title: Personalized Stem Cell Therapy to Correct Corneal Defects Due to a Unique Homozygous-Heterozygous Mosaicism of Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.
abstract: : 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.
  
  
  
  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.
authors.affiliation: Fondazione Banca degli Occhi del Veneto, Venice, Italy.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Barbaro
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Annamaria Assunta
authors.initials: AA
authors.lastname: Nasti
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Raffa
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Migliorati
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Nespeca
authors.affiliation: Fondazione Banca degli Occhi del Veneto, Venice, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Ferrari
authors.affiliation: Department of Biology, University of Padua, Padua, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Palumbo
authors.affiliation: Fondazione Banca degli Occhi del Veneto, Venice, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Bertolin
authors.affiliation: Fondazione Banca degli Occhi del Veneto, Venice, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Breda
authors.affiliation: Department of Neuroscience, University of Naples Federico II, Naples, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Miceli
authors.affiliation: Department of Biology, University of Padua, Padua, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Russo
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Luciana
authors.initials: L
authors.lastname: Caenazzo
authors.affiliation: Fondazione Banca degli Occhi del Veneto, Venice, Italy.
authors.forename: Diego
authors.initials: D
authors.lastname: Ponzin
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Palù
authors.affiliation: Department of Molecular Medicine, University of Padua, Padua, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Parolin
authors.affiliation: Fondazione Banca degli Occhi del Veneto, Venice, Italy Department of Molecular Medicine, University of Padua, Padua, Italy enzo.diiorio@fbov.it.
authors.forename: Enzo
authors.initials: E
authors.lastname: Di Iorio
mesh_terms: D016475:3T3 Cells; D000293:Adolescent; D000818:Animals; D016022:Case-Control Studies; D002971:Cleft Lip / Q000150:complications / Q000175:diagnosis / Q000235:genetics*; D002972:Cleft Palate / Q000150:complications / Q000175:diagnosis / Q000235:genetics*; D018920:Coculture Techniques; D003316:Corneal Diseases / Q000175:diagnosis / Q000235:genetics / Q000601:surgery*; D016039:Corneal Transplantation / Q000379:methods*; D004252:DNA Mutational Analysis; D004476:Ectodermal Dysplasia / Q000150:complications / Q000175:diagnosis / Q000235:genetics*; D061252:Feeder Cells; D005260:Female; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D051379:Mice; D009030:Mosaicism; D009061:Mouth Mucosa / Q000166:cytology / Q000378:metabolism / Q000637:transplantation*; D020125:Mutation, Missense; D018579:Patient Selection; D010641:Phenotype; D057285:Precision Medicine / Q000379:methods*; D011237:Predictive Value of Tests; D033581:Stem Cell Transplantation / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection; D014182:Transplantation, Autologous; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins
keywords: Cell therapy; Ectrodactyly-ectodermal dysplasia-clefting syndrome; Gene conversion; Mosaicism; p63