Spectrum of p63 Mutations in a Selected Patient
Cohort Affected With Ankyloblepharon-Ectodermal
Defects-Cleft Lip/Palate Syndrome (AEC)
Tuula Rinne, Emine Bolat, Rowdy Meijer, Hans Scheffer, and Hans van Bokhoven*
Department of Human Genetics, Radboud University Medical Centre Nijmegen, Nijmegen, The Netherlands
Received 24 August 2008; Accepted 4 February 2009
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
Mutations in the AEC and Rapp Hodgkin syndromes cluster in
the 30end of the p63 gene. Previously reported mutations are
mainly missense and frameshift mutations in exons 13 and 14,
affecting the p63a-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 Ectoder-
mal Dysplasias. Nineteen patients underwent full clinical eval-
uations 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 muta-
sequences, which encode the TI domain. The effects of the
mutations in the SAM and TI domains are poorly understood
isoform for the AEC/RHS phenotype. ? 2009 Wiley-Liss, Inc.
Key words: p63; TP63; ectodermal dysplasia; AEC; RHS; SAM-
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 syn-
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 character-
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.
p63 gene. These mutations are mainly missense and frameshift
mutations in exons 13 and 14, affecting the p63a-specific sterile
alpha motive (SAM) and transactivation inhibitory (TI) domains.
a-tail of the p63 gene, we have recently discovered three novel
mutations in the 50end of the p63 gene [Rinneet al., 2008].Two of
these novel mutations only affect the DNp63-isoforms (not the
Grant sponsor: National Foundation for Ectodermal Dysplasias; Grant
sponsor: NIH/NIAMS/NORD; Grant sponsor: European Union Sixth
Framework program EpiStem project; Grant number: LSHB-CT-2005-
Hans van Bokhoven, Department of Human Genetics, 588, Radboud
University Nijmegen Medical Centre, Box 9101, 6500 HB Nijmegen,
The Netherlands. E-mail: firstname.lastname@example.org
Published online 12 August 2009 in Wiley InterScience
How to Cite this Article:
Rinne T, Bolat E, Meijer R, Scheffer H, van
Bokhoven H. 2009. Spectrum of p63
with ankyloblepharon-ectodermal defects-
cleft lip/palate syndrome (AEC).
Am J Med Genet Part A 149A:1948–1951.
? 2009 Wiley-Liss, Inc.
longer TAp63-isoforms), indicating that the specific disruption of
the DNp63a-isoform is the key to the AEC and RHS syndrome
MATERIALS AND METHODS
The National Foundation for Ectodermal Dysplasias convened the
International Research Symposium for ankyloblepharon-ectoder-
mal defects-cleft lip/palate (AEC) syndrome at Texas Children’s
Hospitalin Houston, TX, with financialsupport through the NIH.
Nineteen patients with a suspected diagnosis of AEC syndrome
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,
In total, 24 participants from 12 different families had genomic
DNA analysis to assess for mutations in the p63 gene. Since all
13, and 14 in the p63 gene, these exons were investigated first by
direct sequencing. In case no mutation was identified, all other
described elsewhere [van Bokhoven et al., 2001].
RESULTS AND DISCUSSION
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
The number of solved patients from this NFED cohort is
remarkably high, as our previous studies showed causative p63
mutationsin only?75% ofpatientswithanAEC/RHSlikepheno-
type. 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
p.Ile537Thr, p.Asp544Tyr, p.Asp544Val, p.Leu545Pro, p.Pro551-
the coding region of the SAM domain (Fig. 1). Such mutations are
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.,
Rinne et al., 2007]. However, two of these missense mutations
(p.Asp544Tyr, p.Asp544Val) are flanking the intron 13–exon 14
boundaryandmay causeasplice sitedefect ofexon 14.Inpatient1
the first nucleotide of codon 544, guanine at nucleotide position
1,630 is changed to thymine; and in patient 15, the second nucleo-
tide 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
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.
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 muta-
tions are deletions in the TI domain causing a frameshift and an
extended protein product [van Bokhoven et al., 2001; van Bok-
hoven and Brunner, 2002; Bougeard et al., 2003; Dianzani et al.,
The newly identified p.Arg616fsX665 mutation has a similar pre-
dicted effect. More surprising is the detection of two missense
mutations, p.Arg598Leu and a p.Asp601Val, which are located in
in the TI domain. Including this study, a total of 38 different
mutations in AEC/RHS has been reported. Mutations are
clustered in the DN-specific amino-terminus and a-specific
carboxy-terminus of the p63 protein, which points towards a
critical role of DNp63a isoform for the AEC/RHS phenotype and
TABLE I. p63 Mutations Identified in 24 Patients of NFED Cohort
SAM, sterile alpha motif; TI, transactivation inhibitory domain.
aThis mutation likely affects the splicing of exon 14.
bNovel p63 mutations, which are not published before.
RINNE ET AL.
pathogenesis. Moreover the expression pattern in epithelial tissues
of DNp63a is in agreement with the phenotypic malformations in
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
the p63a protein [Yang et al., 1998; King et al., 2003; Candi et al.,
causing mutations in the 50end of the p63 gene lose the activator
function of DNp63a, and have even dominant negative activity
against the wild typep63 protein [Rinne etal., 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 conse-
quences affecting the same pathway and causing the same disease.
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.
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 labo-
EpiStem Project (LSHB-CT-2005-019067).
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FIG. 1. Multiple sequence alignment ofp63a-tail represents the highly conserved SAM and TIdomains. Thenine 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
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