A Novel Van der Woude Syndrome-Causing IRF6 Variant Is Subject to Incomplete Non-sense-Mediated mRNA Decay Affecting the Phenotype of Keratinocytes

Abstract

Van der Woude syndrome (VWS) is a genetic syndrome that leads to typical phenotypic traits, including lower lip pits and cleft lip/palate (CLP). The majority of VWS-affected patients harbor a pathogenic variant in the gene encoding for the transcription factor interferon regulatory factor 6 (IRF6), a crucial regulator of orofacial development, epidermal differentiation and tissue repair. However, most of the underlying mechanisms leading from pathogenic IRF6 gene variants to phenotypes observed in VWS remain poorly understood and elusive. The availability of one VWS individual within our cohort of CLP patients allowed us to identify a novel VWS-causing IRF6 variant and to functionally characterize it. Using VWS patient-derived keratinocytes, we reveal that most of the mutated IRF6_VWS transcripts are subject to a nonsense mediated mRNA decay mechanism, resulting in IRF6 haploinsufficiency. While moderate levels of IRF6_VWS remain detectable in the VWS keratinocytes, our data illustrate that the IRF6_VWS protein, which lacks part of its protein-binding domain and its whole C-terminus, is noticeably less stable than its wild-type counterpart. Still, it maintains transcription factor function. As we report and characterize a so far undescribed VWS-causing IRF6 variant, our results shed light on the physiological as well as pathological role of IRF6 in keratinocytes. This acquired knowledge is essential for a better understanding of the molecular mechanisms leading to VWS and CLP.

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“…Here, we show that in keratinocytes derived from the VWS2, both IRF6 protein as well as mRNA levels are comparable to VWS1, but robustly decreased when compared to non-syndromic (pathogenic variants in the known VWS-associated genes IRF6 , GRHL3 , and NME1 have been excluded by whole exome sequencing) CLP- and healthy lip-derived keratinocytes. These data fit to the assumed notion that VWS-causing IRF6 mutations often result in haploinsufficiency of IRF6 ( de Lima et al, 2009 ; Degen et al, 2020 ). Therefore, without knowledge of the underlying gene defect causing VWS in the second individual, we can speculate that the affected individual harbors a pathogenic IRF6 variant ( Figure 5A , In vitro characteristics).…”

“…In vitro characteristics : (A) qPCR and immunoblot analyses show that the levels of IRF6 in VWS1 and VWS2 keratinocytes are significantly decreased compared to the levels in 4 non-syndromic [wild-type for the VWS-associated genes IRF6 (NM_006147.4), GRHL3 (NM_198173), and NME1 (NM_198175)] CLP and one healthy lip keratinocyte cell cultures. These results strongly suggest that the VWS2 individual harbors an IRF6 variant [this has been confirmed for VWS1 by genetic analysis ( Degen et al, 2020 )] (left). Additionally, fibroblasts isolated from the scar tissue of VWS2 showed higher expression of α -SMA and Collagen type III (COL3) mRNA levels compared to fibroblasts isolated from the same donor but from different origins (gingiva, lower lip, and “lip pits,” right).…”

“…Cellular and molecular analyses of the patient-derived cells clearly demonstrated proof-of-concept: the original hallmarks of the syndrome are retained in the cells in vitro and represent promising study tools. Using the isolated keratinocytes (VWS1) we were able to confirm and thoroughly characterize a novel IRF6 variant (c961_965delGTGTAinsC), which has been initially sequence-identified by the Division of Human Genetics, Children’s Hospital Bern ( Degen et al, 2020 ). Here, we show that in keratinocytes derived from the VWS2, both IRF6 protein as well as mRNA levels are comparable to VWS1, but robustly decreased when compared to non-syndromic (pathogenic variants in the known VWS-associated genes IRF6 , GRHL3 , and NME1 have been excluded by whole exome sequencing) CLP- and healthy lip-derived keratinocytes.…”

“…Thanks to these approaches, hundreds of genes and variants have been found to be linked to specific CFAs. However, scientific activities also envision to understand how certain gene mutations affect the behavior of cells and to establish genotype-phenotype correlations ( Degen et al, 2020 ). Immortalized cell lines usually are used in such studies.…”

“…The use of postnatal tissues to study CFAs that arise early in development also comes with challenges and assumptions regarding the retention of the original tissue characteristics. However, increasing evidence shows the persistence of various cellular properties postnatally in tissues as well as in patient-derived cells ( Beyeler et al, 2014 ; Hixon et al, 2017 ; Degen et al, 2018 , 2020 ). In this regard, it is also noteworthy to mention that often it is very ambitious and difficult to have access to proper healthy control tissue and/or cells, which ideally should be age- and anatomically matched to the studied condition ( Degen et al, 2020 ).…”