Orofacial clefts (OFCs), which include non-syndromic cleft lip with or without cleft palate (CL/P), are among the most common birth defects in humans, affecting approximately 1 in 700 newborns. CL/P is phenotypically heterogeneous and has a complex etiology caused by genetic and environmental factors. Previous genome-wide association studies (GWASs) have identified at least 15 risk loci for CL/P. As these loci do not account for all of the genetic variance of CL/P, we hypothesized the existence of additional risk loci. We conducted a multiethnic GWAS in 6480 participants (823 unrelated cases, 1700 unrelated controls and 1319 case-parent trios) with European, Asian, African and Central and South American ancestry. Our GWAS revealed novel associations on 2p24 near FAM49A, a gene of unknown function (P = 4.22 × 10), and 19q13 near RHPN2, a gene involved in organizing the actin cytoskeleton (P = 4.17 × 10). Other regions reaching genome-wide significance were 1p36 (PAX7), 1p22 (ARHGAP29), 1q32 (IRF6), 8q24 and 17p13 (NTN1), all reported in previous GWASs. Stratification by ancestry group revealed a novel association with a region on 17q23 (P = 2.92 × 10) among individuals with European ancestry. This region included several promising candidates including TANC2, an oncogene required for development, and DCAF7, a scaffolding protein required for craniofacial development. In the Central and South American ancestry group, significant associations with loci previously identified in Asian or European ancestry groups reflected their admixed ancestry. In summary, we have identified novel CL/P risk loci and suggest new genes involved in craniofacial development, confirming the highly heterogeneous etiology of OFCs.

Cleft palate (CP) is a common birth defect occurring in 1 in 2,500 live births. Approximately half of infants with CP have a syndromic form, exhibiting other physical and cognitive disabilities. The other half have nonsyndromic CP, and to date, few genes associated with risk for nonsyndromic CP have been characterized. To identify such risk factors, we performed a genome-wide association study of this disorder. We discovered a genome-wide significant association with a missense variant in GRHL3 (p.Thr454Met [c.1361C>T]; rs41268753; p = 4.08 × 10(-9)) and replicated the result in an independent sample of case and control subjects. In both the discovery and replication samples, rs41268753 conferred increased risk for CP (OR = 8.3, 95% CI 4.1-16.8; OR = 2.16, 95% CI 1.43-3.27, respectively). In luciferase transactivation assays, p.Thr454Met had about one-third of the activity of wild-type GRHL3, and in zebrafish embryos, perturbed periderm development. We conclude that this mutation is an etiologic variant for nonsyndromic CP and is one of few functional variants identified to date for nonsyndromic orofacial clefting. This finding advances our understanding of the genetic basis of craniofacial development and might ultimately lead to improvements in recurrence risk prediction, treatment, and prognosis.

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Non-syndromic (NS) cleft lip with or without cleft palate (CL/P) is a common disorder with a strong genetic underpinning. Genome-wide association studies have detected common variants associated with this disorder, but a large portion of the genetic risk for NSCL/P is conferred by unidentified rare sequence variants. Mutations in IRF6 (Interferon Regulatory Factor 6) and GRHL3 (Grainyhead-like 3) cause Van der Woude syndrome, which includes CL/P. Both genes encode members of a regulatory network governing periderm differentiation in model organisms. Here, we report that Krüppel-like factor 17 (Klf17), like Grhl3, acts downstream of Irf6 in this network in zebrafish periderm. Although Klf17 expression is absent from mammalian oral epithelium, a close homologue, Klf4, is expressed in this tissue and is required for the differentiation of epidermis. Chromosome configuration capture and reporter assays indicated that IRF6 directly regulates an oral-epithelium enhancer of KLF4. To test whether rare missense variants of KLF4 contribute risk for NSCL/P, we sequenced KLF4 in approximately 1000 NSCL/P cases and 300 controls. By one statistical test, missense variants of KLF4 as a group were enriched in cases versus controls. Moreover, two patient-derived KLF4 variants disrupted periderm differentiation upon forced expression in zebrafish embryos, suggesting that they have dominant-negative effect. These results indicate that rare NSCL/P risk variants can be found in members of the gene regulatory network governing periderm differentiation.

Orofacial clefts (OFCs) are congenital dysmorphologies of the human face and oral cavity, with a global incidence of 1 per 700 live births. These anomalies exhibit a multifactorial pattern of inheritance, with genetic and environmental factors both playing crucial roles. Many loci have been implicated in the etiology of nonsyndromic cleft lip with or without cleft palate (NSCL/P) in populations of Asian and European ancestries, through genome-wide association studies and candidate gene studies. However, few populations of African descent have been studied to date. Here, the authors show evidence of an association of some loci with NSCL/P and nonsyndromic cleft palate only (NSCPO) in cohorts from Africa (Ghana, Ethiopia, and Nigeria). The authors genotyped 48 single-nucleotide polymorphisms that were selected from previous genome-wide association studies and candidate gene studies. These markers were successfully genotyped on 701 NSCL/P and 163 NSCPO cases, 1,070 unaffected relatives, and 1,078 unrelated controls. The authors also directly sequenced 7 genes in 184 nonsyndromic OFC (NSOFC) cases and 96 controls from Ghana. Population-specific associations were observed in the casecontrol analyses of the subpopulations, with West African subpopulations (Ghana and Nigeria) showing a similar pattern of associations. In meta-analyses of the case-control cohort, PAX7 (rs742071, P = 5.10 × 10 -3 ), 8q24 (rs987525, P = 1.22 × 10 -3 ), and VAX1 (rs7078160, P = 0.04) were nominally associated with NSCL/P, and MSX1 (rs115200552, P = 0.01), TULP4 (rs651333, P = 0.04), CRISPLD2 (rs4783099, P = 0.02), and NOG1 (rs17760296, P = 0.04) were nominally associated with NSCPO. Moreover, 7 loci exhibited evidence of threshold overtransmission in NSOFC cases through the transmission disequilibrium test and through analyses of the family-based association for disease traits. Through DNA sequencing, the authors also identified 2 novel, rare, potentially pathogenic variants (p.Asn323Asp and p.Lys426IlefsTer6) in ARHGAP29. In conclusion, the authors have shown evidence for the association of many loci with NSCL/P and NSCPO. To the best of this knowledge, this study is the first to demonstrate any of these association signals in any African population.

A two-year retrospective survey in southern Ethiopia revealed that 49 amputations had been performed, 25 of which were for gangrene following tight splintage applied by traditional bone setters. The aim of this study was to determine if it was possible to reduce this incidence of gangrene by offering one-day instructional courses to bone setters. In ten separate one-day courses 112 tradition healers attended. In addition, two-day courses were given to local health assistants, who also received written instructions for the safe care of fractures. A two-year prospective study revealed a marked reduction in amputations, from 49 to 25, with only seven rather than 25 being required for gangrene. We found that it is possible to educate traditional healers so that fewer gangrenous limbs require to be amputated.

Nonsyndromic clefts of the lip and palate [NSCLP] are complex genetic traits. Together, they are classified as one of the most common birth defects with a prevalence of 1/700 live births. Genome-wide association studies [GWAS] for non-syndromic cleft lip with or without cleft palate [NSCL[P]] revealed significant association for common single nucleotide polymorphisms near genes involved in craniofacial development i.e. MAFB, PAX7, VAX1, ARHGAP29 (ABCA4 locus), and IRF6. Sequencing of protein coding regions of the NSCL[P] GWAS candidate genes or adjacent genes suggest a role for rare functional variants. Replication studies in the African population did not observe any significant association with the GWAS candidate genes. On the other hand, the role of rare functional variants in GWAS candidate genes has not been evaluated in the African population. We obtained saliva samples from case triads in Nigeria and Ethiopia for Sanger sequencing of the GWAS candidate genes [MAFB, PAX7, VAX1, ARHGAP29, and IRF6] in order to identify rare functional variants. A total of 220 African samples [140 Nigerians and 80 Ethiopians] were sequenced and we found the following new rare variants— p.His165Asn in the MAFB gene, p.Asp428Asn in the PAX7, a splice-site variant that creates a new donor splice-site in PAX7. We also found three previously reported missense variants p.Gly466Ser in PAX7; p.Leu913Ser and Arg955His in ARHGAP29. No de novo mutations were found. Future genome-wide association and sequencing studies should be conducted using samples from Africa in order to identify new molecular genetic factors that contribute to the etiology of NSCLP.

Orofacial clefts (OFC) are complex genetic traits that are often classified as syndromic or nonsyndromic clefts. Currently, there are over 500 types of syndromic clefts in the Online Mendelian Inheritance in Man (OMIM) database, of which Van der Woude syndrome (VWS) is one of the most common (accounting for 2% of all OFC). Popliteal pterygium syndrome (PPS) is considered to be a more severe form of VWS. Mutations in the IRF6 gene have been reported worldwide to cause VWS and PPS. Here, we report studies of families with VWS and PPS in sub-Saharan Africa. We screened the DNA of eight families with VWS and one family with PPS from Nigeria and Ethiopia by Sanger sequencing of the most commonly affected exons in IRF6 (exons 3, 4, 7, and 9). For the VWS families, we found a novel nonsense variant in exon 4 (p.Lys66X), a novel splice-site variant in exon 4 (p.Pro126Pro), a novel missense variant in exon 4 (p.Phe230Leu), a previously reported splice-site variant in exon 7 that changes the acceptor splice site, and a known missense variant in exon 7 (p.Leu251Pro). A previously known missense variant was found in exon 4 (p.Arg84His) in the PPS family. All the mutations segregate in the families. Our data confirm the presence of IRF6-related VWS and PPS in sub-Saharan Africa and highlights the importance of screening for novel mutations in known genes when studying diverse global populations. This is important for counseling and prenatal diagnosis for high-risk families.