- Email: [email protected]
Dlx6 pathway
Gene 497 (2012) 292–297
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
Rapp–Hodgkin syndrome and SHFM1 patients: Delineating the p63–Dlx5/Dlx6 pathway Ascensión Vera-Carbonell a, María Rosa Moya-Quiles b, María Ballesta-Martínez c, Vanesa López-González c, Juan Antonio Bafallíu a, Encarna Guillén-Navarro c, Isabel López-Expósito a,⁎ a b c
Sección de Citogenética, Centro de Bioquímica y Genética Clínica, Hospital U. “Virgen de la Arrixaca”, El Palmar, Murcia, Spain Sección de Genética Molecular, Centro de Bioquímica y Genética Clínica, Hospital U. “Virgen de la Arrixaca”, El Palmar, Murcia, Spain Unidad de Genética Médica y Dismorfología. Servicio de Pediatría, Hospital U. “Virgen de la Arrixaca”, El Palmar, Murcia, Spain
a r t i c l e
i n f o
Article history: Accepted 29 January 2012 Available online 9 February 2012 Keywords: Rapp–Hodgkin syndrome Split hand/foot malformation p63-mutation analysis Array-CGH analysis
a b s t r a c t Rapp–Hodgkin Syndrome (RHS) is a genetic disorder resulting from mutations in the TP63 gene encoding p63 transcription factor. p63 is directly associated with a cis-regulatory element on chromosome 7q21 that controls the expression of DLX5 and DLX6 genes which are involved in craniofacial abnormalities and ectrodactyly or split hand/foot malformation (SHFM). Chromosomal deletions on 7q21 locus can result in loss of DXL5/DLX6 and/or in loss/disruption of cis-regulatory elements, at which p63 binds. We report two patients that have in common a p63–Dlx5/Dlx6 pathway dysregulation. One showed growth retardation, craniofacial dysmorphism, syndactyly, developmental delay and a de novo deletion (~ 8.5 Mb) on chromosome 7q21.13–q21.3, including DLX5 and DLX6. The second patient with a clinical diagnosis of RHS showed a de novo heterozygous missense mutation, c. 401G > A (p.G134D), in TP63 (exon 4). Our findings may contribute to a greater understanding of the pathogenic mechanisms underlying disorders caused by TP63 mutations. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The complex human development process results from gradients of signaling molecules with precisely regulated temporal and spatial patterns and it is therefore important that developmental genes are subject to strict regulation. The p63, Dlx5 and Dlx6 proteins, encoded by the TP63, DLX5 and DLX6 genes, respectively, are transcription factors essential for epidermal morphogenesis and limb development. Animal models have shown that p63 −/− mice are born alive but show striking defects in development; their skin does not progress from the early stages of development, lacking stratification as well as expression of
Abbreviations: TP63, tumor protein p63; DLX, distal-less homeobox; AER, apical ectodermal ridge; DBD, DNA-binding domain; ISO, isomerization; TA, transactivation; SAM, sterile alpha motif; TI, transactivation inhibitory; EEC, ectrodactyly–ectodermal dysplasia–cleft lip/palate; AEC, ankyloblepharon–ectodermal dysplasia–cleft lip/palate; RHS, Rapp–Hodgkin syndrome; LMS, limb–mammary syndrome; ADULT, acro-dermatoungual-lacrimal-tooth; SHFM, split hand/foot malformation; DSS1, deleted split hand/ split-foot 1; SHFM-BS, SHFM-binding site; OFC, occipito-frontal head circumference; CT, computed tomography; MRI, magnetic resonance imaging; PCR, Polymerase Chain Reaction; GTG, Giemsa-Trypsin-G bands; PHA, phytohemagglutinin; CGH, Comparative Genomic Hybridization; dCTP, deoxycytidine triphosphate; ADM, Aberration Detection Method; bp, base pair; kb, kilobase; Mb, megabase; NCBI, National Center for Biotechnology Information; IRTKS, insulin-receptor tyrosine kinase substrate; inv, inversion; del, deletion. ⁎ Corresponding author. Tel.: + 34 968381269; fax: + 34 968 889861. E-mail address: [email protected] (I. López-Expósito). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.01.088
differentiation markers, and the mammary glands, hair follicles and teeth are absent (Mills et al., 1999; Yang et al., 1999). Moreover, Dlx5/Dlx6 −/− results in inner ear and severe limb malformations and craniofacial and axial skeletal defects (Robledo et al., 2002). p63 is directly associated with the DLX5 and DLX6 promoters and regulates expression of both genes in mice (Lo Iacono et al., 2008), suggesting that p63 and the Dlx5/Dlx6 locus are involved in the same signaling pathway. The DLX5 and DLX6 genes encode homeobox-containing proteins and are usually co-expressed in a unique spatial and temporal pattern in the apical ectodermal ridge (AER), which is critical for normal limb development (Duijf et al., 2003; Ferrari et al., 1995). The TP63 gene, a member of the p53 family, as a result of the alternative usage of two promoters and a complex alternative splicing, encodes at least six different p63 protein isoforms (TAp63α, TAp63β, TAp63γ, ΔNp63α, ΔNp63β and ΔNp63γ). Of these, the transactivation (TA) isoforms contain an N-terminal TA domain, which is absent in the ΔN isoforms (Yang et al., 1998). In ΔNp63 isoforms, an additional TA2 domain has been recognized as responsible for ΔN-specific transcriptional activities distinct from that of TA isoforms (Dohn et al., 2001; Ghioni et al., 2002). All p63 isoforms contain a DNA binding domain (DBD) and a tetramerization domain (ISO). The extended 3′coding sequences of the α isotypes of p63 encodes a sterile-α-motif (SAM) domain and a transactivation inhibitory (TI) domain. The modular structure of p63 reflects the functional differences between p63 isoforms. Thus, TAp63 isotypes act in a p53-like fashion
A. Vera-Carbonell et al. / Gene 497 (2012) 292–297
displaying anti-proliferative, pro-apoptotic and tumor-suppressive functions while ΔNp63 isotypes act as mitogenic, anti-apoptotic and oncogenic regulators (Yang et al., 1998). By expressing TAp63 and ΔNp63 isoforms, p63 has the ability to regulate a number of genes with diverse roles and possesses opposing regulatory effects depending on the form used (Murray-Zmijewski et al., 2006). In humans, mutations in the TP63 gene have been found in a number of dominantly inherited congenital disorders, including EEC syndrome (ectrodactyly, ectodermal dysplasia, and cleft lip/palate, MIM 604292), ankyloblepharon–ectodermal dysplasia–clefting syndrome (AEC, MIM 106260), Rapp–Hodgkin syndrome (RHS, MIM 129400), limb–mammary syndrome (LMS, MIM 603543), acrodermato-ungual-lacrimal-tooth syndrome (ADULT, MIM 103285), and split hand/foot malformation 4 (SHFM4, MIM 605289) (Celli et al., 1999; Rinne et al., 2007; van Bokhoven et al., 2001). These syndromes share several phenotypic features, consisting of ectodermal dysplasia, orofacial clefting and limb malformations, that can be related to alterations of a tightly controlled balance between proliferation and differentiation of precursor cells during the development of ectodermal-derived epithelia and organs (Rinne et al., 2007). The ectodermal dysplasia manifests as defects in skin, hair, teeth, nails and exocrine glands. The orofacial clefting is mainly cleft lip and/or cleft palate and the ectrodactyly (also known as split-hand/foot malformation, SHFM) is characterized by a deficiency of the central rays of the hands and/or feet, resulting in missing or malformed digits, and may present with syndactyly, median clefts of the hands and feet and aplasia and/or hypoplasia of the phalanges, metacarpals and metatarsals (Elliott et al., 2005). Besides TP63 located on chromosome 3q27, five other loci have been considered in relation to syndromic and non-syndromic SHFM: SHFM1 on chromosome 7q21, SHFM2 on Xq26, SHFM3 on 10q24, SHFM5 on 2q31 and SHFM6 on 12q13 (for review see Saitsu et al., 2009). SHFM1 is associated with deletions of variable extent on chromosome 7q21, minimally including the DSS1 gene and the distallessrelated homeogenes DLX5 and DLX6 (Crackower et al., 1996; Scherer et al., 1994a, 1994b). Studies in mutant mice with double knock-out of DLX5 and DLX6 have shown ectrodactyly whereas DSS1expression was normal in these mutants (Robledo et al., 2002) and therefore a concomitant haploinsufficiency of DLX5 and DLX6 has been proposed as the causative mechanism in humans. Since translocations or inversions with breakpoints within the SHFM1 critical region (~0.9 Mb) (Wieland et al., 2004) were identified in patients affected with ectrodactyly, a ‘position effect’ mutagenic mechanism (Scherer et al., 1994a) or ‘functional haploinsufficiency’ (van Silfhout et al., 2009) for SHFM1 was proposed. It seems that these breaks could separate DLX5 and DLX6 from a regulatory element, which was proposed to be located at least 800 kb centromeric of DLX5/DLX6 (van Silfhout et al., 2009). Recently, Kouwenhoven et al. (2010), working with human primary keratinocyte cultures and animal models, have identified an enhancer element referred to as “SHFM1-BS1” in the SHFM1 critical region on chromosome 7q21, located more that 250 kb centromeric of DLX5/ DLX6 genes and which controls expression of DLX6 and possibly DLX5 in limb development. The identification of a microdeletion in a patient with SHFM, including the enhancer element but not the DLX5/DLX6 genes, has allowed them to conclude that this regulatory element acts as an enhancer for expression of these genes mediated by p63 during embryonic limb development and that p63 may regulate DLX5/DLX6 expression from a distance, perhaps by a looping mechanism. Therefore, the loss or disruption of this cis-regulatory element has been proposed as a novel pathogenic mechanism in SHFM1. Together, these data link the pathogenesis of two genetically different groups of developmental diseases into a common molecular pathway. Here we report two patients, one with Rapp–Hodgkin syndrome caused by a mutation on TP63 gene and another with SHFM1 and a deletion on chromosome 7q21, to illustrate the clinical and molecular etiology of RHS and SHFM1 in the context of the p63–
293
Dlx5/Dlx6 pathway. In addition, we compare the clinical phenotype of these patients with others previously reported. 2. Methods 2.1. Molecular genetic analysis Genomic DNA was isolated from peripheral blood, according to established protocols. The TP63 exons with intron–exon boundaries were amplified using previously published primers (Leoyklang et al., 2006). PCR fragments were sequenced using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3130 Genetic Analyser (Applied Biosystems). The position of mutations corresponds to the coding sequence for the original published TA-p63α isotype (GenBank accession AF075430). 2.2. Cytogenetic and array-CGH analysis High-resolution GTG banded chromosomes obtained from PHAstimulated peripheral lymphocytes were analyzed. Oligo array-CGH analysis was performed with the Agilent Human Genome CGH 400K microarray (Agilent Technologies, Santa Clara, CA, USA). This array spans the entire human genome at a median probe spacing of ~5.3 kb. Samples of 1 μg of genomic DNA from the patient and a reference normal genomic DNA male (Promega, Madison, WI) were differentially labeled by random priming with Cy5-dCTP and Cy3-dCTP. The hybridization was carried out according to the manufacturer's protocol. Microarray data were extracted and visualized using Feature Extraction software v10.7 and analyzed using Genomic Workbench software v5.0 (Agilent Technologies). Copy number altered regions were detected using the Z-score (set as 4) and ADM-2 (set as 6) statistics provided by DNA Analytics. 3. Results 3.1. Patient 1 Patient 1 was a 15-month-old boy who was referred for cytogenetic analysis because of developmental delay, growth retardation, dysmorphic features and limb anomalies. He was the second child of a young and non-consanguineous couple. During pregnancy there was a threat of premature birth at six months gestation and intrauterine growth retardation was detected at eight months by ultrasound. The baby was born at 38-weeks gestation with a weight of 2.330 kg (b3rd centile), length of 46 cm (3rd centile) and occipito-frontal head circumference (OFC) of 32 cm (2nd centile). Apgar scores were 9 and 10 at 1 and 5 min, respectively. Physical examination demonstrated several dysmorphic facial features including high forehead, epicanthal folds, short nose with anteverted nares, downturned corners of the mouth, high palate and low-set ears, as well as mild right foot ectrodactyly (syndactyly of 1st–2nd toes, absence of distal phalanges of 3rd toe and defective alignment of 4th and 5th toes) (Fig. S1a,b). He also had mild global hypotonia. At 15 months of age, all his measurements were well below the 3rd centile and the intermamillary distance was slightly increased with a lower and inverted right nipple. At 30 months, psychomotor developmental and language delay were diagnosed (sitting up at 16 months and independent walking at 27 months). Initially, hearing impairment was suspected, but cranial computed tomography (CT) scan was normal. Results of additional investigations including audiometric and ophthalmological examinations, abdominal ultrasound, magnetic resonance imaging (MRI) scan of the brain and echocardiogram were all normal. GTG banded chromosomal analysis revealed a de novo interstitial deletion on the long arm of one chromosome 7 (Fig. 1a). The karyotype was 46,XY,del(7)(q21.1q21.3). Additional high-resolution oligo arrayCGH analysis of peripheral blood from this patient confirmed the
294
A. Vera-Carbonell et al. / Gene 497 (2012) 292–297
Fig. 1. Partial karyotype and microarray of Patient 1 and sequencing analysis of the TP63 gene of Patient 2. a: Chromosomes 7 with GTG bands of Patient 1 (right: normal, left: deleted). b: Oligo array-CGH log 2 ratio plot of whole chromosome 7 with ideogram illustrating deletion and enlargement of the deleted region, also of Patient 1. c: Electrophoregram of Patient 2 shows the c.401G>A (p.G134D) mutation in exon 4 of the TP63 gene (above), which is not present in the control (below).
deletion on chromosome 7q21.13–q21.3 (chr7:89,378,731-97,857,149 bp on the March 2006 assembly of the human genome: NCBI36/hg18). The size of the deleted region was estimated to be 8.478 Mb, including DSS1, DLX5 and DLX6 among more than fifty genes (Fig. 1b). Analysis of TP63 mutations was normal (data not shown). 3.2. Patient 2 Patient 2 was another male referred as a newborn for assessment of multiple congenital anomalies. He was the second child of healthy and non-consanguineous parents. At 24-weeks gestation, ultrasound evaluation revealed bilateral anotia and left hydronephrosis with normal karyotype on amniotic fluid. He was born after an uncomplicated 40-week gestation by vaginal delivery. At birth, his weight was 3.450 kg (50th centile), length 52 cm (75th-90th centile) and OFC 36 cm (75th centile). On examination, he showed right sided facial hypoplasia, left ptosis, absent lacrimal puncta, bilateral microtia with atretic ear canals, left mandibular pit, hypoplastic ala nasi, maxillary hypoplasia, retrognathia, cleft uvula, bilateral fifth finger clinodactyly and convex nails. He required feeding by nasogastric tube during the first eight months of life. At 19 months, sparse fine hair, eyebrows and eyelashes, short palpebral fissures with irregular borders, hypodontia with small and conical teeth and velopharyngeal incompetence with normal laryngeal structures were noted (Fig. S1c,d). CT scan revealed bilateral external auditory canal atresia and hypoplasia of the tympanic cavity and ossicular chain and a normal cochlea. He had severe conductive deafness and hearing aids were implanted at three months. Abdominal ultrasound confirmed the left kidney hydronephrosis and a right ureteric stenosis, which was surgically repaired at four months of age, together with a
nasolacrimal duct stenosis. At 35 months, his weight was 12.500 kg (10th centile), height 90.5 cm (3th–10th centile) and OFC 48.5 cm (3th–10th centile). To date, his psychomotor development is normal. Sequencing analysis revealed a heterozygous missense mutation in exon 4 of TP63 gene; the mutation c. 401G > A resulting in a glycine to aspartic acid amino acid substitution in position 134 of the p63 protein (p.G134D), just in front of the DNA binding domain (DBD) (Fig. 1c). This mutation was not present in the parents, thus appearing to have arised de novo. 4. Discussion We have described two patients with genetic aberrations of the same p63–Dlx5/Dlx6 pathway, one with a deletion involving DLX5/ DLX6 genes and the other with a mutation of the TP63 gene. The patient with the deletion showed typical features of SHFM1 and that with the TP63 mutation showed clinical features suggestive of RHS without SHFM (Table 1). Mutations in RHS/AEC syndromes are clustered in the 3′end of the TP63 gene. These are mainly missense and frameshift mutations affecting the p63α-specific SAM and TI domains and are thought to have either dominant-negative or gain-of-function effects (Rinne et al., 2008, 2009). More recently, three novel pathogenic mutations in the N-terminus of ΔNp63 isoform have been reported in RHS/AEC-like patients; the mutations p.Gln9fsX23 and p.Gln11X in the alternative exon 3′ and p.Gln16X in exon 4. The first two are present in only the ΔNp63 isoforms whereas the latter is present in both ΔNp63 and TAp63 isoforms. All these mutations lead to a premature termination
A. Vera-Carbonell et al. / Gene 497 (2012) 292–297 Table 1 Major clinical characteristics of patients with similar 7q21.1q21.3 deletion and the same mutation p.G134D. Deletion 7q21.1q21.3
Mutation p.G134D
Haberlandt Our Our patient 1 patient 2 Age (at description) Growth retardation Craniofacial abnormalities Arched eyebrows Micrognatia/retrognatia Cleft lip/palate/bifid uvula Low set dysplastic ears Digital abnormalities Ectrodactyly Braquidactyly Syndactyly/camptodactyly Ectodermal abnormalities Sparse hair Nail dystrophy Anodontia/hipodontia Lacrimal duct obstruction Dry skin Neurological abnormalities Hypotonia Developmental delay/mental retardation Ear abnormalities/deafness Renal/genitourinary abnormalities
Slavotinek
2 years +
3 years +
16 months 15 years − ?
+ + + +
− − − +
+ + + +
+ − − −
+ − −
+ − +
− − −
− + +
+ ? + ? −
− − − − −
+ + + + +
+ + + + +
− +
+ +
− −
− −
+ +
− −
+ +
− ?
codon but, surprisingly, in keratinocytes from these patients these mutant alleles still produce a p63-related protein by re-initiation of translation at the next ATG codon after the premature termination codon. This causes a deletion of 25 amino acids in the ΔNp63 isoforms, nullifying the TA2 domain, thus suggesting a crucial role for this part of p63 in the pathogenesis of RHS/AEC syndromes (Rinne et al., 2008). Since RHS/AEC is a dominantly inherited disease, it has been proposed that mutations in the 5′end of the TP63 gene lose the activator function of ΔNp63α and have even dominant negative activity against the wild type p63 protein (Rinne et al., 2009). Molecular analysis for the TP63 gene in our Patient 2 identified the missense mutation c.401G > A (p.G134D) in heterozygosity. This falls in the TP63 proline-rich domain that connects the TA and DBD domains at the N-terminus of ΔNp63 and TAp63 isoforms (Slavotinek et al., 2005) and is involved in regulating transcriptional activity (Helton et al., 2008). To our knowledge, this mutation has
295
not been previously reported in RHS/AEC-like patients and it is the first missense mutation reported in the N-terminus of p63 in patients with these syndromes. Interestingly, it was previously identified in one case of ADULT syndrome (Slavotinek et al., 2005) and in another of LMS syndrome (Rinne et al., 2007). RHS and AEC syndromes are variable manifestations of the same clinical entity (Bertola et al., 2004; Rinne et al., 2007) and among their symptoms, the limb anomalies are typical, being most commonly syndactyly of fingers and toes (reported in 30% of RHS patients), but camptodactyly of hands has been reported and 12% of individuals presented with ectrodactyly of the hands and feet (McGrath et al., 2001; Sutton et al., 2009). Importantly, in our patient with RHS, only bilateral fifth finger clinodactyly was observed, consistent with the almost complete absence of limb malformations in this syndrome (Rinne et al., 2007). However, in the patient with ADULT syndrome bearing the mutation p.G134D, the hands showed ulnar ray hypoplasia with bilateral fifth finger brachydactyly and camptodactyly (Slavotinek et al., 2005) (Table 1). These fifth finger brachydactyly and camptodactyly have not previously been reported in ADULT syndrome (Slavotinek et al., 2005). For the patient with LMS syndrome and this mutation, no clinical information was available. In addition, four other mutations have been reported in this region; the mutation G76W in the ΔN-specific TA2 domain in a large LMS family (van Bokhoven and Brunner, 2002), S90W and P127L in the TP63 proline-rich domain in two LMS and ADULT families, respectively (Rinne et al., 2007; van Zelst-Stams and van Steensel, 2009), and the splicing mutation 3′ss intron 4 in SHFM4 (van Bokhoven et al., 2001). The mutation G134D identified in our second patient may also enhance transcriptional activity, as previously reported for the mutation P127L (van Zelst-Stams and van Steensel, 2009). Since p63 is a transcription factor, the molecular basis of these defects most likely resides in the inability of mutated p63 proteins to properly activate/repress expression of target genes, which are beginning to be identified (Di Constanzo et al., 2009; Jamora et al., 2003; Moretti et al., 2010). Indeed, phenotypic defects found in some patients with TP63 mutations are common in those affected by SHFM1, which is caused by loss/misregulation of p63 target genes such as DLX5 and DLX6, as previously reported (Kouwenhoven et al., 2010) and which is again illustrated by the characterization of the chromosomal 7q21 deletion by array-CGH in our first patient who had clinical features consistent with SHFM1. He had a de novo deletion 7q21.13–q21.3 and the size of the deletion was ~8.478 Mb (from
Fig. 2. Schematic representation of the region 7q21. The figure shows the partial chromosomal 7 ideogram and the distance in Mb from p-telomere. The region of interest has been amplified (red) and shows the positions of DLX5, DLX6 and DSS1 genes consistent with the UCSC Genome Browser (version march 2006) and the SHFM1-BS1 enhancer according to Kouwenhoven et al. (2010). Also, the location of our deletion (Patient 1) and of previously reported cases is shown (grey and red shading = deletion, red line = break).
296
A. Vera-Carbonell et al. / Gene 497 (2012) 292–297
89378731 to 97857149 pb), including DSS1, DLX5 and DLX6 among more than fifty genes (Fig. 2). The most distal breakpoint of our deletion was mapped to 1.367 and 1.381 Mb telomeric to the DLX5 and DLX6 genes, respectively, and it disrupted the IRTKS gene. Previously reported patients with SHFM1 showed significant variability in clinical presentation. As well as the widely variable pattern of limb deformities in each case, additional anomalies and developmental delay/mental retardation were present. General growth impairment, microcephaly, hearing loss and dysmorphic face are the most common features although others, including cleft palate and abnormal teeth are sometimes seen. The feet are involved more often than the hands and hearing loss and mental retardation are present in 30% of SHFM1 patients (Elliott and Evans, 2006). The phenotype of Patient 1 showed some of these features including growth retardation, developmental delay and dysmorphic features, as well as a mild right foot ectrodactyly, although deafness was not a feature. It seems likely that his limb anomalies were due to loss of DLX5/DLX6 genes and regulatory elements located within the deletion, as the causative role of DSS1gene seems less clear and it has not been demonstrated. A very similar deletion to that of our patient has been reported by Haberlandt et al. (2001) and the patient with SHFM had profound sensorineural hearing loss resulting from Mondini dysplasia as well as features of ectodermal hypoplasia and a submucous cleft palate (Fig. 2) (Table 1). The smallest deletion (~0.9 Mb), including loss of DSS1 and DLX5/DLX6 genes, has been reported by Wieland et al. (2004) and the patient showed ectrodactyly of the left hand and both feet, with typical lobster claw and the right hand had syndactyly of the 3rd and 4th digits. Further dysmorphic features and profound deafness, due to malformation of the inner ear, were also observed (Fig. 2). Recent reports using microarray analysis and breakpoint cloning have detected various microdeletions at the chromosomal 7q21.3 region, without loss of DLX5/DLX6 genes, causing some of these above-mentioned features. Brown et al. (2010) reported a microdeletion of 5115 bp (located 65 and 80 kb centromeric to the DLX6 and DLX5, respectively) in a family whose affected members had an inv(7)(q21.3q35). All had hearing loss due to abnormalities of the inner ear but none showed limb abnormalities. Saitsu et al. (2009) reported a complex rearrangement in a patient with bilateral split-foot malformation and hearing loss. This well-characterized rearrangement detected a chromosomal 7q21.3 break, mapped to 258 and 272 kb centromeric to the DLX6 and DLX5, respectively, in addition to a microdeletion of 806 kb located 750 kb telomeric to the 7q21.3 breakpoint. Finally, the patient reported by Kouwenhoven et al. (2010) with a 880 kb deletion showed only bilateral foot anomalies and no other phenotypic features. Surprisingly, in this deletion, the DLX5 and DLX6 genes were intact, containing only the regulatory element SHFM1-BS1 identified as a p63 binding site (Fig. 2). Therefore, the phenotypic variability observed among patients with chromosomal aberrations at the 7q21 region shows that the mechanisms that control the tissue-specific expression of DLX5/DLX6 during development are not yet clear. On the other hand, recent studies have provided some insight into the differential regulation of target genes by mutant p63 proteins. Thus, whereas p63 proteins carrying AEC mutations can activate the DLX5 and DLX6 promoters, p63 proteins carrying EEC or SHFM4 mutations cannot, potentially providing an explanation for the lack of limb abnormalities in patients with AEC (Lo Iacono et al., 2008). These data may explain the almost complete absence of limb malformations in our patient with Rapp–Hodgkin syndrome, in which this mutation may likewise enhance transcriptional activity of p63 on DLX5 and DLX6 promoters. However, the above-mentioned studies did not determine whether mutant p63 proteins affect the function of wild-type proteins when they are co-expressed. Besides, coexpression of the ΔN and TAp63 isotypes occurs in normal tissues (Nylander et al., 2002) and, in a given cellular context, the relative ratios of these isoforms could ultimately affect biological outcome
(King et al., 2006). Thus, the function of mutant p63 proteins in the cellular context in which they are normally expressed is largely unknown. Furthermore, the molecular basis for preferential target gene activation by different mutant p63 proteins also remains unclear. Supplementary materials related to this article can be found online at doi:10.1016/j.gene.2012.01.088. 5. Conclusion The phenotypic characteristics associated with these two distinct genetic aberrations, commonly related to the same functional development pathway, show the clinical complexity of these heterogeneous disorders and highlight the necessity to apply different diagnostic strategies, including array-CGH and p63-mutations analysis, to patients with split-hand/foot malformation and/or ectodermal dysplasias. Further contributions of molecular analysis to new patients will allow us to progress in elucidating the genetic basis of many of these clinically or functionally related disorders which may be important for future therapeutic options and genetic counseling. References Bertola, D.R., Kim, C.A., Albano, L.M., Scheffer, H., Meijer, R., van Bokhoven, H., 2004. Molecular evidence that AEC syndrome and Rapp–Hodgkin syndrome are variable expression of a single genetic disorder. Clin. Genet. 66, 79–80. Brown, K.K., et al., 2010. Deletion of an enhancer near DLX5 and DLX6 in a family with hearing loss, craniofacial defects, and an inv(7)(q21.3q35). Hum. Genet. 127, 19–31. Celli, J., et al., 1999. Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 99, 143–153. Crackower, M.A., et al., 1996. Characterization of the split hand/split foot malformation locus SHFM1 at 7q21.3–q22.1 and analysis of a candidate gene for its expression during limb development. Hum. Mol. Genet. 5, 571–579. Di Constanzo, A., et al., 2009. Homeodomain protein Dlx3 induces phosphorilationdependent p63 degradation. Cell Cycle 8, 1185–1195. Dohn, M., Zhang, S., Chen, X., 2001. p63α and ΔNp63α can induce cell cycle arrest and apoptosis and differentially regulate p53 target genes. Oncogene 20, 3193–3205. Duijf, P.H.G., van Bokhoven, H., Brunner, H.G., 2003. Pathogenesis of split-hand/splitfoot malformation. Hum. Mol. Genet. 12, R51–R60. Elliott, A.M., Evans, J.A., Chudley, A.E., 2005. Split hand foot malformation (SHFM). Clin. Genet. 68, 501–505. Elliott, A.M., Evans, J.A., 2006. Genotype–phenotype correlations in mapped split hand foot malformations (SHFM) patients. Am. J. Med. Genet. Part A 140A, 1419–1427. Ferrari, D., et al., 1995. The expression pattern of the Distal-less homeobox-containing gene Dlx-5 in the developing chick limb bud suggests its involvement in apical ectodermal ridge activity, pattern formation, and cartilage differentiation. Mech. Dev. 52, 257–264. Ghioni, P., Bolognese, F., Duijf, P.H.G., van Bokhoven, H., Mantovani, R., Guerrini, L., 2002. Complex transcriptional effects of p63 isoforms: identification of novel activation and repression domains. Mol. Cell. Biol. 22, 8659–8668. Haberlandt, E., et al., 2001. Split hand/split foot malformation associated with sensorineural deafness, inner and middle ear malformation, hypodontia, congenital vertical talus, and deletion of eight microsatellite markers in 7q21.1–q21.3. J. Med. Genet. 38, 405–409. Helton, E.S., Zhang, J., Chen, X., 2008. The proline-rich domain in p63 is necessary for the transcriptional and apoptosis-inducing activities of TAp63. Oncogene 27, 2843–2850. Jamora, C., DasGupta, R., Kocieniewski, P., Fuchs, E., 2003. Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 422, 317–322. King, K.E., et al., 2006. Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis. Carcinogenesis 27, 53–63. Kouwenhoven, E.N., et al., 2010. Genome-wide profiling of p63 DNA-binding sites identifies an element that regulates gene expression during limb development in the 7q21 SHFM1 locus. PLoS Genet. 6, e1001065. Leoyklang, P., Siriwan, P., Shotelersuk, V., 2006. A mutation of the p63 gene in nonsyndromic cleft lip. J. Med. Genet. 43, e28. Lo Iacono, N., et al., 2008. Regulation of Dlx5 and Dlx6 gene expresión by p63 is involved in EEC and SHFM congenital limb defects. Development 135, 1377–1388. McGrath, J.A., et al., 2001. Hay–Wells syndrome is caused by heterozygous missense mutations in the Sam domain of p63. Hum. Mol. Genet. 10, 221–229. Mills, A., Zhenh, B., Wang, X., Vogel, H., Roop, D., Bradley, A., 1999. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398, 708–713. Moretti, F., et al., 2010. A regulatory feedback loop involving p63 and IRF6 links the pathogenesis of 2 genetically different human ectodermal dysplasias. J. Clin. Invest. 120, 1570–1577. Murray-Zmijewski, F., Lane, D.P., Bourdon, J.C., 2006. p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress. Cell Death Differ. 13, 92–972.
A. Vera-Carbonell et al. / Gene 497 (2012) 292–297 Nylander, K., et al., 2002. Differential expression of p63 isoforms in normal tissues and neoplastic cells. J. Pathol. 198, 417–427. Rinne, T., Brunner, H.G., van Bokhoven, H., 2007. p63-associated disorders. Cell Cycle 6, 262–268. Rinne, T., et al., 2008. A novel translation re-initiation mechanism for the p63 gene revealed by amino-terminal truncating mutations in Rapp–Hodgkin/Hay–Wellslike syndromes. Hum. Mol. Genet. 17, 1968–1977. 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. Robledo, R.F., Rajan, L., Li, X., Lufkin, T., 2002. The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development. Genes Dev. 16, 1089–1101. Saitsu, H., et al., 2009. Characterization of the complex 7q21.3 rearrangement in a patient with bilateral split-foot malformation and hearing loss. Am. J. Med. Genet. Part A 149A, 1224–1230. Scherer, S.W., et al., 1994a. Physical mapping of the split hand/split foot locus on chromosome 7 and implication in syndromic ectrodactyly. Hum. Mol. Genet. 3, 1345–1354. Scherer, S.W., et al., 1994b. Fine mapping of the autosomal dominant split hand/split foot locus on chromosome 7, band q21.3–q22.1. Am. J. Hum. Genet. 55, 12–20. Slavotinek, A.M., Tanaka, J., Winder, A., Vargervik, K., Haggstrom, A., Bamshad, M., 2005. Acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome: report of a child
297
with phenotypic overlap with ulnar–mammary syndrome and a new mutation in TP63. Am. J. Med. Genet. 138A, 146–149. Sutton, V.R., Plunkett, K., Dang, D.X., Lewis, R.A., Bree, A.F., Bacino, C.A., 2009. Craniofacial and anthropometric phenotype in ankyloblepharon–ectodermal defects–cleft lip/palate syndrome (Hay–Wells syndrome) in a cohort of 17 patients. Am. J. Med. Genet. Part A 149A, 1916–1921. van Bokhoven, H., et al., 2001. p63 gene mutations in EEC syndrome, limb–mammary syndrome, and isolated split hand-split foot malformations suggest a genotype– phenotype correlation. Am. J. Hum. Genet. 69, 481–492. van Bokhoven, H., Brunner, H.G., 2002. Splitting p63. Am. J. Hum. Genet. 71, 1–13. van Silfhout, A.T., et al., 2009. Split hand/foot malformation due to chromosome 7q aberrations (SHFM1): additional support for functional haploinsufficiency as the causative mechanism. Eur. J. Hum. Genet. 17, 1432–1438. van Zelst-Stams, W.A.G., van Steensel, M.A.M., 2009. A novel TP63 mutation in family with ADULT syndrome presenting with eczema and hypothelia. Am. J. Med. Genet. 149A, 1558–1560. Wieland, I., Muschke, P., Jakubiczka, S., Volleth, M., Freigang, B., Wieacker, P.F., 2004. Refinement of the deletion in 7q21.3 associated with split hand/foot malformation type 1 and Mondini dysplasia. J. Med. Genet. 41, e54. Yang, A., et al., 1998. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating death-inducing, and dominant-negative activities. Mol. Cell 2, 305–316. Yang, A., et al., 1999. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398, 714–718.
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
Rapp–Hodgkin syndrome and SHFM1 patients: Delineating the p63–Dlx5/Dlx6 pathway Ascensión Vera-Carbonell a, María Rosa Moya-Quiles b, María Ballesta-Martínez c, Vanesa López-González c, Juan Antonio Bafallíu a, Encarna Guillén-Navarro c, Isabel López-Expósito a,⁎ a b c
Sección de Citogenética, Centro de Bioquímica y Genética Clínica, Hospital U. “Virgen de la Arrixaca”, El Palmar, Murcia, Spain Sección de Genética Molecular, Centro de Bioquímica y Genética Clínica, Hospital U. “Virgen de la Arrixaca”, El Palmar, Murcia, Spain Unidad de Genética Médica y Dismorfología. Servicio de Pediatría, Hospital U. “Virgen de la Arrixaca”, El Palmar, Murcia, Spain
a r t i c l e
i n f o
Article history: Accepted 29 January 2012 Available online 9 February 2012 Keywords: Rapp–Hodgkin syndrome Split hand/foot malformation p63-mutation analysis Array-CGH analysis
a b s t r a c t Rapp–Hodgkin Syndrome (RHS) is a genetic disorder resulting from mutations in the TP63 gene encoding p63 transcription factor. p63 is directly associated with a cis-regulatory element on chromosome 7q21 that controls the expression of DLX5 and DLX6 genes which are involved in craniofacial abnormalities and ectrodactyly or split hand/foot malformation (SHFM). Chromosomal deletions on 7q21 locus can result in loss of DXL5/DLX6 and/or in loss/disruption of cis-regulatory elements, at which p63 binds. We report two patients that have in common a p63–Dlx5/Dlx6 pathway dysregulation. One showed growth retardation, craniofacial dysmorphism, syndactyly, developmental delay and a de novo deletion (~ 8.5 Mb) on chromosome 7q21.13–q21.3, including DLX5 and DLX6. The second patient with a clinical diagnosis of RHS showed a de novo heterozygous missense mutation, c. 401G > A (p.G134D), in TP63 (exon 4). Our findings may contribute to a greater understanding of the pathogenic mechanisms underlying disorders caused by TP63 mutations. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The complex human development process results from gradients of signaling molecules with precisely regulated temporal and spatial patterns and it is therefore important that developmental genes are subject to strict regulation. The p63, Dlx5 and Dlx6 proteins, encoded by the TP63, DLX5 and DLX6 genes, respectively, are transcription factors essential for epidermal morphogenesis and limb development. Animal models have shown that p63 −/− mice are born alive but show striking defects in development; their skin does not progress from the early stages of development, lacking stratification as well as expression of
Abbreviations: TP63, tumor protein p63; DLX, distal-less homeobox; AER, apical ectodermal ridge; DBD, DNA-binding domain; ISO, isomerization; TA, transactivation; SAM, sterile alpha motif; TI, transactivation inhibitory; EEC, ectrodactyly–ectodermal dysplasia–cleft lip/palate; AEC, ankyloblepharon–ectodermal dysplasia–cleft lip/palate; RHS, Rapp–Hodgkin syndrome; LMS, limb–mammary syndrome; ADULT, acro-dermatoungual-lacrimal-tooth; SHFM, split hand/foot malformation; DSS1, deleted split hand/ split-foot 1; SHFM-BS, SHFM-binding site; OFC, occipito-frontal head circumference; CT, computed tomography; MRI, magnetic resonance imaging; PCR, Polymerase Chain Reaction; GTG, Giemsa-Trypsin-G bands; PHA, phytohemagglutinin; CGH, Comparative Genomic Hybridization; dCTP, deoxycytidine triphosphate; ADM, Aberration Detection Method; bp, base pair; kb, kilobase; Mb, megabase; NCBI, National Center for Biotechnology Information; IRTKS, insulin-receptor tyrosine kinase substrate; inv, inversion; del, deletion. ⁎ Corresponding author. Tel.: + 34 968381269; fax: + 34 968 889861. E-mail address: [email protected] (I. López-Expósito). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.01.088
differentiation markers, and the mammary glands, hair follicles and teeth are absent (Mills et al., 1999; Yang et al., 1999). Moreover, Dlx5/Dlx6 −/− results in inner ear and severe limb malformations and craniofacial and axial skeletal defects (Robledo et al., 2002). p63 is directly associated with the DLX5 and DLX6 promoters and regulates expression of both genes in mice (Lo Iacono et al., 2008), suggesting that p63 and the Dlx5/Dlx6 locus are involved in the same signaling pathway. The DLX5 and DLX6 genes encode homeobox-containing proteins and are usually co-expressed in a unique spatial and temporal pattern in the apical ectodermal ridge (AER), which is critical for normal limb development (Duijf et al., 2003; Ferrari et al., 1995). The TP63 gene, a member of the p53 family, as a result of the alternative usage of two promoters and a complex alternative splicing, encodes at least six different p63 protein isoforms (TAp63α, TAp63β, TAp63γ, ΔNp63α, ΔNp63β and ΔNp63γ). Of these, the transactivation (TA) isoforms contain an N-terminal TA domain, which is absent in the ΔN isoforms (Yang et al., 1998). In ΔNp63 isoforms, an additional TA2 domain has been recognized as responsible for ΔN-specific transcriptional activities distinct from that of TA isoforms (Dohn et al., 2001; Ghioni et al., 2002). All p63 isoforms contain a DNA binding domain (DBD) and a tetramerization domain (ISO). The extended 3′coding sequences of the α isotypes of p63 encodes a sterile-α-motif (SAM) domain and a transactivation inhibitory (TI) domain. The modular structure of p63 reflects the functional differences between p63 isoforms. Thus, TAp63 isotypes act in a p53-like fashion
A. Vera-Carbonell et al. / Gene 497 (2012) 292–297
displaying anti-proliferative, pro-apoptotic and tumor-suppressive functions while ΔNp63 isotypes act as mitogenic, anti-apoptotic and oncogenic regulators (Yang et al., 1998). By expressing TAp63 and ΔNp63 isoforms, p63 has the ability to regulate a number of genes with diverse roles and possesses opposing regulatory effects depending on the form used (Murray-Zmijewski et al., 2006). In humans, mutations in the TP63 gene have been found in a number of dominantly inherited congenital disorders, including EEC syndrome (ectrodactyly, ectodermal dysplasia, and cleft lip/palate, MIM 604292), ankyloblepharon–ectodermal dysplasia–clefting syndrome (AEC, MIM 106260), Rapp–Hodgkin syndrome (RHS, MIM 129400), limb–mammary syndrome (LMS, MIM 603543), acrodermato-ungual-lacrimal-tooth syndrome (ADULT, MIM 103285), and split hand/foot malformation 4 (SHFM4, MIM 605289) (Celli et al., 1999; Rinne et al., 2007; van Bokhoven et al., 2001). These syndromes share several phenotypic features, consisting of ectodermal dysplasia, orofacial clefting and limb malformations, that can be related to alterations of a tightly controlled balance between proliferation and differentiation of precursor cells during the development of ectodermal-derived epithelia and organs (Rinne et al., 2007). The ectodermal dysplasia manifests as defects in skin, hair, teeth, nails and exocrine glands. The orofacial clefting is mainly cleft lip and/or cleft palate and the ectrodactyly (also known as split-hand/foot malformation, SHFM) is characterized by a deficiency of the central rays of the hands and/or feet, resulting in missing or malformed digits, and may present with syndactyly, median clefts of the hands and feet and aplasia and/or hypoplasia of the phalanges, metacarpals and metatarsals (Elliott et al., 2005). Besides TP63 located on chromosome 3q27, five other loci have been considered in relation to syndromic and non-syndromic SHFM: SHFM1 on chromosome 7q21, SHFM2 on Xq26, SHFM3 on 10q24, SHFM5 on 2q31 and SHFM6 on 12q13 (for review see Saitsu et al., 2009). SHFM1 is associated with deletions of variable extent on chromosome 7q21, minimally including the DSS1 gene and the distallessrelated homeogenes DLX5 and DLX6 (Crackower et al., 1996; Scherer et al., 1994a, 1994b). Studies in mutant mice with double knock-out of DLX5 and DLX6 have shown ectrodactyly whereas DSS1expression was normal in these mutants (Robledo et al., 2002) and therefore a concomitant haploinsufficiency of DLX5 and DLX6 has been proposed as the causative mechanism in humans. Since translocations or inversions with breakpoints within the SHFM1 critical region (~0.9 Mb) (Wieland et al., 2004) were identified in patients affected with ectrodactyly, a ‘position effect’ mutagenic mechanism (Scherer et al., 1994a) or ‘functional haploinsufficiency’ (van Silfhout et al., 2009) for SHFM1 was proposed. It seems that these breaks could separate DLX5 and DLX6 from a regulatory element, which was proposed to be located at least 800 kb centromeric of DLX5/DLX6 (van Silfhout et al., 2009). Recently, Kouwenhoven et al. (2010), working with human primary keratinocyte cultures and animal models, have identified an enhancer element referred to as “SHFM1-BS1” in the SHFM1 critical region on chromosome 7q21, located more that 250 kb centromeric of DLX5/ DLX6 genes and which controls expression of DLX6 and possibly DLX5 in limb development. The identification of a microdeletion in a patient with SHFM, including the enhancer element but not the DLX5/DLX6 genes, has allowed them to conclude that this regulatory element acts as an enhancer for expression of these genes mediated by p63 during embryonic limb development and that p63 may regulate DLX5/DLX6 expression from a distance, perhaps by a looping mechanism. Therefore, the loss or disruption of this cis-regulatory element has been proposed as a novel pathogenic mechanism in SHFM1. Together, these data link the pathogenesis of two genetically different groups of developmental diseases into a common molecular pathway. Here we report two patients, one with Rapp–Hodgkin syndrome caused by a mutation on TP63 gene and another with SHFM1 and a deletion on chromosome 7q21, to illustrate the clinical and molecular etiology of RHS and SHFM1 in the context of the p63–
293
Dlx5/Dlx6 pathway. In addition, we compare the clinical phenotype of these patients with others previously reported. 2. Methods 2.1. Molecular genetic analysis Genomic DNA was isolated from peripheral blood, according to established protocols. The TP63 exons with intron–exon boundaries were amplified using previously published primers (Leoyklang et al., 2006). PCR fragments were sequenced using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3130 Genetic Analyser (Applied Biosystems). The position of mutations corresponds to the coding sequence for the original published TA-p63α isotype (GenBank accession AF075430). 2.2. Cytogenetic and array-CGH analysis High-resolution GTG banded chromosomes obtained from PHAstimulated peripheral lymphocytes were analyzed. Oligo array-CGH analysis was performed with the Agilent Human Genome CGH 400K microarray (Agilent Technologies, Santa Clara, CA, USA). This array spans the entire human genome at a median probe spacing of ~5.3 kb. Samples of 1 μg of genomic DNA from the patient and a reference normal genomic DNA male (Promega, Madison, WI) were differentially labeled by random priming with Cy5-dCTP and Cy3-dCTP. The hybridization was carried out according to the manufacturer's protocol. Microarray data were extracted and visualized using Feature Extraction software v10.7 and analyzed using Genomic Workbench software v5.0 (Agilent Technologies). Copy number altered regions were detected using the Z-score (set as 4) and ADM-2 (set as 6) statistics provided by DNA Analytics. 3. Results 3.1. Patient 1 Patient 1 was a 15-month-old boy who was referred for cytogenetic analysis because of developmental delay, growth retardation, dysmorphic features and limb anomalies. He was the second child of a young and non-consanguineous couple. During pregnancy there was a threat of premature birth at six months gestation and intrauterine growth retardation was detected at eight months by ultrasound. The baby was born at 38-weeks gestation with a weight of 2.330 kg (b3rd centile), length of 46 cm (3rd centile) and occipito-frontal head circumference (OFC) of 32 cm (2nd centile). Apgar scores were 9 and 10 at 1 and 5 min, respectively. Physical examination demonstrated several dysmorphic facial features including high forehead, epicanthal folds, short nose with anteverted nares, downturned corners of the mouth, high palate and low-set ears, as well as mild right foot ectrodactyly (syndactyly of 1st–2nd toes, absence of distal phalanges of 3rd toe and defective alignment of 4th and 5th toes) (Fig. S1a,b). He also had mild global hypotonia. At 15 months of age, all his measurements were well below the 3rd centile and the intermamillary distance was slightly increased with a lower and inverted right nipple. At 30 months, psychomotor developmental and language delay were diagnosed (sitting up at 16 months and independent walking at 27 months). Initially, hearing impairment was suspected, but cranial computed tomography (CT) scan was normal. Results of additional investigations including audiometric and ophthalmological examinations, abdominal ultrasound, magnetic resonance imaging (MRI) scan of the brain and echocardiogram were all normal. GTG banded chromosomal analysis revealed a de novo interstitial deletion on the long arm of one chromosome 7 (Fig. 1a). The karyotype was 46,XY,del(7)(q21.1q21.3). Additional high-resolution oligo arrayCGH analysis of peripheral blood from this patient confirmed the
294
A. Vera-Carbonell et al. / Gene 497 (2012) 292–297
Fig. 1. Partial karyotype and microarray of Patient 1 and sequencing analysis of the TP63 gene of Patient 2. a: Chromosomes 7 with GTG bands of Patient 1 (right: normal, left: deleted). b: Oligo array-CGH log 2 ratio plot of whole chromosome 7 with ideogram illustrating deletion and enlargement of the deleted region, also of Patient 1. c: Electrophoregram of Patient 2 shows the c.401G>A (p.G134D) mutation in exon 4 of the TP63 gene (above), which is not present in the control (below).
deletion on chromosome 7q21.13–q21.3 (chr7:89,378,731-97,857,149 bp on the March 2006 assembly of the human genome: NCBI36/hg18). The size of the deleted region was estimated to be 8.478 Mb, including DSS1, DLX5 and DLX6 among more than fifty genes (Fig. 1b). Analysis of TP63 mutations was normal (data not shown). 3.2. Patient 2 Patient 2 was another male referred as a newborn for assessment of multiple congenital anomalies. He was the second child of healthy and non-consanguineous parents. At 24-weeks gestation, ultrasound evaluation revealed bilateral anotia and left hydronephrosis with normal karyotype on amniotic fluid. He was born after an uncomplicated 40-week gestation by vaginal delivery. At birth, his weight was 3.450 kg (50th centile), length 52 cm (75th-90th centile) and OFC 36 cm (75th centile). On examination, he showed right sided facial hypoplasia, left ptosis, absent lacrimal puncta, bilateral microtia with atretic ear canals, left mandibular pit, hypoplastic ala nasi, maxillary hypoplasia, retrognathia, cleft uvula, bilateral fifth finger clinodactyly and convex nails. He required feeding by nasogastric tube during the first eight months of life. At 19 months, sparse fine hair, eyebrows and eyelashes, short palpebral fissures with irregular borders, hypodontia with small and conical teeth and velopharyngeal incompetence with normal laryngeal structures were noted (Fig. S1c,d). CT scan revealed bilateral external auditory canal atresia and hypoplasia of the tympanic cavity and ossicular chain and a normal cochlea. He had severe conductive deafness and hearing aids were implanted at three months. Abdominal ultrasound confirmed the left kidney hydronephrosis and a right ureteric stenosis, which was surgically repaired at four months of age, together with a
nasolacrimal duct stenosis. At 35 months, his weight was 12.500 kg (10th centile), height 90.5 cm (3th–10th centile) and OFC 48.5 cm (3th–10th centile). To date, his psychomotor development is normal. Sequencing analysis revealed a heterozygous missense mutation in exon 4 of TP63 gene; the mutation c. 401G > A resulting in a glycine to aspartic acid amino acid substitution in position 134 of the p63 protein (p.G134D), just in front of the DNA binding domain (DBD) (Fig. 1c). This mutation was not present in the parents, thus appearing to have arised de novo. 4. Discussion We have described two patients with genetic aberrations of the same p63–Dlx5/Dlx6 pathway, one with a deletion involving DLX5/ DLX6 genes and the other with a mutation of the TP63 gene. The patient with the deletion showed typical features of SHFM1 and that with the TP63 mutation showed clinical features suggestive of RHS without SHFM (Table 1). Mutations in RHS/AEC syndromes are clustered in the 3′end of the TP63 gene. These are mainly missense and frameshift mutations affecting the p63α-specific SAM and TI domains and are thought to have either dominant-negative or gain-of-function effects (Rinne et al., 2008, 2009). More recently, three novel pathogenic mutations in the N-terminus of ΔNp63 isoform have been reported in RHS/AEC-like patients; the mutations p.Gln9fsX23 and p.Gln11X in the alternative exon 3′ and p.Gln16X in exon 4. The first two are present in only the ΔNp63 isoforms whereas the latter is present in both ΔNp63 and TAp63 isoforms. All these mutations lead to a premature termination
A. Vera-Carbonell et al. / Gene 497 (2012) 292–297 Table 1 Major clinical characteristics of patients with similar 7q21.1q21.3 deletion and the same mutation p.G134D. Deletion 7q21.1q21.3
Mutation p.G134D
Haberlandt Our Our patient 1 patient 2 Age (at description) Growth retardation Craniofacial abnormalities Arched eyebrows Micrognatia/retrognatia Cleft lip/palate/bifid uvula Low set dysplastic ears Digital abnormalities Ectrodactyly Braquidactyly Syndactyly/camptodactyly Ectodermal abnormalities Sparse hair Nail dystrophy Anodontia/hipodontia Lacrimal duct obstruction Dry skin Neurological abnormalities Hypotonia Developmental delay/mental retardation Ear abnormalities/deafness Renal/genitourinary abnormalities
Slavotinek
2 years +
3 years +
16 months 15 years − ?
+ + + +
− − − +
+ + + +
+ − − −
+ − −
+ − +
− − −
− + +
+ ? + ? −
− − − − −
+ + + + +
+ + + + +
− +
+ +
− −
− −
+ +
− −
+ +
− ?
codon but, surprisingly, in keratinocytes from these patients these mutant alleles still produce a p63-related protein by re-initiation of translation at the next ATG codon after the premature termination codon. This causes a deletion of 25 amino acids in the ΔNp63 isoforms, nullifying the TA2 domain, thus suggesting a crucial role for this part of p63 in the pathogenesis of RHS/AEC syndromes (Rinne et al., 2008). Since RHS/AEC is a dominantly inherited disease, it has been proposed that mutations in the 5′end of the TP63 gene lose the activator function of ΔNp63α and have even dominant negative activity against the wild type p63 protein (Rinne et al., 2009). Molecular analysis for the TP63 gene in our Patient 2 identified the missense mutation c.401G > A (p.G134D) in heterozygosity. This falls in the TP63 proline-rich domain that connects the TA and DBD domains at the N-terminus of ΔNp63 and TAp63 isoforms (Slavotinek et al., 2005) and is involved in regulating transcriptional activity (Helton et al., 2008). To our knowledge, this mutation has
295
not been previously reported in RHS/AEC-like patients and it is the first missense mutation reported in the N-terminus of p63 in patients with these syndromes. Interestingly, it was previously identified in one case of ADULT syndrome (Slavotinek et al., 2005) and in another of LMS syndrome (Rinne et al., 2007). RHS and AEC syndromes are variable manifestations of the same clinical entity (Bertola et al., 2004; Rinne et al., 2007) and among their symptoms, the limb anomalies are typical, being most commonly syndactyly of fingers and toes (reported in 30% of RHS patients), but camptodactyly of hands has been reported and 12% of individuals presented with ectrodactyly of the hands and feet (McGrath et al., 2001; Sutton et al., 2009). Importantly, in our patient with RHS, only bilateral fifth finger clinodactyly was observed, consistent with the almost complete absence of limb malformations in this syndrome (Rinne et al., 2007). However, in the patient with ADULT syndrome bearing the mutation p.G134D, the hands showed ulnar ray hypoplasia with bilateral fifth finger brachydactyly and camptodactyly (Slavotinek et al., 2005) (Table 1). These fifth finger brachydactyly and camptodactyly have not previously been reported in ADULT syndrome (Slavotinek et al., 2005). For the patient with LMS syndrome and this mutation, no clinical information was available. In addition, four other mutations have been reported in this region; the mutation G76W in the ΔN-specific TA2 domain in a large LMS family (van Bokhoven and Brunner, 2002), S90W and P127L in the TP63 proline-rich domain in two LMS and ADULT families, respectively (Rinne et al., 2007; van Zelst-Stams and van Steensel, 2009), and the splicing mutation 3′ss intron 4 in SHFM4 (van Bokhoven et al., 2001). The mutation G134D identified in our second patient may also enhance transcriptional activity, as previously reported for the mutation P127L (van Zelst-Stams and van Steensel, 2009). Since p63 is a transcription factor, the molecular basis of these defects most likely resides in the inability of mutated p63 proteins to properly activate/repress expression of target genes, which are beginning to be identified (Di Constanzo et al., 2009; Jamora et al., 2003; Moretti et al., 2010). Indeed, phenotypic defects found in some patients with TP63 mutations are common in those affected by SHFM1, which is caused by loss/misregulation of p63 target genes such as DLX5 and DLX6, as previously reported (Kouwenhoven et al., 2010) and which is again illustrated by the characterization of the chromosomal 7q21 deletion by array-CGH in our first patient who had clinical features consistent with SHFM1. He had a de novo deletion 7q21.13–q21.3 and the size of the deletion was ~8.478 Mb (from
Fig. 2. Schematic representation of the region 7q21. The figure shows the partial chromosomal 7 ideogram and the distance in Mb from p-telomere. The region of interest has been amplified (red) and shows the positions of DLX5, DLX6 and DSS1 genes consistent with the UCSC Genome Browser (version march 2006) and the SHFM1-BS1 enhancer according to Kouwenhoven et al. (2010). Also, the location of our deletion (Patient 1) and of previously reported cases is shown (grey and red shading = deletion, red line = break).
296
A. Vera-Carbonell et al. / Gene 497 (2012) 292–297
89378731 to 97857149 pb), including DSS1, DLX5 and DLX6 among more than fifty genes (Fig. 2). The most distal breakpoint of our deletion was mapped to 1.367 and 1.381 Mb telomeric to the DLX5 and DLX6 genes, respectively, and it disrupted the IRTKS gene. Previously reported patients with SHFM1 showed significant variability in clinical presentation. As well as the widely variable pattern of limb deformities in each case, additional anomalies and developmental delay/mental retardation were present. General growth impairment, microcephaly, hearing loss and dysmorphic face are the most common features although others, including cleft palate and abnormal teeth are sometimes seen. The feet are involved more often than the hands and hearing loss and mental retardation are present in 30% of SHFM1 patients (Elliott and Evans, 2006). The phenotype of Patient 1 showed some of these features including growth retardation, developmental delay and dysmorphic features, as well as a mild right foot ectrodactyly, although deafness was not a feature. It seems likely that his limb anomalies were due to loss of DLX5/DLX6 genes and regulatory elements located within the deletion, as the causative role of DSS1gene seems less clear and it has not been demonstrated. A very similar deletion to that of our patient has been reported by Haberlandt et al. (2001) and the patient with SHFM had profound sensorineural hearing loss resulting from Mondini dysplasia as well as features of ectodermal hypoplasia and a submucous cleft palate (Fig. 2) (Table 1). The smallest deletion (~0.9 Mb), including loss of DSS1 and DLX5/DLX6 genes, has been reported by Wieland et al. (2004) and the patient showed ectrodactyly of the left hand and both feet, with typical lobster claw and the right hand had syndactyly of the 3rd and 4th digits. Further dysmorphic features and profound deafness, due to malformation of the inner ear, were also observed (Fig. 2). Recent reports using microarray analysis and breakpoint cloning have detected various microdeletions at the chromosomal 7q21.3 region, without loss of DLX5/DLX6 genes, causing some of these above-mentioned features. Brown et al. (2010) reported a microdeletion of 5115 bp (located 65 and 80 kb centromeric to the DLX6 and DLX5, respectively) in a family whose affected members had an inv(7)(q21.3q35). All had hearing loss due to abnormalities of the inner ear but none showed limb abnormalities. Saitsu et al. (2009) reported a complex rearrangement in a patient with bilateral split-foot malformation and hearing loss. This well-characterized rearrangement detected a chromosomal 7q21.3 break, mapped to 258 and 272 kb centromeric to the DLX6 and DLX5, respectively, in addition to a microdeletion of 806 kb located 750 kb telomeric to the 7q21.3 breakpoint. Finally, the patient reported by Kouwenhoven et al. (2010) with a 880 kb deletion showed only bilateral foot anomalies and no other phenotypic features. Surprisingly, in this deletion, the DLX5 and DLX6 genes were intact, containing only the regulatory element SHFM1-BS1 identified as a p63 binding site (Fig. 2). Therefore, the phenotypic variability observed among patients with chromosomal aberrations at the 7q21 region shows that the mechanisms that control the tissue-specific expression of DLX5/DLX6 during development are not yet clear. On the other hand, recent studies have provided some insight into the differential regulation of target genes by mutant p63 proteins. Thus, whereas p63 proteins carrying AEC mutations can activate the DLX5 and DLX6 promoters, p63 proteins carrying EEC or SHFM4 mutations cannot, potentially providing an explanation for the lack of limb abnormalities in patients with AEC (Lo Iacono et al., 2008). These data may explain the almost complete absence of limb malformations in our patient with Rapp–Hodgkin syndrome, in which this mutation may likewise enhance transcriptional activity of p63 on DLX5 and DLX6 promoters. However, the above-mentioned studies did not determine whether mutant p63 proteins affect the function of wild-type proteins when they are co-expressed. Besides, coexpression of the ΔN and TAp63 isotypes occurs in normal tissues (Nylander et al., 2002) and, in a given cellular context, the relative ratios of these isoforms could ultimately affect biological outcome
(King et al., 2006). Thus, the function of mutant p63 proteins in the cellular context in which they are normally expressed is largely unknown. Furthermore, the molecular basis for preferential target gene activation by different mutant p63 proteins also remains unclear. Supplementary materials related to this article can be found online at doi:10.1016/j.gene.2012.01.088. 5. Conclusion The phenotypic characteristics associated with these two distinct genetic aberrations, commonly related to the same functional development pathway, show the clinical complexity of these heterogeneous disorders and highlight the necessity to apply different diagnostic strategies, including array-CGH and p63-mutations analysis, to patients with split-hand/foot malformation and/or ectodermal dysplasias. Further contributions of molecular analysis to new patients will allow us to progress in elucidating the genetic basis of many of these clinically or functionally related disorders which may be important for future therapeutic options and genetic counseling. References Bertola, D.R., Kim, C.A., Albano, L.M., Scheffer, H., Meijer, R., van Bokhoven, H., 2004. Molecular evidence that AEC syndrome and Rapp–Hodgkin syndrome are variable expression of a single genetic disorder. Clin. Genet. 66, 79–80. Brown, K.K., et al., 2010. Deletion of an enhancer near DLX5 and DLX6 in a family with hearing loss, craniofacial defects, and an inv(7)(q21.3q35). Hum. Genet. 127, 19–31. Celli, J., et al., 1999. Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome. Cell 99, 143–153. Crackower, M.A., et al., 1996. Characterization of the split hand/split foot malformation locus SHFM1 at 7q21.3–q22.1 and analysis of a candidate gene for its expression during limb development. Hum. Mol. Genet. 5, 571–579. Di Constanzo, A., et al., 2009. Homeodomain protein Dlx3 induces phosphorilationdependent p63 degradation. Cell Cycle 8, 1185–1195. Dohn, M., Zhang, S., Chen, X., 2001. p63α and ΔNp63α can induce cell cycle arrest and apoptosis and differentially regulate p53 target genes. Oncogene 20, 3193–3205. Duijf, P.H.G., van Bokhoven, H., Brunner, H.G., 2003. Pathogenesis of split-hand/splitfoot malformation. Hum. Mol. Genet. 12, R51–R60. Elliott, A.M., Evans, J.A., Chudley, A.E., 2005. Split hand foot malformation (SHFM). Clin. Genet. 68, 501–505. Elliott, A.M., Evans, J.A., 2006. Genotype–phenotype correlations in mapped split hand foot malformations (SHFM) patients. Am. J. Med. Genet. Part A 140A, 1419–1427. Ferrari, D., et al., 1995. The expression pattern of the Distal-less homeobox-containing gene Dlx-5 in the developing chick limb bud suggests its involvement in apical ectodermal ridge activity, pattern formation, and cartilage differentiation. Mech. Dev. 52, 257–264. Ghioni, P., Bolognese, F., Duijf, P.H.G., van Bokhoven, H., Mantovani, R., Guerrini, L., 2002. Complex transcriptional effects of p63 isoforms: identification of novel activation and repression domains. Mol. Cell. Biol. 22, 8659–8668. Haberlandt, E., et al., 2001. Split hand/split foot malformation associated with sensorineural deafness, inner and middle ear malformation, hypodontia, congenital vertical talus, and deletion of eight microsatellite markers in 7q21.1–q21.3. J. Med. Genet. 38, 405–409. Helton, E.S., Zhang, J., Chen, X., 2008. The proline-rich domain in p63 is necessary for the transcriptional and apoptosis-inducing activities of TAp63. Oncogene 27, 2843–2850. Jamora, C., DasGupta, R., Kocieniewski, P., Fuchs, E., 2003. Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 422, 317–322. King, K.E., et al., 2006. Unique domain functions of p63 isotypes that differentially regulate distinct aspects of epidermal homeostasis. Carcinogenesis 27, 53–63. Kouwenhoven, E.N., et al., 2010. Genome-wide profiling of p63 DNA-binding sites identifies an element that regulates gene expression during limb development in the 7q21 SHFM1 locus. PLoS Genet. 6, e1001065. Leoyklang, P., Siriwan, P., Shotelersuk, V., 2006. A mutation of the p63 gene in nonsyndromic cleft lip. J. Med. Genet. 43, e28. Lo Iacono, N., et al., 2008. Regulation of Dlx5 and Dlx6 gene expresión by p63 is involved in EEC and SHFM congenital limb defects. Development 135, 1377–1388. McGrath, J.A., et al., 2001. Hay–Wells syndrome is caused by heterozygous missense mutations in the Sam domain of p63. Hum. Mol. Genet. 10, 221–229. Mills, A., Zhenh, B., Wang, X., Vogel, H., Roop, D., Bradley, A., 1999. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398, 708–713. Moretti, F., et al., 2010. A regulatory feedback loop involving p63 and IRF6 links the pathogenesis of 2 genetically different human ectodermal dysplasias. J. Clin. Invest. 120, 1570–1577. Murray-Zmijewski, F., Lane, D.P., Bourdon, J.C., 2006. p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress. Cell Death Differ. 13, 92–972.
A. Vera-Carbonell et al. / Gene 497 (2012) 292–297 Nylander, K., et al., 2002. Differential expression of p63 isoforms in normal tissues and neoplastic cells. J. Pathol. 198, 417–427. Rinne, T., Brunner, H.G., van Bokhoven, H., 2007. p63-associated disorders. Cell Cycle 6, 262–268. Rinne, T., et al., 2008. A novel translation re-initiation mechanism for the p63 gene revealed by amino-terminal truncating mutations in Rapp–Hodgkin/Hay–Wellslike syndromes. Hum. Mol. Genet. 17, 1968–1977. 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. Robledo, R.F., Rajan, L., Li, X., Lufkin, T., 2002. The Dlx5 and Dlx6 homeobox genes are essential for craniofacial, axial, and appendicular skeletal development. Genes Dev. 16, 1089–1101. Saitsu, H., et al., 2009. Characterization of the complex 7q21.3 rearrangement in a patient with bilateral split-foot malformation and hearing loss. Am. J. Med. Genet. Part A 149A, 1224–1230. Scherer, S.W., et al., 1994a. Physical mapping of the split hand/split foot locus on chromosome 7 and implication in syndromic ectrodactyly. Hum. Mol. Genet. 3, 1345–1354. Scherer, S.W., et al., 1994b. Fine mapping of the autosomal dominant split hand/split foot locus on chromosome 7, band q21.3–q22.1. Am. J. Hum. Genet. 55, 12–20. Slavotinek, A.M., Tanaka, J., Winder, A., Vargervik, K., Haggstrom, A., Bamshad, M., 2005. Acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome: report of a child
297
with phenotypic overlap with ulnar–mammary syndrome and a new mutation in TP63. Am. J. Med. Genet. 138A, 146–149. Sutton, V.R., Plunkett, K., Dang, D.X., Lewis, R.A., Bree, A.F., Bacino, C.A., 2009. Craniofacial and anthropometric phenotype in ankyloblepharon–ectodermal defects–cleft lip/palate syndrome (Hay–Wells syndrome) in a cohort of 17 patients. Am. J. Med. Genet. Part A 149A, 1916–1921. van Bokhoven, H., et al., 2001. p63 gene mutations in EEC syndrome, limb–mammary syndrome, and isolated split hand-split foot malformations suggest a genotype– phenotype correlation. Am. J. Hum. Genet. 69, 481–492. van Bokhoven, H., Brunner, H.G., 2002. Splitting p63. Am. J. Hum. Genet. 71, 1–13. van Silfhout, A.T., et al., 2009. Split hand/foot malformation due to chromosome 7q aberrations (SHFM1): additional support for functional haploinsufficiency as the causative mechanism. Eur. J. Hum. Genet. 17, 1432–1438. van Zelst-Stams, W.A.G., van Steensel, M.A.M., 2009. A novel TP63 mutation in family with ADULT syndrome presenting with eczema and hypothelia. Am. J. Med. Genet. 149A, 1558–1560. Wieland, I., Muschke, P., Jakubiczka, S., Volleth, M., Freigang, B., Wieacker, P.F., 2004. Refinement of the deletion in 7q21.3 associated with split hand/foot malformation type 1 and Mondini dysplasia. J. Med. Genet. 41, e54. Yang, A., et al., 1998. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating death-inducing, and dominant-negative activities. Mol. Cell 2, 305–316. Yang, A., et al., 1999. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398, 714–718.