uORF-creating mutations in Van der Woude syndrome: why it is important to study 5’UTRs

International audienceBackground/Objectives:Van der Woude syndrome (VWS, MIM 119300) is an autosomal dominant cleft lip and/or palate with typical lower lip pits. Most patients carry loss-of-function mutations in IRF6. Upstream open reading frame (uORF)-creating mutations have been reported in four VWS patients. Pathogenic uORFcreating mutations are mostly out-of-frame upstream start codons (uAUG) in the 5’UTR. We searched for IRF6 uORF mutations and assessed the ability to predict the pathogenicity of uORFcreating variations of 5 prediction tools.Methods:We analyzed IRF6 UTR and coding regions in 68 VWS probands. By using a set of 44 reference genes, we assessed 5 in silico tools predicting the probability of ATGs to initiate translation: NetStart, ATGpr, TIS Miner, AltORFev, TIS Predictor. We then assessed the potential pathogenicity of all the theoretical uORFs in IRF6 5’UTR.Results:We have identified two novel uORF-creating mutations (c.-141C>T and c.-162C>T), representing 3% (2/68) of the probands. The 7 carriers of the two families had typical VWS signs. Our in silico analyses revealed a higher accuracy for AI-based tools over those based on Kozak consensus scoring. There are 28 theoretical uAUG-creating SNVs in IRF6 5’UTR. With AI-based tools, the six uAUG identified in VWS patients have high translation initiation site scores; 3 to 4 of the theoretical uAUG-creating SNVs had high scores and could correspond to pathogenic mutations. For the dozen of theoretical SNVs with intermediate scores, predicting pathogenicity remains challenging.Conclusion:As untranslated regions are frequently understudied in NGS strategies, uORF-creating mutations may be underdiagnosed in VWS and in human pathology in general

uORF-creating-mutations in Van der Woude syndrome: why it is important to study 5’UTRs

International audienceThe Van der Woude syndrome (VWS, MIM 119300), is an autosomal dominant disorder characterized by cleft lip with or without cleft palate, or isolated cleft palate, due to loss-of-function mutations in IRF6 (Interferon Regulatory Factor-6) or GRHL3 (Grainyhead-Like Transcription Factor 3). Most patients show pits on their lower lips. In VWS, most IRF6 alterations are premature stop codons or missense mutations. Pathogenic upstream open reading frame (uORF) mutations are characterized by an out-of-frame upstream start codon (uAUG) located in the 5’UTR and leading to a premature stop codon. uORFs overlap the usual ATG start codon and have a minimum length of 9 nucleotides. We assessed the main features of six uORF-creating mutations identified in VWS patients (two in the lab and four previously described). We also determined all the theoretical SNVs located in IRF6 5’UTR (NM_006147.4) that could create an uORFs and we assessed their potential pathogenicity based on Kozak site in silico prediction.Only four uORF-creating mutations in IRF6 have been previously associated with VWS to date. We report here two novel mutations creating out-of-frame uAUGs (c.-141C>T p.? and c.-162C>T p.?) that probably reduce IRF6 expression. In our lab, IRF6 mutations are found in about 80% of families with VWS and uORFs-creating mutations represent of 3.2% of them (2/63). Previous studies identifying uORFs-creating mutations did not provide detailed phenotypic data. In our group, in the 6 heterozygotes for c.-141C>T, three had a cleft lip with or without cleft palate and three had only a bifid uvula. The patient heterozygote for c.-162C>T had a posterior cleft with an ankyloblepharon.Most genes naturally have uORFs in their 5’UTR region, nonetheless we observed that there were no physiological uORF in IRF6. All 6 uORFs identified in VWS had the same termination codon that occurs in exon 3 (56 nucleotides after the usual ATG). To go further, we searched for all potential uAUG-creating SNV in IRF6 5’UTR. We identified 41 of them, including the 6 identified in patients. Except one, none of them is present in the gnomAD control population. Using a machine-learning-based tool (TIS Predictor, biorxiv.org/content/10.1101/2021.08.17.456657v1, 0 to 1 score range), we then assessed each Kozak similarity score. All 6 uORFs identified in patients had high scores comprised between 0.71 and 0.83, compared to 0.81 for the usual ATG. Among the 35 theoretical SNVs creating an uAUG, 10 may create a potential but weak Kozak site (score range: 0.50-0.71).In conclusion, uORF-creating mutations can thus be responsible for typical VWS. As untranslated regions are not included in most captured regions in high throughput sequencing strategies, this category of variants may be underdiagnosed in VWS and in human pathology in general