A promising stem cells strategy to correct corneal defects due to a unique homozygous missense mutation p.R311K in the Tp63 gene causing dominant EEC syndrome.
Chiar., mo (supervisor)
Di Iorio, Enzo (supervisor)
Nasti, Annamaria A. (supervisor)
Hassan, Nada Tarek (supervisor)
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Background Ectrodactyly - Ectodermal dysplasia - Clefting (EEC) syndrome is a rare autosomal dominant inherited disease. To date, approximately 40 different Tp63 mutations have been identified, all reported as heterozygous mutations. There is no definitive cure available to counteract and resolve the progressive corneal degeneration resulted from the premature ageing of limbal epithelial stem cells (LESC). However, oral mucosal epithelial stem cells (OMESCs) represent a valid alternative capable of regenerating the corneal epithelium and provide an attractive therapeutic avenue. Our group has recently discovered R311K-∆Np63 mutation, a unique homozygous mutation causing EEC syndrome with a mosaic pattern. The survival of the patient was intriguing as homozygous dominant mutations are lethal. Unlike other EEC mutations, R311K-∆Np63 keeps the binding ability to DNA that can make it a milder mutation if raised in heterozygous form. Aim of research The research focus on studying the R311K-∆Np63 mutation, its effect on protein expression, its transactivation activity and comparing it with other heterozygous mutations (R311G-, R304Q- and R279H∆Np63). The final goal of the research was to find a therapy for the ocular impairment of our EEC case by isolating and expanding ex vivo the heterozygous epithelial stem cells, identified by means of DNA and RNA characterization, from OMESCs biopsy. Cultured autologous OMESCs can represent an effective treatment to reconstruct the ocular surface and partially restore the ocular function in our case. Materials and methods R311K-∆Np63 mutation was studied by testing their protein expression in transfected HEK293T cells by western blot and by measuring their transactivation activity by Luciferase reporter assay on K14 promoter, as it contains p63 binding sites and because the protein expression is ∆Νp63 dependent. Individual cells were isolated from a primary cell culture obtained of OMESCs biopsy from the patient by clonal analysis, expanded ex vivo to be used for CFE analysis and molecular characterization. DNA was extracted with Chelex resin, purified by ethanol precipitation and amplified by PCR to be sequenced by Sanger Method. RNA was extracted by Triazol, purified from possible DNA contaminants by RNAeasy (Biorad microkit), quantified, tested for integrity and quality by agarose gel electrophoresis, reverse-transcribed to cDNA, and then amplified to be analyzed by absolute real time PCR. Results The expression of R311K∆Np63 is increased in ectopic system of expression and Luciferase reporter assay revealed a severe inhibition in the transactivation activity of the K14 promoter in case of homozygously mimicking conditions in transfected HEK293T cells and a significant less severe inhibition in the transactivation activity of the same promoter in heterozygously mimicking transfected cells. Only 20% of isolated single cells of the primary cell culture of the OMESCs biopsy were able to grow into full colonies. These colonies were found heterozygous for the mutation R311K∆Np63. CFE assay for all colonies revealed the presence of 24 holoclones that, when analyzed by qPCR analysis, showed similar ∆Np63α expression level to LESC (previously used cells to successively correct limbal stem cell deficiency- LSCD) suggesting the possibility to use these holoclones to correct LSCD in our case. Conclusion Cultured autologous OMESCs, obtained by selected R311K-N∆p63 heterozygous stem cells, could be effective as personalized therapy to reconstruct the ocular surface of this particular and unique case of EEC syndrome.