Although the aetiology of non-syndromic orofacial clefts (nsOFCs) is usually multifactorial, syndromic OFCs (syOFCs) are often caused by single mutations in known genes. Some syndromes, e.g., Van der Woude syndrome (VWS1; VWS2) and X-linked cleft palate with or without ankyloglossia (CPX), show only minor clinical signs in addition to OFC and are sometimes difficult to differentiate from nsOFCs. We recruited 34 Slovenian multi-case families with apparent nsOFCs (isolated OFCs or OFCs with minor additional facial signs). First, we examined IRF6, GRHL3, and TBX22 by Sanger or whole exome sequencing to identify VWS and CPX families. Next, we examined 72 additional nsOFC genes in the remaining families. Variant validation and co-segregation analysis were performed for each identified variant using Sanger sequencing, real-time quantitative PCR and microarray-based comparative genomic hybridization. We identified six disease-causing variants (three novel) in IRF6, GRHL3, and TBX22 in 21% of families with apparent nsOFCs, suggesting that our sequencing approach is useful for distinguishing syOFCs from nsOFCs. The novel variants, a frameshift variant in exon 7 of IRF6, a splice-altering variant in GRHL3, and a deletion of the coding exons of TBX22, indicate VWS1, VWS2, and CPX, respectively. We also identified five rare variants in nsOFC genes in families without VWS or CPX, but they could not be conclusively linked to nsOFC.
Keywords: GRHL3; IRF6; TBX22; Van der Woude syndrome; X-linked cleft palate with or without ankyloglossia; family study; genetics; non-syndromic orofacial cleft; whole exome sequencing.