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.

Digital 3D photogrammetry with the Genex camera system is sufficiently precise and accurate for the anthropometric needs of most medical and craniofacial research designs.

Isolated or nonsyndromic cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex etiology. A 10-cM genome scan of 388 extended multiplex families with CL/P from seven diverse populations (2,551 genotyped individuals) revealed CL/P genes in six chromosomal regions, including a novel region at 9q21 (heterogeneity LOD score [HLOD]=6.6). In addition, meta-analyses with the addition of results from 186 more families (six populations; 1,033 genotyped individuals) showed genomewide significance for 10 more regions, including another novel region at 2q32-35 (P=.0004). These are the first genomewide significant linkage results ever reported for CL/P, and they represent an unprecedented demonstration of the power of linkage analysis to detect multiple genes simultaneously for a complex disorder.

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.

Cleft lip with or without cleft palate (CL/P) is a complex trait with evidence that the clinical spectrum includes both microform and subepithelial lip defects. We identified missense and nonsense mutations in the BMP4 gene in 1 of 30 cases of microform clefts, 2 of 87 cases with subepithelial defects in the orbicularis oris muscle (OOM), 5 of 968 cases of overt CL/P, and 0 of 529 controls. These results provide confirmation that microforms and subepithelial OOM defects are part of the spectrum of CL/P and should be considered during clinical evaluation of families with clefts. Furthermore, we suggest a role for BMP4 in wound healing.

The Pittsburgh Oral-Facial Cleft study was begun in 1993 with the primary goal of identifying genes involved in nonsyndromic orofacial clefts in a variety of populations worldwide. Based on the results from a number of pilot studies and preliminary genetic analyses, a new research focus was added to the Pittsburgh Oral-Facial Cleft study in 1999: to elucidate the role that associated phenotypic features play in the familial transmission patterns of orofacial clefts in order to expand the definition of the nonsyndromic cleft phenotype. The purpose of this paper is to provide a comprehensive review of phenotypic features associated with nonsyndromic orofacial clefts. These features include fluctuating and directional asymmetry, non-right-handedness, dermatoglyphic patterns, craniofacial morphology, orbicularis oris muscle defects, dental anomalies, structural brain and vertebral anomalies, minor physical anomalies, and velopharyngeal incompetence.

Objective-Various lines of evidence suggest that face shape may be a predisposing factor for nonsyndromic cleft lip with or without cleft palate (CL/P). In the present study, 3D surface imaging and statistical shape analysis were used to evaluate face shape differences between the unaffected (non-cleft) parents of individuals with CL/P and unrelated controls.Methods-Sixteen facial landmarks were collected from 3D captures of 80 unaffected parents and 80 matched controls. Prior to analysis, each unaffected parent was assigned to a subgroup on the basis of prior family history (positive or negative). A geometric morphometric approach was utilized to scale and superimpose the landmark coordinate data (Procrustes analysis), test for omnibus group differences in face shape, and uncover specific modes of shape variation capable of discriminating unaffected parents from controls.Results-Significant disparity in face shape was observed between unaffected parents and controls (p < 0.01). Notably, these changes were specific to parents with a positive family history of CL/P. Shape changes associated with CL/P predisposition included marked flattening of the facial profile (midface retrusion), reduced upper facial height, increased lower facial height and excess interorbital width. Additionally, a sex-specific pattern of parent-control difference was evident in the transverse dimensions of the nasolabial complex.Conclusions-The faces of unaffected parents from multiplex cleft families display meaningful shape differences compared with the general population. Quantitative assessment of the facial phenotype in cleft families may enhance efforts to discover the root causes of CL/P.

Nonsyndromic cleft lip+/-cleft palate is a complex disease with a wide phenotypic spectrum; occult defects of the superior orbicularis oris muscle may represent the mildest subclinical form of the lip portion of the phenotype. This study used high-resolution ultrasonography to compare the frequency of discontinuities in the OO muscle in 525 unaffected relatives of individuals with nonsyndromic cleft lip+/-cleft palate versus 257 unaffected controls. OO muscle discontinuities were observed in 54 (10.3%) of the non-cleft relatives, compared to 15 (5.8%) of the controls-a statistically significant increase (P=0.04). Male relatives had a significantly higher rate of discontinuities than male controls (12.0% vs. 3.2%; P=0.01); female relatives also had a higher rate of discontinuities than female controls, but the increase was not statistically significant (8.9% vs. 7.4%; P=0.56). These data confirm the hypothesis that subepithelial OO muscle defects are a mild manifestation of the cleft lip phenotype. Identification of subepithelial OO muscle defects may be important in a clinical setting, as a means of providing more accurate recurrence risk estimates to relatives in cleft families. Furthermore, the expansion of the cleft lip+/-cleft palate phenotypic spectrum should improve the power of genetic studies.