Sequencing the GRHL3 Coding Region Reveals Rare Truncating Mutations and a Common Susceptibility Variant for Nonsyndromic Cleft Palate

Abstract

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO.

Figure 1

Analysis of the Effect of rs41268753 on the Disorganized Protein State for GRHL3 Using DISOPRED3, the intrinsic disorder profile for GRHL3 was generated for the wild-type variant (A) and the variant version p.Thr454Met (B). Protein-binding sites are also indicated within disordered regions. The black line indicates the variant position around which a set of 19 consecutive amino acids (positions 443 to 461) are newly annotated as “disordered” or “disordered protein binding,” suggesting an increase of binding capacity for that region.

Figure 2

A GRHL3 Mutation in a Family with Three Individuals Affected by CPO, Two of Them with Asymmetrical Lower Lips, which Might Represent a Subtle VWS Symptom (A) In BN-813-III-7 we identified c.1285+2delT, predicted to affect a splice donor site. This variant was also identified in the half-sister (BN-813-III-8) and in the mother (BN-813-II-5), but was not present in the half-sister’s father (BN-813-II-6). No DNA from BN-813-I-1 or BN-813-I-2 was available for analysis. Both half-sisters had a complete cleft of the hard and soft palate. Of note, the mother was initially reported as unaffected, but at clinical re-examination was found to have a broad uvula and had nasal speech that was strongly suggestive of a submucous cleft palate. None of the three affected individuals showed the VWS typical lip pits. (B and C) Both half-sisters BN-813-III-7 (B) and BN-813-III-8 (C) showed slightly asymmetrical lower lips with elevations on the left side when opening their mouths. Given their molecular diagnosis, these elevations might a posteriori be interpreted as a very subtle form of either lower lip pits or transverse sulci of the lips, symptoms occasionally described in VWS.