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Cristina Has

Publications and source records attributed to Cristina Has.

13 recordsLinked to original sources
PubMed2006

Molecular basis of inherited skin-blistering disorders, and therapeutic implications.

Epidermolysis bullosa (EB) and associated skin-fragility syndromes are a group of inherited skin diseases characterised by trauma-induced blistering of the skin and mucous membranes. Mutations in at least 14 distinct genes encoding molecular components of the epidermis or the dermal-epidermal junction (DEJ) can cause blistering skin diseases that differ by clinical presentation and severity of the symptoms. Despite great advances in discerning the genetic basis of this group of diseases, the molecular pathways leading to symptoms are not yet fully understood. Unravelling these pathways by molecular analysis of the structure and in vitro assessment of functional properties of the human proteins involved, combined with genetic models in lower organisms, should pave the way for specific cures for inherited skin fragility.

Animals↗
PubMed2006

Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes.

A novel family of focal adhesion proteins, the kindlins, is involved in attachment of the actin cytoskeleton to the plasma membrane and in integrin-mediated cellular processes. Deficiency of kindlin-1, as a result of loss-of-function mutations in the KIND1 gene, causes Kindler syndrome, an autosomal recessive genodermatosis characterized by skin blistering, progressive skin atrophy, photosensitivity and, occasionally, carcinogenesis. Here we characterized authentic and recombinantly expressed kindlin-1 and show that it is localized in basal epidermal keratinocytes in a polar fashion, close to the cell surface facing the basement membrane, in the areas between the hemidesmosomes. We identified two forms of kindlin-1 in keratinocytes, with apparent molecular masses of 78 and 74 kDa, corresponding to phosphorylated and desphosphorylated forms of the protein. In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis. In vitro, deficiency of kindlin-1 in keratinocytes leads to strongly reduced cell proliferation, decreased adhesion, undirected motility, and intense protrusion activity of the plasma membrane. Taken together, these results show that kindlin-1 plays a role in keratinocyte adhesion, polarization, proliferation, and migration. It is involved in organization and anchorage of the actin cytoskeleton to integrin-associated signaling platforms.

Animals↗
PubMed2006

Molecular and diagnostic aspects of genetic skin fragility.

Genetic syndromes with skin fragility represent a heterogeneous group of very rare disorders caused by mutations in genes encoding proteins or protein subunits important for the mechanical resistance of keratinocytes and for cell-cell or cell-extracellular matrix adhesion. The common symptoms are skin blistering or peeling, with various degrees of severity and distribution, ranging from localized to generalized forms. Associated features include involvement of skin annexes, mucous membranes, teeth, muscles or the digestive tract. Morphological investigation of skin samples provides evidence for the tissue level of blister formation, while immunostainings may reveal defective proteins, providing clues concerning the genetic origin of the disease. Extensive mutation analysis and subsequent identification of new gene defects provide accurate diagnostics, and lead to better understanding of the functions of the respective proteins, with the potential for new therapeutic strategies.

Cell Adhesion↗
PubMed2006

C-terminal truncation impairs glycosylation of transmembrane collagen XVII and leads to intracellular accumulation.

Collagen XVII, a type II transmembrane protein in hemidesmosomes, is involved in the anchorage of stratified epithelia to the underlying mesenchyme. Its functions are regulated by ectodomain shedding, and its genetic defects lead to epidermal detachment in junctional epidermolysis bullosa (JEB), a heritable skin fragility syndrome, but the molecular disease mechanisms remain elusive. Here we used a spontaneously occurring homozygous COL17A1 deletion mutant in JEB to discern glycosylation of collagen XVII. The mutation truncated the distal ectodomain and positioned the only N-glycosylation site 34 amino acids from the newly formed C terminus, which impaired efficient N-glycosylation. Immunofluorescence staining of authentic JEB keratinocytes and of COS-7 cells transfected with the mutant indicated intracellular accumulation of collagen XVII precursor molecules. Cell surface biotinylation and quantification of ectodomain shedding demonstrated that only about 15% of the truncated collagen XVII reached the cell surface. The cell surface-associated molecules were N-glycosylated in a normal manner, in contrast to the molecules retained within the cells, indicating that N-glycosylation of the ectodomain is required for targeting of collagen XVII to the plasma membrane and that reduced accessibility of the N-glycosylation site negatively regulates this process. Functional consequences of the strong reduction of collagen XVII on the cell surface included scattered deposition of cell adhesion molecule laminin 5 into the extracellular environment and, as a consequence of faulty collagen XVII-laminin ligand interactions, aberrant motility of the mutant cells.

Amino Acid Sequence↗
PubMed2006

Molecular basis of Kindler syndrome in Italy: novel and recurrent Alu/Alu recombination, splice site, nonsense, and frameshift mutations in the KIND1 gene.

Kindler syndrome (KS) is a rare autosomal recessive disorder characterized by skin blistering in childhood followed by photosensitivity and progressive poikiloderma. Most cases of KS result from mutations in the KIND1 gene encoding kindlin-1, a component of focal adhesions in keratinocytes. Here, we report novel and recurrent KIND1 gene mutations in nine unrelated Italian KS individuals. A novel genomic deletion of approximately 3.9 kb was identified in four patients originating from the same Italian region. This mutation deletes exons 10 and 11 from the KIND1 mRNA leading to a truncated kindlin-1. The deletion breakpoint was embedded in AluSx repeats, specifically in identical 30-bp sequences, suggesting Alu-mediated homologous recombination as the pathogenic mechanism. KIND1 haplotype analysis demonstrated that patients with this large deletion were ancestrally related. Five additional mutations were disclosed, two of which were novel. To date, four recurrent mutations have been identified in Italian patients accounting for approximately approximately 75% of KS alleles in this population. The abundance of repetitive elements in intronic regions of KIND1, together with the identification of a large deletion, suggests that genomic rearrangements could be responsible for a significant proportion of KS cases. This finding has implications for optimal KIND1 mutational screening in KS individuals.

Adolescent↗
PubMed2006

Expanding the COL7A1 mutation database: novel and recurrent mutations and unusual genotype-phenotype constellations in 41 patients with dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB), a heterogeneous hereditary skin disorder characterized by trauma-induced blistering and scarring, affects thousands of families worldwide. The clinical manifestations extend from minor nail dystrophy to severe life-threatening blistering, making early molecular diagnosis and prognostication of utmost importance for the affected families. DEB is caused by mutations in the COL7A1 gene encoding collagen VII in the skin. Molecular diagnostics and genotype-phenotype correlations in DEB remain complex owing to the gene structure, large variety of mutations, high rate of novel mutations, complex protein structure and assembly, and the heterogeneity of phenotypes. Here, we report an efficient strategy for COL7A1 mutation detection using direct automated DNA sequencing and implementation of software tools. With this approach, COL7A1 mutations of 41 DEB families were disclosed. Twenty-four mutations were novel and two recurrent. Elucidation of biological consequences of the mutations helped define disease mechanisms, but also revealed several unusual genotypic and/or phenotypic constellations, which impeded the diagnostics and prognostication. In addition, the studies disclosed a de novo mutation in recessive DEB and two new polymorphisms in the COL7A1 gene.

Collagen Type VII↗
PubMed2005

Molecular mechanisms of junctional epidermolysis bullosa: Col 15 domain mutations decrease the thermal stability of collagen XVII.

Mutations in the collagen XVII gene, COL17A1, are associated with junctional epidermolysis bullosa. Most COL17A1 mutations lead to a premature termination codon (PTC), whereas only a few mutations result in amino acid substitutions or deletions. We describe here two novel glycine substitutions, G609D and G612R, and a splice site mutation resulting in a deletion of three Gly-X-Y amino acid triplets. In order to investigate the molecular pathomechanisms of non-PTC mutations, G609D and G612R and two previously known substitutions, G627V and G633, and deletion of the amino acids 779-787 were introduced into recombinant collagen XVII. The thermal stability of the mutated collagens was assessed using trypsin digestions at incremental temperatures. All the four glycine substitutions significantly destabilized the ectodomain of collagen XVII, which manifested as 16 degrees C-20 degrees C lower T(m) (midpoint of the helix-to-coil transition). These results were supported by secondary structure predictions, which suggested interruptions of the collagenous triple helix within the largest collagenous domain, Col15. In contrast, deletion of the three full Gly-X-Y triplets, amino acids 779-787, had no overall effect on the stability of the ectodomain, as the deletion was in register with the triplet structure and also generated compensatory changes in the NC15 domain.

Adult↗
PubMed2004

Dystroglycan in skin and cutaneous cells: beta-subunit is shed from the cell surface.

In skin, hemidesmosomal protein complexes attach the epidermis to the dermis and are critical for stable connection of the basal epithelial cell cytoskeleton with the basement membrane (BM). In muscle, a similar supramolecular aggregate, the dystrophin glycoprotein complex links the inside of muscle cells with the BM. A component of the muscle complex, dystroglycan (DG), also occurs in epithelia. In this study, we characterized the expression and biochemical properties of authentic and recombinant DG in human skin and cutaneous cells in vitro. We show that DG is present at the epidermal BM zone, and it is produced by both keratinocytes and fibroblasts in vitro. The biosynthetic precursor is efficiently processed to the alpha- and beta-DG subunits; and, in addition, a distinct extracellular segment of the transmembranous beta-subunit is shed from the cell surface by metalloproteinases. Shedding of the beta-subunit releases the alpha-subunit from the DG complex on the cell surface into the extracellular space. The shedding is enhanced by IL-1beta and phorbol esters, and inhibited by metalloproteinase inhibitors. Deficiency of perlecan, a major ligand of alpha-DG, enhanced shedding suggesting that lack of a binding partner destabilizes the epithelial DG complex and makes it accessible to proteolytic processing.

Cell Line↗
PubMed2002

Gas chromatography-mass spectrometry and molecular genetic studies in families with the Conradi-Hünermann-Happle syndrome.

The Conradi-Hünermann-Happle syndrome is an X-linked dominant disease that is due to mutations in the gene for emopamil binding protein. Emopamil binding protein is a Delta8-Delta7 sterol isomerase and plays a pivotal role in the final steps of cholesterol biosynthesis. We wanted to know to what extent this X-linked dominant enzyme defect has functional consequences at the biochemical level and whether it is possible to predict the clinical phenotype from serum sterol measurements. Therefore we performed sterol biochemical studies in 11 Conradi-Hünermann-Happle syndrome families and compared the results obtained to the clinical and molecular genetic findings. To assess disease severity a score considering bone and skin involvement and further features was used. For evaluation of the functional consequences we studied serum samples using gas chromatography-mass spectrometry analysis. For mutation screening we analyzed the emopamil binding protein gene using polymerase chain reaction, heteroduplex analysis of all exons, direct sequencing, and restriction enzyme analysis. Mutations in the emopamil binding protein gene were found in all 11 families including seven novel mutations affecting exons 2, 4, and 5. Gas chromatography-mass spectrometry analysis revealed markedly elevated levels of 8-dehydrocholesterol and of cholest-8(9)-en-3beta-ol and helped to identify somatic mosaicism in a clinically unaffected man. The extent of the metabolic alterations in the serum, however, do not allow prediction of the clinical phenotype, nor the genotype. This lack of correlation may be due to differences in X-inactivation between different tissues of the same patient and/or loss of the mutant clone by outgrowth of proficient clones after some time.

Adolescent↗
PubMedDate not supplied

Recurring staphylococcal scalded skin syndrome-like bullous mastocytosis: the utility of cytodiagnosis and the rapid regression with steroids.

We report a male infant with onset of an extensive bullous eruption at the age of 45 days. Staphylococcal scalded skin syndrome (SSSS) was suspected. Bullous mastocytosis was diagnosed by cytodiagnosis and confirmed by histologic examination. Three serious relapses were noted in a 2-year follow-up, and SSSS was again suspected because of high fever and leukocytosis with neutrophilia in an infectious context. Cytodiagnosis revealed the presence of mast cells and permitted rapid diagnosis of recurrences of bullous mastocytosis. Systemic corticotherapy dramatically improved the cutaneous lesions and general symptoms. This case report emphasizes the utility of cytodiagnosis in extensive blistering diseases in infancy and the possibility of obtaining rapid healing by using steroids.

Diagnosis, Differential↗