Epidemiologia descritiva e implicações do gene IRF6 em uma série de casos de fendas orofaciais típicas

Abstract

Background: Typical orofacial clefts (TOC) are among the most frequent human malformations, with a prevalence rate of 1:700 to 1000 live births. IRF6 gene (Interferon Regulatory Factor 6) has been worldwide implicated in cases of familial non-syndromic TOC and in the Van der Woude syndrome (VWS). Aim: To describe the epidemiology and the implications of IRF6 gene in a series of non-syndromic TOC and VWS from the Ambulatório de Genética Craniofacial do Serviço de Genética Clínica do Hospital Universitário Professor Alberto Antunes da Universidade Federal de Alagoas (SGCHUPAA-UFAL). Patients and methods: From 2009 to 2021, 375 patients entered the Alagoas database, 255 (71.6%) of whom, presenting non-syndromic TOC, were selected for sociodemographic, clinical, and familial profiling. Information was extracted from the Brazilian Database on Craniofacial Anomalies and tabulated using Excel. Fisher test and Qui-square were used for statistics with a p-value of 0,05 through Epi infoTM. The IRF6 gene was selected for molecular analysis of 21 multiplex families and the single case of VWS of the sample. Six families presenting parent-child transmission were studied through whole exome sequencing in collaboration with the State University of Campinas. The remaining families as well as the patient diagnosed with VWS were studied through Sanger sequencing at our laboratory at SGC-HUPAA-UFAL. The inhouse experiments comprised: Phenolic DNA extraction, the polymerase chain reaction of all exons and splice sites, purification, quantification, and sequencing reaction. Software Chromas was used for electropherogram analysis and the CLC Sequencing Viewer for sequences alignment. Identified variants were checked against free access databases. In silico predictive analyses using software PROVEAN, SIFT, PolyPhen2, Mutation Taster, Align GVGD e MutPred-2 were used for the classification purpose of the new variants. The amino acid conservation among mammalians analysis was through Clustal Omega, and the protein modeling through Swissmodel and PyMol software. Results: Among 255 patients with non-syndromic TOC, the male biologic sex (55,8%), and the unilateral (74,2%), left-sided (51,0%) cleft lip and palate (51,0%) were predominant. These results corroborate the literature. There were 97 (38,6%) familial cases, almost twice the expected frequency. Cleft lip with/without cleft palate (p=0,14), parental consanguinity (p=0,02) and twining (p=0,008) were significantly high in familial cases compared to sporadic. The IRF6 complete sequencing did reveal no pathogenic variants among the six parent-child transmission families. Sanger sequencing of the remaining families is not finished, and, up to now, no pathogenic variants have been recognized. The clinical diagnosis of VWS was based on the typical association between cleft lip and palate, and paramedian lower-lip small mounds. The novel p.Phe266Ser (c.797T>C) heterozygous (TTT/TCT) variant was identified through Sanger sequencing. It has achieved pathogenic scores in all prediction software used. According to Clustal Omega, the amino acid phenylalanine is highly conserved, and the parental sequencing showed this is a de novo variant. According to the model obtained, the phenylalanine-serine exchange disrupts the IRF6 protein. Conclusions: Apart from the high rate of familial cases and consanguinity, the sociodemographic and clinical profile of non-syndromic TOC corroborates the literature. Up to now, no IRF6 pathogenic variant was found among 27 families presenting nonsyndromic TOC, including the six families with parent-child transmission for which the IRF6 sequencing is finished. The novel p.Phe266Ser variant, present in the single case of VWS in this series, was classified as pathogenic and de novo.