BACKGROUND: Three different homozygous loss-of-function mutations of the Forkhead box E1 (FOXE1) gene have been associated with syndromic cleft palate. Here, we screened the entire promoter region to identify the variations in significant consensus motifs affecting FOXE1 transcription. METHOD: Genomic DNAs of 35 cleft palate patients, 10 of whom with CHARGE association, 80 unrelated healthy people and 80 unaffected first-degree relatives were analysed by automatic sequencing. The Transcription Element Search System program was employed to identify transcription factor binding sites. The protein-DNA complexes were observed using DNA band-shift assays and oligonucleotide competition analyses. Real-time PCR was used to estimate FOXE1 expression at mRNA level. RESULTS: In 11 non-syndromic cleft palate patients, a novel non-coding polymorphism (C-->G) in the 5'-untranslated region of FOXE1 was found. The variation fell into a putative consensus sequence for the transcription factor MYF-5 and completely impaired the ability of MYF-5 to bind to its motif, as shown by EMSA experiments. As a consequence, a significantly reduced FOXE1 mRNA expression was observed. CONCLUSIONS: In 45% of non-syndromic cleft palate patients, a novel homozygous polymorphism that prevented the binding of MYF-5 to FOXE1 promoter and affected the FOXE1 expression was found. As recent data show the role of MYF-5 in the muscle-dependent craniofacial skeletal development and in the fusion of primary palate and secondary palate, the results reported here strongly suggest a more significant involvement of this factor in the cleft palate onset.
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