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An etiologic regulatory mutation in IRF6 with loss- and gain-of-function effects
Journal article   Open access   Peer reviewed

An etiologic regulatory mutation in IRF6 with loss- and gain-of-function effects

Walid D Fakhouri, Fedik Rahimov, Catia Attanasio, Evelyn N Kouwenhoven, Renata L Ferreira De Lima, Temis Maria Felix, Larissa Nitschke, David Huver, Julie Barrons, Youssef A Kousa, …
Human molecular genetics, Vol.23(10), pp.2711-2720
05/15/2014
DOI: 10.1093/hmg/ddt664
PMCID: PMC3990169
PMID: 24442519
url
https://doi.org/10.1093/hmg/ddt664View
Published (Version of record) Open Access

Abstract

DNA variation in Interferon Regulatory Factor 6 (IRF6) causes Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate (CLP). However, an etiologic variant in IRF6 has been found in only 70% of VWS families. To test whether DNA variants in regulatory elements cause VWS, we sequenced three conserved elements near IRF6 in 70 VWS families that lack an etiologic mutation within IRF6 exons. A rare mutation (350dupA) was found in a conserved IRF6 enhancer element (MCS9.7) in a Brazilian family. The 350dupA mutation abrogated the binding of p63 and E47 transcription factors to cis-overlapping motifs, and significantly disrupted enhancer activity in human cell cultures. Moreover, using a transgenic assay in mice, the 350dupA mutation disrupted the activation of MCS9.7 enhancer element and led to failure of lacZ expression in all head and neck pharyngeal arches. Interestingly, disruption of the p63 Motif1 and/or E47 binding sites by nucleotide substitution did not fully recapitulate the effect of the 350dupA mutation. Rather, we recognized that the 350dupA created a CAAAGT motif, a binding site for Lef1 protein. We showed that Lef1 binds to the mutated site and that overexpression of Lef1/β-Catenin chimeric protein repressed MCS9.7-350dupA enhancer activity. In conclusion, our data strongly suggest that 350dupA variant is an etiologic mutation in VWS patients and disrupts enhancer activity by a loss- and gain-of-function mechanism, and thus support the rationale for additional screening for regulatory mutations in patients with CLP.
 Lip - abnormalities Humans Interferon Regulatory Factors - metabolism Male Cleft Palate - genetics Case-Control Studies DNA Mutational Analysis Base Sequence HEK293 Cells Transcription Factor 3 - metabolism Female Binding Sites Abnormalities, Multiple - genetics Tumor Suppressor Proteins - metabolism Genetic Association Studies Gene Expression Regulation Interferon Regulatory Factors - genetics Transcription Factors - metabolism Point Mutation Cleft Lip - genetics Enhancer Elements, Genetic Pedigree Cysts - genetics Cell Line, Tumor Protein Binding

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