Phenotypic variability in TP63-related disorders: comparative analysis of the novel T258I missense mutation and R279C/R340G mutations

p63-related disorders comprehend various symptoms affecting skin, hair, teeth, nails, exocrine glands and ocular ectodermal derivatives. The three main manifestations of p63 related disorders are ectodermal dysplasia, orofacial clefting and limb malformations. These clinical manifestations are common in five p63-related syndromes: Ectrodactyly, Ectodermal Dysplasia and Cleft Lip Palate Syndrome (EEC), Ankyloblepharon-Ectodermal Defects-Cleft Lip/Palate syndrome (AEC), Limb Mammary Syndrome (LMS), Acro Dermato-Ungual-Lacrimal-Tooth Syndrome (ADULT) and Rapp-Hodgkin Syndrome (RHS). Additionally, p63 mutations can cause two non-syndromic human disorders: split hand/foot malformations (SHFM4) and non-syndromic cleft lip (NSCL). These disorders are caused by heterozygous mutations in the TP63 gene, fundamental for epidermal, skin and ectodermal tissues differentiation. This study focuses on two cases: a first patient bearing a severe phenotypic spectrum due to two p63 variants (Arg279Cys reported as pathogenic, and Arg340Gly, a novel missense mutation) and a second patient showing a mild phenotypic spectrum (carrying Thr258Ile, a novel uncharacterized missense variant). Patient n.1 is affected by EEC syndrome, presenting a severe monodactyly affecting both hands and feet, hypopigmented skin and scalp hair, and limbal stem cell deficiency in both eyes. Patient n.2, carries the Thr258Ile mutation, presents hypopigmented scalp hair, hypodontia with dental agenesis and generalized xerosis. The aim of the study is to characterize the novel variants: the Thr258Ile, the Arg340Gly and the Arg279Cys/Arg340Gly. Their position and a first evaluation on their pathogenicity was assessed using in silico predictors. One of the most important things to find out was the allelic phase of the two arginine variants, which could produce a single complex allele or a compound heterozygous cell configuration. Once determined that each of these configurations existed in the same patient, identifying the presence of a cellular mosaicism, the clonogenic ability and cell doubling potential of these cells was tested compared to controls. Eventually, protein functionality was performed, using in vitro assays spanning from protein localization to tetramerization (Bioluminescence Resonance Emission Transfer, BRET) promoter induction (k14 promoter induction) or inhibition (p53 responsive element inhibition) measured through luciferase-based assays.