The Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominantly inherited disease characterized by multi-organ dysfunctions including ectrodactyly, ectodermal dysplasia, facial clefting (mostly in the form of cleft lip and palate) and urogenital abnormalities in 13 out of 25 cases. Hypohidrosis is variable, the hair is sparse, fair and dry and eyebrows and lashes are often absent. The teeth are small and hypodontia and anodontia occur. The nails are thin, brittle and ridged, and tear duct abnormalities are common. While in childhood clefting and hand deformities are the main clinical features, during early adulthood ocular problems become the predominant clinical aspect of EEC syndrome. Patients often show ocular surface alterations such as recurrent blepharitis and conjunctivitis, superficial microlesions of the cornea, spontaneous corneal perforation and ulceration, defective regeneration and poor reepithelialization following trauma or penetrating keratoplasty (PK). They are characterised by dense vascularized corneal pannus, leading to progressive corneal clouding and eventually severe visual impairment. Alterations leading to these symptoms are: (1) the atresia of the lacrimal duct system and the absence of meibomian glands causing tear film instability and (2) the epithelial defects of the cornea caused by mutations in the p63 gene. While tear film instability and dry eye symptoms can be overcome with tear supplements, no curative treatments are currently available for the epithelial defects.


The EEC syndrome is caused by mutations in the p63 gene, an important transcription factor during embryogenesis (for the normal development of orofacial ectoderma and limbs) and for stem cell differentiation in stratified epithelia. Mutations in the p63 gene can cause at least 5 different sindrome. The rare combination of ectrodactyly (lobster-claw deformity), ectodermal dysplasia and cleft lip with or without cleft palate has historically been described as EEC syndrome. The EEC syndrome is mainly caused by point mutations in the DNA binding domain of the p63 gene. Thirty-four different mutations have so far been reported, and at least 20 different amino acids are involved. Only 2 mutations are outside the DNA binding domain: one insertion (1572 InsA) and one point mutation (L563P) in the Sterile A Motif (SAM) domain. Five frequently mutated amino acids (R204, R227, R279, R280 and R304) were found in the EEC population, all located in the CpG islands. These 5 mutations

account for almost 90% of all the EEC syndrome patients. Mutations found in the p63 gene of EEC patients appear therefore to impair p63 protein binding to DNA. Even if EEC mutations have dominant negative effects, recent genotype-phenotype analyses for the five hot-spot mutations revealed significant differences between the corresponding phenotypes. For instance while cleft lip/palate is present in the R304 mutated population (80%), R227 patients seldom have cleft lip/palate. Syndactyly is completely absent in the R227 population, while 30-60% of the other hot-spot mutation population have syndactyly. Genito-urinary defects are frequently observed in R227 patients (40%), but less in other patients. It thus seems that these hot-spot mutations might exert specific modifying effects as, for example, on promoters for p63 transcriptional target genes. All patients with R304Q, R279H, R279S, R311G, H208R, R280C, S272N and R311G show serious ocular problems and progressive loss of visual capacity. Most of cases are sporadic, related to de novo mutations arising during early-stage development. Familial cases show an autosomic dominant inheritance with variable penetrance. The incidence and prevalence of the EEC syndrome in the Italian and European populations are unknown. Male and females are equally affected.


“p63” is a transcription factor belonging to the same family that includes p53 and p73. Whereas p53 plays a well-established role in tumor suppression, p63 and p73 play unique roles in morphogenesis. The p63 gene generates six isoforms. The transactivating (TA) and the deltaN isoforms. In both cases, alternative splicing gives rise to 3 different C termini, designated alpha, beta and gamma. In human corneal epithelia, deltaNp63alpha is the major p63 isoform expressed and it is necessary for the maintenance of the proliferative potential of limbal stem cells and essential for regenerative proliferation in the ocular surface. Limbal-corneal keratinocytes express not only deltaNp63alpha but also the deltaNp63beta and deltaNp63gamma isoforms. However, while expression of deltaNp63alpha is restricted to the limbal stem cell compartment, the expression of deltaNp63beta and deltaNp63gamma correlates with limbal cell migration, corneal wound healing and corneal differentiation. deltaNp63alpha is expressed in a small amount of undifferentiated and small cells (stem cells). The percentage of these cells in primary cultures ranges between 3% and 8% and decreases progressively both during clonal conversion (the transition from holoclones to paraclones) and serial propagation of stem cell in vitro (life span). Patients with the EEC syndrome are characterised by dense vascularized corneal pannus, leading to progressive corneal clouding and eventually severe visual impairment. Two are the causes that we believe could lead to these alterations and symptoms:

(1) the atresia of the lacrimal duct system and the absence of meibomian glands causing tear film instability, thus eventually leading to epithelial defects;

(2) the epithelial defects of the cornea caused by mutations in the p63 gene.

We believe that for several reasons the latter is more likely to be true. In fact:

  1. therapies based on treatments with tear supplements are only palliative. They reduce the dry eye symptoms and tear film instability, but do not stop the progression of the disease;
  2. common therapeutic strategies, such as PK, are not a solution: as soon as the donor epithelium is exhausted conjunctivalisation occurs again;
  3. previous data from us and other groups show that the ocular problems present in patients with EEC syndrome are linked to the loss of corneal epithelial stem cells. Ophthalmologic and genetic analyses on  patients affected by EEC show evident alterations of visual ability due to recurrent corneal epithelial defects, such as corneal ulceration and scars, presence of corneal pannus, neovascularization, inflammation and blepharitis. For all of them the major cause of visual morbidity was limbal stem cell failure which resulted in a progressive degeneration of corneal epithelial tissues, an event generally reaching its peak around 30 years. Declining visual acuity was accompanied by a partial or complete degeneration of the palisades of Vogt, the niche of corneal stem cells.

Because of all these reasons, our hypothesis is that the recurrent epithelial breakdown observed in patients affected by EEC syndrome reflects a diminished capability of limbal stem cells to regenerate the corneal epithelium and that this is caused by mutations in the p63 gene.


Fondazione Banca degli Occhi del Veneto and the University of Padua have long been working on this project. The main objective would be to evaluate whether allele-specific p63 gene silencing approaches could lead to correction of the genetic defect underlying EEC syndrome in corneal stem cells and path the way for the therapeutic transplantation of genetically corrected corneal stem cells. Gene therapy (through gene silencing techniques) could really represent the only alternative for these patients. Common therapeutic strategies, such as PK, are not a solution since in case of limbal stem cell deficiency as soon as the donor epithelium is exhausted conjunctivalisation occurs again. Similarly, autologous cultured limbal stem cell transplantation (cell therapy) cannot be a definitive cure for EEC patients, since p63 mutations severely compromise the ability of autologous stem cells to regenerate the corneal epithelium.


Preliminary data and results have been published in peer-reviewed journals:


  • 1.       Barbaro V, Confalonieri L, Vallini I, Ferrari S, Ponzin D, Mantero G, Willoughby CE, Parekh M, Di Iorio E. Development of an allele-specific real-time PCR assay for discrimination and quantification of p63 R279H mutation in EEC syndrome. The Journal of Molecular Diagnostics 2012; 14 (1): 38-45. DOI: 10.1016/j.jmoldx.2011.07.008
  • 2.      Di Iorio E, Kaye SB, Ponzin D, Barbaro V, Ferrari S, Böhm E, Nardiello P, Castaldo G, McGrath JA, Willoughby CE. Limbal Stem Cell Deficiency and Ocular Phenotype in Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome caused by p63 Mutations. Ophthalmology 2012; 119 (1): 74-83.
  • 3.      Barbaro V, Nardiello P, Castaldo G, Willoughby CE, Ferrari S, Ponzin D, Amato F, Bonifazi E, Parekh M, Calistri A, Parolin C, Di Iorio E. A novel de novo missense mutation in TP63 underlying germline mosaicism in AEC syndrome: implications for recurrence risk and prenatal diagnosis. American Journal of Medical Genetics Part A 2012; 158A (8): 1957-1961. DOI: 10.1002/ajmg.a.35414.

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