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Common variants in interferon regulatory factor 6 ( IRF6) have been associated with nonsyndromic cleft lip with or without cleft palate (NSCL/P) as well as with tooth agenesis (TA). These variants contribute a small risk towards the 2 congenital conditions and explain only a small percentage of heritability. On the other hand, many IRF6 mutations are known to be a monogenic cause of disease for syndromic orofacial clefting (OFC). We hypothesize that IRF6 mutations in some rare instances could also cause nonsyndromic OFC. To find novel rare variants in IRF6 responsible for nonsyndromic OFC and TA, we performed targeted multiplex sequencing using molecular inversion probes (MIPs) in 1,072 OFC patients, 67 TA patients, and 706 controls. We identified 3 potentially pathogenic de novo mutations in OFC patients. In addition, 3 rare missense variants were identified, for which pathogenicity could not unequivocally be shown, as all variants were either inherited from an unaffected parent or the parental DNA was not available. Retrospective investigation of the patients with these variants revealed the presence of lip pits in one of the patients with a de novo mutation suggesting a Van der Woude syndrome (VWS) phenotype, whereas, in other patients, no lip pits were identified. [ABSTRACT FROM AUTHOR]

Summary Background Congenital atrichia is a rare autosomal recessive form of isolated alopecia which is caused by mutations in the human hairless ( HR) gene. Patients are born with normal hair that is shed almost completely and irreversibly during the first weeks of life. Objectives To investigate the molecular genetic basis of congenital atrichia in two patients, and to analyse the functional consequences of one newly identified and all seven previously identified HR splice site mutations using a minigene assay. Methods Molecular analysis of the HR gene was performed by direct DNA sequencing. To analyse the functional consequences of the splice site mutations, the respective sequences were cloned into a vector which allows directed splicing. After transfection of COS7 cells, isolation of RNA and cDNA synthesis, sequencing was performed to analyse the products. Results Two novel mutations were identified: an insertion in exon 2 (c.485insT; p.C162LfsX17), and a splice site mutation (c.2847-1G>A). In vitro analysis revealed aberrant splicing for all eight of the investigated HR splice site mutations. Comparison with the results of two biocomputational programs (neural network splice server and CRYP-SKIP) and calculation of consensus values revealed that the predictions of these two programs were consistent in only five and two of the eight mutations, respectively. Conclusions This is the first report to analyse the consequences of HR splice site mutations using a cell-based in vitro assay. The results highlight the importance of performing splicing experiments to clarify the consequences of putative splice site mutations. [ABSTRACT FROM AUTHOR]