Biomedical subjects

R A Eady

Publications and source records attributed to R A Eady.

At least 73 records · Page 4Linked to original sources

PubMed1997

Recessive epidermolysis bullosa simplex associated with plectin mutations: infantile respiratory complications in two unrelated cases.

Plectin is a 500 kDa protein involved in cytoskeleton-plasma membrane attachment with a wide tissue distribution including cutaneous and airway epithelia, muscle and neuronal tissue. Recently, mutations in the gene encoding plectin (PLEC1) have been implicated in the pathogenesis of an autosomal recessive variant of epidermolysis bullosa simplex in which cutaneous blistering starting in the neonatal period is associated with muscular dystrophy in later life. In this study, we report two unrelated patients, both of consanguineous parentage, who presented with cutaneous blistering and a hoarse cry from birth. Both experienced inspiratory stridor and respiratory distress, necessitating emergency tracheostomy in one case. Immunoreactivity to monoclonal antibodies against plectin was absent or markedly reduced in skin biopsies from both patients. Electron microscopy revealed a low intraepidermal plane of cleavage and hypoplastic hemidesmosomes with a reduced association with keratin intermediate filaments. Direct sequencing of PLEC1 in each case demonstrated two novel homozygous frameshift deletion mutations, 5069del19 and 5905del2, which both create downstream premature termination codons. Although currently neither patient has symptoms of muscle disease, the identification of mutations in PLEC1 may be predictive for the future development of muscular dystrophy. Recessive epidermolysis bullosa simplex resulting from abnormalities in plectin should be considered in the differential diagnosis blistering, hoarseness and stridor in infancy.

Child, Preschool↗

PubMed1996

Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization.

Plectin is a widely expressed high molecular weight protein that is involved in cytoskeleton-membrane attachment in epithelial cells, muscle, and other tissues. The human autosomal recessive disorder epidermolysis bullosa with muscular dystrophy (MD-EBS) shows epidermal blister formation at the level of the hemidesmosome and is associated with a myopathy of unknown etiology. Here, plectin was found to be absent in skin and cultured keratinocytes from an MD-EBS patient by immunofluorescence and immunoprecipitation, suggesting that plectin is a candidate gene/protein system for MD-EBS mutation. The 14800-bp human plectin cDNA was cloned and sequenced. The predicted 518-kD polypeptide has homology to the actin-binding domain of the dystrophin family at the amino terminus, a central rod domain, and homology to the intermediate filament-associated protein desmoplakin at the carboxyl terminus. The corresponding human gene (PLEC1), consisting of 33 exons spanning >26 kb of genomic DNA was cloned, sequenced, and mapped to chromosomal band 8q24. Homozygosity by descent was observed in the consanguineous MD-EBS family with intragenic plectin polymorphisms. Direct sequencing of PCR-amplified plectin cDNA from the patient's keratinocytes revealed a homozygous 8-bp deletion in exon 32 causing a frameshift and a premature termination codon 42 bp downstream. The clinically unaffected parents of the proband were found to be heterozygous carriers of the mutation. These results establish the molecular basis of MD-EBS in this family and clearly demonstrate the important structural role for plectin in cytoskeleton-membrane adherence in both skin and muscle.

Actins↗

PubMed1996

Morphology of the keratin filament network in palm and sole skin: evidence for site-dependent features based on stereological analysis.

Ridged or glabrous skin of palms and soles has a specialized function and can be preferentially involved in various disorders of keratinization. To better define the morphological features of ridged skin, we carried out a qualitative and quantitative (stereological) analysis of normal epidermis from the palm and sole of four subjects. Skin from the upper arm was examined for control purposes. The study focused on the appearance and arrangement of the keratin filament network in relation to epidermal differentiation. Whereas palm and sole epidermis was essentially similar both qualitatively and quantitatively, it differed markedly from the epidermis from the arm. The volume density of keratin filaments was significantly higher (P < 0.03) in all subcorneal layers of the palm and sole compared with the arm. The volume density of the keratin filaments increased markedly from the basal to the upper spinous layer of ridged skin and they formed denser aggregates in the upper spinous and granular layers, providing an extensive matrix for the deposition of keratohyalin. The presence of dense keratin aggregates appeared to be a distinct ultrastructural feature of human ridged skin. Such keratin aggregates have not been described in normal skin from other sites, but showed some resemblance to the keratin clumps seen in non-ridged skin of patients with the Dowling-Meara form of epidermolysis bullosa simplex.

Adult↗

PubMed1996

Hemidesmosome ontogeny in digit skin of the human fetus.

Hemidesmosomes are junctional complexes involved in the attachment of epidermal basal keratinocytes to the basement membrane. To try to understand better the sequence of events in the morphogenesis of hemidesmosomes, we undertook an ultrastructural analysis of hemidesmosome formation in fetal and neonatal digit skin. Hemidesmosomes, defined as membrane-associated densities or plaques, were counted and scored for three morphological characteristics: (1) the presence of a sub-basal dense plate, (2) association with anchoring filaments within the lamina lucida and (3) contacts with intermediate filaments. No hemidesmosomes were evident at 7 weeks' gestational age. Between 9 and 15 weeks the number of hemidesmosomes increased by about fourfold (from 20.6 +/- 3.8 (SD) to 95.5 +/- 8.4 per 40 micro m of basal cell plasma membrane; P < 0.01). The association of hemidesmosomes with intermediate filaments and anchoring filaments also increased after 15 weeks (P < 0.05). Early attachment plaques first appeared as triangular focal densities on the basal plasma membrane with the appearance of sub-basal dense plates, which later became both larger and more electron dense. By 15 weeks, an inner plaque could be distinguished from the outer plaque, which coincided with a closer association with intermediate filaments. Hemidesmosomes appeared fully developed by 15 weeks' gestation. This study illustrates the structural relationship of hemidesmosomes to both intra- and extracellular filaments, suggesting close functional interactions. The complexity of the hemidesmosome plaque is also revealed early during development.

Desmosomes↗

PubMed1996

Plectin deficiency results in muscular dystrophy with epidermolysis bullosa.

We report that mutation in the gene for plectin, a cytoskeleton-membrane anchorage protein, is a cause of autosomal recessive muscular dystrophy associated with skin blistering (epidermolysis bullosa simplex). The evidence comes from absence of plectin by antibody staining in affected individuals from four families, supportive genetic analysis (localization of the human plectin gene to chromosome 8q24.13-qter and evidence for disease segregation with markers in this region) and finally the identification of a homozygous frameshift mutation detected in plectin cDNA. Absence of the large multifunctional cytoskeleton protein plectin can simultaneously account for structural failure in both muscle and skin.

Amino Acid Sequence↗

PubMed1996

Gene targeting at the mouse cytokeratin 10 locus: severe skin fragility and changes of cytokeratin expression in the epidermis.

Bullous congenital ichthyosiform erythroderma (BCIE) is a dominantly inherited blistering skin disorder caused by point mutations in the suprabasal cytokeratins 1 or 10. Targeting the murine cytokeratin 10 gene in ES cells resulted in mice with different phenotypes in the homozygotes and heterozygotes; both of which exhibit similarities to specific clinical characteristics of BCIE. Homozygotes suffered from severe skin fragility and died shortly after birth. Heterozygotes were apparently unaffected at birth, but developed hyperkeratosis with age. In both genotypes, aggregation of cytokeratin intermediate filaments, changes in cytokeratin expression, and alterations in the program of epidermal differentiation were observed. In addition we demonstrate, for the first time, the existence of the murine equivalent of human cytokeratin 16.

Animals↗

PubMed1996

Clinicopathological correlations of compound heterozygous COL7A1 mutations in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa is an inherited mechano-bullous disorder of skin and mucous membranes. Ultrastructurally, the disease is characterized by abnormalities of anchoring fibrils, attachment structures below the epidermal basement membrane, composed of type VII collagen. Mutations in the type VII collagen gene (COL7A1) have been shown conclusively to underlie dystrophic epidermolysis bullosa. Since there is variation of the phenotype, accompanied by heterogeneous anchoring fibril morphology and type VII collagen immunostaining, it is conceivable that different types and combinations of COL7A1 mutations correlate with different phenotypes. We therefore screened recessive dystrophic epidermolysis bullosa patients for COL7A1 mutations. Three unrelated patients showed the same premature termination codon mutation in exon 13 of one allele, yet they were all compound heterozygotes, each having a different mutation in the second allele. The first patient had a premature termination codon within the collagenous region of COL7A1 associated with severe disease, absent anchoring fibrils and undetectable type VII collagen immunostaining. The second had a premature termination codon in the non-collagenous NC-2 region associated with severe disease, wispy anchoring fibrils, and patchy type VII collagen immunostaining. The third had a glycine-to-aspartic acid substitution within the collagenous region, associated with milder disease, no identifiable anchoring fibrils, but near normal type VII collagen immunostaining. We conclude that the nature and position of mutations within COL7A1 correlate with specific disease features and may provide an insight into the molecular mechanisms of anchoring fibril formation and epidermal-dermal adhesion.

Adult↗

PubMed1996

Compound heterozygosity for nonsense ans missense mutations in the LAMB3 gene in nonlethal junctional epidermolysis bullosa.

Mutations in the genes encoding laminin 5 (LAMA3, LAMB3, and LAMC2) have been delineated in the autosomal recessive blistering skin disorder, junctional epidermolysis bullosa, particularly in the lethal (Herlitz) variant. In this study, we searched for mutations in these genes in two patients with nonlethal forms of junctional epidermolysis bullosa using polymerase chain reaction amplification of genomic DA, followed by heteroduplex analysis and direct automated nucleotide sequencing. Both patients were found to be compound heterozygotes for the same nonsense mutation on one LAMB3 allele, and different missense mutations on the other LAMB3 allele. The combination of a nonsense and a missense mutation in the LAMB3 gene appears to be important in determining the milder clinical phenotype in some cases of the nonlethal forms of junctional epidermolysis bullosa involving abnormalities in laminin 5.

Aged↗

PubMed1996

Compound heterozygosity for nonsense and missense mutations in the LAMB3 gene in nonlethal junctional epidermolysis bullosa.

Mutations in the genes encoding laminin 5 (LAMA3, LAMB3, and LAMC2) have been delineated in the autosomal recessive blistering skin disorder, junctional epidermolysis bullosa, particularly in the lethal (Herlitz) variant. In this study, we searched for mutations in these genes in two patients with nonlethal forms of junctional epidermolysis bullosa using polymerase chain reaction amplification of genomic DNA, followed by heteroduplex analysis and direct automated nucleotide sequencing. Both patients were found to be compound heterozygotes for the same nonsense mutation on one LAMB3 allele, and different missense mutations on the other LAMB3 allele. The combination of nonsense and a missense mutation in the LAMB3 gene appears to be important in determining the milder clinical phenotype in some cases of the nonlethal forms of junctional epidermolysis bullosa involving abnormalities in laminin 5.

Aged↗

PubMed1996

First trimester DNA-based exclusion of recessive dystrophic epidermolysis bullosa from chorionic villus sampling.

A 28-year-old woman, who previously had had a child affected with the hereditary blistering skin disorder, recessive dystrophic epidermolysis bullosa, presented at 7 weeks' gestation for prenatal diagnosis. Genomic DNA, obtained from her, her husband (who is a first cousin), their unaffected child, and their previously affected child, was used to screen all 118 exons of the type VII collagen gene (COL7A1) by polymerase chain reaction (PCR) amplification followed by heteroduplex analysis of the PCR products. Established common polymorphisms within the NC-1 region of COL7A1 were informative for both the normal maternal and paternal alleles. In addition, a putative homozygous mutation, a G to C transversion at nucleotide position 7708, was identified in the affected child. This substitution converts a glycine residue (GGT) within the Gly-X-Y region of the type VII collagen triple helix into an arginine residue (CGT), and leads to the creation of a new MnlI restriction site. Both parents and the healthy sibling were shown to be clinically normal heterozygous carriers of this mutation. A chorionic villus biopsy was performed at 10 weeks' gestation and DNA was extracted from the villi. Assessment of informative intragenic markers, and the putative mutation, revealed that the fetus had inherited both the normal maternal and paternal COL7A1 alleles. Thus, first trimester DNA-based prenatal diagnosis predicts that this child is neither affected with recessive dystrophic epidermolysis bullosa, nor is an unaffected carrier of this genodermatosis.

Base Sequence↗

PubMed1996

Immunoelectron microscopic analysis of cornified cell envelope formation in normal and psoriatic epidermis.

The cornified cell envelope (CE) is an insoluble, highly resistant structure formed beneath the plasma membrane of differentiating keratinocytes. It consists of various crosslinked precursor proteins, including involucrin and loricrin. However, neither the normal assembly process of CE nor its alteration in skin disorders has been fully characterized. In this study we analyzed CE formation in normal skin and in lesional psoriatic skin by immunoelectron microscopy. In the superficial granular cells of normal epidermis, involucrin labeling was detected along the plasma membrane, whereas loricrin staining was mostly distributed diffusely within the cells, occasionally with some additional granular aggregates within the cytoplasm and nucleus. Loricrin was also present predominantly on the desmosomal attachment plaques, colocalizing with desmoglein. In the cornified cells, involucrin labeling was reduced, whereas loricrin labeling was retained and continuously decorated the CEs, with relative sparing of the desmosomal areas. In typical psoriatic epidermis, involucrin staining was very intense but loricrin labeling was markedly reduced. The involucrin-positive CEs were formed precociously and further maturation of CE did not occur. These results suggest that formation of CE occurs sequentially, initially involving involucrin deposition and subsequently involving loricrin incorporation. Psoriatic epidermis demonstrates a lack of proper CE maturation.

Aged↗

PubMed1996

Compound heterozygosity for a dominant glycine substitution and a recessive internal duplication mutation in the type XVII collagen gene results in junctional epidermolysis bullosa and abnormal dentition.

Junctional epidermolysis bullosa is a heterogeneous autosomal recessively inherited blistering skin disorder associated with fragility at the dermal-epidermal junction. Previously, mutations in this condition have been described in the three genes for the anchoring filament protein laminin 5 (LAMA3, LAMB3, and LAMC2), in the gene encoding the hemidesmosome-associated beta4 integrin (ITGB4), and in the gene for the hemidesmosomal protein type XVII collagen (COL17A1/BPAG2). In this study, we report a patient with a form of junctional epidermolysis bullosa with skin fragility and dental anomalies who is a compound heterozygote for a novel combination of mutations, ie, a glycine substitution mutation in one allele and an internal duplication in the other allele of COL17A1. The patient also has two offspring, both of whom have inherited the glycine substitution mutation, whereas the other COL17A1 allele is normal. The latter individuals show no evidence of skin fragility but have marked dental abnormalities with enamel hypoplasia and pitting. The clinical phenotype of junctional epidermolysis bullosa in the proband in this family probably arises due to a combination of the glycine substitution and the internal duplication in COL17A1, whereas the dental abnormalities of her offspring may be the result of the glycine substitution in COL17A1 alone, resulting in this dominantly inherited clinical phenotype.

Base Sequence↗

PubMed1995

A homozygous nonsense mutation in the alpha 3 chain gene of laminin 5 (LAMA3) in Herlitz junctional epidermolysis bullosa: prenatal exclusion in a fetus at risk.

Mutations in the three genes (LAMA3, LAMB3, and LAMC2) that encode the three chains (alpha 3, beta 3, and gamma 2, respectively) of laminin 5, a protein involved in epidermal-dermal adhesion, have been established as the genetic basis for the inherited blistering skin disorder, Herlitz junctional epidermolysis bullosa (H-JEB). In this study, we performed mutational analysis on genomic DNA from a child with H-JEB and identified a nonsense mutation in the alpha 3 chain gene (LAMA3) consisting of a homozygous C-to-T transition resulting in a premature termination codon (CGA-->TGA) on both alleles. The parents were shown to be heterozygous carriers of the same mutation. Direct mutation analysis was used to perform DNA-based prenatal diagnosis from a chorionic villus biopsy at 10 weeks' gestation in a subsequent pregnancy. The fetus was predicted to be genotypically normal with respect to the LAMA3 mutation.

Base Sequence↗

PubMed1995

Genetic basis of lethal junctional epidermolysis bullosa in an affected fetus: implications for prenatal diagnosis in one family.

Fetal skin biopsy at 20 weeks' gestation in a woman at risk for a child with the lethal skin-blistering disorder junctional epidermolysis bullosa (Herlitz) confirmed an affected fetus. Genomic DNA from the aborted fetus was examined for mutations in laminin 5, a macromolecule involved in adhesion at the dermal-epidermal junction, and a candidate protein in this condition. Polymerase chain reaction (PCR) amplification of exon 10 and parts of the flanking introns of the gene encoding the beta 3 chain of laminin 5 (LAMB3) and subsequent analysis by agarose gel electrophoresis showed a more slowly migrating band in the affected fetus compared with the normal control. Nucleotide sequencing of the abnormal PCR product revealed a homozygous 77 bp duplication within the exon, resulting in a premature termination codon 250 bp downstream from the 3' end of the duplication. Maternal DNA was heterozygous for the mutant and wild-type alleles. These findings illustrate the genetic basis of the skin disease in this case and also offer the prospects of a simple, rapid, and reliable first-trimester DNA-based prenatal, or even preimplantation, diagnostic test for future pregnancies in this family.

Adult↗

PubMed1995

Altered distribution of keratinization markers in epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis (EH) is a genetic disorder of keratins associated with epidermal differentiation. Affected individuals carry gene mutations for conserved sequences of keratins K1 or K10. The structural alterations of tonofilaments in EH seem to be a direct consequence of the keratin gene mutations. EH epidermis, however, shows many other unexplained abnormalities including acanthosis, hypergranulosis, and hyperkeratosis. To further elucidate the pathogenetic mechanism of EH, we studied distribution patterns of other keratinization-associated molecules including involucrin, small proline-rich protein (SPRR) 1, loricrin and trichohyalin in the skin of four patients by light and electron microscopic immunohistochemistry in conjunction with conventional transmission electron microscopy. The middle to upper epidermal cells showed moderate to strong immunoreactivities to involucrin, SPRR1 and loricrin antibodies. Both intracellular staining and cell peripheral staining was seen for involucrin and SPRR1 antibodies. Loricrin labelling was prematurely associated with the plasma membrane of granular cells, possibly relating to abnormal keratin filament aggregation and cellular vacuolization. Some loricrin labelling was localized on the keratin aggregates, suggesting intermolecular associations between keratin and loricrin. Trichohyalin, hardly detectable in normal epidermis, was present in some granular and cornified cells in EH in association with keratin filaments, suggesting that it may function as an intermediate filament-associated protein. While cornified cell envelopes were intensely labelled only with loricrin antibodies in normal skin, they were immunoreactive to involucrin, SPRR1 and loricrin antibodies in EH. Sequential change in electron density of the cornified cell envelopes, a constant feature in normal skin, was often absent in EH. These results suggest an altered assembly process of cornified cell envelopes in EH.

Adolescent↗

PubMed1995

Keratin 16 and keratin 17 mutations cause pachyonychia congenita.

Pachyonychia congenita (PC) is a group of autosomal dominant disorders characterized by dystrophic nails and other ectodermal aberrations. A gene for Jackson-Lawler PC was recently mapped to the type I keratin cluster on 17q. Here, we show that a heterozygous missense mutation in the helix initiation motif of K17 (Asn92Asp) co-segregates with the disease in this kindred. We also show that Jadassohn-Lewandowsky PC is caused by a heterozygous missense mutation in the helix initiation peptide of K16 (Leu130Pro). The known expression patterns of these keratins in epidermal structures correlates with the specific abnormalities observed in each form of PC.

Amino Acid Sequence↗

PubMed1995

Mutations in the 180-kD bullous pemphigoid antigen (BPAG2), a hemidesmosomal transmembrane collagen (COL17A1), in generalized atrophic benign epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a heterogeneous autosomal recessively inherited blistering skin disorder associated with fragility at the dermal-epidermal junction. Characteristic ultrastructural findings in JEB are abnormalities in the hemidesmosome-anchoring filament complexes. These focal attachment structures, which extend from the intracellular compartment of the basal keratinocytes to the underlying basement membrane, have been shown to be hypoplastic or rudimentary in different forms of JEB. Previously, in different JEB phenotypes, mutations have been found in the three genes for the anchoring filament component laminin 5 (LAMA3, LAMB3, and LAMC2) and in the gene for the hemidesmosome-associated integrin beta 4 subunit. Here, we describe the first mutations in the gene encoding the 180-kD bullous pemphigoid antigen (BPAG2), a transmembranous hemidesmosomal collagen, also known as type XVII collagen (COL17A1). The patient is affected with generalized atrophic benign epidermolysis bullosa (GABEB), a rare variant of JEB, and is a compound heterozygote for premature termination codons on both alleles. These novel findings emphasize the molecular heterogeneity of this group of genodermatoses, and attest to the importance of BPAG2 in maintaining adhesion between the epidermis and the dermis.

Adolescent↗

PubMed1995

A homozygous nonsense mutation in the alpha 3 chain gene of laminin 5 (LAMA3) in lethal (Herlitz) junctional epidermolysis bullosa.

The inherited mechanobullous disorder, junctional epidermolysis bullosa (JEB), is characterized by extensive blistering and erosions of the skin and mucous membranes. The diagnostic hallmarks of JEB include ultrastructural abnormalities in the hemidesmosomes of the cutaneous basement membrane zone, as well as an absence of staining with antibodies against the anchoring filament protein, laminin 5. Therefore, the three genes encoding alpha 3, beta 3 and gamma 2 chains of laminin 5, known as LAMA3, LAMB3 and LAMC2, are candidate genes for JEB. We have previously demonstrated mutations in the LAMB3 and LAMC2 genes in several families with JEB. We initiated mutation analysis from an affected child by PCR amplification of individual LAMA3 exons, followed by heteroduplex analysis. Nucleotide sequencing of heteroduplexes identified a homozygous nonsense mutation within domain I/II of the alpha 3 chain. These findings provide the first evidence that nonsense mutations within the LAMA3 gene are also involved in the pathogenesis of JEB, and indicate that mutations of all three genes of laminin 5 can result in the JEB phenotype.

Base Sequence↗