Congenital lower lip sinuses are rare developmental anomalies, most commonly associated with Van der Woude syndrome. Isolated bilateral nonsyndromic presentations are extremely uncommon.[1,2] A 13-year-old boy presented with bilateral lower lip pits since birth, associated with intermittent mucous discharge. Clinical examination showed two symmetric pits near the midline at the vermilion border, without cleft lip, cleft palate, dental anomalies, limb defects, or family history [Figure 1a]. Magnetic resonance imaging of the face showed no deep extension or associated craniofacial abnormalities. Bilateral excision was performed using rhomboid incisions [Figure 1b]. Each sinus tract was superficial, blind-ending, and non-communicating [Figure 1c]. Histopathology confirmed that the sinus tracts were lined by stratified squamous epithelium with minor salivary glands. The postoperative course was uneventful, with excellent cosmetic outcome and no recurrence at 6 months [Figure 1d].Figure 1: (a) Preoperative photograph showing bilateral congenital lower lip sinuses with mucous discharge. (b) Intraoperative marking with rhomboid incisions surrounding the pits. (c) Complete excision of the sinus tracts along with the surrounding abnormal tissue. (d) Postoperative photograph at 6-month follow-up demonstrating excellent cosmetic healing without recurrenceDeclaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Orofacial clefts (OFCs) are one of the most common structural birth defects, with the prevalence of OFC varying across populations, and studies on the causes of OFCs in diverse populations are necessary, but still limited. We analyzed whole genome sequencing data on 419 parent–child trios from the Philippines, a population with a particularly high rate of OFC. To identify novel genes for OFCs, we studied both common variation and de novo variants (DNVs). We identified a significant enrichment in both loss-of-function (N = 62; P = 8.34 × 10−5) and protein-altering DNVs (N = 394; P = 1.49 × 10−7) among OFC probands. Among the genes individually enriched for DNVs was GRHL2 (P = 6.60 × 10−6), where there were two DNVs, a stop-gain and a frameshift deletion. We then queried OFC trios from other cohorts in the Gabriella Miller Kids First program (total N = 1254) and GeneMatcher and identified an 89 kb de novo deletion in GRHL2 and a de novo 8q22.3 microdeletion with one breakpoint in GRHL2. Additionally, within the common variant analyses we found significant gene x gene interactions with GRHL2. GRHL2 is a conserved transcription factor involved in embryonic development, with truncating mutations causing autosomal dominant progressive hearing loss and missense variants causing autosomal recessive ectodermal dysplasia. Heterozygous variation in its homolog, GRHL3, causes Van der Woude syndrome and isolated cleft palate. Additionally, mice deficient for either Grhl2 or Grhl3 have craniofacial anomalies, including facial and palatal clefts, strongly supporting GRHL2 as a risk locus for OFCs.

Rare variants in PRKCI cause Van der Woude syndrome and other features of peridermopathy.

Van der Woude Syndrome (VWS) is an autosomal dominant condition characterized by a cleft lip and/or palate along with pits on the lower lip. These pits are associated with salivary glands, leading to clinical manifestations such as secretions and, in some cases, infections, which may influence the surgical approach. The goal of treatment is to improve cosmetic appearance and to repair the pits, thereby preventing recurrent infections. This study aims to emphasize the prevalence and significance of this syndrome among individuals with cleft lip and/or palate. In this study, the frequency and epidemiological characteristics of VWS among patients with cleft lip and/or palate were examined through a single-center retrospective analysis conducted between 2016 and 2021. A total of 1633 patients were included in this study, of whom 21 were identified as having VWS. The prevalence of VWS among patients with cleft lip and/or palate was found to be 1.3%. The average number of surgeries was 2.1 for patients without VWS and 2.8 for VWS patients. The female-to-male ratio among VWS patients was 1.6. The most common associated condition was bilateral cleft lip and palate. Secondary lip surgeries were more frequent in patients with VWS than without VWS. Since the repair of the pits on the lip is also necessary in VWS, surgical treatment is more complex compared with patients with cleft lip and palate alone. Achieving satisfactory functional and cosmetic outcomes in patients with Van der Woude Syndrome requires more surgical interventions than in patients without VWS.

Grainyhead-like transcription factor 3 (GRHL3) directs surface ectoderm differentiation under the control of the canonical Wnt/β-catenin pathway. However, the molecular mechanisms that control nuclear GRHL3 expression through β-catenin are not fully understood. Here, we show that the essential for mitotic growth 1 (EMG1) protein constitutes a protein complex with GRHL3, and that EMG1 is required for correct nuclear localization of GRHL3, and for activation of the canonical Wnt signaling pathway. Conditional knockout mutation of Emg1 in the GRHL3-positive surface ectoderm causes neural tube defects at the level of the spinal cord, i.e. spina bifida. Additionally, the severity of compound mutant phenotypes of Emg1 and Grhl3 indicates that they interact genetically in neurulation and palate development. These lines of evidence demonstrate that EMG1 cooperates with GRHL3 in β-catenin-mediated surface ectoderm differentiation. Since the EMG1 mutation causes Bowen-Conradi syndrome and the GRHL3 mutation causes Van der Woude syndrome 2, both of which are associated with neural tube dysplasia and cleft palate, our study will help to improve our understanding of the pathogenic mechanisms of these two human genetic diseases.

This case report describes a rare occurrence of non-syndromic lower lip pits. The 16-year-old female patient had bilateral pits on the lower lip vermilion, without any other associated abnormalities, which sets this case apart from the syndromic lip pits seen in Van der Woude syndrome. The report outlines the clinical features, diagnostic evaluation, and chosen treatment approach, emphasizing the importance of recognizing and distinguishing isolated lip pits from syndromic presentations.

Van der Woude syndrome is synonymous with a severe phenotypic expression of cleft lip and palate. Traditionally, the severity of unilateral cleft lip has been associated to the width of a cleft. The authors examine the presentation of the unilateral cleft lip in Van der Woude syndrome, with a focus on the lateral lip element. This is a retrospective, observational study of patients with a unilateral cleft lip and palate, a diagnosis of Van der Woude syndrome and preoperative clinical images treated at The Royal Children's Hospital, Melbourne, Australia; Royal Victoria Infirmary, Newcastle upon Tyne, UK; or Menino Jesus Municipal Hospital, São Paulo, Brazil. Patient images were classified into different levels of lateral element hypoplasia according to the Melbourne Classification. Eight patients with Van der Woude syndrome and unilateral cleft lip and palate were identified across the 3 hospital sites. All 8 patients had left-sided unilateral cleft lip and palate and Melbourne Classification type 2 tissue hypoplasia, with shortened vertical lip and red vermillion height. This cohort of children with unilateral cleft lip and palate in the setting of Van der Woude syndrome presented with a left-sided preponderance and type 2 hypoplasia of the lateral lip element. It is important to observe the presence of hypoplasia in the lateral lip element when attempting to balance any cleft lip repair. Technical nuances are presented, which may assist in improving appearance outcomes.

Van der Woude syndrome (VWS) is an autosomal dominant disorder characterized by lower lip pits and orofacial clefts (OFCs). With a prevalence of approximately 1 in 35,000 live births, it is the most common form of syndromic clefting and may account for ~2% of all OFCs. The majority of VWS is attributed to genetic variants in IRF6 (~70%) or GRHL3 (~5%), leaving up to 25% of individuals with VWS without a molecular diagnosis. Both IRF6 and GRHL3 function in a transcriptional regulatory network governing differentiation of periderm, a single layer of epithelial cells that prevents pathological adhesions during palatogenesis. Disruption of this layer results in a spectrum of phenotypes ranging from lip pits and OFCs to severe pterygia and other congenital anomalies that can be incompatible with life. Understanding the mechanisms of peridermopathies is vital in improving health outcomes for affected individuals. We reasoned that genes encoding additional members of the periderm gene regulatory network, including kinases acting upstream of IRF6 (i.e., atypical protein kinase C family members, RIPK4, and CHUK), are candidates to harbor variants resulting in VWS. Consistent with this prediction, we identified 6 de novo variants (DNs) and 11 rare variants in PRKCI, an atypical protein kinase C, in 17 individuals with clinical features consistent with syndromic OFCs and peridermopathies. Of the identified DNs, 4 were identical p.(Asn383Ser) variants in unrelated individuals with syndromic OFCs, indicating a likely hotspot mutation. We also performed functional validation of 12 variants using the enveloping layer in zebrafish embryos, a structure analogous to the periderm. Three patient-specific alleles (p.Arg130His, p.(Asn383Ser), and p.Leu385Phe) were confirmed to be loss-of-function variants. In summary, we identified PRKCI as a novel causal gene for VWS and syndromic OFC with other features of peridermopathies.

Rare heterozygous variants in IRF6 (interferon regulatoryfactor-6) gene cause van der Woude syndrome 1 (VWS1) or Popliteal Pterygiumsyndrome, two forms of syndromic cleft lip/palate (CLP) that present with avariety of congenital malformations due to impairment ectodermal homeostasis.These malformations include, in addition to CLP, lip pits, pterygia, andintraoral and eyelid fibrous bands. Amniotic band sequence (ABS) is a rarecondition of unknown genetic etiology that involves a range of congenitalanomalies caused by the entanglement of fibrous bands, which disrupt fetal bodyparts. However, ABS co-occurs with CLP and other malformations that cannot beexplained by this mechanism. Therefore, investigating the genetic relationshipbetween ABS and CLP may provide clues regardind the genes involved in theseconditions. Here, we report a case of a girl diagnosed with VWS1, autism,intellectual disability, and congenital right limb anomalies compatible withABS. Molecular analysis revealed a novel, rare heterozygous missense variant inIRF6 (NM_006147.3:c.970T>C) located in exon 7, inheritedfrom her father. This variant results in the replacement of serine by proline atposition 324 of the IRF6 protein with potentially deleterious effects. Thisreport expands the mutational landscape of IRF6 and providesfurther support for a possible link between the genetics of CLP and ABS.

Orofacial clefts (OC) are a congenital anomaly typically classified as syndromic or non-syndromic. Among the syndromic cases, Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are primarily caused by pathogenic variants in IRF6, which encodes a transcriptional factor essential for orofacial development. Our aim is to identify and characterize IRF6 variants in two syndromic OC cases and assess their structural and functional consequences through computational modeling. Sequencing of IRF6 exons 3, 4, 7, and 9 was conducted in one proband with VWS and one proband with PPS, as well as their parents. Structural impacts were measured through comparative modeling based on the IRF4-DNA complex, followed by molecular dynamics simulations. Binding free energies and protein-DNA interactions were assessed using MM/GBSA calculations and hydrogen bond occupancy analysis. We identified two pathogenic variants: NP_006138.1:p.(Ala16Val) in a VWS proband and NP_006138.1:p.(Arg84Cys) in a PPS proband, both confirmed as de novo. Variants were classified as pathogenic by the American College of Medical Genetics and Genomics criteria, and by most pathogenicity predictors. Molecular dynamics simulations showed that NP_006138.1:p.(Ala16Val) induces long-range allosteric effects with increased structural fluctuations, while NP_006138.1:p.(Arg84Cys) directly disrupts critical DNA-binding interactions. MM/GBSA analysis demonstrated reduced DNA-binding affinity for both variants, with NP_006138.1:p.(Arg84Cys) showing the most severe electrostatic disruption. Our computational analyses suggest that pathogenic IRF6 variants may impact DNA binding through distinct molecular mechanisms: NP_006138.1:p.(Ala16Val) potentially through allosteric conformational changes and NP_006138.1:p.(Arg84Cys) likely through direct disruption of the protein-DNA interface.

Background: Van der Woude syndrome (VWS) is a rare developmental malformation, characterized by pits in the lower lip. Van der Woude syndrome is an autosomal dominant craniofacial syndrome with various expression: lower lip pits, cleft lip with or without cleft palate, syngnathia, hypoodontia, and ankyloglossia. Extra-oral abnormalities findings can be found: syndactily, corpus callosum dysgenesis, megacolon, ventricular septal defect and genital abnormality. Methods: We reported a case of 5-month-old male with rare expression of VWS: bilateral cleft lip and palate, syngnathia, lower lip pits, ptosis of upper left eyelid and macropenis. Results: We perform surgery to release the fibrous band to achieve satisfactory maximum mouth opening. Next we perform cheiloplasty and lower lip pit removal. Conclusion: Proper surgical intervention in VWS patients can improve feeding and prevent further temporomandibular complications. Careful examination of patients with cleft lip and lower lip pit should be done to avoid misdiagnosis.

ObjectiveVan der Woude Syndrome (VWS) presents with combinations of lip pits (LP) and cleft lip and/or cleft palate (CL/P, CPO). VWS phenotypic heterogeneity even amongst relatives, suggests that epigenetic factors may act as modifiers. IRF6, causal for 70% of VWS cases, and TP63 interact in a regulatory loop coordinating epithelial proliferation and differentiation in palatogenesis. We hypothesize that differential DNA methylation within IRF6 and TP63 regulatory regions underlie VWS phenotypic discordance.MethodsDNA methylation of CpG sites in IRF6 and TP63 promoters and in an IRF6 enhancer element was compared amongst blood or saliva DNA samples of 78 unrelated cases. Analyses were done separately for blood and saliva, within each sex and in combination, and to address cleft type (CL/P ± LP vs. CPO ± LP) and phenotypic severity (any cleft + LP vs. any cleft only).ResultsFor cleft type, blood samples showed higher IRF6 and TP63 promoter methylation on males with CPO ± LP compared to CL/P ± LP and on individuals with CPO ± LP compared to those with CL/P ± LP, respectively. Saliva samples showed higher IRF6 enhancer methylation on individuals with CPO ± LP compared to CL/P ± LP and contrary to above, lower TP63 promoter methylation on CPO ± LP compared to CL/P ± LP. For phenotypic severity, blood samples showed no differences; however, saliva samples showed higher IRF6 promoter methylation in individuals with any cleft + LP compared to those without lip pits.ConclusionWe observed differential methylation in IRF6 and TP63 regulatory regions associated with cleft type and phenotypic severity, indicating that epigenetic changes in IRF6 and TP63 can contribute to phenotypic heterogeneity in VWS.

ABSTRACT Context: Craniofacial anomalies (CFAs) are rare congenital anomalies that have a profound impact on social acceptance. Most of the syndromic CFAs are genetic in origin and are the result of alteration in single or multiple genes inherited from parents or de novo. Aims: The main aim of this study was to explore the phenotypic variations and the mutation profiles in the various CFA. Subjects and Methods: This was a retrospective study where records of 20 patients were obtained from electronic medical records for analysis. The numbers provided do not accurately reflect the true prevalence as they only encompass cases referred to our department for testing. Results: The median age in this cohort was 1 year with males 60% and females 40%. Out of 20 cases, 8 cases were classified as CFA-associated craniosynostosis and 12 with orofacial clefts. Disease-causing genes identified were FGFR2, RTTN, ASXL3, IRF6, TP63, POLR1D, TCOF1, KMT2D, KDM6A, LARP1, and C5orf42. Most of these craniofacial syndromes were predominantly autosomal dominant, sporadic, and de novo. We had three autosomal recessive cases (RTTN, LARP1, and C5orf42) and one X-linked dominant case (KDM6A). Two novel variants were identified, one for Van der Woude syndrome and another for Crouzon’s syndrome. Conclusions: Although these syndromes are rare in occurrence, the present study provides a detailed phenotypic spectrum and causative mutation in various CFAs in Indian patients. This is crucial for understanding the genetic basis of these conditions and can potentially lead to advancements in diagnosis, treatment, and appropriate genetic counseling.

Congenital lip pits are characterized by sinuses or fistulas in the lips that can occur in isolation or as part of a genetic disorder. A 6-year-old girl with a right upper lip lesion present at birth presented with recurrent swelling and occasional erythema. Examination revealed a mildly swollen punctum at the right upper wet/dry vermillion with expressible serous drainage. There were no other phenotypic or cognitive concerns. The lesion was surgically excised using vertical wedge resection. The postoperative course showed well well-healed incision. The pathology report confirmed a lip pit. The family was referred to genetics for further evaluation. Van der Woude syndrome (VWS) is a genetic disorder associated with abnormal development of the paramedian lip. Most congenital lip pits are primarily found on the lower lips, with paramedian lip pits being the most common. Upper lateral lip pits with or without accompanying lip pits are considerably rarer. Though VWS is commonly associated with mutations in the interferon regulatory factor 6 or grainyhead-like protein 3 genes, ~25% of affected individuals lack an identified genetic etiology. A high index of suspicion for VWS is warranted if lip pits are present in the absence of other phenotypic abnormalities and should prompt genetic testing for interferon regulatory factor 6 and grainyhead-like protein 3 mutations. Multidisciplinary teams should consider patient self-esteem, quality of life, and potential family planning when deciding on surgical intervention for lip pits. Surgical management of pits should entail tissue-preserving techniques such as vertical wedge resection and inverted T-lip reduction to prevent whistle-lip deformity.

Several mutations in the IRF6 gene have been identified as a causative link to VWS. In this investigation, whole-exome sequencing (WES) and Sanger sequencing of a three-generation pedigree with an autosomal-dominant inheritance pattern affected by VWS identified a unique stop-gain mutation-c.748C>T:p.R250X-in the IRF6 gene that co-segregated exclusively with the disease phenotype. Immunofluorescence analysis revealed that the IRF6-p.R250X mutation predominantly shifted its localization from the nucleus to the cytoplasm. WES and protein interaction analyses were conducted to understand this mutation's role in the pathogenesis of VWS. Using LC-MS/MS, we found that this mutation led to a reduction in the binding of IRF6 to histone modification-associated proteins (NAA10, SNRPN, NAP1L1). Furthermore, RNA-seq results show that the mutation resulted in a downregulation of TGFβ2-AS1 expression. The findings highlight the mutation's influence on TGFβ2-AS1 and its subsequent effects on the phosphorylation of SMAD2/3, which are critical in maxillofacial development, particularly the palate. These insights contribute to a deeper understanding of VWS's molecular underpinnings and might inform future therapeutic strategies.

Treating lower lip fistulas in Van der Woude syndrome using inverted‐T lip reduction: An experience

Hospitalizations from Birth to 28 Years in a Population Cohort of Individuals Born with Five Rare Craniofacial Anomalies in Western Australia

The purpose of this study is to analyze the clinical characteristics of patients with Van der Woude syndrome (VWS) and to detect variations in each patient. Finally, the combination of genotype and phenotype can make a clear diagnosis of VWS patients with different phenotype penetrance. Five Chinese VWS pedigree were enrolled. Whole exome sequencing of the proband was performed, and the potential pathogenic variation was further verified by Sanger sequencing in the patient and their parents. The human mutant IRF6 coding sequence was generated from the human full-length IRF6 plasmid by site-directed mutagenesis and cloned into the GV658 vector, RT-qPCR and Western blot were used to detect the expression of IRF6. We found one de novo nonsense variation (p. Gln118Ter) and three novel missense variations (p. Gly301Glu, p. Gly267Ala, and p. Glu404Gly) co-segregated with VWS. RT-qPCR analysis revealed that p. Glu404Gly significantly reduced the expression level of IRF6 mRNA. Western blot of cell lysates confirmed that IRF6 p. Glu404Gly abundance levels were lower than those for IRF6 wild type. This discovery of the novel variation ( IRF6 p. Glu404Gly) expands the spectrum of known variations in VWS in Chinese humans. Genetic results combined with clinical phenotypes and differential diagnosis points from other diseases can make a definitive diagnosis and provide genetic counseling for families.

BackgroundTo date, there are over 320 variants identified in the IRF6 gene that cause Van der Woude syndrome or popliteal pterygium syndrome. We sequenced this gene in a South African orofacial cleft cohort to identify the causal IRF6 variants in our population.MethodSaliva samples from 100 patients with syndromic and non‐syndromic CL ± P were collected. Patients were recruited from the cleft clinics at two public, tertiary hospitals in Durban, South Africa (SA), namely Inkosi Albert Luthuli Central Hospital (IALCH) and KwaZulu‐Natal Children's Hospital (KZNCH). We prospectively sequenced the exons of IRF6 in 100 orofacial cleft cases, and where possible, we also sequenced the parents of the individuals to determine the segregation pattern.ResultsTwo variants were identified; one novel (p.Cys114Tyr) and one known (p.Arg84His) missense variant in IRF6 gene were identified. The patient with the p.Cys114Tyr variant was non‐syndromic with no clinical VWS phenotype expected of individuals with IRF6 coding variants, and the patient with the p.Arg84His had phenotypic features of popliteal pterygium syndrome. The p.Arg84His variant segregated in the family, with the father also being affected.ConclusionsThis study provides evidence that IRF6 variants are found in the South African population. Genetic counselling is essential for affected families, particularly in the absence of a known clinical phenotype since it helps with the plans for future pregnancies.

Although the aetiology of non-syndromic orofacial clefts (nsOFCs) is usually multifactorial, syndromic OFCs (syOFCs) are often caused by single mutations in known genes. Some syndromes, e.g., Van der Woude syndrome (VWS1; VWS2) and X-linked cleft palate with or without ankyloglossia (CPX), show only minor clinical signs in addition to OFC and are sometimes difficult to differentiate from nsOFCs. We recruited 34 Slovenian multi-case families with apparent nsOFCs (isolated OFCs or OFCs with minor additional facial signs). First, we examined IRF6, GRHL3, and TBX22 by Sanger or whole exome sequencing to identify VWS and CPX families. Next, we examined 72 additional nsOFC genes in the remaining families. Variant validation and co-segregation analysis were performed for each identified variant using Sanger sequencing, real-time quantitative PCR and microarray-based comparative genomic hybridization. We identified six disease-causing variants (three novel) in IRF6, GRHL3, and TBX22 in 21% of families with apparent nsOFCs, suggesting that our sequencing approach is useful for distinguishing syOFCs from nsOFCs. The novel variants, a frameshift variant in exon 7 of IRF6, a splice-altering variant in GRHL3, and a deletion of the coding exons of TBX22, indicate VWS1, VWS2, and CPX, respectively. We also identified five rare variants in nsOFC genes in families without VWS or CPX, but they could not be conclusively linked to nsOFC.