p63 in corneal and epidermal differentiation.

The transcription factor p63, belonging to the p53 family, is considered the master regulator of epidermal differentiation, skin, and in general of the differentiation of ectodermal tissues. Mutations in TP63 gene cause several rare ectodermal dysplasia disorders that refers to epidermal structural abnormalities and ocular surface disease, such as Ectrodactyly Ectodermal Dysplasia Clefting (EEC) syndrome. In this review, we discuss the key roles of p63 in keratinocytes and corneal epithelial differentiation, highlighting the function of the ΔNp63α isoform in driving limbal stem cell and epithelial stem cells commitment. We have summarized the specific ocular phenotypes observed in the TP63-mutation derived EEC syndrome, discussing the current and novel therapeutic strategies for the management of the ocular manifestations in EEC syndrome.

Dermatological manifestations in cardiofaciocutaneous syndrome: a prospective multicentric study of 45 mutation-positive patients.

BACKGROUND: Data on dermatological manifestations of cardiofaciocutaneous syndrome (CFCS) remain heterogeneous and almost without expert dermatological classification. OBJECTIVES: To describe the dermatological manifestations of CFCS; to compare them with the literature findings; to assess those discriminating CFCS from other RASopathies, including Noonan syndrome (NS) and Costello syndrome (CS); and to test for dermatological phenotype-genotype correlations. METHODS: We performed a 4-year, large, prospective, multicentric, collaborative dermatological and genetic study. RESULTS: Forty-five patients were enrolled. Hair abnormalities were ubiquitous, including scarcity or absence of eyebrows and wavy or curly hair in 73% and 69% of patients, respectively. Keratosis pilaris (KP), ulerythema ophryogenes (UO), palmoplantar hyperkeratosis (PPHK) and multiple melanocytic naevi (MMN; over 50 naevi) were noted in 82%, 44%, 27% and 29% of patients, respectively. Scarcity or absence of eyebrows, association of UO and PPHK, diffuse KP and MMN best differentiated CFCS from NS and CS. Oral acitretin may be highly beneficial for therapeutic management of PPHK, whereas treatment of UO by topical sirolimus 1% failed. No significant dermatological phenotype-genotype correlation was determined. CONCLUSIONS: A thorough knowledge of CFCS skin manifestations would help in making a positive diagnosis and differentiating CFCS from CS and NS.

Topical Leptospermum Honey in the Management of Aplasia Cutis Congenita in Neonates: A Case Study.

BACKGROUND: Aplasia cutis congenita (ACC) is an uncommon, heterogeneous group of disorders characterized by focal or widespread absence of skin from certain parts of the body. Complications range from 20% to 50%; they are potentially life-threatening. There is no consensus on best treatment of ACC. We report on successful closure of aplasia cutis lesions using topical active Leptospermum honey (ALH). CASES: This article describes a case of a full-term neonate with a large ACC lesion. A conservative approach was taken in the care of this lesion, in accordance with appropriate wound care principles and the care of this medically fragile neonate. This included applying topical ALH twice a day and covering defects with a secure dressing. All lesions progressed to complete closure. Time to closure was either similar or shorter than published data. CONCLUSIONS: Our experience with these cases suggests that topical ALH may be an effective natural treatment option for neonates with ACC. This conservative management led to wound closure without topical or systemic antibiotics or prolonged hospital stay.

Topical sucralfate cream treatment for aplasia cutis congenita with dystrophic epidermolysis bullosa: a case study.

Bart syndrome consists of aplasia cutis congenita (ACC) and dominant or recessive dystrophic epidermolysis bullosa (DEB), associated with skin fragility and nail dysplasia. ACC in DEB is thought to be caused by trauma, the most cited cause being in utero formation of bullae consequent to friction of the limbs. Epidermolysis bullosa (EB) refers to a hereditary mechanobullous disease following trauma, characterised by formation of blisters on the skin and mucous membranes. There are four categories of the disease, including epidermolysis bullosa simplex, junctional epidermolysis bullosa, dystrophic epidermolysis bullosa and Kindler syndrome. Infection, sepsis and death may occur as a consequence of generalised blistering with complication. We present the case of a newborn diagnosed with DEB and whose lesions became almost fully epithelialised after treatment with 10% topical sucralfate.

Topical cetirizine and oral vitamin D: a valid treatment for hypotrichosis caused by ectodermal dysplasia.

BACKGROUND: Ectodermal dysplasia is a clinically and genetically heterogeneous group of inherited disorders characterized by abnormal development of two or more of the following ectodermal-derived structures: hair, teeth, nails and sweat glands. The hair is the most frequently affected structure. Hair shaft abnormalities are of great concern to these patients, but no effective treatments are available. METHODS: We describe three girls with congenital hypotrichosis (9, 5 and 6 years old) caused by ectodermal dysplasia treated with topical cetirizine solution (2 mL. once daily) and oral vitamin D supplementation (1000 IU daily). RESULTS: After 6 months of treatment, the density of hair on the scalp increased in all patients. The vellus hair was replaced by terminal hair. Hair regrowth was evaluated both from the clinical and trichoscopic point of view. CONCLUSION: We propose a combination of topical cetirizine and oral vitamin D as a rational treatment of choice in congenital hypotrichosis caused by ectodermal dysplasia.

Conservative Treatment of Large Aplasia Cutis Congenita of the Scalp With Bone Defect With Basic Fibroblast Growth Factor Application.

The object of this report is to share our experience of conservative management of giant aplasia cutis congenita (ACC) of the scalp with the topical application of basic fibroblast growth factor (bFGF). Complete epithelialization of the 9 × 8 cm sized defect was achieved in 33 weeks. Careful conservative management could eliminate the requirement of surgery for giant ACC defects of the scalp with bone defects and should be tried if surgery is thought to be risky or has consecutive morbidity. Topical bFGF application seems to accelerate healing, also providing a better epithelium for later reconstructive treatments and its usage could be standardized in the future.

New BRAF knockin mice provide a pathogenetic mechanism of developmental defects and a therapeutic approach in cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome is one of the 'RASopathies', a group of phenotypically overlapping syndromes caused by germline mutations that encode components of the RAS-MAPK pathway. Germline mutations in BRAF cause CFC syndrome, which is characterized by heart defects, distinctive facial features and ectodermal abnormalities. To define the pathogenesis and to develop a potential therapeutic approach in CFC syndrome, we here generated new knockin mice (here Braf(Q241R/+)) expressing the Braf Q241R mutation, which corresponds to the most frequent mutation in CFC syndrome, Q257R. Braf(Q241R/+) mice manifested embryonic/neonatal lethality, showing liver necrosis, edema and craniofacial abnormalities. Histological analysis revealed multiple heart defects, including cardiomegaly, enlarged cardiac valves, ventricular noncompaction and ventricular septal defects. Braf(Q241R/+) embryos also showed massively distended jugular lymphatic sacs and subcutaneous lymphatic vessels, demonstrating lymphatic defects in RASopathy knockin mice for the first time. Prenatal treatment with a MEK inhibitor, PD0325901, rescued the embryonic lethality with amelioration of craniofacial abnormalities and edema in Braf(Q241R/+) embryos. Unexpectedly, one surviving pup was obtained after treatment with a histone 3 demethylase inhibitor, GSK-J4, or NCDM-32b. Combination treatment with PD0325901 and GSK-J4 further increased the rescue from embryonic lethality, ameliorating enlarged cardiac valves. These results suggest that our new Braf knockin mice recapitulate major features of RASopathies and that epigenetic modulation as well as the inhibition of the ERK pathway will be a potential therapeutic strategy for the treatment of CFC syndrome.

Clinical Report: Cognitive decline in a patient with Cardiofaciocutaneous syndrome.

Cardiofaciocutaneous Syndrome (CFCS) is a rare genetic syndrome caused by mutations in one of four genes: BRAF, MAP2K1, MAP2K2, and KRAS. There is tremendous phenotypic heterogeneity in patients with CFCS and so confirmation of diagnosis requires genetic testing. Neurologic and/or cognitive symptoms are present in almost all CFCS individuals. Little is known about cognitive function in older patients with CFCS. In this report, we present the cognitive, neuropsychiatric, and imaging findings of a patient diagnosed with CFCS who after having remained stable developed progressive cognitive/behavioral and motor decline.

Interferon-gamma reduces the proliferation of M. tuberculosis within macrophages from a patient with a novel hypomorphic NEMO mutation.

X-linked ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by mutations in the nuclear factor-kappa B essential modulator (NEMO) gene. Here, we report the clinical and genetic features of a XL-EDA-ID patient who developed bacillus Calmette-Guérin infection. Patient lymphocytes failed to degrade IκB-α, and sequencing of NEMO identified the novel mutation c.1238A>C/p.H413P. Furthermore, patient monocyte-derived macrophages ingested Mycobacterium tuberculosis normally, but failed to control the intracellular proliferation of bacilli, a defect which was improved in the presence of interferon-gamma (IFN-γ). This work expands the genetic spectrum of XL-EDA-ID and demonstrates improvement in macrophage function in a NEMO-deficient patient by IFN-γ.

High-Potency Topical Steroids: An Effective Therapy for Chronic Scalp Inflammation in Rapp-Hodgkin Ectodermal Dysplasia.

Chronic erosive pustular dermatitis with a predilection for the scalp is a hallmark of ectodermal dysplasias (EDs) caused by mutations in TP63, including Rapp-Hodgkin and Hay-Wells EDs. It is among the most troublesome and symptomatic complications and is typically refractory to classic wound care approaches. We report two cases of Rapp-Hodgkin ED with refractory scalp erosions that markedly improved with the use of potent topical steroids. We also note marked similarities between this scalp inflammation and "erosive pustular dermatosis of the scalp," a condition more typically found in elderly individuals with severe scalp sun damage, and speculate about possible shared pathogenetic mechanisms.

APR-246/PRIMA-1(MET) rescues epidermal differentiation in skin keratinocytes derived from EEC syndrome patients with p63 mutations.

p53 and p63 share extensive sequence and structure homology. p53 is frequently mutated in cancer, whereas mutations in p63 cause developmental disorders manifested in ectodermal dysplasia, limb defects, and orofacial clefting. We have established primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations as an in vitro human model to study the disease mechanism in the skin of EEC patients. We show that these patient keratinocytes cultured either in submerged 2D cultures or in 3D skin equivalents have impaired epidermal differentiation and stratification. Treatment of these patient keratinocytes with the mutant p53-targeting compound APR-246/PRIMA-1(MET) (p53 reactivation and induction of massive apoptosis) that has been successfully tested in a phase I/II clinical trial in cancer patients partially but consistently rescued morphological features and gene expression during epidermal stratification in both 2D and 3D models. This rescue coincides with restoration of p63 target-gene expression. Our data show that EEC patient keratinocytes with p63 mutations can be used for characterization of the abnormal molecular circuitry in patient skin and may open possibilities for the design of novel pharmacological treatment strategies for patients with mutant p63-associated developmental abnormalities.

Impaired epithelial differentiation of induced pluripotent stem cells from ectodermal dysplasia-related patients is rescued by the small compound APR-246/PRIMA-1MET.

Ectodermal dysplasia is a group of congenital syndromes affecting a variety of ectodermal derivatives. Among them, ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome is caused by single point mutations in the p63 gene, which controls epidermal development and homeostasis. Phenotypic defects of the EEC syndrome include skin defects and limbal stem-cell deficiency. In this study, we designed a unique cellular model that recapitulated major embryonic defects related to EEC. Fibroblasts from healthy donors and EEC patients carrying two different point mutations in the DNA binding domain of p63 were reprogrammed into induced pluripotent stem cell (iPSC) lines. EEC-iPSC from both patients showed early ectodermal commitment into K18(+) cells but failed to further differentiate into K14(+) cells (epidermis/limbus) or K3/K12(+) cells (corneal epithelium). APR-246 (PRIMA-1(MET)), a small compound that restores functionality of mutant p53 in human tumor cells, could revert corneal epithelial lineage commitment and reinstate a normal p63-related signaling pathway. This study illustrates the relevance of iPSC for p63 related disorders and paves the way for future therapy of EEC.

Costello and cardio-facio-cutaneous syndromes: Moving toward clinical trials in RASopathies.

The RASopathies, one of the largest groups of multiple congenital anomaly syndromes known, are caused by germline mutations in various genes encoding components of the Ras/mitogen-activated protein kinase (MAPK) pathway. The RASopathies have many overlapping characteristics, including craniofacial manifestations, cardiac malformations, cutaneous, musculoskeletal, gastrointestinal, and ocular abnormalities, neurocognitive impairment, hypotonia, and an increased risk of developing cancer. Costello syndrome (CS) and cardio-facio-cutaneous (CFC) syndrome are two of the more rare RASopathies. CS is caused by activating mutations in HRAS, and CFC is caused by dysregulation of signaling in the Ras/MAPK pathway due to mutations in BRAF, MEK1, or MEK2. The Ras/MAPK pathway, which has been well-studied in cancer, is an attractive target for inhibition in the treatment of various malignancies utilizing small molecule therapeutics that specifically inhibit the pathway. With many inhibitors of the Ras/MAPK pathway in clinical trials, the notion of using these molecules to ameliorate developmental defects in CS and CFC is under consideration. CS and CFC, like other syndromes in their class, have a progressive phenotype and may be amenable to inhibition or normalization of signaling.

Permanent correction of an inherited ectodermal dysplasia with recombinant EDA.

X-linked hypohidrotic ectodermal dysplasia (XLHED; OMIM 305100) is a genetic disorder characterized by absence or deficient function of hair, teeth and sweat glands. Affected children may experience life-threatening high fever resulting from reduced ability to sweat. Mice with the Tabby phenotype share many symptoms with human XLHED patients because both phenotypes are caused by mutations of the syntenic ectodysplasin A gene (Eda) on the X chromosome. Two main splice variants of Eda, encoding EDA1 and EDA2, engage the tumor necrosis factor (TNF) family receptors EDAR and XEDAR, respectively. The EDA1 protein, acting through EDAR, is essential for proper formation of skin appendages; the functions of EDA2 and XEDAR are not known. EDA1 must be proteolytically processed to a soluble form to be active. Here, we show that treatment of pregnant Tabby mice with a recombinant form of EDA1, engineered to cross the placental barrier, permanently rescues the Tabby phenotype in the offspring. Notably, sweat glands can also be induced by EDA1 after birth. This is the first example of a developmental genetic defect that can be permanently corrected by short-term treatment with a recombinant protein.

Methods for the Administration of EDAR Pathway Modulators in Mice.

Genetic deficiency of ectodysplasin A (EDA) causes X-linked hypohidrotic ectodermal dysplasia, a congenital condition characterized by the absence or abnormal formation of sweat glands, teeth, and several skin appendages. Stimulation of the EDA receptor (EDAR) with agonists in the form of recombinant EDA or anti-EDAR antibodies can compensate for the absence of Eda in a mouse model of Eda deficiency, provided that agonists are administered in a timely manner during fetal development. Here we provide detailed protocols for the administration of EDAR agonists or antagonists, or other proteins, by the intravenous, intraperitoneal, and intra-amniotic routes as well as protocols to collect blood, to visualize sweat gland function, and to prepare skulls in mice.

Topical ropivacaine for analgesia of aplasia cutis congenita in newborns with hereditary epidermolysis bullosa.

Aplasia cutis congenita (ACC) in patients with hereditary epidermolysis bullosa (EB) is often associated with major pain. We report our experience with using topical ropivacaine during dressing in newborns with ACC. Eight full-term newborns with EB and ACC were hospitalized in a neonatal intensive care unit for severe pain during dressing despite the use of paracetamol, opioids (n = 8) or ketamine (n = 7). Topical xylocaine was poorly tolerated and not effective. Ropivacaine 2 mg/ml was used directly in contact with the ACC, with a maximum 1 mg/kg/day, which enabled care without the child crying. No immediate or late systemic toxicity was observed. Topical ropivacaine 0.2% appears to be an interesting topical analgesic, with good clinical tolerance and rapid action, in newborns with ACC and EB. These data need to be confirmed in a prospective study including pharmacokinetics evaluations.

Improvement of epidermal covering on AEC patients with severe skin erosions by PRIMA-1MET/APR-246.

P63 is a major transcription factor regulating skin development and homeostasis. It controls many genes involved in cell proliferation, adhesion, and early differentiation. P63 is mutated in several rare syndromes called p63-related ectodermal dysplasia syndromes (ED). The main forms are EEC and AEC syndromes due to p63 missense mutations on the DBD and SAM domains, respectively. ED patients display many developmental defects, including ectrodactyly, clef/lip palate, and ectodermal dysplasia, while AEC patients suffer from severe skin erosions that not always heal. We have previously showed that ED-derived iPSC display altered epidermal commitment. P63 belongs to the p53 gene family sharing similar structural domains. We found that ED-iPSC epidermal commitment can be rescued by a p53-reactivating compounds called PRIMA-1MET, also named APR-246 and currently used in anticancer clinical trials. Here, we established primary epidermal culture from two AEC children (S.F. and Y.M.) suffering from persistent skin erosions at age of 9 and 15, respectively. These patients carry missense mutations on the SAM domain (I576T and I537T). We found that primary keratinocytes (KCs) isolated from these AEC patients underwent altered epidermal differentiation that was rescued by PRIMA-1MET treatment. It prompted us to formulate the compound onto a cream that was topically applied on the right hand of one patient and on the scalp of the second patient. In both cases, the daily treatment allowed re-epithelialization of the eroded skin and a drastic loss of pain after few weeks, improving quality of life. Normally, mutant p63 exerts a dominant-negative effect, mainly through the formation of aggregate with WT p63 and p73. PRIMA-1MET did not reduce protein aggregation while enhancing cell differentiation, suggesting that PRIMA-1MET targets cell differentiation and not p63 activity directly. In conclusion, we propose that repurposing of the antitumoral PRIMA-1MET compound could become a general treatment of AEC skin erosions.

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice.

BACKGROUND: Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as "leaky" hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels. METHODS: We employed the antibody to treat Cx30A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca2+ imaging and ATP release assay in vitro. FINDINGS: Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels. INTERPRETATION: Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. FUND: Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.