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.

Whole exome sequencing identifies mutations in 10% of patients with familial non-syndromic cleft lip and/or palate in genes mutated in well-known syndromes

“…Our results, as well as previously published data including IRF6 ,20 42  MSX1 ,43  TBX22 44 and GRHL3 ,41 substantiate evidence for Mendelian transmission (with incomplete penetrance) of aetiological variants in ‘syndrome-causing genes’ in subclinical or non-syndromic cleft families. Therefore, custom-designed panels including CL/P-syndrome-causing genes should be used in diagnostic screens when a particular syndrome is suspected and also for families with nsCL/P.…”

Genome-wide meta-analyses of nonsyndromic orofacial clefts identify novel associations between FOXE1 and all orofacial clefts, and TP63 and cleft lip with or without cleft palate

“…The first study, despite interrogating 400 CP case-parent trios, did not identify any statistically significant SNP main effects (Beaty et al 2011). The second study identified a single locus associated with CP, but this association signal was limited to European populations because of very low frequencies of the risk allele in other populations (Leslie et al 2016b; Mangold et al 2016). For both CL/P and CP, the identified risk loci only account for a modest portion of the genetic variance of OFCs, suggesting that additional genetic risk factors may be involved.…”

Rare deleterious variants in GRHL3 are associated with human spina bifida

“…While these data confirm an important role of Grhl3 in the pathogenesis of NTDs in mouse models, its role in the human NTD pathology remained unclear (Ting et al., ). To date, variants in GRHL3 in humans have been associated only with a minority (5%) of cases presenting Van Der Woude syndrome 2 (VWS2; MIM# 606713) (the most common syndromic form of cleft lip and palate (Peyrard‐Janvid et al., ) and recently with nonsyndromic cleft palate (Leslie et al., ; Mangold et al., )).…”