Case study of patient affected by the mosaic form of de-novo p63 related EEC syndrome

The Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare genetic disorder that is inherited in an autosomal dominant manner, resulting in ocular morbidity, hand and foot malformations. The leading cause of visual failure in EEC syndrome is limbal stem cell deficiency (LSCD) and it has been reported that p63 mutations are related to the diminished ability of limbal epithelial stem cells (LESCs) to regenerate fully stratified corneal epithelia. Therapeutic strategies based on allele-specific (AS) gene silencing through interfering RNA (RNAi), specifically inhibits the expression of disease-associated alleles without suppressing the expression of the corresponding wild-type ones, aiming to phenotypically correct the stem cell population and restore function. Many studies have shown that oral mucosal epithelial stem cells (OMESCs) are a feasible alternative to limbal transplants and an ideal substitute of LESCs for ocular surface reconstruction. In OMESCs, the phenotypic correction of mutant p63 using small interfering RNA (siRNA) mediated AS silencing restored full thickness stratification and differentiation of the epithelia. Being R279H and R304Q (arginine residues) mutational hotspots for EEC syndrome, R279C (pathogenic) and R340G (likely pathogenic) are the mutations found in a new patient. The aim of this study is to assess the position and pathogenicity of these de-novo mutations in this unique EEC patient. The sequencing analysis showed that the two variants are not “cis-acting” and the presence of the wild type allele likely confirms the possibility of a mosaicism. The interaction between p63 and the mutant isoforms and pathogenicity of these variants will be characterized by means of Bioluminescence Resonance Energy Transfer and dual- luciferase reporter assays. Finally, as potential treatment strategy, Gapmers sequences have been designed to bind to the mutant allele, induce the allele-specific silencing of target mRNA and its degradation and selectively suppress the expression of the mutant transcripts.