ABSTRACT Objectives Clefts of the lip, alveolus and/or palate (CLA/P) are the most common craniofacial congenital malformations in humans. These oral clefts can be divided into non-syndromic (isolated) and syndromic forms. Many cleft-related syndromes are clinically variable and genetically heterogeneous, making it challenging to distinguish syndromic from non-syndromic cases. Recognition of syndromic/genetic causes is important for personalized tailored care, identification of (unrecognized) co-morbidities and accurate genetic counseling. Therefore, Next Generation Sequencing (NGS) based targeted gene panel testing is increasingly implemented in diagnostics of CLA/P patients. In this retrospective study we assess the yield of NGS gene panel testing in a cohort of CLA/P cases. Methods Whole exome sequencing followed by variant detection and interpretation in an a-priori selected set of genes associated with CLA/P phenotypes was performed in 212 unrelated CLA/P patients after genetic counseling between 2015 and 2020. Medical records including family history and results of additional genetic tests were evaluated. Results In 24 CLA/P cases (11.3 %) a pathogenic genetic variant was identified. Twenty out of these 24 had a genetic syndrome requiring specific monitoring and follow-up. Six of these 24 cases (25%) were presumed to be isolated CLA/P cases prior to testing, corresponding to 2,8% of the total cohort. In eight CLA/P cases (3.8%) without a diagnosis after NGS based gene panel testing, a molecular diagnosis was established by additional genetic analyses (e.g., SNP array, single gene testing, trio WES). Conclusion This study illustrates NGS based gene panel testing as a powerful diagnostic tool in the diagnostic work-up of CLA/P patients. Also, in apparently isolated cases and non- familial cases a genetic diagnose could be identified. Early diagnosis facilitates personalized care for patients and accurate genetic counseling of their families.

ABSTRACT Objectives Clefts of the lip, alveolus and/or palate (CLA/P) are the most common craniofacial congenital malformations in humans. These oral clefts can be divided into non-syndromic (isolated) and syndromic forms. Many cleft-related syndromes are clinically variable and genetically heterogeneous, making it challenging to distinguish syndromic from non-syndromic cases. Recognition of syndromic/genetic causes is important for personalized tailored care, identification of (unrecognized) co-morbidities and accurate genetic counseling. Therefore, Next Generation Sequencing (NGS) based targeted gene panel testing is increasingly implemented in diagnostics of CLA/P patients. In this retrospective study we assess the yield of NGS gene panel testing in a cohort of CLA/P cases. Methods Whole exome sequencing followed by variant detection and interpretation in an a-priori selected set of genes associated with CLA/P phenotypes was performed in 212 unrelated CLA/P patients after genetic counseling between 2015 and 2020. Medical records including family history and results of additional genetic tests were evaluated. Results In 24 CLA/P cases (11.3 %) a pathogenic genetic variant was identified. Twenty out of these 24 had a genetic syndrome requiring specific monitoring and follow-up. Six of these 24 cases (25%) were presumed to be isolated CLA/P cases prior to testing, corresponding to 2,8% of the total cohort. In eight CLA/P cases (3.8%) without a diagnosis after NGS based gene panel testing, a molecular diagnosis was established by additional genetic analyses (e.g., SNP array, single gene testing, trio WES). Conclusion This study illustrates NGS based gene panel testing as a powerful diagnostic tool in the diagnostic work-up of CLA/P patients. Also, in apparently isolated cases and non- familial cases a genetic diagnose could be identified. Early diagnosis facilitates personalized care for patients and accurate genetic counseling of their families.