Shared genetic risk between major orofacial cleft phenotypes in an African population.

Nonsyndromic orofacial clefts (NSOFCs) represent a large proportion (70%-80%) of all OFCs. They can be broadly categorized into nonsyndromic cleft lip with or without cleft palate (NSCL/P) and nonsyndromic cleft palate only (NSCPO). Although NSCL/P and NSCPO are considered etiologically distinct, recent evidence suggests the presence of shared genetic risks. Thus, we investigated the genetic overlap between NSCL/P and NSCPO using African genome-wide association study (GWAS) data on NSOFCs. These data consist of 814 NSCL/P, 205 NSCPO cases, and 2159 unrelated controls. We generated common single-nucleotide variants (SNVs) association summary statistics separately for each phenotype (NSCL/P and NSCPO) under an additive genetic model. Subsequently, we employed the pleiotropic analysis under the composite null (PLACO) method to test for genetic overlap. Our analysis identified two loci with genome-wide significance (rs181737795 [p = 2.58E-08] and rs2221169 [p = 4.5E-08]) and one locus with marginal significance (rs187523265 [p = 5.22E-08]). Using mouse transcriptomics data and information from genetic phenotype databases, we identified MDN1, MAP3k7, KMT2A, ARCN1, and VADC2 as top candidate genes for the associated SNVs. These findings enhance our understanding of genetic variants associated with NSOFCs and identify potential candidate genes for further exploration.

Perceptions and beliefs of community gatekeepers about genomic risk information in African cleft research.

BACKGROUND: A fundamental ethical issue in African genomics research is how socio-cultural factors impact perspectives, acceptance, and utility of genomic information, especially in stigmatizing conditions like orofacial clefts (OFCs). Previous research has shown that gatekeepers (e.g., religious, political, family or community leaders) wield considerable influence on the decision-making capabilities of their members, including health issues. Thus, their perspectives can inform the design of engagement strategies and increase exposure to the benefits of genomics testing/research. This is especially important for Africans underrepresented in genomic research. Our study aims to investigate the perspectives of gatekeepers concerning genomic risk information (GRI) in the presence of OFCs in a sub-Saharan African cohort. METHODS: Twenty-five focus group discussions (FGDs) consisting of 214 gatekeepers (religious, community, ethnic leaders, and traditional birth attendants) in Lagos, Nigeria, explored the opinions of participants on genomic risk information (GRI), OFC experience, and the possibility of involvement in collaborative decision-making in Lagos, Nigeria. Transcripts generated from audio recordings were coded and analyzed in NVivo using thematic analysis. RESULTS: Three main themes-knowledge, beliefs, and willingness to act-emerged from exploring the perspective of gatekeepers about GRI in this group. We observed mixed opinions regarding the acceptance of GRI. Many participants believed their role is to guide and support members when they receive results; this is based on the level of trust their members have in them. However, participants felt they would need to be trained by medical experts to do this. Also, religious and cultural beliefs were crucial to determining participants' understanding of OFCs and the acceptance and utilization of GRI. CONCLUSIONS: Incorporating cultural sensitivity into public engagement could help develop appropriate strategies to manage conflicting ideologies surrounding genomic information in African communities. This will allow for more widespread access to the advances in genomics research in underrepresented populations. We also recommend a synergistic relationship between community health specialists/scientists, and community leaders, including spiritual providers to better understand and utilize GRI.

Parents and Provider Perspectives on the Return of Genomic Findings for Cleft Families in Africa.

BACKGROUND: Inadequate knowledge among health care providers (HCPs) and parents of affected children limits the understanding and utility of secondary genetic findings (SFs) in under-represented populations in genomics research. SFs arise from deep DNA sequencing done for research or diagnostic purposes and may burden patients and their families despite their potential health importance. This study aims to evaluate the perspective of both groups regarding SFs and their choices in the return of results from genetic testing in the context of orofacial clefts. METHODS: Using an online survey, we evaluated the experiences of 252 HCPs and 197 parents across participating cleft clinics in Ghana and Nigeria toward the return of SFs across several domains. RESULTS: Only 1.6% of the HCPs felt they had an expert understanding of when and how to incorporate genomic medicine into practice, while 50.0% agreed that all SFs should be returned to patients. About 95.4% of parents were willing to receive all the information from genetic testing (including SFs), while the majority cited physicians as their primary information source (64%). CONCLUSIONS: Overall, parents and providers were aware that genetic testing could help in the clinical management of diseases. However, they cited a lack of knowledge about genomic medicine, uncertain clinical utility, and lack of available learning resources as barriers. The knowledge gained from this study will assist with developing guidelines and policies to guide providers on the return of SFs in sub-Saharan Africa and across the continent.

Trio-based GWAS identifies novel associations and subtype-specific risk factors for cleft palate.

Cleft palate (CP) is one of the most common craniofacial birth defects; however, there are relatively few established genetic risk factors associated with its occurrence despite high heritability. Historically, CP has been studied as a single phenotype, although it manifests across a spectrum of defects involving the hard and/or soft palate. We performed a genome-wide association study using transmission disequilibrium tests of 435 case-parent trios to evaluate broad risks for any cleft palate (ACP) (n = 435), and subtype-specific risks for any cleft soft palate (CSP), (n = 259) and any cleft hard palate (CHP) (n = 125). We identified a single genome-wide significant locus at 9q33.3 (lead SNP rs7035976, p = 4.24 × 10-8) associated with CHP. One gene at this locus, angiopoietin-like 2 (ANGPTL2), plays a role in osteoblast differentiation. It is expressed both in craniofacial tissue of human embryos and developing mouse palatal shelves. We found 19 additional loci reaching suggestive significance (p < 5 × 10-6), of which only one overlapped between groups (chromosome 17q24.2, ACP and CSP). Odds ratios for the 20 loci were most similar across all 3 groups for SNPs associated with the ACP group, but more distinct when comparing SNPs associated with either subtype. We also found nominal evidence of replication (p < 0.05) for 22 SNPs previously associated with orofacial clefts. Our study to evaluate CP risks in the context of its subtypes and we provide newly reported associations affecting the broad risk for CP as well as evidence of subtype-specific risks.

Damaging Mutations in AFDN Contribute to Risk of Nonsyndromic Cleft Lip With or Without Cleft Palate.

Novel or rare damaging mutations have been implicated in the developmental pathogenesis of nonsyndromic cleft lip with or without cleft palate (nsCL ± P). Thus, we investigated the human genome for high-impact mutations that could explain the risk of nsCL ± P in our cohorts.We conducted next-generation sequencing (NGS) analysis of 130 nsCL ± P case-parent African trios to identify pathogenic variants that contribute to the risk of clefting. We replicated this analysis using whole-exome sequence data from a Brazilian nsCL ± P cohort. Computational analyses were then used to predict the mechanism by which these variants could result in increased risks for nsCL ± P.We discovered damaging mutations within the AFDN gene, a cell adhesion molecule (CAMs) that was previously shown to contribute to cleft palate in mice. These mutations include p.Met1164Ile, p.Thr453Asn, p.Pro1638Ala, p.Arg669Gln, p.Ala1717Val, and p.Arg1596His. We also discovered a novel splicing p.Leu1588Leu mutation in this protein. Computational analysis suggests that these amino acid changes affect the interactions with other cleft-associated genes including nectins (PVRL1, PVRL2, PVRL3, and PVRL4) CDH1, CTNNA1, and CTNND1.This is the first report on the contribution of AFDN to the risk for nsCL ± P in humans. AFDN encodes AFADIN, an important CAM that forms calcium-independent complexes with nectins 1 and 4 (encoded by the genes PVRL1 and PVRL4). This discovery shows the power of NGS analysis of multiethnic cleft samples in combination with a computational approach in the understanding of the pathogenesis of nsCL ± P.

Investigating the relationship between cancer and orofacial clefts using GWAS significant loci for cancers: A case-control and case-triad study.

Background: Several population-based case-control studies have reported concurrent presentation of cancer and congenital malformations. Many associations have been made between oral clefting and cancers, though some of these results are conflicting. Some studies have reported an increased risk of cancer among 1st-degree relatives of cleft cases and vice versa, and also an excess risk of cancers of the breast, lung, and brain among those with oral clefts. This study aimed to determine if the genetic polymorphisms found in some cancers are also associated with orofacial cleft in an African cohort. Methods: The study was a case-control and case-triad study in which cases were 400 individuals clinically diagnosed with non-syndromic cleft lip and/or palate (CL/P), while controls were 450 individuals without CL/P. Samples were obtained from three African countries while DNA extraction, PCR, and genotyping were carried out at the University of Iowa, US. Eleven SNPs in genes coding for SWI/SNF subunits and 13 GWAS significant SNPs for cancers associated with orofacial cleft were selected. Case-control analysis, transmission disequilibrium test (TDT), and DFAM to combine the parent-offspring trio data and unrelated case/control data in a single analysis were carried out using PLINK. Results: For the case-control analyses that included all the clefts and for the CLP subtype, none of the SNPs were statistically significant. Statistically increased risk for the following SNPs rs34775372 (p = 0.02; OR = 1.54, CI:1.07-2.22), rs55658222 (p = 0.009; OR = 2.64, CI:1.28-5.45) and rs72728755 (p = 0.02; OR=2.27, CI:1.17-4.45) was observed with the CL only sub-group. None of these were significant after Bonferoni correction. In the TDT analyses, a significantly reduced risk with rs10941679 (p = 0.003; OR = 0.43, CI:0.24-0.75) was observed and this was significant after Bonferroni correction. The rs10941679 was also significant (p = 0.003) in the DFAM analyses as well even after Bonferroni correction. Conclusion: The results from this study represent an important starting point for understanding the concurrent presentation of some cancers in orofacial clefts, and cancer risks in cleft patients. The associations observed warrant further investigation in a larger cohort and will set the stage for a more mechanistic approach toward understanding the risk for cancers in families with clefts.

Whole-genome sequencing reveals de-novo mutations associated with nonsyndromic cleft lip/palate.

The majority (85%) of nonsyndromic cleft lip with or without cleft palate (nsCL/P) cases occur sporadically, suggesting a role for de novo mutations (DNMs) in the etiology of nsCL/P. To identify high impact protein-altering DNMs that contribute to the risk of nsCL/P, we conducted whole-genome sequencing (WGS) analyses in 130 African case-parent trios (affected probands and unaffected parents). We identified 162 high confidence protein-altering DNMs some of which are based on available evidence, contribute to the risk of nsCL/P. These include novel protein-truncating DNMs in the ACTL6A, ARHGAP10, MINK1, TMEM5 and TTN genes; as well as missense variants in ACAN, DHRS3, DLX6, EPHB2, FKBP10, KMT2D, RECQL4, SEMA3C, SEMA4D, SHH, TP63, and TULP4. Many of these protein-altering DNMs were predicted to be pathogenic. Analysis using mouse transcriptomics data showed that some of these genes are expressed during the development of primary and secondary palate. Gene-set enrichment analysis of the protein-altering DNMs identified palatal development and neural crest migration among the few processes that were significantly enriched. These processes are directly involved in the etiopathogenesis of clefting. The analysis of the coding sequence in the WGS data provides more evidence of the opportunity for novel findings in the African genome.

Novel GRHL3 Variants in a South African Cohort With Cleft Lip and Palate.

OBJECTIVE: The etiology of cleft palate (CP) is poorly understood compared with that of cleft lip with or without palate (CL ± P). Recently, variants in Grainyhead like transcription factor 3 (GRHL3) were reported to be associated with a risk for CP in European and some African populations including Nigeria, Ghana, and Ethiopia. In order to identify genetic variants that may further explain the etiology of CP, we sequenced GRHL3 in a South African population to determine if rare variants in GRHL3 are associated with the presence of syndromic or nonsyndromic CP. DESIGN: We sequenced the exons of GRHL3 in 100 cases and where possible, we sequenced the parents of the individuals to determine the segregation pattern and presence of de novo variants. SETTING: The cleft clinics from 2 public, tertiary hospitals in Durban, South Africa (SA), namely Inkosi Albert Luthuli Central Hospital and KwaZulu-Natal Children's Hospital. PATIENTS, PARTICIPANTS: One hundred patients with CL ± P and their parents. INTERVENTIONS: Saliva samples were collected. MAIN OUTCOME MEASURES: To ascertain the genetic variants in the GRHL3 gene in patients with CL ± P in SA. RESULTS: Five variants in GRHL3 were observed; 3 were novel and 2 were known variants. The novel variants were intronic variants (c.1062 + 77A>G and c.627 + 1G>A) and missense variant (p.Asp169Gly). CONCLUSIONS: This study provides further evidence that variants in GRHL3 contribute to the risk of nonsyndromic CP in African populations, specifically, in the South African population.

Variant analyses of candidate genes in orofacial clefts in multi-ethnic populations.

OBJECTIVES: Cleft lip with/without cleft palate and cleft palate only is congenital birth defects where the upper lip and/or palate fail to fuse properly during embryonic facial development. Affecting ~1.2/1000 live births worldwide, these orofacial clefts impose significant social and financial burdens on affected individuals and their families. Orofacial clefts have a complex etiology resulting from genetic variants combined with environmental covariates. Recent genome-wide association studies and whole-exome sequencing for orofacial clefts identified significant genetic associations and variants in several genes. Of these, we investigated the role of common/rare variants in SHH, RORA, MRPL53, ACVR1, and GDF11. MATERIALS AND METHODS: We sequenced these five genes in 1255 multi-ethnic cleft lip with/without palate and cleft palate only samples in order to find variants that may provide potential explanations for the missing heritability of orofacial clefts. Rare and novel variants were further analyzed using in silico predictive tools. RESULTS: Ninteen total variants of interest were found, with variant types including stop-gain, missense, synonymous, intronic, and splice-site variants. Of these, 3 novel missense variants were found, one in SHH, one in RORA, and one in GDF11. CONCLUSION: This study provides evidence that variants in SHH, RORA, MRPL53, ACVR1, and GDF11 may contribute to risk of orofacial clefts in various populations.

Genome-Wide Scan for Parent-of-Origin Effects in a sub-Saharan African Cohort With Nonsyndromic Cleft Lip and/or Cleft Palate (CL/P).

OBJECTIVE: Nonsyndromic cleft lip and/or cleft palate (NSCL/P) have multifactorial etiology where genetic factors, gene-environment interactions, stochastic factors, gene-gene interactions, and parent-of-origin effects (POEs) play cardinal roles. POEs arise when the parental origin of alleles differentially impacts the phenotype of the offspring. The aim of this study was to identify POEs that can increase risk for NSCL/P in humans using a genome-wide dataset. METHODS: The samples (174 case-parent trios from Ghana, Ethiopia, and Nigeria) included in this study were from the African only genome wide association studies (GWAS) that was published in 2019. Genotyping of individual DNA using over 2 million multiethnic and African ancestry-specific single-nucleotide polymorphisms from the Illumina Multi-Ethnic Genotyping Array v2 15070954 A2 (genome build GRCh37/hg19) was done at the Center for Inherited Diseases Research. After quality control checks, PLINK was employed to carry out POE analysis employing the pooled subphenotypes of NSCL/P. RESULTS: We observed possible hints of POEs at a cluster of genes at a 1 mega base pair window at the major histocompatibility complex class 1 locus on chromosome 6, as well as at other loci encompassing candidate genes such as ASB18, ANKEF1, AGAP1, GABRD, HHAT, CCT7, DNMT3A, EPHA7, FOXO3, lncRNAs, microRNA, antisense RNAs, ZNRD1, ZFAT, and ZBTB16. CONCLUSION: Findings from our study suggest that some loci may increase the risk for NSCL/P through POEs. Additional studies are required to confirm these suggestive loci in NSCL/P etiology.

Co-occurrence of orofacial clefts and clubfoot phenotypes in a sub-Saharan African cohort: Whole-exome sequencing implicates multiple syndromes and genes.

BACKGROUND: Orofacial clefts (OFCs) are congenital malformations of the face and palate, with an incidence of 1 per 700 live births. Clubfoot or congenital talipes equinovarus (CTEV) is a three-dimensional abnormality of the leg, ankle, and feet that leads to the anomalous positioning of foot and ankle joints and has an incidence of 1 per 1000 live births. OFCs and CTEV may occur together or separately in certain genetic syndromes in addition to other congenital abnormalities. Here, we sought to decipher the genetic etiology of OFC and CTEV that occurred together in six probands. METHODS: At the time of recruitment, the most clinically obvious congenital anomalies in these individuals were the OFC and CTEV. We carried out whole-exome sequencing (WES) on DNA samples from probands and available parents employing the Agilent SureSelect XT kit and Illumina HiSeq2500 platform, followed by bioinformatics analyses. WES variants were validated by clinical Sanger Sequencing. RESULTS: Of the six probands, we observed probable pathogenic genetic variants in four. In three probands with probable pathogenic genetic variants, each individual had variants in three different genes, whereas one proband had probable pathogenic variant in just one gene. In one proband, we observed variants in DIS3L2, a gene associated with Perlman syndrome. A second proband had variants in EPG5 (associated with Vici Syndrome), BARX1 and MKI67, while another proband had potentially etiologic variants in FRAS1 (associated with Fraser Syndrome 1), TCOF1 (associated with Treacher Collins Syndrome 1) and MKI67. The last proband had variants in FRAS1, PRDM16 (associated with Cardiomyopathy, dilated, 1LL/Left ventricular noncompaction 8) and CHD7 (associated with CHARGE syndrome/Hypogonadotropic hypogonadism 5 with or without anosmia). CONCLUSION: Our results suggest that clubfoot and OFCs are two congenital abnormalities that can co-occur in certain individuals with varying genetic causes and expressivity, warranting the need for deep phenotyping.

Non-random distribution of deleterious mutations in the DNA and protein-binding domains of IRF6 are associated with Van Der Woude syndrome.

BACKGROUND: The development of the face occurs during the early days of intrauterine life by the formation of facial processes from the first Pharyngeal arch. Derangement in these well-organized fusion events results in Orofacial clefts (OFC). Van der Woude syndrome (VWS) is one of the most common causes of syndromic cleft lip and/or palate accounting for 2% of all cases. Mutations in the IRF6 gene account for 70% of cases with the majority of these mutations located in the DNA-binding (exon 3, 4) or protein-binding domains (exon 7-9). The current study was designed to update the list of IRF6 variants reported for VWS by compiling all the published mutations from 2013 to date as well as including the previously unreported VWS cases from Africa and Puerto Rico. METHODS: We used PubMed with the search terms; "Van der Woude syndrome," "Popliteal pterygium syndrome," "IRF6," and "Orofacial cleft" to identify eligible studies. We compiled the CADD score for all the mutations to determine the percentage of deleterious variants. RESULTS: Twenty-one new mutations were identified from nine papers. The majority of these mutations were in exon 4. Mutations in exon 3 and 4 had CADD scores between 20 and 30 and mutations in exon 7-9 had CADD scores between 30 and 40. The presence of higher CADD scores in the protein-binding domain (exon 7-9) further confirms the crucial role played by this domain in the function of IRF6. In the new cases, we identified five IRF6 mutations, three novel missense mutations (p.Phe36Tyr, p.Lys109Thr, and p.Gln438Leu), and two previously reported nonsense mutations (p.Ser424*and p.Arg250*). CONCLUSION: Mutations in the protein and DNA-binding domains of IRF6 ranked among the top 0.1% and 1% most deleterious genetic mutations, respectively. Overall, these findings expand the range of VWS mutations and are important for diagnostic and counseling purposes.

Six2 regulates Pax9 expression, palatogenesis and craniofacial bone formation.

In this study, we investigated the role of the transcription factor Six2 in palate development. Six2 was selected using the SysFACE tool to predict genes from the 2p21 locus, a region associated with clefting in humans by GWAS, that are likely to be involved in palatogenesis. We functionally validated the predicted role of Six2 in palatogenesis by showing that 22% of Six2 null embryos develop cleft palate. Six2 contributes to palatogenesis by promoting mesenchymal cell proliferation and regulating bone formation. The clefting phenotype in Six2-/- embryos is similar to Pax9 null embryos, so we examined the functional relationship of these two genes. Mechanistically, SIX2 binds to a PAX9 5' upstream regulatory element and activates PAX9 expression. In addition, we identified a human SIX2 coding variant (p.Gly264Glu) in a proband with cleft palate. We show this missense mutation affects the stability of the SIX2 protein and leads to decreased PAX9 expression. The low penetrance of clefting in the Six2 null mouse combined with the mutation in one patient with cleft palate underscores the potential combinatorial interactions of other genes in clefting. Our study demonstrates that Six2 interacts with the developmental gene regulatory network in the developing palate.

Missense Pathogenic variants in KIF4A Affect Dental Morphogenesis Resulting in X-linked Taurodontism, Microdontia and Dens-Invaginatus.

The etiology of dental anomalies is multifactorial; and genetic and environmental factors that affect the dental lamina have been implicated. We investigated two families of European ancestry in which males were affected by taurodontism, microdontia and dens invaginatus. In both families, males were related to each other via unaffected females. A linkage analysis was conducted in a New Zealand family, followed by exome sequencing and focused analysis of the X-chromosome. In a US family, exome sequencing of the X-chromosome was followed by Sanger sequencing to conduct segregation analyses. We identified two independent missense variants in KIF4A that segregate in affected males and female carriers. The variant in a New Zealand family (p.Asp371His) predicts the substitution of a residue in the motor domain of the protein while the one in a US family (p.Arg771Lys) predicts the substitution of a residue in the domain that interacts with Protein Regulator of Cytokinesis 1 (PRC1). We demonstrated that the gene is expressed in the developing tooth bud during development, and that the p.Arg771Lys variant influences cell migration in an in vitro assay. These data implicate missense variations in KIF4A in a pathogenic mechanism that causes taurodontism, microdontia and dens invaginatus phenotypes.

Genomic analyses in African populations identify novel risk loci for cleft palate.

Orofacial clefts are common developmental disorders that pose significant clinical, economical and psychological problems. We conducted genome-wide association analyses for cleft palate only (CPO) and cleft lip with or without palate (CL/P) with ~17 million markers in sub-Saharan Africans. After replication and combined analyses, we identified novel loci for CPO at or near genome-wide significance on chromosomes 2 (near CTNNA2) and 19 (near SULT2A1). In situ hybridization of Sult2a1 in mice showed expression of SULT2A1 in mesenchymal cells in palate, palatal rugae and palatal epithelium in the fused palate. The previously reported 8q24 was the most significant locus for CL/P in our study, and we replicated several previously reported loci including PAX7 and VAX1.

Identification of paternal uniparental disomy on chromosome 22 and a de novo deletion on chromosome 18 in individuals with orofacial clefts.

BACKGROUND: Orofacial clefts are the most common malformations of the head and neck region. Genetic and environmental factors have been implicated in the etiology of these traits. METHODS: We recently conducted genotyping of individuals from the African population using the multiethnic genotyping array (MEGA) to identify common genetic variation associated with nonsyndromic orofacial clefts. The data cleaning of this dataset allowed for screening of annotated sex versus genetic sex, confirmation of identify by descent and identification of large chromosomal anomalies. RESULTS: We identified the first reported orofacial cleft case associated with paternal uniparental disomy (patUPD) on chromosome 22. We also identified a de novo deletion on chromosome 18. In addition to chromosomal anomalies, we identified cases with molecular karyotypes suggesting Klinefelter syndrome, Turner syndrome and Triple X syndrome. CONCLUSION: Observations from our study support the need for genetic testing when clinically indicated in order to exclude chromosomal anomalies associated with clefting. The identification of these chromosomal anomalies and sex aneuploidies is important in genetic counseling for families that are at risk. Clinicians should share any identified genetic findings and place them in context for the families during routine clinical visits and evaluations.

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

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. 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.Birth Defects Research 109:27-37, 2017. © 2016 Wiley Periodicals, Inc.