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J Uitto

Publications and source records attributed to J Uitto.

At least 19 recordsLinked to original sources
PubMed2007

Plectin defects in epidermolysis bullosa simplex with muscular dystrophy.

Epidermolysis bullosa simplex with muscular dystrophy (EBS-MD, MIM 226670) is caused by plectin defects. We performed mutational analysis and immunohistochemistry using EBS-MD (n = 3 cases) and control skeletal muscle to determine pathogenesis. Mutational analysis revealed a novel homozygous plectin-exon32 rod domain mutation (R2465X). All plectin/HD1-121 antibodies stained the control skeletal muscle membrane. However, plectin antibodies stained the cytoplasm of type II control muscle fibers (as confirmed by ATPase staining), whereas HD1-121 stained the cytoplasm of type I fibers. EBS-MD samples lacked membrane (n = 3) but retained cytoplasmic HD1-121 (n = 1) and plectin staining in type II fibers (n = 3). Ultrastructurally, EBS-MD demonstrated widening and vacuolization adjacent to the membrane and disorganization of Z-lines (n = 2 of 3) compared to controls (n = 5). Control muscle immunogold labeling colocalized plectin and desmin to filamentous bridges between Z-lines and the membrane that were disrupted in EBS-MD muscle. We conclude that fiber-specific plectin expression is associated with the desmin-cytoskeleton, Z-lines, and crucially myocyte membrane linkage, analogous to hemidesmosomes in skin.

Adult↗
PubMed2006

Epidermolysis bullosa. I. Molecular genetics of the junctional and hemidesmosomal variants.

INTRODUCTION: Epidermolysis bullosa (EB), a group of autosomal heritable blistering diseases, is characterised by extensive phenotypic variability with considerable morbidity and mortality. EB is classified into distinct subtypes depending on the location of blistering within the cutaneous dermoepidermal basement membrane zone. Ten genes are known to harbour mutations in the major types of EB, and the level of expression of these genes within the cutaneous basement membrane zone and in extracutaneous tissues, as well as the types and combinations of the mutations, explain in general terms the phenotypic variability. METHODS: The DebRA Molecular Diagnostics Laboratory, established in 1996 and supported in part by the patient advocacy organisation DebRA of America, has analysed over 1000 families with different forms of EB. RESULTS: In total, 265 cases were submitted with the preliminary diagnosis of junctional or hemidesmosomal forms of EB. We found 393 mutant alleles in seven different genes, with 173 of the mutations being distinct and 71 previously unpublished. DISCUSSION: These findings attest to the clinical and molecular heterogeneity of the junctional and hemidesmosomal subtypes of EB. The results also reveal exceptions to the general rules on genotype-phenotype correlations, unusual phenotypes, and surprising genetics. Collectively, mutation analysis in different forms of EB provides the basis for improved classification with prognostic implications and for prenatal and preimplantation diagnosis in families at risk for recurrence of EB.

Basement Membrane↗
PubMed2005

Progress in epidermolysis bullosa: the phenotypic spectrum of plectin mutations.

Plectin, a large multidomain adhesive protein with versatile binding functions, is expressed in a number of tissues and cell types. In the skin, plectin is a critical component of hemidesmosomes, interacting with keratin intermediate filaments and beta4 integrin. Mutations in the plectin gene (PLEC1) result in fragility of skin, demonstrating blister formation at the level of hemidesmosomes. These blistering disorders belong to the spectrum of epidermolysis bullosa (EB) phenotypes, and three distinct variants because of plectin mutations have been identified. First, EB with muscular dystrophy, an autosomal recessive syndrome, is frequently caused by premature termination codon-causing mutations leading to the absence of plectin both in the skin and in the muscle. Second, a heterozygous missense mutation (R2110W) in PLEC1 has been documented in patients with EB simplex of the Ogna type, a rare autosomal dominant disorder. Finally, recent studies have disclosed plectin mutations in patients with EB with pyloric atresia, an autosomal recessive syndrome, frequently with lethal consequences. Collectively, these observations attest to the phenotypic spectrum of plectin mutations, and provide the basis for accurate genetic counselling with prognostic implications, as well as for prenatal diagnosis in families at the risk of recurrence of the disease.

Animals↗
PubMed2003

Novel member of the mouse desmoglein gene family: Dsg1-beta.

Desmosomes are major intercellular adhesion junctions that provide stable cell-cell contacts and mechanical strength to epithelial tissues by anchoring cytokeratin intermediate filaments of adjacent cells. Desmogleins (Dsg) are transmembrane core components of the desmosomes, and belong to the cadherin supergene family of calcium-dependent adhesion molecules. Currently, there are three known isoforms of Dsgs (Dsg1, Dsg2, and Dsg3), encoded by distinct genes that are differentially expressed to determine their tissue specificity and differentiation state of epithelial cells. In this study, we cloned a novel mouse desmoglein gene sharing high homology to both mouse and human Dsg1. We propose to designate the previously published mouse Dsg1 gene as Dsg1-alpha and the new gene as Dsg1-beta. Analysis of intron/exon organization of the Dsg1-alpha and Dsg1-beta genes revealed significant conservation. The full-length mouse Dsg1-beta cDNA contains an open reading frame of 3180 bp encoding a precursor protein of 1060 amino acids. Dsg1-beta protein shares 94% and 76% identity with mouse Dsg1-alpha and human DSG1, respectively. RT-PCR using a multitissue cDNA panel demonstrated that while Dsg1-alpha mRNA was expressed in 15- to 17-day-old embryos and adult spleen and testis, Dsg1-beta mRNA was detected in 17-day-old embryos only. To assess subcellular localization, a FLAG-tagged expression construct of Dsg1-beta was transiently expressed in epithelial HaCaT cells. Dsg1-beta-FLAG was found at the cell-cell border and was recognized by the anti-Dsg1/Dsg2 antibody DG3.10. In summary, we have cloned and characterized a novel member of the mouse desmoglein gene family, Dsg1-beta.

Amino Acid Sequence↗
PubMed2003

Divergent effects of two sequence variants of GJB3 (G12D and R32W) on the function of connexin 31 in vitro.

Recently, we identified several missense mutations of the connexin gene GJB3 encoding connexin 31 (Cx31) in erythrokeratodermia variabilis (EKV), an autosomal dominant skin disorder. These mutations include G12D, which replaces a conserved glycine residue in the amino-terminus of Cx31 and is associated with a severe EKV phenotype. In contrast, the biologic relevance of the GJB3 sequence variant R32W located in the first transmembrane domain of Cx31 is disputed. To examine the effects of these sequence variants on Cx31 biogenesis and gap junction activity we expressed wild type and mutant Cx31-Flag constructs in HeLa cells. Using immunostaining, all expression variants were detected in the cytoplasm and in a punctate pattern at the cell surface, indicating that G12D and R32W did not interfere with either protein synthesis or transport to the cell membrane. Similarly, oligomerization into hemichannels appeared not impaired when expressing either Cx31 mutant as assessed by size exclusion chromatography, immunoblotting and immunostaining. However, dye transfer experiments and monitoring of intracellular calcium levels in response to serum stimulation revealed that G12D-Cx31 did not form functional gap junction channels, probably due to incorrect assembly or altered properties of Cx31 channels. In contrast, intercellular coupling between cells expressing R32W-Cx31 was comparable to that of wtCx31, suggesting that R32W is a functionally inconsequential polymorphism of Cx31.

Base Sequence↗
PubMed2003

A novel homozygous nonsense deletion/insertion mutation in the keratin 14 gene (Y248X; 744delC/insAG) causes recessive epidermolysis bullosa simplex type Köbner.

We report the sixth case of a human keratin 14 'knockout' mutation resulting in recessive epidermolysis bullosa simplex (EBS). A novel, homozygous nonsense mutation resulting from a deletion/insertion mutation (744delC/insAG) leads to a premature termination codon in the KRT14 gene (Y248X). The patient suffers from generalized cutaneous blistering since birth, mild nail dystrophy, involvement of mucous membranes and multiple epidermolysis bullosa naevi. The clinical variability noted in K14-deficient EBS patients suggests phenotypic modulation by additional genetic and/or epigenetic factors.

Child↗
PubMed2003

Relevance of differential immunogenicity of human and mouse recombinant desmoglein-3 for the induction of acantholytic autoantibodies in mice.

Pemphigus vulgaris (PV) is an antibody-mediated autoimmune disease of the skin and mucous membranes. Desmoglein-3 (dsg-3) expressed in the suprabasal layer of the skin serves as an autoantigen in PV. Passive transfer of sera, either from patients with PV or from experimental animals immunized with a recombinant human dsg3 (hdsg3) into neonatal BALB/c mice results in blister formation, suggesting strongly that there is significant cross-reactivity between the mouse dsg3 (mdsg3) and the hdsg3. However, efforts to induce disease in adult mice through active immunization using hdsg-3 have not been successful, suggesting that the epitopes required for the induction of pathogenic antibodies in adult mice might not be present in hdsg3. Therefore, in this study, we expressed a full-length mdsg3 in insect cells and compared its serological reactivity with that of the hdsg3 using species specific polyclonal sera and a panel of seven monoclonal antibodies (MoAbs) with unique binding specificities to hdsg3. Studies using sera demonstrated a considerable cross-reactivity, while studies using MoAbs exhibited specific epitope differences between the two proteins. Because of these differences, we reasoned that immunization with mdsg3 might induce disease in adult mice. Immunization of four strains of mice (i.e. BALB/c, DBA/1, HRS/J and SJL/J) with mdsg3 resulted in considerable antibody response, but failed to induce lesions. However, sera from immunized BALB/c mice induced acantholysis of neonatal mouse skin in vitro. These studies indicated that our inability to induce lesions in adult mice through active immunization is not due to differences in the ability of mouse and human dsg3 to induce acantholytic antibodies, but due probably to structural differences between adult and neonatal mouse skin. Alternatively, immunization with a combination of dsg3 protein along with other proteins might be necessary to induce pemphigus disease in adult mice. Nevertheless, our current studies show that molecular mechanisms leading to the production of acantholytic antibodies in mice can now be studied using homologous mdsg3.

Animals↗
PubMed2002

Intrinsic aging vs. photoaging: a comparative histopathological, immunohistochemical, and ultrastructural study of skin.

Cutaneous aging is a complex biological phenomenon affecting the different constituents of the skin. To compare the effects of intrinsic and extrinsic aging processes, a total of 83 biopsies were collected from sun-exposed and protected skin of healthy volunteers representing decades from the 1st to the 9th (6-84 years of age). Routine histopathology coupled with computer-assisted image analysis was used to assess epidermal changes. Immunoperoxidase techniques with antibodies against type I and type III collagens and elastin were used to quantitatively evaluate changes in collagen and elastic fibers and their ultrastructure was examined by transmission electron microscopy. Epidermal thickness was found to be constant in different decades in both sun-exposed and protected skin; however, it was significantly greater in sun-exposed skin (P = 0.0001). In protected skin, type I and III collagen staining was altered only after the 8th decade, while in sun-exposed skin the relative staining intensity significantly decreased from 82.5% and 80.4% in the 1st decade to 53.2% and 44.1% in the 9th decade, respectively (P = 0.0004 and 0.0008). In facial skin the collagen fiber architecture appeared disorganized after the 4th decade. The staining intensity of elastin in protected skin significantly decreased from 49.2% in the 1st decade to 30.4% in the 9th decade (P = 0.05), whereas in sun-exposed skin the intensity gradually increased from 56.5% in the 1st decade to 75.2% in the 9th decade (P = 0.001). The accumulated elastin in facial skin was morphologically abnormal and appeared to occupy the areas of lost collagen. Collectively, the aging processes, whether intrinsic or extrinsic, have both quantitative and qualitative effects on collagen and elastic fibers in the skin.

Adolescent↗
PubMed2001

Differential expression and subcellular distribution of the mouse metastasis-associated proteins Mta1 and Mta3.

The human metastasis-associated gene (MTA1) is overexpressed in cell lines and tissues representing metastatic tumors. Here we report cloning of the mouse Mta1 as well as a novel structurally related mouse gene, Mta3. The mouse Mta1 protein shares 94 and 59% homology to the human MTA1 and mouse Mta3 proteins, respectively. Northern blotting analysis using an Mta1 cDNA probe revealed a prevalent 3 kb hybridization signal in all mouse tissues except the skeletal muscle while a smaller approximately 1.0 kb mRNA product was also detected in the heart. Mta3 transcripts (approximately 2 kb) were detected in most tissues with an additional approximately 6.2 kb signal detected in the brain. In vitro transcription/translation of the full-length Mta1 and Mta3 cDNAs generated products of the expected molecular masses, i.e. 80 and 60 kDa, respectively. To assess subcellular localization, green fluorescence protein (GFP)-tagged expression constructs of Mta1 and Mta3 and various deletion constructs of GFP-Mta1 were transiently expressed in Balb/MK keratinocytes. GFP-Mta1 was found exclusively in the nucleus while GFP-Mta3 was present in both the nucleus and cytoplasm. Compared to Mta3, the carboxy terminal end of Mta1 contains an additional nuclear localization signal (NLS) and a proline-rich Src homology 3 (SH3) ligand. The results of transient expression experiments of various Mta1 fragments containing these domains in different combinations indicated that nuclear localization of Mta1 depended on the presence of at least one NLS and one SH3 binding site. These SH3 ligands appeared to be functional as they facilitated interaction with the adaptor protein, Grb2, and the Src-family tyrosine kinase, Fyn.

Amino Acid Sequence↗
PubMed2001

Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner.

Smad proteins transduce signals from TGF-beta receptors and regulate transcription of target genes either directly or in combination with other sequence-specific transcription factors. AP-1 sites and their cognate transcription factors also play important roles in the gene regulatory activities of TGF-beta. In this report, we have investigated the functional interactions of the Smad and AP-1 transcription factors. We demonstrate that Smad and AP-1 complexes specifically bind to their cognate cis-elements and do not interact with each other on-DNA, whereas off-DNA interactions occur between Smad3 and both c-Jun and JunB. Using both artificial constructs specific for either the Smad or AP-1 signaling pathways or natural promoters known to be TGF-beta-responsive, we have determined that Jun family members downregulate Smad3-mediated gene transactivation whereas AP-1-dependent promoters are synergistically activated by Smad3 and Jun proteins. We propose a model where the presence of Smad- and/or AP-1-specific cis-elements within TGF-beta-responsive genes allows dynamic modulation of gene expression, in contrast to the existing model where interactions between Smad and AP-1 proteins are merely an on/off mechanism to regulate TGF-beta/Smad targets.

Activin Receptors, Type I↗
PubMed2001

Compound heterozygosity for a recurrent 16.5-kb Alu-mediated deletion mutation and single-base-pair substitutions in the ABCC6 gene results in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder affecting the elastic structures in the skin, eyes, and cardiovascular system, with considerable morbidity and mortality. Recently, mutations in the ABCC6 gene (also referred to as "MRP6" or "eMOAT") encoding multidrug-resistance protein 6 (MRP6), a putative transmembrane ABC transporter protein of unknown function, have been disclosed. Most of the genetic lesions delineated thus far consist of single-base-pair substitutions resulting in nonsense, missense, or splice-site mutations. In this study, we examined four multiplex families with PXE inherited in an autosomal recessive pattern. In each family, the proband was a compound heterozygote for a single-base-pair-substitution mutation and a novel, approximately 16.5-kb deletion mutation spanning the site of the single-base-pair substitution in trans. The deletion mutation was shown to extend from intron 22 to intron 29, resulting in out-of-frame deletion of 1,213 nucleotides from the corresponding mRNA and causing elimination of 505 amino acids from the MRP6 polypeptide. The deletion breakpoints were precisely the same in all four families, which were of different ethnic backgrounds, and haplotype analysis by 13 microsatellite markers suggested that the deletion had occurred independently. Deletion breakpoints within introns 22 and 29 were embedded within AluSx repeat sequences, specifically in a 16-bp segment of DNA, suggesting Alu-mediated homologous recombination as a mechanism.

ATP-Binding Cassette Transporters↗
PubMed2001

Molecular genetics of heritable blistering disorders.

Over the past decade, there has been tremendous progress in understanding the genetic basis of different forms of genodermatoses. Specifically, with the advent of technologies in molecular biology in general, an increasingly large number of gene defects have been identified in different genodermatoses, and mutations are now known to occur in more than 100 distinct genes in such a manner that the genetic lesions explain the spectrum of phenotypic manifestations encountered in these diseases.

Basement Membrane↗
PubMed2001

Identification of ABCC6 pseudogenes on human chromosome 16p: implications for mutation detection in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE), a heritable disorder affecting the skin, eyes, and the cardiovascular system, has recently been linked to mutations in the ABCC6 gene on chromosome 16p13.1. The original mutation detection strategy employed by us consisted of the amplification of each exon of the ABCC6 gene with primer pairs placed on the flanking introns, followed by heteroduplex scanning and direct nucleotide sequencing. However, this approach suggested the presence of multiple copies of the 5'-region of the gene when total genomic DNA was used as a template. In this study, we have identified two pseudogenes containing sequences highly homologous to the 5'-end of ABCC6. First, by the use of allele-specific polymerase chain reaction (PCR), two bacterial artificial chromosome (BAC) clones containing a putative pseudogene of ABCC6, designated as ABCC6-psi 1, were isolated from the human BAC library. Sequence analysis of ABCC6-psi 1 revealed it to be a truncated copy of ABCC6, which contains the upstream region and exon 1 through intron 9 of the gene. Secondly, a homology search of a high-throughput sequence database revealed the presence of another truncated copy of ABCC6, which was designated as ABCC6-psi 2, and which was shown to harbor upstream sequences and a segment spanning exon 1 through intron 4 of ABCC6. In addition to several nucleotide differences in the flanking introns and the upstream region, both pseudogenes contain several nucleotide changes in the exonic sequences, including stop codon mutations, which complicate mutation analysis in patients with PXE. Nucleotide differences in flanking introns between these two pseudogenes and ABCC6 allowed us to design allele-specific primers that eliminated the amplification of both pseudogene sequences by PCR and provided reliable amplification of ABCC6-specific sequences only. The use of allele-specific PCR has revealed, thus far, two novel 5'-end PXE mutations, 179del9 and T364R in exons 2 and 9, respectively, and several polymorphisms within the upstream region and exons 1-9 of ABCC6. These strategies facilitate comprehensive analysis of ABCC6 for mutations in PXE.

Alleles↗
PubMed2001

A compound heterozygous one amino-acid insertion/nonsense mutation in the plectin gene causes epidermolysis bullosa simplex with plectin deficiency.

Plectin is a cytoskeleton linker protein expressed in a variety of tissues including skin, muscle, and nerves. Mutations in its gene are associated with epidermolysis bullosa simplex with late-onset muscular dystrophy. Whereas in most of these patients the pathogenic events are mediated by nonsense-mediated mRNA decay, the consequences of an in-frame mutation are less clear. We analyzed a patient with compound heterozygosity for a 3-bp insertion at position 1287 leading to the insertion of leucine as well as the missense mutation Q1518X leading to a stop codon. The presence of plectin mRNA was demonstrated by a RNase protection assay. However, a marked reduction of plectin protein was found using immunofluorescence microscopy of the patient's skin and Western blot analysis of the patient's cultured keratinocytes. The loss of plectin protein was associated with morphological alterations in plectin-containing structures of the dermo-epidermal junction, in skeletal muscle, and in nerves as detected by electron microscopy. In an in vitro overlay assay using recombinant plectin peptides spanning exons 2 to 15 the insertion of leucine resulted in markedly increased self-aggregation of plectin peptides. These results describe for the first time the functional consequences of an in-frame insertion mutation in humans.

Base Sequence↗
PubMed2001

Molecular genetics of pseudoxanthoma elasticum: a metabolic disorder at the environment-genome interface?

Pseudoxanthoma elasticum (PXE) is a relatively rare heritable disorder affecting the skin, eyes and cardiovascular system, with considerable morbidity and mortality. The disease affects the elastic fibers of affected organs, which become progressively calcified. Thus, PXE has been considered as a prototypic heritable connective tissue disorder affecting the elastic fiber system. Recently, PXE has been linked to mutations in the MRP6/ABCC6 gene, a member of the ABC transporter family, expressed primarily in the liver and the kidneys. This information, together with clinical observations suggesting environmental, hormonal and/or dietary modulation of the disease, raises the intriguing possibility that PXE is a primary metabolic disorder at the environment-genome interface.

ATP-Binding Cassette Transporters↗
PubMed2001

Darier disease--novel mutations in ATP2A2 and genotype-phenotype correlation.

Darier disease (DD) is with a frequency of up to 1 in 36,000 a relatively common genodermatosis with autosomal dominant inheritance and late age of onset. The progressive skin manifestations are variable, but often debilitating and disfiguring, and may be associated with a wide range of neuropsychiatric problems, such as epilepsy and depression. On histology, acantholysis and dyskeratosis are prominent findings, implicating impaired functionality of desmosomes. Recently, mutations in the ATP2A2 gene encoding SERCA2, a calcium pump of the endo/sacrcoplasmic reticulum, have been identified as the molecular basis of DD. This slow-twitched calcium ATPase has two splice variants, one of which is highly expressed in epidermis, and maintains low intracellular calcium levels by facilitating transport of cytosolic calcium into the endoplasmic reticulum. Thus, it may confer a direct effect on the established calcium-dependent assembly of desmosomes. We screened ATP2A2 in a cohort of 24 DD families using conformation sensitive gel electrophoresis and direct sequencing, and detected 14 distinct mutations, 9 of which were novel. The mutational spectrum included 9 missense mutations, 1 nonsense mutation, 3 small in-frame deletions, and a 19-basepair insertion. Mutations were scattered over the entire gene with a slight preponderance in the first 8 exons, and affected exclusively residues conserved among all SERCAs. In addition, we found 2 silent polymorphisms, 1 of which occurred in 4 unrelated families. Comparison of molecular data and phenotypic features, such as severity and type of disease, occurrence of mucosal involvement, or association with neuropsychiatric disorders, did not reveal an obvious genotype-phenotype correlation in our cohort.

Calcium-Transporting ATPases↗