Publications by authors named "Paola Fortugno"

35 Publications

Biallelic mutations in RNF220 cause laminopathies featuring leukodystrophy, ataxia and deafness.

Brain 2021 May 8. Epub 2021 May 8.

Genetics and Rare Diseases Research Division, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy.

Leukodystrophies are a heterogeneous group of rare inherited disorders that involve preferentially the white matter of the central nervous system (CNS). These conditions are characterized by a primary glial cell and myelin sheath pathology of variable etiology, which causes secondary axonal degeneration, generally emerging with disease progression. Whole exome sequencing performed in 5 large consanguineous nuclear families allowed to identify homozygosity for two recurrent missense variants affecting highly conserved residues of RNF220 as the causative event underlying a novel form of leukodystrophy with ataxia and sensorineural deafness. We report on two homozygous missense variants (p.R363Q and p.R365Q) in the ubiquitin E3 ligase RNF220 as the cause underlying a novel form of leukodystrophy with ataxia and sensorineural deafness having fibrotic cardiomyopathy and hepatopathy as associated features, in seven consanguineous families. Mass spectrometry analysis identified lamin B1 as RNF220 binding protein and co-immunoprecipitation experiments demonstrated reduced binding of both RNF220 mutants to lamin B1. We demonstrate that RNF220 silencing in Drosophila melanogaster specifically affects proper localization of lamin Dm0, the fly lamin B1 orthologue, promotes its aggregation, and causes a neurodegenerative phenotype, strongly supporting the functional link between RNF220 and lamin B1. Finally, we demonstrate that RNF220 plays a crucial role in the maintenance of nuclear morphology: mutations primary skin fibroblasts determine nuclear abnormalities such as blebs, herniations and invaginations, which are typically observed in cells of patients affected by laminopathies. Overall, our data identify RNF220 as a gene implicated in leukodystrophy with ataxia and sensorineural deafness, and document a critical role of RNF220 in the regulation of nuclear lamina. Our findings provide further evidence on the direct link between nuclear lamina dysfunction and neurodegeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/brain/awab185DOI Listing
May 2021

Pathogenic variants in CDH11 impair cell adhesion and cause Teebi hypertelorism syndrome.

Hum Genet 2021 Apr 3. Epub 2021 Apr 3.

Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Teebi hypertelorism syndrome (THS; OMIM 145420) is a rare craniofacial disorder characterized by hypertelorism, prominent forehead, short nose with broad or depressed nasal root. Some cases of THS have been attributed to SPECC1L variants. Homozygous variants in CDH11 truncating the transmembrane and intracellular domains have been implicated in Elsahy-Waters syndrome (EWS; OMIM 211380) with hypertelorism. We report THS due to CDH11 heterozygous missense variants on 19 subjects from 9 families. All affected residues in the extracellular region of Cadherin-11 (CHD11) are highly conserved across vertebrate species and classical cadherins. Six of the variants that cluster around the EC2-EC3 and EC3-EC4 linker regions are predicted to affect Ca binding that is required for cadherin stability. Two of the additional variants [c.164G > C, p.(Trp55Ser) and c.418G > A, p.(Glu140Lys)] are also notable as they are predicted to directly affect trans-homodimer formation. Immunohistochemical study demonstrates that CDH11 is strongly expressed in human facial mesenchyme. Using multiple functional assays, we show that five variants from the EC1, EC2-EC3 linker, and EC3 regions significantly reduced the cell-substrate trans adhesion activity and one variant from EC3-EC4 linker results in changes in cell morphology, focal adhesion, and migration, suggesting dominant negative effect. Characteristic features in this cohort included depressed nasal root, cardiac and umbilical defects. These features distinguished this phenotype from that seen in SPECC1L-related hypertelorism syndrome and CDH11-related EWS. Our results demonstrate heterozygous variants in CDH11, which decrease cell-cell adhesion and increase cell migratory behavior, cause a form of THS, as termed CDH11-related THS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00439-021-02274-3DOI Listing
April 2021

Measles skin rash: Infection of lymphoid and myeloid cells in the dermis precedes viral dissemination to the epidermis.

PLoS Pathog 2020 10 8;16(10):e1008253. Epub 2020 Oct 8.

Department of Viroscience, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.

Measles is characterized by fever and a maculopapular skin rash, which is accompanied by immune clearance of measles virus (MV)-infected cells. Histopathological analyses of skin biopsies from humans and non-human primates (NHPs) with measles rash have identified MV-infected keratinocytes and mononuclear cells in the epidermis, around hair follicles and near sebaceous glands. Here, we address the pathogenesis of measles skin rash by combining data from experimentally infected NHPs, ex vivo infection of human skin sheets and in vitro infection of primary human keratinocytes. Analysis of NHP skin samples collected at different time points following MV inoculation demonstrated that infection in the skin precedes onset of rash by several days. MV infection was detected in lymphoid and myeloid cells in the dermis before dissemination to the epidermal leukocytes and keratinocytes. These data were in good concordance with ex vivo MV infections of human skin sheets, in which dermal cells were more targeted than the epidermal cells. To address viral dissemination to the epidermis and to determine whether the dissemination is receptor-dependent, we performed experimental infections of primary keratinocytes collected from healthy donors. These experiments demonstrated that MV infection of keratinocytes is mainly nectin-4-dependent, and differentiated keratinocytes, which express higher levels of nectin-4, are more susceptible to MV infection than proliferating keratinocytes. Based on these data, we propose a model to explain measles skin rash: migrating MV-infected lymphocytes initiate the infection of dermal skin-resident CD150+ immune cells. The infection is subsequently disseminated from the dermal papillae to nectin-4+ keratinocytes in the basal epidermis. Lateral spread of MV infection is observed in the superficial epidermis, most likely due to the higher level of nectin-4 expression on differentiated keratinocytes. Finally, MV-infected cells are cleared by infiltrating immune cells, causing hyperemia and edema, which give the appearance of morbilliform skin rash.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1008253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575069PMC
October 2020

Multiple Skin Squamous Cell Carcinomas in Junctional Epidermolysis Bullosa Due to Altered Laminin-332 Function.

Int J Mol Sci 2020 Feb 20;21(4). Epub 2020 Feb 20.

Laboratory of Molecular and Cell Biology, IDI-IRCCS, via Monti di Creta 104, 00167 Rome, Italy.

Variably reduced expression of the basement membrane component laminin-332 (α3aβ3γ2) causes junctional epidermolysis bullosa generalized intermediate (JEB-GI), a skin fragility disorder with an increased susceptibility to squamous cell carcinoma (SCC) development in adulthood. Laminin-332 is highly expressed in several types of epithelial tumors and is central to signaling pathways that promote SCC tumorigenesis. However, laminin-332 mutations and expression in individuals affected by JEB-GI and suffering from recurrent SCCs have been poorly characterized. We studied a JEB-GI patient who developed over a hundred primary cutaneous SCCs. Molecular analysis combined with gene expression studies in patient skin and primary keratinocytes revealed that the patient is a functional hemizygous for the p.Cys1171* mutant allele which is transcribed in a stable mRNA encoding for a β3 chain shortened of the last two C-terminal amino acids (Cys1171-Lys1172). The lack of the Cys1171 residue involved in the C-terminal disulphide bond to γ2 chain did not prevent assembly, secretion, and proteolytic processing of the heterotrimeric molecule. Immunohistochemistry of SCC specimens revealed accumulation of mutant laminin-332 at the epithelial-stromal interface of invasive front. We conclude that the C-terminal disulphide bond is a structural element crucial for laminin-332 adhesion function in-vivo. By saving laminin-332 amount, processing, and signaling role the p.Cys1171* mutation may allow intrinsic pro-tumorigenic properties of the protein to be conveyed, thus contributing to invasiveness and recurrence of SCCs in this patient.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21041426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073068PMC
February 2020

Genotypic Categorization of Loeys-Dietz Syndrome Based on 24 Novel Families and Literature Data.

Genes (Basel) 2019 09 28;10(10). Epub 2019 Sep 28.

Human Genetics Institute, Department of Life, Health, and Environmental Sciences, University of L'Aquila, 67100 L'Aquila, Italy.

Loeys-Dietz syndrome (LDS) is a connective tissue disorder first described in 2005 featuring aortic/arterial aneurysms, dissections, and tortuosity associated with craniofacial, osteoarticular, musculoskeletal, and cutaneous manifestations. Heterozygous mutations in 6 genes (), encoding components of the TGF-β pathway, cause LDS. Such genetic heterogeneity mirrors broad phenotypic variability with significant differences, especially in terms of the age of onset, penetrance, and severity of life-threatening vascular manifestations and multiorgan involvement, indicating the need to obtain genotype-to-phenotype correlations for personalized management and counseling. Herein, we report on a cohort of 34 LDS patients from 24 families all receiving a molecular diagnosis. Fifteen variants were novel, affecting the (6), (6), (2), and (1) genes. Clinical features were scored for each distinct gene and matched with literature data to strengthen genotype-phenotype correlations such as more severe vascular manifestations in -related LDS. Additional features included spontaneous pneumothorax in -related LDS and cervical spine instability in -related LDS. Our study broadens the clinical and molecular spectrum of LDS and indicates that a phenotypic continuum emerges as more patients are described, although genotype-phenotype correlations may still contribute to clinical management.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/genes10100764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826414PMC
September 2019

Recessive mutations in the neuronal isoforms of DST, encoding dystonin, lead to abnormal actin cytoskeleton organization and HSAN type VI.

Hum Mutat 2019 01 18;40(1):106-114. Epub 2018 Nov 18.

Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Rome, Italy.

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders, characterized by a progressive sensory neuropathy often complicated by ulcers and amputations, with variable motor and autonomic involvement. Several pathways have been implicated in the pathogenesis of neuronal degeneration in HSAN, while recent observations point to an emerging role of cytoskeleton organization and function. Here, we report novel biallelic mutations in the DST gene encoding dystonin, a large cytolinker protein of the plakin family, in an adult form of HSAN type VI. Affected individuals harbored the premature termination codon variant p.(Lys4330*) in trans with the p.(Ala203Glu) change affecting a highly conserved residue in an isoform-specific N-terminal region of dystonin. Functional studies showed defects in actin cytoskeleton organization and consequent delayed cell adhesion, spreading and migration, while recombinant p.Ala203Glu dystonin loses the ability to bind actin. Our data aid in the clinical and molecular delineation of HSAN-VI and suggest a central role for cell-motility and cytoskeletal defects in its pathogenesis possibly interfering with the neuronal outgrowth and guidance processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.23678DOI Listing
January 2019

Microprocessor-dependent processing of splice site overlapping microRNA exons does not result in changes in alternative splicing.

RNA 2018 09 12;24(9):1158-1171. Epub 2018 Jun 12.

Human Molecular Genetics, International Centre for Genetic Engineering and Biotechnology, 34149 Trieste, Italy.

MicroRNAs are found throughout the genome and are processed by the microprocessor complex (MPC) from longer precursors. Some precursor miRNAs overlap intron:exon junctions. These plice site verlapping microRNAs (SO-miRNAs) are mostly located in coding genes. It has been intimated, in the rarer examples of SO-miRNAs in noncoding RNAs, that the competition between the spliceosome and the MPC modulates alternative splicing. However, the effect of this overlap on coding transcripts is unknown. Unexpectedly, we show that neither Drosha silencing nor SF3b1 silencing changed the inclusion ratio of SO-miRNA exons. Two SO-miRNAs, located in genes that code for basal membrane proteins, are known to inhibit proliferation in primary keratinocytes. These SO-miRNAs were up-regulated during differentiation and the host mRNAs were down-regulated, but again there was no change in inclusion ratio of the SO-miRNA exons. Interestingly, Drosha silencing increased nascent RNA density, on chromatin, downstream from SO-miRNA exons. Overall our data suggest a novel mechanism for regulating gene expression in which MPC-dependent cleavage of SO-miRNA exons could cause premature transcriptional termination of coding genes rather than affecting alternative splicing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.063438.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097652PMC
September 2018

Epidermolysis Bullosa (EB) Acquisita in an Adult Patient with Previously Unrecognized Mild Dystrophic EB and Biallelic COL7A1 Mutations.

Acta Derm Venereol 2018 Apr;98(4):411-415

Laboratory of Molecular and Cell Biology, Instituto Dermopatico dell'Immacolata-IRCCS, Via dei Monti di Creta, 104, IT-00167 Rome, Italy.

Circulating anti-type VII collagen autoantibodies are frequently detected in patients with recessive dystrophic epidermolysis bullosa (RDEB). However, evidence supporting their pathogenic role in inducing epidermolysis bullosa acquisita (EBA) has been provided for only one individual with dominant dystrophic epidermolysis bullosa (DDEB). We describe here a patient who presented with dystrophic toenails since early childhood and developed trauma-induced skin blisters and oral erosions at age 26 years. Direct immunofluorescence showed IgG deposits with a u-serrated pattern along the cutaneous basement membrane zone, while no change in the expression of collagen VII could be detected by antigen mapping. High-titre anti-collagen VII antibodies were detected by enzyme-linked immunoassay (ELISA). In parallel, sequencing of epidermolysis bullosa (EB) genes identified compound heterozygous COL7A1 missense c.410G>A (p.Arg137Gln) and splicing c.3674C>T (p.Ala1225_Gln1241del) mutations, previously unrecognized in dystrophic epidermolysis bullosa (DEB). Thus, our patient had RDEB "nails-only" and developed mechanobullous EBA in adulthood. These data support a pathogenic role of circulating autoantibodies to collagen VII in inducing EBA in selected patients with DEB. Unforeseen worsening of skin symptoms in DEB should prompt laboratory investigations for EBA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2340/00015555-2851DOI Listing
April 2018

Structural Defects of Laminin β3 N-terminus Underlie Junctional Epidermolysis Bullosa with Altered Granulation Tissue Response.

Acta Derm Venereol 2016 Nov;96(7):954-958

Dermatology Division, Bambino Gesù Children's Hospital-IRCCS, P.zza Sant'Onofrio, 4, IT-00165 Rome, Italy.

Mutations in the laminin-332 (α3Aβ3γ2) genes cause junctional epidermolysis bullosa (JEB), a recessively inherited disease characterized by blistering and altered wound repair. In addition, specific mutations that affect the N-terminus of the α3A chain cause a JEB-related non-blistering condition characterized by chronic production of granulation tissue, suggesting a critical role of this region in epithelial-mesenchymal communication. We report here a 9-year-old patient with JEB with a few long-standing skin ulcers with prominent granulation tissue in the absence of active blistering. He bears a homozygous missense mutation, p.Gly254Asp, within the first laminin epidermal growth factor-like (LE) repeat of the β3 short arm. We show that p.Gly254Asp causes mis-folding of the LE motif, leading to reduced secretion of laminin-332 and structural alterations of the cutaneous basement membrane zone. These findings demonstrate, in a patient in vivo, that the β3 short arm is also involved in the outcome of the granulation tissue response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2340/00015555-2439DOI Listing
November 2016

A compound synonymous mutation c.474G>A with p.Arg578X mutation in SPINK5 causes splicing disorder and mild phenotype in Netherton syndrome.

Exp Dermatol 2016 07 20;25(7):568-70. Epub 2016 May 20.

Department of Dermatology, Kurume University School of Medicine, and Kurume University Institute of Cutaneous Cell Biology, Fukuoka, Japan.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/exd.13011DOI Listing
July 2016

Betapapillomavirus in multiple non-melanoma skin cancers of Netherton syndrome: Case report and published work review.

J Dermatol 2015 Aug 28;42(8):786-94. Epub 2015 Apr 28.

Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata-IRCCS, Rome, Italy.

Netherton syndrome (NS) is a rare genetic disease presenting with ichthyosiform erythroderma, hair alterations and atopy. NS is due to SPINK5 gene mutations, which cause absent or decreased expression of the encoded protein lymphoepithelial Kazal-type-related inhibitor (LEKTI) in all stratified epithelia. We report a 43-year-old man affected with NS, who developed several squamous and basal cell carcinomas on the face, ears and scalp and papillomatous lesions of hips, groin and genitoanal area. Molecular analysis of the SPINK5 gene revealed homozygosity for the recurrent mutation c.238dupG. Human papillomavirus (HPV) DNA detection and genotyping on patient skin carcinomas and hyperplastic lesions found betapapillomavirus DNA in 10 of 12 (83%) carcinomas and in a hip papilloma, with multiple betapapillomavirus types being identified. Immunohistochemistry showed upregulated expression of p16(INK4a) protein in nine of 12 (75%) patient carcinomas, in line with findings reported in HPV-related cancers. LEKTI and filaggrin immunostaining was strongly decreased in patient skin. A published work search for NS cases with skin cancers and HPV infection identified 15 NS patients, five of them showing mucosal or cutaneous HPV infection. Overall, our results confirm the increased susceptibility to skin carcinomas of some NS patients and provide further evidence of an association between HPV and non-melanoma skin cancers in NS. The highly impaired skin barrier function, hallmark of NS, could facilitate HPV infection, in turn increasing the risk for cancer development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1346-8138.12913DOI Listing
August 2015

Ichthyosis Linearis Circumflexa as the Only Clinical Manifestation of Netherton Syndrome.

Acta Derm Venereol 2015 Jul;95(6):720-4

Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata (IDI)-IRCCS, 00167 Rome, Italy.

Ichthyosis linearis circumflexa (ILC) presents as serpiginous and migratory erythematous patches with double-edged scales. ILC is rarely an isolated skin manifestation, but most commonly a part of Netherton syndrome (NS). NS is caused by SPINK5 mutations, which lead to absent or sometimes reduced expression of the serine protease inhibitor LEKTI. NS is characterised by congenital ichthyosiform erytroderma, trichorrhexis invaginata (TI) and atopy. We report 2 children who presented since the first months of life cheek erythema followed by the appearance of sparse ILC lesions on the face, trunk and proximal extremities. Erythroderma at birth, TI and atopy were absent. LEKTI immunoreactivity was reduced in patient epidermis, and serine protease activity was modestly increased, while desmoglein-1 expression remained unaffected. SPINK5 mutation and expression analysis in patient keratinocytes revealed compound heterozygous splicing variants, which allowed residual LEKTI secretion. Our results show that ILC can be the only clinical manifestation of NS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2340/00015555-2075DOI Listing
July 2015

Exon-Specific U1s Correct SPINK5 Exon 11 Skipping Caused by a Synonymous Substitution that Affects a Bifunctional Splicing Regulatory Element.

Hum Mutat 2015 May 19;36(5):504-12. Epub 2015 Mar 19.

International Centre for Genetic Engineering and Biotechnology (ICGEB), Human Molecular Genetics, Trieste, Italy.

The c.891C>T synonymous transition in SPINK5 induces exon 11 (E11) skipping and causes Netherton syndrome (NS). Using a specific RNA-protein interaction assay followed by mass spectrometry analysis along with silencing and overexpression of splicing factors, we showed that this mutation affects an exonic bifunctional splicing regulatory element composed by two partially overlapping silencer and enhancer sequences, recognized by hnRNPA1 and Tra2β splicing factors, respectively. The C-to-T substitution concomitantly increases hnRNPA1 and weakens Tra2β-binding sites, leading to pathological E11 skipping. In hybrid minigenes, exon-specific U1 small nuclear RNAs (ExSpe U1s) that target by complementarity intronic sequences downstream of the donor splice site rescued the E11 skipping defect caused by the c.891C>T mutation. ExSpe U1 lentiviral-mediated transduction of primary NS keratinocytes from a patient bearing the mutation recovered the correct full-length SPINK5 mRNA and the corresponding functional lympho-epithelial Kazal-type related inhibitor protein in a dose-dependent manner. This study documents the reliability of a mutation-specific, ExSpe U1-based, splicing therapy for a relatively large subset of European NS patients. Usage of ExSpe U1 may represent a general approach for correction of splicing defects affecting skin disease genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/humu.22762DOI Listing
May 2015

Reference genes for gene expression analysis in proliferating and differentiating human keratinocytes.

Exp Dermatol 2015 Apr;24(4):314-6

Institute of Molecular Genetics CNR, Pavia, Italy.

Abnormalities in keratinocyte growth and differentiation have a pathogenic significance in many skin disorders and result in gene expression alterations detectable by quantitative real-time RT-PCR (qRT-PCR). Relative quantification based on endogenous control (EC) genes is the commonly adopted approach, and the use of multiple reference genes from independent pathways is considered a best practice guideline, unless fully validated EC genes are available. The literature on optimal reference genes during in vitro calcium-induced differentiation of normal human epidermal keratinocytes (NHEK) is inconsistent. In many studies, the expression of target genes is compared to that of housekeeping genes whose expression, however, significantly varies during keratinocyte differentiation. Here, we report the results of our investigations on the expression stability of 15 candidate EC genes, including those commonly used as reference in expression analysis by qRT-PCR, during NHEK calcium-induced differentiation. We demonstrate that YWHAZ and UBC are extremely stable genes, and therefore, they represent optimal EC genes for expression studies in proliferating and calcium-induced differentiating NHEK. Furthermore, we demonstrate that YWHAZ/14-3-3-zeta is a suitable reference for quantitative comparison of both transcript and protein levels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/exd.12657DOI Listing
April 2015

TFIIH-dependent MMP-1 overexpression in trichothiodystrophy leads to extracellular matrix alterations in patient skin.

Proc Natl Acad Sci U S A 2015 Feb 20;112(5):1499-504. Epub 2015 Jan 20.

Istituto di Genetica Molecolare Consiglio Nazionale delle Ricerche, Pavia 27100, Italy;

Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH result in distinct clinical entities, including the cancer-prone xeroderma pigmentosum (XP) and the multisystem disorder trichothiodystrophy (TTD), which share only cutaneous photosensitivity. Gene-expression profiles of primary dermal fibroblasts revealed overexpression of matrix metalloproteinase 1 (MMP-1), the gene encoding the metalloproteinase that degrades the interstitial collagens of the extracellular matrix (ECM), in TTD patients mutated in XPD compared with their healthy parents. The defect is observed in TTD and not in XP and is specific for fibroblasts, which are the main producers of dermal ECM. MMP-1 transcriptional up-regulation in TTD is caused by an erroneous signaling mediated by retinoic acid receptors on the MMP-1 promoter and leads to hypersecretion of active MMP-1 enzyme and degradation of collagen type I in the ECM of cell/tissue systems and TTD patient skin. In agreement with the well-known role of ECM in eliciting signaling events controlling cell behavior and tissue homeostasis, ECM alterations in TTD were shown to impact on the migration and wound-healing properties of patient dermal fibroblasts. The presence of a specific inhibitor of MMP activity was sufficient to restore normal cell migration, thus providing a potential approach for therapeutic strategies. This study highlights the relevance of ECM anomalies in TTD pathogenesis and in the phenotypic differences between TTD and XP.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1416181112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321311PMC
February 2015

Kindler syndrome with severe mucosal involvement in a large Palestinian pedigree.

Eur J Dermatol 2015 Jan-Feb;25(1):14-9

Dermatology Section, Shifa Hospital, Shifa Street, Gaza, Gaza Strip.

Background: Kindler syndrome (KS) is a rare autosomal recessive disease of skin fragility, photosensitivity and progressive poikiloderma. Mucous membranes may also be involved. KS is caused by mutations in the FERMT1 gene encoding kindlin-1.

Objectives: We report the clinical and molecular features of the largest kindred with KS to date, comprising 18 affected family members (age range: 12-63 years) from the Gaza Strip.

Materials And Methods: All the affected family members were clinically examined. In addition a skin biopsy for immunofluorescence testing was obtained from the index case. Molecular analysis of the FERMT1 gene was performed on genomic DNA extracted from peripheral blood of 5 patients.

Results: All patients presented skin and eye photosensitivity, cutaneous atrophy, dyschromia and poikiloderma, oral cavity involvement, dysphagia and constipation with anal fissures. In addition, nail dystrophy and digit webbing were observed in most of them. Ocular manifestations detected in all patients comprised ectropion and keratoconjunctivitis, with early development of symblepharon in 17 out of 18 cases and blindness in one. Of note, 17 out of 18 affected family members also suffered from urethral strictures since childhood. Diagnosis was supported by immunofluorescence findings and definitely confirmed by FERMT1 sequencing which identified the homozygous frame-shift mutation c.137_140delTAGT.

Conclusions: The high rate of mucosal involvement, its early onset and progressive course are noticeable features of our kindred. Also noteworthy is the lack of muco-cutaneous malignancies, despite the sunny habitat.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1684/ejd.2014.2457DOI Listing
January 2016

Whole-exome sequencing in patients with ichthyosis reveals modifiers associated with increased IgE levels and allergic sensitizations.

J Allergy Clin Immunol 2015 Jan 11;135(1):280-3. Epub 2014 Nov 11.

Department of Dermatology, Medical Center, University of Freiburg, Freiburg, Germany. Electronic address:

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaci.2014.09.042DOI Listing
January 2015

p63-dependent and independent mechanisms of nectin-1 and nectin-4 regulation in the epidermis.

Exp Dermatol 2015 Feb;24(2):114-9

CEINGE Biotecnologie Avanzate, Napoli, Italy; Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Napoli, Italy.

Nectins are immunoglobulin-like cell adhesion molecules mainly localized in adherens junctions. The transcription factor p63 is a master regulator of gene expression in stratified epithelia and controls several molecular processes. As mutations in the Pvrl1 and Pvrl4 genes encoding for nectins cause genetic disorders with phenotypes similar to p63-related syndromes, we investigated whether these proteins might be under p63 transcriptional control. Here, we show that in p63-null skin, Pvrl1 gene expression is strongly reduced, whereas Pvrl4 expression is unaffected. In human and mouse primary keratinocytes p63 depletion leads to a specific downregulation of the Pvrl1 gene. Consistent with a direct regulation, chromatin immunoprecipitation experiments (ChIP) indicate that p63 binds to two conserved intronic Pvrl1 enhancer regions. Ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome is a rare autosomal dominant disorder, caused by mutations in p63 gene, mainly characterized by skin fragility. To test whether nectins may be affected in AEC syndrome, their expression was measured in keratinocytes obtained from patients with AEC or from a conditional mouse model for AEC syndrome. Pvrl1 expression was reduced in AEC keratinocytes, consistent with impaired p63 function. Surprisingly, Pvrl4 expression was similarly affected, in parallel with decreased expression of the transcription factor Irf6. Consistent with the well-characterized role of Irf6 in keratinocyte differentiation and its strong downregulation in AEC syndrome, Irf6 depletion caused reduced expression of Pvrl4 in wild-type keratinocytes. Taken together, our results indicate that Pvrl1 is a bona fide target gene of the transcription factor p63, whereas Pvrl4 regulation is linked to epidermal differentiation and is under Irf6 control.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/exd.12593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4329386PMC
February 2015

Early immunopathological diagnosis of ichthyosis with confetti in two sporadic cases with new mutations in keratin 10.

Acta Derm Venereol 2014 Sep;94(5):579-82

Dermatology Division, Bambino Gesù Children's Hospital-IRCCS, P.zza Sant'Onofrio, 4, IT-00165 Rome, Italy.

Ichthyosis with confetti (IC) is a severe non-syndromic ichthyosis due to heterozygous mutations in the KRT10 gene. The disease manifests at birth with erythroderma and scaling and is characterised by the gradual development of numerous confetti-like spots of normal skin. Diagnosis of IC is frequently delayed until adolescence or even adulthood. We report 2 young children who were first diagnosed as having congenital ichthyosiform erythroderma. However, the development of thick, confluent hyperkeratotic plaques together with the histopathological finding of keratinocyte vacuolisation in the suprabasal epidermis evoked IC. Immunofluorescence analysis showed a highly reduced keratin 10 expression within the cytoplasm of suprabasal keratinocytes and its characteristic mislocalisation to the nuclei. The diagnosis was confirmed by the identification of 2 previously unreported mutations in intron 6 and exon 7 of KRT10. Careful clinical examination then showed the presence of the first spots of normal skin in both patients at the age of 2.5 and 5 years, respectively. These cases point to the usefulness of immunofluorescence analysis of keratin 10 expression for an early diagnosis of IC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2340/00015555-1796DOI Listing
September 2014

Nectin-4 mutations causing ectodermal dysplasia with syndactyly perturb the rac1 pathway and the kinetics of adherens junction formation.

J Invest Dermatol 2014 Aug 27;134(8):2146-2153. Epub 2014 Feb 27.

Department of Medical, Oral and Biotechnological Sciences, Gabriele D'Annunzio University of Chieti-Pescara, Chieti, Italy; Medical Genetics Unit, Policlinico Tor Vergata University Hospital, Rome, Italy. Electronic address:

Defective nectin-1 and -4 have been implicated in ectodermal dysplasia (ED) syndromes with variably associated features including orofacial and limb defects. In particular, nectin-1 mutations cause cleft lip/palate ED (CLPED1; OMIM#225060), whereas defective nectin-4 is associated with ED-syndactyly syndrome (EDSS1; OMIM#613573). Although the broad phenotypic overlap suggests a common mode of action of nectin-1 and -4, little is known about the pathogenic mechanisms involved. We report the identification of, to our knowledge, a previously undescribed nectin-4 homozygous p.Val242Met missense mutation in a patient with EDSS1. We used patient skin biopsy and primary keratinocytes, as well as nectin-4 ectopic expression in epithelial cell lines, to characterize functional consequences of p.Val242Met and p.Thr185Met mutations, the latter previously identified in compound heterozygosity with a truncating mutation. We show that nectin-4-altered expression perturbs nectin-1 clustering at keratinocyte contact sites and delays, but does not impede cell-cell aggregation and cadherin recruitment at adherens junctions (AJs). Moreover, trans-interaction of nectin-1 and -4 induces the activation of Rac1, a member of the Rho family of small GTPases, and regulates E-cadherin-mediated cell-cell adhesion. These data outline a synergistic action of nectin-1 and -4 in the early steps of AJ formation and implicate this interaction in modulating the Rac1 signaling pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2014.119DOI Listing
August 2014

Lethal Netherton syndrome due to homozygous p.Arg371X mutation in SPINK5.

Pediatr Dermatol 2013 Jul-Aug;30(4):e65-7. Epub 2013 Jan 17.

Dermatology Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.

Here we report a lethal case of Netherton syndrome presenting with neurologic complications, hypernatremic dehydration, failure to thrive, and episodes of sepsis. Molecular analysis of the serine protease inhibitor Kazal-type 5 gene identified a homozygous mutation (c.1111C>T, p.Arg371X). This case highlights the importance of early diagnosis to start appropriate care in a timely fashion and prevent disease complications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/pde.12076DOI Listing
February 2014

The 420K LEKTI variant alters LEKTI proteolytic activation and results in protease deregulation: implications for atopic dermatitis.

Hum Mol Genet 2012 Oct 23;21(19):4187-200. Epub 2012 Jun 23.

Dermatology Unit, Bambino Gesú Children's Hospital IRCCS, Rome 00165, Italy

Lymphoepithelial Kazal-type related inhibitor (LEKTI) is a multidomain serine protease inhibitor which plays a central role in skin permeability barrier and allergy. Loss-of-function mutations in the LEKTI encoding gene SPINK5 cause Netherton syndrome, a rare and severe genetic skin disease with a profound skin barrier defect and atopic manifestations. Several studies also reported genetic association between the multifactorial disease atopic dermatitis (AD) and a frequent and non-conservative LEKTI variant, E420K, in different populations. Here, we provide evidence that the 420K variant impacts on LEKTI function by increasing the likelihood of furin-dependent LEKTI precursor cleavage within the linker region D6-D7. This results in the reversal of the cleavage priorities for LEKTI proteolytic activation and prevents the formation of the LEKTI fragment D6D9 known to display the strongest inhibitory activity against kallikrein (KLK) 5-mediated desmoglein-1 (DSG1) degradation. Using in situ and gel zymographies, we show that the modification of the subtle balance in LEKTI inhibitory fragments leads to enhanced KLK5, KLK7 and elastase-2 (ELA-2) activities in 420KK epidermis. By immunohistochemistry and western blot analyses, we found that increased epidermal protease activity correlates with reduced DSG1 protein expression and accelerated profilaggrin proteolysis. All changes determined by the presence of residue 420K within the LEKTI sequence likely contribute to defective skin barrier permeability. Remarkably, LEKTI 420KK epidermis displays an increased expression of the proallergic cytokine thymic stromal lymphopoietin (TSLP). This is the first functional evidence supporting association studies which identified the 420K LEKTI variant as a predisposing factor to AD, in combination with other genetic and environmental factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/dds243DOI Listing
October 2012

A synonymous mutation in SPINK5 exon 11 causes Netherton syndrome by altering exonic splicing regulatory elements.

J Hum Genet 2012 May 1;57(5):311-5. Epub 2012 Mar 1.

Dermatology Unit, Ospedale Pediatrico Bambino Gesù-IRCCS, Rome, Italy.

Netherton syndrome (NS) is a rare, life-threatening ichthyosiform syndrome caused by recessive loss-of-function mutations in SPINK5 gene encoding lymphoepithelial Kazal-type-related inhibitor (LEKTI), a serine protease inhibitor expressed in the most differentiated epidermal layers and crucial for skin barrier function. We report the functional characterization of a previously unrecognized synonymous variant, c.891C>T (p.Cys297Cys), identified in the SPINK5 exon 11 of an NS patient. We demonstrated that the c.891C>T mutation is associated with abnormal pre-mRNA splicing and residual LEKTI expression in the patient's keratinocytes. Subsequent minigene splicing assays and in silico predictions confirmed the direct role of the synonymous mutation in inhibiting exon 11 inclusion by a mechanism that involves the activity of exonic regulatory sequences, namely splicing enhancer and silencer. However, this deleterious effect was not complete and a residual amount of normal mRNA and LEKTI protein could be detected, correlating with the relatively mild patient's phenotype. Our study represents the first identification of a disease-causing SPINK5 mutation that alters splicing without affecting canonical splice sites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jhg.2012.22DOI Listing
May 2012

Proteolytic activation cascade of the Netherton syndrome-defective protein, LEKTI, in the epidermis: implications for skin homeostasis.

J Invest Dermatol 2011 Nov 23;131(11):2223-32. Epub 2011 Jun 23.

Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata IDI-IRCCS, Rome, Italy.

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is the defective protein of the ichthyosiform condition Netherton syndrome (NS). Strongly expressed in the most differentiated epidermal layers, LEKTI is a serine protease inhibitor synthesized as three different high-molecular-weight precursors, which are rapidly processed into shorter fragments and secreted extracellularly. LEKTI polypeptides interact with several proteases to regulate skin barrier homeostasis as well as inflammatory and/or immunoallergic responses. Here, by combining antibody mapping, N-terminal sequencing, and site-specific mutagenesis, we defined the amino-acid sequence of most of the LEKTI polypeptides physiologically generated in human epidermis. We also identified three processing intermediates not described so far. Hence, a proteolytic cascade model for LEKTI activation is proposed. We then pinpointed the most effective fragments against the desquamation-related kallikreins (KLKs) and we proved that LEKTI is involved in stratum corneum shedding as some of its polypeptides inhibit the KLK-mediated proteolysis of desmoglein-1. Finally, we quantified the individual LEKTI fragments in the uppermost epidermis, showing that the ratios between LEKTI polypeptides and active KLK5 are compatible with a fine-tuned inhibition. These findings are relevant both to the understanding of skin homeostasis regulation and to the design of novel therapeutic strategies for NS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2011.174DOI Listing
November 2011

Mutations in PVRL4, encoding cell adhesion molecule nectin-4, cause ectodermal dysplasia-syndactyly syndrome.

Am J Hum Genet 2010 Aug;87(2):265-73

Department of Biomedical Sciences, Gabriele d'Annunzio University, Chieti, Italy.

Ectodermal dysplasias form a large disease family with more than 200 members. The combination of hair and tooth abnormalities, alopecia, and cutaneous syndactyly is characteristic of ectodermal dysplasia-syndactyly syndrome (EDSS). We used a homozygosity mapping approach to map the EDSS locus to 1q23 in a consanguineous Algerian family. By candidate gene analysis, we identified a homozygous mutation in the PVRL4 gene that not only evoked an amino acid change but also led to exon skipping. In an Italian family with two siblings affected by EDSS, we further detected a missense and a frameshift mutation. PVRL4 encodes for nectin-4, a cell adhesion molecule mainly implicated in the formation of cadherin-based adherens junctions. We demonstrated high nectin-4 expression in hair follicle structures, as well as in the separating digits of murine embryos, the tissues mainly affected by the EDSS phenotype. In patient keratinocytes, mutated nectin-4 lost its capability to bind nectin-1. Additionally, in discrete structures of the hair follicle, we found alterations of the membrane localization of nectin-afadin and cadherin-catenin complexes, which are essential for adherens junction formation, and we found reorganization of actin cytoskeleton. Together with cleft lip and/or palate ectodermal dysplasia (CLPED1, or Zlotogora-Ogur syndrome) due to an impaired function of nectin-1, EDSS is the second known "nectinopathy" caused by mutations in a nectin adhesion molecule.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2010.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2917716PMC
August 2010

Intracellular targets of RGDS peptide in melanoma cells.

Mol Cancer 2010 Apr 22;9:84. Epub 2010 Apr 22.

Laboratorio Patologia Vascolare, Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Roma, Italy.

Background: RGD-motif acts as a specific integrins-ligand and regulates a variety of cell-functions via extracellular action affecting cell-adhesion properties. However, increasing evidence identifies additional RGDS-functions at intracellular level. Previous reports show RGDS-internalization in endothelial cells, cardiomyocytes and lymphocytes, indicating intracellular targets such as caspase-8 and caspase-9, and suggest RGDS specific activity at cytoplasmic level. Given the role RGDS-peptides play in controlling proliferation and apoptosis in several cell types, investigating intracellular targets of RGDS in melanoma cells may un-reveal novel molecular targets and key pathways, potentially useful for a more effective approach to melanoma treatment.

Results: In the present study we show for the first time that RGDS-peptide is internalized in melanoma cells in a time-dependent way and exerts strong anti-proliferative and pro-apoptotic effects independently from its extracellular anti-adhesive action. RGES control-peptide did not show biological effects, as expected; nevertheless it is internalized, although with slower kinetics. Survivin, a known cell-cycle and survival-regulator is highly expressed in melanoma cells. Co-immunoprecipitation assays in cell lysates and overlay assays with the purified proteins showed that RGDS interacts with survivin, as well as with procaspase-3, -8 and -9. RGDS-peptide binding to survivin was found to be specific, at high affinity (Kd 27.5 muM) and located at the survivin C-terminus. RGDS-survivin interaction appeared to play a key role, since RGDS lost its anti-mitogenic effect in survivin-deprived cells with a specific siRNA.

Conclusions: RGDS inhibits melanoma growth with an adhesion-independent mechanism; it is internalized in melanoma cells and specifically interacts with survivin. The present data may indicate a novel role of RGDS-containing peptides physiologically released from the extracellular matrix and may suggest a possible novel anti-proliferation strategy in melanoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1476-4598-9-84DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2867821PMC
April 2010

Downregulation of DeltaNp63alpha in keratinocytes by p14ARF-mediated SUMO-conjugation and degradation.

Cell Cycle 2009 Nov 1;8(21):3545-51. Epub 2009 Dec 1.

Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli Federico II, Naples, Italy.

The tumor suppressor p14(ARF) inhibits cell growth in response to oncogenic stress in a p53-dependent and independent manner. However, new physiologic roles for ARF activation have been proposed. We have previously demonstrated that ARF interacts with p63, influencing its transcriptional activity. p63 is a member of the p53 family involved in skin and limb development, as well as in the homeostasis of mature epidermis. Here, we show that, in human keratinocytes, as well as in tumor-derived cell lines, ARF targets DeltaNp63alpha, the most abundantly expressed p63 isoform, to proteasomal degradation by stimulating its sumoylation. Interestingly, we have observed an increase of ARF expression in differentiating keratinocytes, that is concomitant to the already described upregulation of SUMO2/3. Remarkably, we found that DeltaNp63alpha is preferentially sumoylated by SUMO2, instead of SUMO1, and p14(ARF) increases the efficiency of this process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4161/cc.8.21.9954DOI Listing
November 2009

Chemerin expression marks early psoriatic skin lesions and correlates with plasmacytoid dendritic cell recruitment.

J Exp Med 2009 Jan 29;206(1):249-58. Epub 2008 Dec 29.

Istituto Dermopatico dell'Immacolata, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 00167 Rome, Italy.

Psoriasis is a type I interferon-driven T cell-mediated disease characterized by the recruitment of plasmacytoid dendritic cells (pDC) into the skin. The molecules involved in pDC accumulation in psoriasis lesions are unknown. Chemerin is the only inflammatory chemotactic factor that is directly active on human blood pDC in vitro. The aim of this study was to evaluate the role of the chemerin/ChemR23 axis in the recruitment of pDC in psoriasis skin. Prepsoriatic skin adjacent to active lesions and early lesions were characterized by a strong expression of chemerin in the dermis and by the presence of CD15(+) neutrophils and CD123(+)/BDCA-2(+)/ChemR23(+) pDC. Conversely, skin from chronic plaques showed low chemerin expression, segregation of neutrophils to epidermal microabscesses, and few pDC in the dermis. Chemerin expression was localized mainly in fibroblasts, mast cells, and endothelial cells. Fibroblasts cultured from skin of psoriatic lesions expressed higher levels of chemerin messenger RNA and protein than fibroblasts from uninvolved psoriatic skin or healthy donors and promoted pDC migration in vitro in a chemerin-dependent manner. Therefore, chemerin expression specifically marks the early phases of evolving skin psoriatic lesions and is temporally strictly associated with pDC. These results support a role for the chemerin/ChemR23 axis in the early phases of psoriasis development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20080129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2626680PMC
January 2009