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

Abstract

Background 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 (CPO). Methods The GEnomeMINIng (GEMINI) pipeline was used to functionally annotate the SNPs, insertions and deletions (In/Del). 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 (dbSNP), Exome Aggregation Consortium (ExAC), and Exome Variant Server (EVS) databases. The bioinformatics tool Systems Tool for Craniofacial Expression-based Gene Discovery (SysFACE) 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 (WT) counterpart, and migration assays revealed that keratinocytes transfected with WT ARHGAP29 migrated faster than counterparts transfected with the p.Ser552Pro ARHGAP29 variant or empty vector (control). Conclusions These findings reveals 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 GDF11 and the extracellular antagonist, Follistatin, as a likely cause of Mendelian forms of orofacial clefting in humans

“…The bioinformatics tool, SysFACE (Systems tool for craniofacial expression-based gene discovery), was used to analyze microarraybased genome-level gene expression profiles from various mouse embryonic orofacial tissues in the FaceBase (https://www.facebase. org) and NCBI GEO repositories (https://www.ncbi.nlm.nih.gov/geo/ ; Lachke et al, 2012;Liu et al, 2017…”

iSyTE 2.0: a database for expression-based gene discovery in the eye

“…We have previously addressed these challenges by developing a strategy termed ‘whole-embryo body (WB) in silico subtraction’ for prioritization of ocular disease genes and making this resource publicly accessible through a web-tool called iSyTE ( i ntegrated Sy stems T ool for E ye gene discovery) ( 1 ). Indeed, in silico subtraction has proven effective to prioritize genes in non-ocular tissues as well ( 3 ).…”

Zebrafish models of orofacial clefts

“…Zebrafish injected with a RNA encoding the reference (wildtype) variant of ARHGAP29 failed to progress through epiboly, possibly because of a disruption in the dynamics of actin polymerization events that are essential for epiboly (Lee, 2014). However, embryos injected with an RNA encoding the p.Ser552Pro variant progressed normally, at the same rate as lacZ-injected controls (Liu et al, 2016a). The inability of the p.Ser552Pro variant to disrupt morphogenesis suggests that it has a lower or different activity than the wild-type ARHGAP29, supporting its candidacy as disease-causing.…”