In January 2024, individuals presenting to the Department of Clinical Genetics at Hong Kong Children’s Hospital with suspected pathogenic variants in the IRF6 or GRHL3 genes were assessed. Eight patients with molecularly confirmed Van der Woude syndrome (VWS) from four unrelated families were identified, aged between 17 and 58 years, with a male-to-female ratio of 5:3. All four index cases had an affected parent. Pedigrees of the four families are shown in online supplementary Figure 1.

Cleft palate was observed in 75% (n=6) of individuals, of whom two had bilateral cleft palate and two had submucous cleft palate. Lower lip pits were present in 62.5% (n=5). Cleft lip and/or alveolus was evident in four patients (50%), usually affecting both sides. Bifid uvula was observed in only one individual, while hypodontia was seen in two. One patient had ankyloglossia and two patients (25%) developed vitiligo.

The clinical findings are summarised in the Table. Clinical photos demonstrating oral findings from Family 4 are shown in the Figure.

A different variant was identified in each of the index patients in our cohort. Three harboured an IRF6 (NM_006147.4) variant, while the remaining index patient had a variant in GRHL3 (NM_198173.3). All variants were classified as likely pathogenic or pathogenic according to the American College of Medical Genetics and Genomics guidelines1. Two of the IRF6 variants were missense variants, while the remaining one was a splice site variant. The only GRHL3 variant identified in our cohort was a nonsense variant. All other variants in our cohort were novel, except IRF6 c.52G>A p.(Val18Met) which has been previously reported.2

The molecular findings of our patients are summarised in the Table and online supplementary Figure 2.

Orofacial cleft is a prevalent congenital defect with an estimated global occurrence of 1 in 500 to 1000 births and a comparatively higher incidence in Asia according to 2019 Global Burden of Disease data3. It is linked to both environmental and genetic factors. Most cases are non-syndromic, presenting a reduced risk of a genetic disorder, with orofacial clefts manifesting as isolated structural anomalies. Nevertheless, syndromic cases comprise approximately 30% of all cases, with an associated higher risk in patients with central cleft lip and/or palate (CL/P) or isolated cleft palate.4 Cases may be familial or non-familial. According to a local study,4 the diagnostic yield of genomic variants, including variants of uncertain significance, for non-syndromic orofacial clefts was only about 4%. In contrast, for syndromic cases, the detection rate of genomic variants is as high as around 70%. Among these, most variants have been detected by karyotyping or chromosomal microarray.5 In view of this finding, genetic testing (ie, chromosomal microarray) is conventionally offered as first-line screening in patients with syndromic orofacial clefts. Whole exome sequencing is less commonly performed in Hong Kong’s public clinical sector, although there is no international consensus. Clinical and molecular findings from our four families with VWS suggest that whole exome sequencing may be helpful in making a genetic diagnosis in patients with orofacial clefts. This is advantageous for both the patient and their family, as reproductive options such as preimplantation genetic diagnosis or prenatal diagnosis can be provided for at-risk individuals.

Van der Woude syndrome has historically been linked to pathogenic variants in IRF6, which encodes a transcription factor essential for the differentiation of skin, as well as breast and oral epithelium. Abnormal differentiation of the epidermis or oral periderm may be implicated in the pathogenesis of CL/P. The subsequent increase in the number of patients with CL/P has been accompanied by the discovery of a second gene, GRHL3. In vivo studies have demonstrated that GRHL3 regulates the epidermal permeability barrier through action downstream of IRF6, explaining the phenotypic convergence.6 Although it has been postulated that GRHL3 is more likely associated with cleft palate and less likely with cleft lip, CL/P and lip pits, the number of affected patients remains too small to draw definitive conclusions about genotype-phenotype correlations. In our cohort, all three individuals with a GRHL3 variant had cleft palate (mostly submucous), no cleft lip, and only one had lower lip pits. These findings appear to align with previous reports.2 4 5

According to the literature, approximately 60% of VWS cases show familial occurrence.7 All index individuals in our cohort had an affected parent. The concurrence of lower lip pits and CL/P has been reported as the most common clinical features among patients with VWS, affecting 80% of cases.8 In total, 75% and 62.5% of our patients had cleft palate or lower lip pits, respectively. Cleft lip and/or alveolus was evident in 50% of individuals. These findings are comparable with figures described in previous studies.2 4 5 Disease-causing variants in both genes have also been associated with dental anomalies, including hypodontia, dental aplasia, and malocclusion. Bifid uvula, hypodontia and ankyloglossia were also observed in our cohort. It is known that individuals with VWS exhibit highly variable expressivity, ranging from isolated lower lip pits to bilateral CL/P. As highlighted by Family 4 in our cohort, lower lip pits were identified only in individual II:2, but not in her elder sibling (II:1) or father (I:1). This again demonstrates the broad intrafamilial variability.

An enriched prevalence of vitiligo was also observed in our cohort. Although not previously reported in patients with VWS, two patients (one harbouring an IRF6 and one a GRHL3 variant) in our cohort had vitiligo. In individual F1 I:1, vitiligo was diagnosed during adulthood; in individual F4 II:2, at 16 years of age. Our observation points to a possible disease association, with the argument that interferon regulatory factors play a significant role in the immune system by functioning as major transcriptional regulators of type I interferon.9 Further research has revealed that IRF6 regulates a subset of toll-like receptor 3 responses in human keratinocytes and may play a role in keratinocyte and/or immune cell functions during cell damage and wound healing. Alternatively, GRHL3 is a transcription factor critical for epidermal differentiation and skin barrier repair. A possible association of dysfunction in interferon regulatory factors or GRHL3 with autoimmune dermatological conditions such as vitiligo cannot be excluded. Further studies are required to confirm a potential correlation.

All IRF6 and GRHL3 variants found in our cohort, except one, were novel. In previous VWS reports, missense variants in IRF6 were mainly clustered in exons 3, 4, 7, and 9, whereas truncating variants were evenly distributed across the whole gene.10 One novel IRF6 variant in our cohort was a missense variant in exon 4, while another was a splice site variant in intron 2. No specific genotype-phenotype correlation was established in our current study due to the limited sample size.

Due to the highly variable expressivity and incomplete penetrance, it is essential for clinicians to remain vigilant in diagnosing individuals with a relatively mild phenotype. Referral to clinical geneticists for consideration of genetic testing is beneficial for affected individuals with familial occurrence of CL/P or when other features suggest a syndromic diagnosis. In view of the limited number of individuals identified in our cohort, future studies may be needed to establish clearer genotype-phenotype genotypephenotype correlations, explore a potential association with vitiligo, and evaluate the diagnostic efficacy of whole exome sequencing.

Concept or design: LT Leung, SKL Ho.
Acquisition of data: WC Yiu, M Ou, JYY Poon, SSW Cheng, IFM Lo, HM Luk.
Analysis or interpretation of data: LT Leung, SKL Ho, WC Yiu.
Drafting of the manuscript: LT Leung, SKL Ho, IFM Lo, HM Luk.
Critical revision of the manuscript for important intellectual content: IFM Lo, HM Luk.

All authors had full access to the data, contributed to the study, approved the final version for publication, and take responsibility for its accuracy and integrity.

All authors disclosed no conflicts of interest.

The authors thank the patients and their family for their support.

This study received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

This study was conducted in accordance with the Declaration of Helsinki. The patients/their legal guardian provided written informed consent for participation and publication of this case report.

The supplementary material was provided by the authors and some information may not have been peer reviewed. Accepted supplementary material will be published as submitted by the authors, without any editing or formatting. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by the Hong Kong Academy of Medicine or the Hong Kong Medical Association. The Hong Kong Academy of Medicine and the Hong Kong Medical Association disclaim all liability and responsibility arising from any reliance placed on the content.

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