Spectrum of p63 mutations in a selected patient cohort affected with ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC)


  • How to cite this article: Rinne T, Bolat E, Meijer R, Scheffer H, van Bokhoven H. 2009. Spectrum of p63 mutations in a selected patient cohort affected with ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC). Am J Med Genet Part A 149A:1948–1951.


Heterozygous mutations in the p63 gene underlie a group of at least seven allelic syndromes, including ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC) and Rapp Hodgkin syndrome (RHS), which involves varying degrees of ectodermal dysplasia, orofacial clefting and limb malformations. Mutations in the AEC and Rapp Hodgkin syndromes cluster in the 3′ end of the p63 gene. Previously reported mutations are mainly missense and frameshift mutations in exons 13 and 14, affecting the p63α-specific SAM (sterile alpha motif) and TI (transactivation inhibitory) domains. A patient cohort affected by AEC syndrome was evaluated during International Research Symposium supported by the National Foundation for Ectodermal Dysplasias. Nineteen patients underwent full clinical evaluations and 18 had findings consistent with a diagnosis of AEC syndrome. These 19 patients, along with 5 additional relatives had genomic DNA analysis. Twenty-one of the 24 participants from 12 families were found to have mutations in the p63 gene. Eleven different mutations were identified; 10 were novel mutations. Eight were missense mutations within the coding region of the SAM domain. Three other mutations were located in exon 14 sequences, which encode the TI domain. The effects of the mutations in the SAM and TI domains are poorly understood and functional studies are required to understand the pathological mechanisms. However, AEC and RHS mutations in the 5′ and 3′ ends of the p63 gene point towards a critical role of the ΔNp63α isoform for the AEC/RHS phenotype. © 2009 Wiley-Liss, Inc.


Heterozygous mutations in the p63 gene (TAp63 mRNA, GenBank AF075430) underlie a group of at least seven human developmental disorders, characterized by different combinations of ectodermal dysplasia, orofacial clefting and limb malformations [Celli et al., 1999; van Bokhoven et al., 1999, 2001; Ianakiev et al., 2000; McGrath et al., 2001; Duijf et al., 2002; Leoyklang et al., 2006]. Manifestations in these syndromes are overlapping, but different enough to be considered discrete syndromes. Some of these syndromes present a strong genotype–phenotype correlation, whereas correlation in the other syndromes is more ambiguous. Hay–Wells syndrome, also known as ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC, OMIM 106260), and Rapp Hodgkin syndrome (RHS, OMIM 129400) are highly similar disorders and possibly variable manifestations of the same clinical entity [Bertola et al., 2004; Rinne et al., 2007]. Typical characteristics for AEC and RHS are ankyloblepharon filiforme adnatum (eye lid fusion), severe skin erosion at birth and abnormal hair (pili torti or pili canaliculi) and the presence of cleft palate with or without cleft lip. Severe limb malformations such as ectrodactyly are less commonly observed in these conditions.

Mutations in AEC and RHS syndromes cluster in the 3′ end of the p63 gene. These mutations are mainly missense and frameshift mutations in exons 13 and 14, affecting the p63α-specific sterile alpha motive (SAM) and transactivation inhibitory (TI) domains. Although, AEC and RHS syndromes have always been linked to the α-tail of the p63 gene, we have recently discovered three novel mutations in the 5′ end of the p63 gene [Rinne et al., 2008]. Two of these novel mutations only affect the ΔNp63-isoforms (not the longer TAp63-isoforms), indicating that the specific disruption of the ΔNp63α-isoform is the key to the AEC and RHS syndrome phenotypes.


The National Foundation for Ectodermal Dysplasias convened the International Research Symposium for ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome at Texas Children's Hospital in Houston, TX, with financial support through the NIH. Nineteen patients with a suspected diagnosis of AEC syndrome underwent full multidisciplinary evaluations via a Baylor College of Medicine IRB-approved protocol. Eighteen of these patients were found to have clinical characteristics consistent with a diagnosis of AEC syndrome; one did not have a phenotype that was consistent with this diagnosis. An additional five relatives also participated, but their participation was limited to laboratory investigation only. In total, 24 participants from 12 different families had genomic DNA analysis to assess for mutations in the p63 gene. Since all known AEC/RHS causing mutations have been found in exons 3′, 4, 13, and 14 in the p63 gene, these exons were investigated first by direct sequencing. In case no mutation was identified, all other exons of the gene were sequenced as well. The mutation analysis was described elsewhere [van Bokhoven et al., 2001].


Mutations were identified in 21 samples (Table I). Three individuals did not have an identified mutation in the p63 gene. Two of these are unaffected relatives, and one is a patient with a phenotype slightly different than AEC/RHS. This individual may have mutation elsewhere in the p63 gene or in another yet unknown ectodermal dysplasia gene.

Table I. p63 Mutations Identified in 24 Patients of NFED Cohort
SampleMutation (DNA)Mutation (protein)ExonDomain
  • SAM, sterile alpha motif; TI, transactivation inhibitory domain.

  • a

    This mutation likely affects the splicing of exon 14.

  • b

    Novel p63 mutations, which are not published before.

8Unaffected family member   
11Unaffected participant   
24Unaffected family member   

The number of solved patients from this NFED cohort is remarkably high, as our previous studies showed causative p63 mutations in only ∼75% of patients with an AEC/RHS like phenotype. Altogether, we found 11 different mutations, of which only one mutation (in patient 21) p.Ile537Thr has been described previously in AEC syndrome families [McGrath et al., 2001; van Bokhoven and Brunner, 2002; Garcia et al., 2007].

Eight of the mutations reported here (p.Phe526Leu, p.Ile537Thr, p.Asp544Tyr, p.Asp544Val, p.Leu545Pro, p.Pro551Leu, p.Gly561Asp and p.Gly561Val) are missense mutations within the coding region of the SAM domain (Fig. 1). Such mutations are characteristic for AEC/RHS, since 57% (16 out of 28) of the known AEC/RHS mutations create amino acid substitutions in the SAM domain [McGrath et al., 2001; van Bokhoven and Brunner, 2002; Kantaputra et al., 2003; Tsutsui et al., 2003; Bertola et al., 2004; Shotelersuk et al., 2005; Payne et al., 2005; Sorasio et al., 2006; Rinne et al., 2007]. However, two of these missense mutations (p.Asp544Tyr, p.Asp544Val) are flanking the intron 13–exon 14 boundary and may cause a splice site defect of exon 14. In patient 1 the first nucleotide of codon 544, guanine at nucleotide position 1,630 is changed to thymine; and in patient 15, the second nucleotide of this codon, adenine at nucleotide position 1,631 is changed to thymine. To predict the influence of these mutations, we tested these sequences in two splicing prediction programs (NetGene2 and Berkeley Drosphila Genome Project). The prediction programs suggest that the c.1630G>T mutation (p.Asp544Tyr) is most likely affecting the acceptor splice site of exon 14, whereas the c.1631A>T mutation (p.Asp544Val) does not. Unfortunately, the consequences on the p63 cDNA level cannot be determined since the patient cDNA is not available.

Figure 1.

Multiple sequence alignment of p63α-tail represents the highly conserved SAM and TI domains. The nine amino acids, which were found to be mutated in this study, are indicated by black arrow and amino acid number. These amino acids are much conserved, which suggest their role as pathogenic mutations. Stars illustrate known missense mutations and dots known deletions or insertions. In total, 18 amino acids are known to be mutated in SAM and TI domains, and seven deletions or insertions are reported.

Three other new mutations are located in exon 14 sequences, which encode the TI domain of the p63 protein (Fig. 1). In total, about 18% (5 out of 28) of previously identified AEC/RHS mutations are deletions in the TI domain causing a frameshift and an extended protein product [van Bokhoven et al., 2001; van Bokhoven and Brunner, 2002; Bougeard et al., 2003; Dianzani et al., 2003; Chan et al., 2005; Rinne et al., 2006, 2007; Kannu et al., 2006]. The newly identified p.Arg616fsX665 mutation has a similar predicted effect. More surprising is the detection of two missense mutations, p.Arg598Leu and a p.Asp601Val, which are located in the middle of the TI domain. These are the first missense mutations in the TI domain. Including this study, a total of 38 different mutations in AEC/RHS has been reported. Mutations are clustered in the ΔN-specific amino-terminus and α-specific carboxy-terminus of the p63 protein, which points towards a critical role of ΔNp63α isoform for the AEC/RHS phenotype and pathogenesis. Moreover the expression pattern in epithelial tissues of ΔNp63α is in agreement with the phenotypic malformations in AEC/RHS patients.

Functional consequences of AEC/RHS mutations have been reported in a few studies. One very likely effect is distorted binding of mutant SAM domain to its interacting proteins and its diverse consequences. It has been reported that SAM domain mutations abolish binding to Apobec-1-binding protein-1 (ABBP1). ABBP1 belongs to the RNA processing machinery and controls splicing of fibroblast growth factor receptor 2 (FGFR2), which is likely to be altered because of mutations in SAM domain [Fomenkov et al., 2003]. Another possible effect is linked to transactivation. Since ΔNp63α protein can act as an activator or a repressor, it is likely that AEC/RHS mutations can alter the repressor or activator function of the p63α protein [Yang et al., 1998; King et al., 2003; Candi et al., 2006; Romano et al., 2007]. Our recent study shows that AEC/RHS causing mutations in the 5′ end of the p63 gene lose the activator function of ΔNp63α, and have even dominant negative activity against the wild type p63 protein [Rinne et al., 2008]. Mutations in the TI domain might also influence the repressor function. While these effects have been shown for some of the known AEC/RHS mutants, other mutations may cause other still unknown consequences affecting the same pathway and causing the same disease.


Online Mendelian Inheritance in Men (OMIM), http://www.ncbi.nlm.nih.gov/Omim/. GenBank, http://ncbi.nlm.nih.gov/GenBank (Accession number is AF075430 in text, Table I and Fig. 1). Splice site prediction sites: NetGene (http://www.cbs.dtu.dk/services/NetGene2/) and BDGP (http://www.fruitfly.org/seq_tools/splice.html).


We thank all the patients for their participation in this study. Special thanks for Mary Fete and Alanna Bree for organization of this symposium. Our participation in this symposium was funded through the National Foundation for Ectodermal Dysplasias and a conference grant from NIH/NIAMS/NORD. Work in our laboratory is supported by European Union Sixth Framework Program EpiStem Project (LSHB-CT-2005-019067).