Exome sequencing provides additional evidence for the involvement of ARHGAP29 in mendelian orofacial clefting and extends the phenotypic spectrum to isolated cleft palate

Recent advances in genomics methodologies, in particular the availability of next-generation sequencing approaches have made it possible to identify risk loci throughout the genome, in particular the exome. In the current study, we present findings from an exome study conducted in five affected individuals of a multiplex family with cleft palate only. Methods: The GEnome MINIng (GEMINI) pipeline was used to functionally annotate the single nucleotide polymorphisms, insertions and deletions. Filtering methods were applied to identify variants that are clinically relevant and present in affected individuals at minor allele frequencies (≥1%) in the 1000 Genomes Project single nucleotide polymorphism database, Exome Aggregation Consortium, and Exome Variant Server databases. The bioinformatics tool Systems Tool for Craniofacial Expression-Based Gene Discovery was used to prioritize cleft candidates in our list of variants, and Sanger sequencing was used to validate the presence of identified variants in affected and unaffected relatives. Results: Our analyses approach narrowed the candidates down to the novel missense variant in ARHGAP29 (GenBank: NM_004815.3, NP_004806.3;c.1654T>C [p.Ser552Pro]. A functional assay in zebrafish embryos showed that the encoded protein lacks the activity possessed by its wild-type counterpart, and migration assays revealed that keratinocytes transfected with wild-type ARHGAP29 migrated faster than counterparts transfected with the p.Ser552Pro ARHGAP29 variant or empty vector (control). Conclusion: These findings reveal ARHGAP29 to be a regulatory protein essential for proper development of the face, identifies an amino acid that is key for this, and provides a potential new diagnostic tool

Mutations in the epithelial cadherin-p120-catenin complex cause Mendelian non-syndromic cleft lip with or without cleft palate

Non-syndromic cleft lip with or without cleft palate (NS-CL/P) is one of the most common human birth defects and is generally considered a complex trait. Despite numerous loci identified by genome-wide association studies, the effect sizes of common variants are relatively small, with much of the presumed genetic contribution remaining elusive. We report exome-sequencing results in 209 people from 72 multi-affected families with pedigree structures consistent with autosomal-dominant inheritance and variable penetrance. Herein, pathogenic variants are described in four genes encoding components of the p120-catenin complex (CTNND1, PLEKHA7, PLEKHA5) and an epithelial splicing regulator (ESRP2), in addition to the known CL/P-associated gene, CDH1, which encodes E-cadherin. The findings were also validated in a second cohort of 497 people with NS-CL/P, comprising small families and singletons with pathogenic variants in these genes identified in 14% of multi-affected families and 2% of the replication cohort of smaller families. Enriched expression of each gene/protein in human and mouse embryonic oro-palatal epithelia, demonstration of functional impact of CTNND1 and ESRP2 variants, and recapitulation of the CL/P spectrum in Ctnnd1 knockout mice support a causative role in CL/P pathogenesis. These data show that primary defects in regulators of epithelial cell adhesion are the most significant contributors to NS-CL/P identified to date and that inherited and de novo single gene variants explain a substantial proportion of NS-CL/P.Liza L. Cox, Timothy C. Cox, Lina M. Moreno Uribe, Ying Zhu, Chika T. Richter, Nichole Nidey, Jennifer M. Standley, Mei Deng, Elizabeth Blue, Jessica X. Chong, Yueqin Yang, Russ P. Carstens, Deepti Anand, Salil A. Lachke, Joshua D. Smith, Michael O. Dorschner, Bruce Bedell, Edwin Kirk, Anne V. Hing, Hanka Venselaar, Luz C. Valencia-Ramirez, Michael J. Bamshad, Ian A. Glass, Jonathan A. Cooper, Eric Haan, Deborah A. Nickerson, Hans van Bokhoven, Huiqing Zhou, Katy N. Krahn, Michael F. Buckley, Jeffrey C. Murray, Andrew C. Lidral, and Tony Rosciol