepe University, Ankara
Source of Support: None, Conflict of Interest: None
Introduction: Van der Woude syndrome (VWS) is characterized by cleft palate, cleft lip, and lower-lip pits. Interferon regulatory factor 6 (IRF6) gene mutations have been reported as the cause of VWS. Subjects and Methods: Full stories of the affected individuals were obtained, and a pedigree analysis that goes back three-generation pedigree was performed. The IRF6 gene Sanger sequence analysis was performed on 43 affected individuals with ages ranging between 1 month and 84 years. Results: Our report comprises the molecular diagnosis of 43 patients with VWS in six different families. The c.841-2A>C mutation and the c.881T>A mutation are novel and have not been reported before. These novel mutations affect exon 7 of the IRF6 gene. Conclusion: The c.841-2A>C mutation is a splice site mutation, which is less common in the IRF6 gene and helps us understand the genomic structure better. A novel c.881T>A mutation was identified in exon 7. Due to variable expression, the same mutation can present itself in different clinical manifestations. However, different mutations in the same gene can also be observed through different phenotypes. Therefore, the clinical manifestations of new mutations can lead us to a better understanding of the causes of the disease. In addition, knowing the molecular cause of the disease is also useful for the surgical interventions.
Keywords: Cleft lip palate and pit surgery, interferon regulatory factor 6, novel mutations, Van der Woude syndrome
|How to cite this article:|
Ceylan AC, Ozgur F, Vargel I. Novel mutations with clinical variability and surgical experience in van der woude syndrome. Turk J Plast Surg 2020;28:176-9
| Introduction|| |
Van der Woude syndrome (VWS) (Online Mendelian Inheritance in Man [OMIM] 119300) is an autosomal dominant clinical condition characterized by cleft palate, cleft lip, and lower-lip pits. VWS is the most common cause of syndromic cleft lip and palate, and it is responsible for 2% of all cleft lip and palate cases. The prevalence rate of cleft lip and palate in Turkey is approximately 0.1%, while the prevalence rate of VWS at birth is 1:35,000. VWS can make itself manifest a variety of phenotypic characteristics and can be observed in a number of individuals in the same family because of its high penetrance.
Interferon regulatory factor 6 (IRF6) gene mutations have been previously reported as the cause of VWS. The IRF6 protein, encoded by the IRF6 gene with 9 exons, has “DNA-binding” and “protein-binding” domains. The third and fourth exons encode the DNA binding domain, whereas the seventh-ninth exons encode the protein-binding domain. IRF6, a transcription factor, plays an important role in midline occlusion during the embryonic period; and more than 300 IRF6 gene mutations have been noted as the cause of the VWS. These mutations are often clustered in exons encoding the functional domains.
Pterygium and ankyloblepharon filiforme adnatum are the conditions that are sometimes diagnosed together with VWS, and they play an important role in the differential diagnosis of the VWS, as the mutations in the IRF6 gene are also associated with the popliteal pterygium syndrome (PPS; OMIM 119500), which shares its orofacial characteristics not only with the VWS but also with popliteal webs, syndactyl, and genital anomalies. However, genotype–phenotype correlation between the IRF6 gene and these mutations is not yet clarified. The prevalence of PPS at birth is 1:300,000. Although the mutations occur within the same gene, the prevalence rates differ.
If the pits on the lower lip are accompanied by additional anomalies such as pterygium and web, the protocol for the surgical treatment needs to be reconsidered and changed. Further, in VWS patients, velopharyngeal insufficiency, hypoplasia of facial midline, middle ear problems, and speech impairment are known problems that might need to be incorporated into the surgical treatment.
Herein, we report our findings after working with 43 VWS patients from six different families, present two novel mutations that have not been published previously, and discuss the surgical and follow-up problems we encountered with these patients.
| Subjects and Methods|| |
Approval of the study came from the Yıldırım Beyazıt University Ethical Committee (No. 26379996/127 dated May 7, 2018).
In addition to physical examination, full stories of the patients were obtained, and a pedigree analysis that goes back three-generation pedigree was performed. Patients were scanned to avoid environmental factors, such as folate antagonists. Available family members of some of the patients were also examined for VWS and PPS phenotypes.
Venous blood was collected from patients with VWS and from available family members for DNA sampling. Mutational analysis was performed using direct genomic sequencing. Primers were designed to cover the full exon and intron/exon boundaries for each exon individually, for exons 1–9 in IRF6 (NM_006147). Primer sequences are available upon request. Polymerase chain reactions have been performed to amplify each of the nine exons individually from the genomic DNA. PCR products were directly sequenced using ABI Prism 3130 Automated Sequencer (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed and then compared to consensus sequences using an NCBI BLAST search (http://blast.ncbi.nlm.nih.gov).
| Results|| |
There were three patients with VWS in three generations of the family A [Figure 1]. Unilateral cleft lip and lower-lip pit were seen in the first and second generations and incomplete cleft palate in the third generation. We performed IRF6 gene sequencing on the patients and observed a novel transition from A to Cat nucleotide substitution affecting the obligatory acceptor splice site of exon 7 (c.841-2A>C). This mutation co-segregated with the status of the affected patients, while the remaining unaffected people showed wild-type sequence. Bioinformatic analyses reveal that the correct splicing of IRF6 mRNA2 is affected by this mutation. Particularly, the c.841-2A>C mutation disrupts the binding to the acceptor splice domain [Table 1].
|Table 1: The type, location, and effect of the mutation in the families in which the interferon regulatory factor 6 mutation is detected|
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There were three patients with VWS in two generations of the family B. Only lower-lip pit was seen in the first generation; whereas unilateral cleft lip, palate, nose deformity, and lower-lip pit were the anomalies observed in the second generation [Figure 1]. Cleft lip repair, cleft palate repair, and pit excision surgeries were performed on the patients sequentially. In the final stage, cleft lip nose deformity was treated, and surgical procedures were added to the treatment for velopharyngeal insufficiency. [Figure 2] shows the patient's condition before and after sequential operations. IRF6 gene sequencing was performed to determine the genetic etiology of the disease. As a result, a novel missense c.881T>A change located in exon 7, resulting in a p. Leu294Gln substitution was identified. The mutation co-segregated with the disease in the family.
|Figure 2: A patient's condition before and after sequential operations (family B). (a) Before operations at 2 months; (b) after cleft lip operation at age 1; (c): after pit surgery at age 6; (d) patient's control at age 10|
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There were 16 patients in four generations of the family C. Unilateral cleft lip, cleft palate, and lower-lip pit were observed in all generations, and one patient in the fourth generation had only lower-lip pit. Seven out of 16 patients had cleft lips, and 12 out of 16 patients had cleft palates [Figure 1]. Both cleft lips and cleft palates were observed in four patients. This was the family where phenotypic variations were most apparent. IRF6 gene sequencing showed a missense c.332A>G change located in exon 4, resulting in a p. Tyr111Cys substitution [Figure 3]. This mutation has previously been discussed as one of the causes of the disease. The mutation co-segregated with the disease in the family.
|Figure 3: The graph showing the exons of the interferon regulatory factor 6 gene. Top of the line shows the mutations detected in the families in this study. The lower half shows the number of mutations that have been reported before|
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There were 10 patients in four generations of the family D. Unilateral cleft lip, cleft palate, and lower-lip pit were seen in all generations, and one patient in the fourth generation had bilateral cleft lip and ankyloblepharon filiforme adnatum. IRF6 gene sequencing showed a missense c.16C>T change located in exon 3, resulting in a p. Arg6Cys substitution [Table 1]. This mutation was previously reported as pathogenic. The mutation co-segregated with the disease in the family.
There were two patients in two generations of the family E. Unilateral cleft lip, palate, and lower-lip pit were seen in both patients. In the proband of the family E, we identified a nonsense c.1271C>G mutation (p. Ser424X). The mutation co-segregated with her father. This mutation has previously been published as one of the causes of the disease.
There were nine patients in four generations of the family F. Unilateral cleft lip, cleft palate, and lower-lip pit were seen in all generations. IRF6 gene sequencing showed a c. 1289_1297 delinsATAACATCG change located in exon 9 and deleted Asp, Asn, and Ile [Table 1]. This deletion has previously been found as pathogenic. The mutation co-segregated with the disease in the family.
Surgical treatment was performed for 12 of 43 patients with stylet in the tractus method for lip pits. In this method, it was done after checking the tract with the help of a stylet.
| Discussion|| |
VWS (OMIM 119300) is a rare autosomal dominant development malformation characterized by lip pits and/or sinuses or conical elevation of lower lip associated with cleft lip and or palate. Mutations in the transcription factor IRF6 gene have been observed in individuals with the VWS., Up to now, approximately 300 different mutations have been identified in the IRF6 gene [Figure 2]. In this article, we present our findings after working with 43 patients with VWS from six different families and argue that two of these families exhibit novel mutations that have not reported before [Table 1]. A novel c.841-2A>C mutation was identified in family A, while a novel c.881T>A mutation was identified in family B. These mutations will provide a better understanding of the phenotypic effect of exon 7 mutations, since, due to variable expression, the same mutation can manifest itself in different clinical conditions. However, different mutations in the same gene can present itself with different phenotypes. Therefore, the clinical manifestations of new mutations can help us understand the causes of the disease better and thus contribute to the genetic counseling.
Mutations are more common in the DNA-binding domain (3th and 4th exons of the gene) and in the protein-binding domain (7th, 8th, and 9th exons of the gene), most of which are missense/nonsense mutations [Figure 3]. Although splice site mutations are not as common, they are nonetheless important as they help us understand the genome structure better. c.841-2A>C is a novel mutation in the splice site region of the seventh exon in our family B. It is listed as a “disease-causing” mutation in several silico databases; however, the seventh pre-exon splice site mutation, which we discuss in this article, has not been reported before. Our observations show us that phenotypic variability is less common in patients from family A and family B. As exon 7 plays a significant role in the “protein-binding domain” of the IRF6 gene, it is considered to be responsible for displaying all the phenotypic features of the syndrome. Accordingly, different exon 7 mutations have been discussed in the literature as well.,, It is important to note that when segregated within the same family, the exon 7 mutations of the IRF6 gene reveal similar findings regarding VWS. However, these same mutations can exhibit different phenotypes in different families. These observations should be kept in mind during genetic counseling.
Phenotypic variations in VWS were seen in our families. These phenotypic variations have also been considered while planning the surgeries. In VWS patients, in addition to the upper lip palate deformities, anomalies in the lower lip and drainage can also be observed. This makes the lower lips more visible and everted and may be retrusive in the development of maxilla in early childhood. Complication regarding maxillary retrusion may require surgery in the early childhood period.
There is no difference in surgical lip palate treatment between nonsyndromic lip palate patients and VWS patients. The techniques used to repair the lip and the palate depend on the experience of the surgeon and the clinical findings regarding the patient. However, surgical instrumentation, preoperative and postoperative care, and anesthesia developments have been performed in the lip repair for up to 3 months and the palate repair for 9 months. Surgical treatment is not necessary if there is no derange in the lower-lip pits. When starting the sinus/pit surgery, to remove the tractus completely, the surgery should be performed after controlling the tractus with the help of a stile. If this pit surgery is done before the age of 2, the lower-lip length is shortened. During cleft lip palate surgery, the upper lip distance also decreases, which leads to a microstomic mouth appearance. Therefore, it is thought to be better to perform the pit surgery around 5–6 years of age. If the entire sinus tract cannot be removed, it may grow into a cyst and bloom inside. Therefore, the surgery needs to be performed with utmost diligence and care.
Twelve of 43 patients in our study needed surgery for lip pits. No deformities were observed after the operation. Stylet in the tractus method made the surgical process easier and reduced the number of postsurgery complications. Different surgical approaches may be necessary if VWS patients also have pterygium and ankyloblepharon filiforme adnatum. The opening of the synapses to prevent the appearance of the exquisite scar is the basic surgical principle.
| Conclusion|| |
Our findings include the molecular diagnosis of 43 patients with VWS syndrome in six different families. The c.841-2A>C mutation and the c.881T>A mutations were novel and have not reported before. The phenotypic effect of these novel mutations is discussed in this study, and the phenotypic diversity of VWS in these patients is also shown. The clinical effect of these new mutations of IRF6 at exon 7 gave us a better understanding for the causes of this disease.
We would like to thank Prof. Nurten Akarsu for her expert advice, continuous support, and her laboratory facilities.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Stuppia L, Capogreco M, Marzo G, La Rovere D, Antonucci I, Gatta V, et al
. Genetics of syndromic and nonsyndromic cleft lip and palate. J Craniofac Surg 2011;22:1722-6.
Vélez A, Alamillos FJ, Deán A, Ruiz-Masera JJ. Congenital lower lip pits (Van der Woude syndrome). J Am Acad Dermatol 1995;32:520-1.
Kondo S, Schutte BC, Richardson RJ, Bjork BC, Knight AS, Watanabe Y, et al
. Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. Nat Genet 2002;32:285-9.
de Lima RL, Hoper SA, Ghassibe M, Cooper ME, Rorick NK, Kondo S, et al
. Prevalence and nonrandom distribution of exonic mutations in interferon regulatory factor 6 in 307 families with Van der Woude syndrome and 37 families with popliteal pterygium syndrome. Genet Med 2009;11:241-7.
Wang X, Liu J, Zhang H, Xiao M, Li J, Yang C, et al
. Novel mutations in the IRF6 gene for Van der Woude syndrome. Hum Genet 2003;113:382-6.
Peyrard-Janvid M, Pegelow M, Koillinen H, Larsson C, Fransson I, Rautio J, et al
. Novel and de novo
mutations of the IRF6 gene detected in patients with Van der Woude or popliteal pterygium syndrome. Eur J Hum Genet 2005;13:1261-7.
Leslie EJ, Standley J, Compton J, Bale S, Schutte BC, Murray JC. Comparative analysis of IRF6 variants in families with Van der Woude syndrome and popliteal pterygium syndrome using public whole-exome databases. Genet Med 2013;15:338-44.
Reardon JB, Brustowicz KA, Marrinan EM, Mulliken JB, Padwa BL. anatomic severity, midfacial growth, and speech outcomes in van der woude/popliteal pterygium syndromes compared to nonsyndromic cleft lip/palate. Cleft Palate Craniofac J 2015;52:676-81.
Richardson S, Khandeparker RV. Management of lip pits in Van der Woude syndrome: A clinical classification with difficulty index. J Oral Maxillofac Surg 2016;74:1849.e1-1849.e10.
Ioannides A, Georgakarakos ND. Management of ankyloblepharon filiforme adnatum. Eye (Lond) 2011;25:823.
[Figure 1], [Figure 2], [Figure 3]