Heterogeneous addiction to transforming growth factor-beta signalling in recessive dystrophic epidermolysis bullosa-associated cutaneous squamous cell carcinoma.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is associated with a high mortality rate due to the development of life-threatening, metastatic cutaneous squamous cell carcinoma (cSCC). Elevated transforming growth factor-beta (TGF-ß) signalling is implicated in cSCC development and progression in patients with RDEB. OBJECTIVES: To determine the effect of exogenous and endogenous TGF-ß signalling in RDEB cSCC with a view to assessing the potential of targeting TGF-ß signalling for RDEB cSCC therapy. METHODS: A panel of 11 patient-derived RDEB cSCC primary tumour keratinocyte cell lines (SCCRDEBs) were tested for their signalling and proliferation responses to exogenous TGF-ß. Their responses to TGF-ß receptor type-1 (TGFBR1) kinase inhibitors [SB-431542 and AZ12601011 (AZA01)] were tested using in vitro proliferation, clonogenicity, migration and three-dimensional invasion assays, and in vivo tumour xenograft assays. RESULTS: All SCCRDEBs responded to exogenous TGF-ß by activation of canonical SMAD signalling and proliferative arrest. Blocking endogenous signalling by treatment with SB-431542 and AZ12601011 significantly inhibited proliferation (seven of 11), clonogenicity (six of 11), migration (eight of 11) and invasion (six of 11) of SCCRDEBs. However, these TGFBR1 kinase inhibitors also promoted proliferation and clonogenicity in two of 11 SCCRDEB cell lines. Pretreatment of in vitro TGFBR1-addicted SCCRDEB70 cells with SB-431542 enhanced overall survival and reduced tumour volume in subcutaneous xenografts but had no effect on nonaddicted SCCRDEB2 cells in these assays. CONCLUSIONS: Targeting TGFBR1 kinase activity may have therapeutic benefit in the majority of RDEB cSCCs. However, the potential tumour suppressive role of TGF-ß signalling in a subset of RDEB cSCCs necessitates biomarker identification to enable patient stratification before clinical intervention.

Seven novel COL7A1 mutations identified in patients with recessive dystrophic epidermolysis bullosa from Mexico.

Recessive dystrophic epidermolysis bullosa (RDEB; OMIM #226600) is one of the most devastating subtypes of epidermolysis bullosa, a group of skin and mucous membrane blistering disorders often associated with extracutaneous manifestations. RDEB is caused by mutations in COL7A1, the gene encoding type VII collagen (C7), and to date over 700 different mutations in the 8835 nucleotides constituting the open reading frame or adjacent exon-intron boundaries of COL7A1 have been described. We used targeted next-generation sequencing to identify seven previously unreported mutations in a cohort of 17 Mexican patients who were diagnosed with RDEB based on clinical presentation and immunoepitope mapping. Our study expands the spectrum of mutations identified in this cohort, including those suitable for emerging therapies reliant on precise genotyping.

High levels of type VII collagen expression in recessive dystrophic epidermolysis bullosa cutaneous squamous cell carcinoma keratinocytes increases PI3K and MAPK signalling, cell migration and invasion.

BACKGROUND: Epidermolysis bullosa is a group of inherited skin fragility diseases varying in severity from mild scarring to infant mortality. Great efforts are being undertaken to develop therapeutic strategies to treat the more pernicious forms of this disease, particularly those associated with recessive, loss-of-function mutations. In such cases significant effort is directed toward delivering recombinant protein at levels sufficient to demonstrate clinical benefit. Recessive dystrophic epidermolysis bullosa (RDEB) predisposes patients to a high incidence of life-threatening cutaneous squamous cell carcinoma (cSCC). Mutations in the gene encoding type VII collagen, COL7A1, are the sole cause of this disease and conflicting reports concerning type VII collagen and COL7A1 in carcinogenesis exist. OBJECTIVES: To investigate potential oncogenic effects of expressing recombinant type VII collagen in patient cells. METHODS: We used retroviral transduction to introduce type VII collagen into keratinocytes derived from patients with and without RDEB. RESULTS: Retroviral expression of type VII collagen in cSCC keratinocytes established from patients with RDEB resulted in increased cell adhesion, migration and invasion coupled with a concurrent increase in PI3K and MAPK signalling. CONCLUSIONS: Our data suggest caution when formulating strategies where delivery of type VII collagen is likely to exceed levels seen under normal physiological conditions in a patient group with a higher inherent risk of developing skin cancer.

Infrared laser pulse triggers increased singlet oxygen production in tumour cells.

Photodynamic therapy (PDT) is a technique developed to treat the ever-increasing global incidence of cancer. This technique utilises singlet oxygen ((1)O2) generation via a laser excited photosensitiser (PS) to kill cancer cells. However, prolonged sensitivity to intensive light (6-8 weeks for lung cancer), relatively low tissue penetration by activating light (630 nm up to 4 mm), and the cost of PS administration can limit progressive PDT applications. The development of quantum-dot laser diodes emitting in the highest absorption region (1268 nm) of triplet oxygen ((3)O2) presents the possibility of inducing apoptosis in tumour cells through direct (3)O2 → (1)O2 transition. Here we demonstrate that a single laser pulse triggers dose-dependent (1)O2 generation in both normal keratinocytes and tumour cells and show that tumour cells yield the highest (1)O2 far beyond the initial laser pulse exposure. Our modelling and experimental results support the development of direct infrared (IR) laser-induced tumour treatment as a promising approach in tumour PDT.

Mutations in AEC syndrome skin reveal a role for p63 in basement membrane adhesion, skin barrier integrity and hair follicle biology.

BACKGROUND: AEC (ankyloblepharon-ectodermal defects-clefting) syndrome is an autosomal dominant ectodermal dysplasia disorder caused by mutations in the transcription factor p63. Clinically, the skin is dry and often fragile; other features can include partial eyelid fusion (ankyloblepharon), hypodontia, orofacial clefting, sparse hair or alopecia, and nail dystrophy. OBJECTIVES: To investigate how p63 gene mutations affect gene and protein expression in AEC syndrome skin. METHODS: We performed microarray analysis on samples of intact and eroded AEC syndrome skin compared with control skin. Changes were verified by quantitative real-time reverse transcription-polymerase chain reaction and, for basal keratinocyte-associated genes, by immunohistochemistry and analysis of microdissected skin. RESULTS: We identified significant upregulation of six genes and downregulation of 69 genes in AEC syndrome skin, with the main changes in genes implicated in epidermal adhesion, skin barrier formation and hair follicle biology. There was reduced expression of genes encoding the basement membrane proteins FRAS1 and collagen VII, as well as the skin barrier-associated small proline-rich proteins 1A and 4, late cornified envelope protein 5A, hornerin, and lipid transporters including ALOX15B. Reduced expression of the hair-associated keratins 25, 27, 31, 33B, 34, 35, 81 and 85 was also noted. We also confirmed similar alterations in gene expression for 26 of the 75 genes in eroded AEC scalp skin. CONCLUSIONS: This study identifies specific changes in skin structural biology and signalling pathways that result from mutant p63 and provides new molecular insight into the AEC syndrome phenotype.

Integrative mRNA profiling comparing cultured primary cells with clinical samples reveals PLK1 and C20orf20 as therapeutic targets in cutaneous squamous cell carcinoma.

Identifying therapeutic targets for cancer treatment relies on consistent changes within particular types or sub-types of malignancy. The ability to define either consistent changes or sub-types of malignancy is often masked by tumor heterogeneity. To elucidate therapeutic targets in cutaneous squamous cell carcinoma (cSCC), the most frequent skin neoplasm with malignant potential, we have developed an integrated approach to gene expression profiling beginning with primary keratinocytes in culture. Candidate drivers of cSCC development were derived by first defining a set of in vitro cancer genes and then comparing their expression in a range of clinical data sets containing normal skin, cSCC and the benign hyper-proliferative condition psoriasis. A small interfering RNA (siRNA) screen of the resulting 21 upregulated genes has yielded targets capable of reducing xenograft tumor volume in vivo. Small-molecule inhibitors for one target, Polo-like kinase-1 (PLK1), are already in clinical trials for other malignancies, and our data show efficacy in cSCC. Another target, C20orf20, is identified as being overexpressed in cSCC, and siRNA-mediated knockdown induces apoptosis in vitro and reduces tumor growth in vivo. Thus, our approach has shown established and uncharacterized drivers of tumorigenesis with potent efficacy as therapeutic targets for the treatment of cSCC.

Hospital readmission among infants with gastroschisis.

OBJECTIVE: Infants with gastroschisis have significant perinatal morbidity including long hospitalizations and feeding intolerance. Two thirds are premature and 20% are growth restricted. Despite these known risk factors for post-natal complications, little is known about readmission for infants with gastroschisis. Our objective was to determine the frequency and indication for hospital readmission after initial discharge among infants with gastroschisis. STUDY DESIGN: Retrospective cohort study. All surviving infants treated for gastroschisis at Cincinnati Children's Hospital Medical Center, born between January 2006 and December 2008 were included. Main outcome measures included the frequency and indication for readmission. Associated neonatal risk factors also were assessed. RESULT: Fifty-eight patients were analyzed. Twenty-three (40%) subjects were readmitted (five with multiple readmissions); 65% of readmissions occurred in the first year and 70% involved complications directly related to gastroschisis. The most common reasons for readmission were bowel obstruction and abdominal distention/pain. Median time to readmission directly related to gastroschisis was 23 weeks (range 5 to 92). All three infants with home parenteral nutrition were readmitted. Readmission was not associated with gestational age, birth weight or length of initial hospitalization. CONCLUSION: Readmission after initial hospitalization is common in gastroschisis patients. Parental counseling should include education regarding the possibility of complications requiring readmission. Determinants of readmission require further study.

Echocardiographic detection of pulmonary hypertension in extremely low birth weight infants with bronchopulmonary dysplasia requiring prolonged positive pressure ventilation.

OBJECTIVE: The goal of this study was to delineate the epidemiology of echocardiographically diagnosed pulmonary hypertension (PH) in extremely low birth weight (ELBW) infants with bronchopulmonary dysplasia (BPD) requiring prolonged positive pressure ventilation (PPV), and to determine the independent relationship between PH and mortality in these patients. STUDY DESIGN: Our retrospective cohort included ELBW infants, with BPD requiring prolonged PPV, hospitalized in Cincinnati, Ohio during 2003-2009, as recorded in the National Institute of Child Health and Human Development Neonatal Research Network Database. Following chart review, a logistic regression model was constructed to understand the contribution of PH to mortality in infants with BPD requiring prolonged PPV. RESULT: We identified 216 patients (19%) with BPD requiring prolonged PPV among 1156 ELBW infants. Of these patients, 41% received echocardiography after 4 weeks of life, with 37% showing evidence of PH. Logistic regression analysis demonstrated that infants with BPD requiring prolonged PPV, with PH detectable by echocardiogram, were four times more likely to die (adjusted odds ratio): 4.6, 95% confidence interval: 1.3-16.5) when compared with infants with BPD requiring prolonged PPV without echocardiographic evidence of PH. CONCLUSION: Pulmonary hypertension appears to be an important, independent determinant of death in infants with BPD requiring prolonged PPV.

Acantholytic ectodermal dysplasia: clinicopathological study of a new desmosomal disorder.

We describe two boys with curly hair, palmoplantar keratoderma and skin fragility who presented clinical and histological features similar, but not identical, to those exhibited by patients with ectodermal dysplasia-skin fragility syndrome (McGrath syndrome) and other genetic desmosomal defects such as Carvajal syndrome and Naxos disease. Clinical features included trauma-induced blisters and erosions, palmoplantar keratoderma and hyperkeratotic, fissured plaques with perioral involvement. The patients had abundant curly scalp hair, and normal eyebrows and eyelashes. Sweating was normal. Nails were normal at birth but subsequently showed secondary dystrophy. Histopathological analysis of the skin demonstrated acantholysis and intercellular widening of the spinous and granular layers in involved regions. No involvement of scalp skin was seen. Desmosomes were markedly reduced in number and poorly developed with no clear insertions of the keratin filaments. The latter were clumped around the nuclei. Immunostaining of patient skin with antibodies raised against key desmosomal proteins demonstrated disrupted expression of desmoplakin, plakoglobin and desmoglein 1. Additional studies of the family history and of the desmoplakin, plakoglobin and desmoglein 1 genotype for both patients may help further elucidate the molecular cause of this variation on ectodermal dysplasia-skin fragility syndrome.

Growth and neurodevelopment at 16 to 24 months of age for infants born with gastroschisis.

OBJECTIVE: To examine growth, neurodevelopment and morbidity in infants with gastroschisis. STUDY DESIGN: We enrolled all infants with gastroschisis treated at the North Carolina Children's Hospital from March 2003 through June 2005. Neonatal data were collected. Medical history, growth and neurodevelopment were assessed at 16 to 24 months. RESULT: Of 24 infants, 17 completed follow-up. Weight and length were below the 10th percentile for five and six infants, respectively. Three infants scored less than 85 on the Bayley Scales of Infant Development, second edition. Small for gestational age (SGA) infants were smaller and had lower neurodevelopmental scores. Fourteen experienced continued bowel dysfunction; nine were rehospitalized. CONCLUSION: One-third of infants with gastroschisis experience growth delay. Infants who are SGA are at higher risk, suggesting that postnatal growth may be influenced by fetal phenomena, and may not be modifiable. Neurodevelopment is not delayed. Continued bowel dysfunction is common.

Unusual molecular findings in Kindler syndrome.

Kindler syndrome (KS) is a rare inherited skin disorder with blistering and poikiloderma as its main clinical features. It is caused by loss-of-function mutations in the C20orf42 (KIND1) gene which encodes kindlin-1, an actin cytoskeleton-focal contact-associated protein which is predominantly expressed in keratinocytes. We investigated the molecular basis of KS in a 16-year-old Indian boy who had additional clinical findings, including scleroatrophic changes of the hands and feet, pseudoainhum and early onset of squamous cell carcinoma on his foot. Immunostaining for kindlin-1 in the patient's skin was completely absent and sequencing of C20orf42 (KIND1) genomic DNA showed a homozygous splice-site mutation at the -6 position, IVS9-6T-->A. Amplification and sequencing of cDNA from the skin revealed aberrant splicing with either deletion of exon 10 or deletion of exons 9, 10 and 11, both of which involve loss of the pleckstrin homology domain of kindlin-1 that is thought to play a role in cytoskeletal attachment and integrin-mediated cell signalling. Pathogenic splice-site mutations at the -6 position are unusual and have rarely been reported for any genetic disorder. Collectively, these findings extend the spectrum of clinical and molecular abnormalities in this rare genodermatosis.

Altered expression of L-arginine metabolism pathway genes in chronic wounds in recessive dystrophic epidermolysis bullosa.

Individuals with the severe, mutilating Hallopeau-Siemens form of recessive dystrophic epidermolysis bullosa (HS-RDEB) have trauma-induced blisters and skin erosions which often progress to wounds that are slow to heal. These chronic wounds cause considerable morbidity and there is an increased risk of squamous cell carcinoma arising in the wound margins. Currently, little is known about the keratinocyte cell biology in these wounds. Therefore, we compared the gene expression profiles of wound edge with nonwounded skin from two individuals with HS-RDEB. Trauma-induced wound sites had been present in both patients for more than 3 months. Hybridizations using DermArray gene expression filters showed relative differences in gene expression between wounded and unwounded skin. Notably, there was a fivefold increase in expression of arginase-1 (ARG1) in the chronic wound samples. Expression of seven other genes relevant to L-arginine metabolism also showed differences greater than twofold. L-arginine is known to have a critical role in the synthesis of nitric oxide as part of normal tissue repair. Although alterations in arginase isoenzymes have been detected previously in other chronic wounds (human and animal models), this is the first study to demonstrate differences in several components of the L-arginine metabolism pathway in chronic wounds, and the first to examine chronic wounds in HS-RDEB. The data show that the cascade of L-arginine metabolites is altered in HS-RDEB and the findings may provide new insight into the pathology of chronic wounds in this genodermatosis.

Striate palmoplantar keratoderma arising from desmoplakin and desmoglein 1 mutations is associated with contrasting perturbations of desmosomes and the keratin filament network.

BACKGROUND: Several hereditary human diseases are now known to be caused by distinct mutations in genes encoding various desmosome components. Although the effects of some of these mutant genes have been analysed by targeted disruption experiments in mouse models, little is known about the cell and tissue changes in affected human patients. OBJECTIVES: To investigate the effects of heterozygous nonsense mutations in desmoplakin (Dp) and desmoglein (Dsg) 1 which cause the autosomal dominant disorder striate palmoplantar keratoderma (SPPK), focusing on changes in desmosome structure and composition and the associated keratin intermediate filament (KIF) network in palm skin, and in cultured keratinocytes generated from the same site. METHODS: We analysed palm and nonpalm skin sections from four SPPK patients with Dp mutations and one patient with a Dsg1 mutation with respect to tissue and subcellular morphologies, and correlated the in vivo and in vitro findings. RESULTS: Using electron microscopy, we found abnormalities of desmosomes and cell-cell adhesion in the suprabasal layers in the epidermis from patients with both Dsg1- and Dp-associated SPPK. These changes were more advanced in skin from patients with Dp mutations. Both Dp and Dsg1 mutations were accompanied by significantly reduced numbers of desmosomes in the suprabasal layers, while decreased desmosome size was evident only in Dsg1-associated SPPK. Confocal microscopy analysis showed marked differences in the expression of keratins and of desmosome components, both between the two types of SPPK, and between SPPK and normal skin. The expression of keratins K5, K14 and K10 was reduced in Dsg1-associated SPPK skin, whereas perinuclear aggregation of keratin filaments was more evident in Dp-associated SPPK. In both types of SPPK upregulation of K16 was pronounced and involucrin labelling was abnormal. CONCLUSIONS: Mutations in Dp and Dsg1 genes causing SPPK may be associated with perturbations in epidermal differentiation accompanied by a marked disruption of several components of the epidermal scaffold including desmosomes and the KIF network.

Plakophilin 1: an important stabilizer of desmosomes.

Desmosomes are cell-cell junctions found primarily in epithelial tissues and particularly in those that frequently undergo mechanical stress. Desmosomal cadherins provide the adhesion for opposing cell membranes and desmosomal plaque proteins link cytoskeletal intermediate filaments to these cadherins. Plakophilin 1 is a desmosomal plaque protein and a member of the armadillo family of structural and signalling proteins. Expressed primarily in the suprabasal layers of stratifying epithelia, plakophilin 1 is absent in patients with the rare autosomal recessive disorder, ectodermal-dysplasia skin-fragility syndrome (OMIM 604536). These patients exhibit skin fragility with trauma induced blistering, especially on the weight-bearing parts of the soles, and thus provide a vivid illustration of the clinical importance of PKP1 in skin physiology. Recent in vitro data also implicate plakophilin 1 in regulating desmosome integrity in response to low calcium concentrations and in altering the migratory properties of wounded keratinocyte sheets in culture. These findings further underscore the significant role of PKP1 in keratinocyte cell biology.

Dermatological applications of DNA array technology.

The Human Genome Project and other large scale sequencing consortia continue to generate huge amounts of DNA sequence data. Despite the identification of specific disease-related genes and genetic markers, we still appear to know little about how diverse gene products actually interact with each other or respond to other chemical or biological stimuli. Such information is of course fundamental to understanding complex disease pathways and biochemical processes and, as such, has spawned new fields of investigative genetics, that of functional genomics and proteomics. DNA array technology is emerging as a powerful, high-throughput and versatile tool that can be applied to the study of functional genomics. This article reviews the methodology involved in array analysis and provides insight into how, as an investigative tool, DNA arrays are becoming increasingly useful in understanding fundamental abnormalities in dermatological disease and also in refining the management of patients with certain skin disorders.

Genotype-phenotype correlation in skin fragility-ectodermal dysplasia syndrome resulting from mutations in plakophilin 1.

We report a 42-year-old Japanese man with an unusual autosomal recessive genodermatosis. The clinical features comprised normal skin at birth, loss of scalp hair at 3-months of age after a febrile illness, progressive nail dystrophy during infancy, palmoplantar keratoderma starting around the age of 18 years and trauma-induced skin fragility and blisters noted from the age of 20 years. Skin biopsy of rubbed non-lesional skin revealed widening of spaces between adjacent keratinocytes from the suprabasal layer upwards. Electron microscopy demonstrated a reduced number of hypoplastic desmosomes. Immunohistochemical labeling showed a reduction in intercellular staining for the desmosome component plakophilin 1. Mutation analysis revealed a homozygous intron 11 donor splice site mutation in the plakophilin 1 gene, 2021+1 G>A (GenBank no. Z34974). RT-PCR, using RNA extracted from the skin biopsy, provided evidence for residual low levels of the full-length wild-type transcript (approximately 8%) as well as multiple other near full-length transcripts, one of which was in frame leading to deletion of 17 amino acids from the 9th arm-repeat unit of the plakophilin 1 tail domain. Thus, the molecular findings help explain the clinical features in the patient, who has a similar but milder phenotype to previously reported patients with skin fragility-ectodermal dysplasia syndrome associated with complete ablation of plakophilin 1 (OMIM 604536). This new 'mitis' phenotype provides further clinicopathological evidence for the role of plakophilin 1 in keratinocyte cell-cell adhesion and ectodermal development.

EEC (Ectrodactyly, Ectodermal dysplasia, Clefting) syndrome: heterozygous mutation in the p63 gene (R279H) and DNA-based prenatal diagnosis.

BACKGROUND: Germline mis-sense mutations in the DNA-binding domain of the p63 gene have recently been established as the molecular basis for the autosomal dominant EEC (Ectrodactyly, Ectodermal dysplasia, Clefting) syndrome. OBJECTIVES: To examine genomic DNA from a 36-year-old woman, her 58-year-old father and her 11-year-old son, all with the EEC syndrome, to determine the inherent p63 mutation and, after genetic counselling, to use knowledge of the mutation to undertake a first-trimester DNA-based prenatal diagnosis in a subsequent pregnancy. METHODS: Fetal DNA was extracted from chorionic villi and used to amplify exon 7 of p63 containing the potential mutation. Direct sequencing and restriction endonuclease digestion (loss of AciI site on mutant allele) were used for DNA-based prenatal diagnosis. RESULTS: We identified a heterozygous arginine to histidine p63 mutation, R279H, in all three affected individuals. Prenatal diagnosis demonstrated a homozygous wild-type sequence predicting an unaffected child: a healthy boy was subsequently born at full-term. CONCLUSIONS: These data expand the p63 gene mutation database and provide the first example of a DNA-based prenatal test in this ectodermal dysplasia syndrome.

Genomic localization, organization and amplification of the human zinc transporter protein gene, ZNT4, and exclusion as a candidate gene in different clinical variants of acrodermatitis enteropathica.

Acrodermatitis enteropathica is an inherited disorder of zinc metabolism, the molecular basis of which is currently unknown. Recent transgenic mouse studies have highlighted the potential significance of certain zinc transport proteins, for example ZnT4, in providing clues to the pathogenesis of zinc-related disorders such as acrodermatitis enteropathica. Specifically, mice of any genotype suckled on ZnT4-deficient mice fail to absorb intestinal zinc and ZnT4-deficient mice also develop dermatitis, alopecia and stunted growth. Therefore, to assess human ZnT4 as a candidate gene/protein in acrodermatitis enteropathica or related disorders, we characterized the intron-exon organization of the human ZNT4 gene, which comprises seven distinct exons spanning approximately 38.7 kb. High-resolution radiation hybrid mapping placed ZNT4 on 15q21.1. We also developed a PCR-based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products. Using this approach, we sequenced DNA from five individuals with acrodermatitis enteropathica; no mutations were identified. Thus, ZNT4 is unlikely to be the correct candidate gene for this disorder. We also identified and characterized two common single nucleotide polymorphisms in exon 5 and in the 3' UTR of ZNT4, which will be useful for future genetic linkage studies in assessing ZNT4 as a candidate gene for other inherited disorders of zinc metabolism.