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Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is caused by heterozygous missense point mutations in the p63 gene, an important transcription factor during embryogenesis and for stem cell differentiation in stratified epithelia. Most of the cases are sporadic, related to de novo mutations arising during early-stage development. Familial cases show an autosomic dominant inheritance. The major cause of visual morbidity is limbal stem cell failure, which develops in the second to third decade of life. Patients often show ocular surface alterations, such as recurrent blepharitis and conjunctivitis, superficial microlesions of the cornea, and spontaneous corneal perforation and ulceration, leading to progressive corneal clouding and eventually visual loss. No definitive cures are currently available, and treatments to alleviate symptoms are only palliative. In this review, we will discuss the proposed therapeutic strategies that have been tested or are under development for the management of the ocular defects in patients affected by EEC syndrome: (i) gene therapy-based approaches by means of Allele-Specific (AS) siRNAs to correct the p63 mutations; (ii) cell therapy-based approaches to replenish the pool of limbal stem cells; and (iii) drug therapy to correct/bypass the genetic defect. However, as the number of patients with EEC syndrome is too limited, further studies are still necessary to prove the effectiveness (and safety) of these innovative therapeutic approaches to counteract the premature differentiation of limbal stem cells.

Human epithelial stem cells (ESCs) are characterized by long-term regenerative properties, much dependent on the tissue of origin and varying during their lifespan. We analysed such variables in cultures of ESCs isolated from the skin, conjunctiva, limbus and oral mucosa of healthy donors and patients affected by ectrodactyly-ectodermal dysplasia-clefting syndrome, a rare genetic disorder caused by mutations in the p63 gene. We cultured cells until exhaustion in the presence or in the absence of DAPT (γ-secretase inhibitor; N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine T-butyl ester). All cells were able to differentiate in vitro but exhibited variable self-renewal potential. In particular, cells carrying p63 mutations stopped prematurely, compared with controls. Importantly, administration of DAPT significantly extended the replicative properties of all stem cells under examination. RNA sequencing analysis revealed that distinct sets of genes were up- or down-regulated during their lifetime, thus allowing to identify druggable gene networks and off-the-shelf compounds potentially dealing with epithelial stem cell senescence. These data will expand our knowledge on the genetic bases of senescence and potentially pave the way to the pharmacological modulation of ageing in epithelial stem cells., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare monogenic disease with autosomal dominant inheritance caused by mutations in the TP63 gene, leading to progressive corneal keratinocyte loss, limbal stem cell deficiency (LSCD), and eventually blindness. Currently, there is no treatment available to cure or slow down the keratinocyte loss. Human oral mucosal epithelial stem cells (hOMESCs), which are a mixed population of keratinocyte precursor stem cells, are used as source of autologous tissue for treatment of bilateral LSCD. However, hOMESCs from EEC patients have a reduced life span due to TP63 mutations and cannot be used for autologous transplantation. Human induced pluripotent stem cells (hiPSCs) represent a potentially unlimited source of autologous limbal stem cell for EEC patients and can be genetically modified by genome editing technologies to correct the disease ex vivo before transplantation. In this study, we describe for the first time the generation of integration-free EEC-hiPSCs from hOMESCs of EEC patients by Sendai virus vector and episomal vector-based reprogramming. The generated hiPSC clones expressed pluripotency markers and were successfully differentiated into derivatives of the three germ layers, as well as toward corneal epithelium. These cells may be used for EEC disease modeling and open perspectives for applications in cell therapy of LSCD.

Oral mucosa epithelial stem cells from a patient affected by Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome carrying the R279H mutation in the TP63 gene were reprogrammed into human induced pluripotent stem cells (hiPSCs) with episomal vectors. The generated UNIPDi003-A-hPSC line retained the mutation of the parental cells and showed a normal karyotype upon long term culture. Analysis of residual transgenes expression showed that the episomal vectors were eliminated from the cell line. UNIPDi003-A-hiPSCs expressed the undifferentiated state marker alkaline phosphatase along with a panel of pluripotency markers, and formed embryoid bodies capable of expressing markers belonging to all the three germ layers., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Unlabelled: : Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches., Significance: This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative capacity, they may be used to successfully correct the corneal defects present in this unique case of ectrodactyly-ectodermal dysplasia-clefting syndrome., (©AlphaMed Press.)

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-ΔNp63α-RFP and R279H-ΔNp63α-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome. Stem Cells 2016;34:1588-1600., (© 2016 AlphaMed Press.)

Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome is a rare autosomal dominant ectodermal dysplasia syndrome. It is caused by heterozygous mutations in TP63, encoding a transcriptional factor of the p53 family. Mutations in TP63, mainly missense in exons 13 and 14 encoding the sterile alpha motif (SAM) and the transactivation inhibitory (TI) domains, account for 99% of mutations in individuals with AEC syndrome. Of these, ≥70% are de novo mutations, present in the affected patient, but not in parents nor in healthy siblings. However, when a mutation appears de novo, it is not possible to differentiate between a sporadic mutation, or germline mosaicism in the parents. In this latter case, there is a risk of having additional affected offspring. We describe two sisters with AEC syndrome, whose parents were unaffected. Both patients carried the heterozygous c.1568T>C substitution in exon 13 of TP63, resulting in a p.L523P change in the SAM domain of the protein. Analyses of DNA from parental blood cells, seminal fluid (from the father) and maternal cells (buccal, vaginal, and cervical) did not reveal the mutation, suggesting that the mosaicism may involve a very low percentage of cells (very low grade somatic mosaicism) or, more likely, maternal gonadal mosaicism. Mosaicism must be considered for the assessment of recurrence risk during genetic counseling in AEC syndrome, and pre-implantation/prenatal genetic diagnosis should be offered to all couples, even when the mutation is apparently de novo., (Copyright © 2012 Wiley Periodicals, Inc.)

Objective: To describe the ocular phenotype in patients with ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome (MIM#604292) and to determine the pathogenic basis of visual morbidity., Design: Retrospective case series., Participants: Nineteen families (23 patients) affected by EEC syndrome from the United Kingdom, Ireland, and Italy., Methods: General medical examination to fulfill the diagnostic criteria for EEC syndrome and determine the phenotypic severity. Mutational analysis of p63 was performed by polymerase chain reaction-based bidirectional Sanger sequencing. All patients with EEC syndrome underwent a complete ophthalmic examination and ocular surface assessment. Limbal stem cell deficiency (LSCD) was diagnosed clinically on the basis of corneal conjunctivalization and anatomy of the limbal palisades of Vogt. Impression cytology using immunofluorescent antibodies was performed in 1 individual. Histologic and immunohistochemical analyses were performed on a corneal button and corneal pannus from 2 EEC patients., Main Outcome Measures: The EEC syndrome phenotypic severity (EEC score), best-corrected Snellen visual acuity (decimal fraction), slit-lamp biomicroscopy, tear function index, tear breakup time, LSCD, p63 DNA sequence variants, impression cytology, and corneal histopathology., Results: Eleven heterozygous missense mutations in the DNA binding domain of p63 were identified in all patients with EEC syndrome. All patients had ocular involvement and the commonest was an anomaly of the meibomian glands and lacrimal drainage system defects. The major cause of visual morbidity was progressive LSCD, which was detected in 61% (14/23). Limbal stem cell deficiency was related to advancing age and caused a progressive keratopathy, resulting in a dense vascularized corneal pannus, and eventually leading to visual impairment. Histologic analysis and impression cytology confirmed LSCD., Conclusions: Heterozygous p63 mutations cause the EEC syndrome and result in visual impairment owing to progressive LSCD. There was no relationship of limbal stem cell failure with the severity of EEC syndrome, as classified by the EEC score, or the underlying molecular defect in p63., Financial Disclosure(s): The authors have no proprietary or commercial interest in any of the materials discussed in this article., (Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

The ectrodactyly-ectodermal dysplasia-clefting syndrome is a rare autosomal dominant disorder caused by heterozygous mutations in the p63 gene, a transcription factor belonging to the p53 family. The majority of cases of ectrodactyly-ectodermal dysplasia syndrome are caused by de novo mutations and are therefore sporadic in approximately 60% of patients. The substitution of arginine to histidine (R279H), due to a c.836G>A mutation in exon 7 of the p63 gene, represents 55% of the identified mutations and is considered a mutational hot spot. A quantitative and sensitive real-time PCR was performed to quantify both wild-type and R279H alleles in DNA extracted from peripheral blood and RNA from cultured epithelial cells. Standard curves were constructed for both wild-type and mutant probes. The sensitivity of the assay was determined by generating serial dilutions of the DNA isolated from heterozygous patients (50% of alleles mutated) with wild-type DNA, thus obtaining decreasing percentages of p63 R279H mutant allele (50%, 37.5%, 25%, 12.5%, 10%, 7.5%, 5%, 2.5%, and 0.0%). The assay detected up to 1% of the mutant p63. The high sensitivity of the assay is of particular relevance to prenatal diagnosis and counseling and to detect therapeutic effects of drug treatment or gene therapy aimed at reducing the amount of mutated p63., (Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)