Biomedical subjects

R A Eady

Publications and source records attributed to R A Eady.

At least 55 records · Page 3Linked to original sources

PubMed1998

Frameshift mutations in the type VII collagen gene (COL7A1) in five Mexican cousins with recessive dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the type VII collagen gene (COL7A1). In this study, we assessed the molecular basis of recessive DEB in five affected individuals from two Mexican families. Both fathers of the affected children were first cousins. Genomic DNA was extracted from peripheral blood samples and assessed for COL7A1 mutations by polymerase chain reaction (PCR) amplification, heteroduplex analysis and direct automated sequencing of PCR products displaying heteroduplex bandshifts. In one family, we identified a homozygous 1 bp insertion of a G nucleotide in exon 19 of COL7A1, designated 2470insG, in three affected sisters. This mutation causes a frameshift and a premature termination codon on both alleles 178 bp downstream from the insertion; both parents were shown to be heterozygous carriers of this mutation. In the second family, the father of the other two affected children was also found to be a heterozygous carrier of this frameshift mutation. In addition, his unrelated partner was shown to be a heterozygous carrier of a different COL7A1 frameshift mutation, an insertion of a T nucleotide in exon 32, designated 3948insT. This mutation also results in a premature termination codon, 126 bp downstream from the insertion. Both affected children were compound heterozygotes for the 2470insG/3948insT mutations in COL7A1. Overall, these molecular findings offer a genetic explanation for the skin fragility in these related Mexican patients with recessive DEB. Immediate benefits from elucidation of the mutations include assessment of carrier status in other members of the family and the feasibility of DNA-based prenatal testing in subsequent pregnancies.

Adolescent↗

PubMed1998

E210K mutation in the gene encoding the beta3 chain of laminin-5 (LAMB3) is predictive of a phenotype of generalized atrophic benign epidermolysis bullosa.

Pathogenetic mutations in the genes encoding the hemidesmosome-anchoring filament complex proteins, laminin-5 and the 180 kDa bullous pemphigoid antigen, have been identified in patients with the inherited mechanobullous disease, junctional epidermolysis bullosa (EB). Furthermore, there is some evidence to suggest that precise definition of the nature of mutations in these genes may correlate to specific phenotypes of disease. We report three junctional EB patients who carry an identical missense mutation, E210K, on one allele of the gene encoding the beta3 subunit chain of laminin-5 (LAMB3) in addition to different nonsense mutations on the second allele. Two of the patients are adults and display a specific phenotype of non-lethal junctional EB known as generalized atrophic benign EB, which is associated with trauma-induced blisters, nail dystrophy and alopecia. As the third patient is a young child with fewer features of this subtype to date, identification of E210K in combination with a nonsense LAMB3 mutation may be predictive of the subsequent development of a generalized atrophic benign EB phenotype both in this child and in other junctional EB patients with the E210K mutation. Identification of this particular mutation has important implications for clinical management and counselling.

Adult↗

PubMed1998

A recurrent glycine substitution mutation, G2043R, in the type VII collagen gene (COL7A1) in dominant dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the type VII collagen gene (COL7A1). Nearly all cases of dominant DEB are caused by glycine substitution mutations occurring within the triple helical region of type VII collagen, and most of the mutations are unique to individual families. In this study, we identified a patient of Hispanic-Mexican origin with a mild form of DEB, which resulted from a de novo dominant glycine substitution, G2043R, in exon 73 of COL7A1. We also investigated a Scottish family with a three-generation pedigree of dominant DEB, in whom the same glycine to arginine substitution mutation was demonstrated. This particular mutation has also been detected previously in three other families with dominant DEB: one Italian, one Hungarian and one Norwegian. Given the widespread geographical distribution of this mutation and the demonstration of its occurrence as a de novo event, G2043R therefore represents the first example of a mutational hotspot in dominant DEB. Interestingly, although both the Mexican and Scottish families we studied had some clinical features in keeping with the Pasini form of the disorder, there was considerable interfamilial variability as well as intrafamilial diversity in the affected individuals.

Adult↗

PubMed1998

Specialized keratin expression pattern in human ridged skin as an adaptation to high physical stress.

We have analysed the expression of keratins in the epidermis of normal human palm and sole skin (ridged skin) using immunohistochemistry and in situ hybridization. The epidermis of human ridged skin expresses a more complex pattern of keratins than thin skin, which is probably due to the greater stress that ridged skin has to withstand. In addition to keratin K9, we document specific expression patterns of keratins K6, K16 and K17 which are suggestive of regional adaptations of this epidermis to a high cell turnover rate. In particular, the sequestered location of nests of K17-positive cells at the bottom of the deep primary epidermal ridges supports the notion of functional heterogeneity of basal cells and suggests that the K17-positive sites may include stem cells. Expression of K6 and K16 in some basal and most suprabasal keratinocytes is compatible with a constitutively high proliferative activity of normal ridged epidermis, but may also reflect different physical properties of the suprabasal cells, in contrast with regions expressing K9. The distinct labelling patterns observed in primary and secondary epidermal ridges as well as epidermal layers above dermal papillae suggest the existence of local microenvironmental niches leading to differences in keratinocyte differentiation.

Adaptation, Physiological↗

PubMed1998

Pyloric atresia-junctional epidermolysis bullosa syndrome: mutations in the integrin beta4 gene (ITGB4) in two unrelated patients with mild disease.

Junctional epidermolysis bullosa associated with pyloric atresia (EB-PA; OMIM 226730) is a rare autosomal recessively inherited disease in which mucocutaneous fragility is associated with gastrointestinal atresia. This disease is usually fatal within the first few weeks or months of life even following surgical correction of the intestinal obstruction. Recently, mutations in the genes encoding the epithelial integrin alpha6beta4 (ITGA6 and ITGB4) have been identified in several patients with EB-PA. We report two unrelated patients with this disease who have survived into early childhood with mild cutaneous involvement, in whom we have identified pathogenetic mutations in ITGB4. The first patient was a compound heterozygote for a splice site mutation in exon 30 (3793 + 1G-to-A) and a non-sense mutation in exon 36 (W1478X), and the second was a compound heterozygote for a missense mutation in exon 3 (C38R) and a 1 bp deletion in exon 36 (4776delG). Although the non-sense and deletion mutations are predicted to result in markedly reduced beta4 integrin mRNA levels, the presence of the missense or splice site mutation on the second allele may enable the synthesis of some functional, albeit perturbed, beta4 polypeptide. Determination of the molecular mechanisms in these two cases increases our understanding of EB-PA and may enable correlation between genotype and phenotype.

Antigens, CD↗

PubMed1998

Focal dermal hypoplasia: report of a case with cutaneous and skeletal manifestations.

Focal dermal hypoplasia is a rare genodermatosis characterized by developmental defects of the skin, resulting in widespread linear lesions of dermal hypoplasia with adipose tissue in the dermis. We describe a 13-year-old girl who has typical cutaneous lesions which have been present since birth; she also has some of the associated dental, nail and skeletal abnormalities, while an X-ray of the long bones osteopathia striata is visible, a feature seen in a high proportion of cases of focal dermal hypoplasia. Eighty-eight per cent of the case reports in the literature are of females and X-linked dominance is the likely mode of inheritance. It has also been proposed that the condition is lethal in homozygous males and the high frequency of miscarriages on the maternal side of this patient's family is consistent with that lethality in males. The literature, particularly with regard to pathogenesis and inheritance, is discussed.

Adipose Tissue↗

PubMed1998

Hemidesmosomes show abnormal association with the keratin filament network in junctional forms of epidermolysis bullosa.

Junctional epidermolysis bullosa is a group of hereditary bullous disorders resulting from defects in several hemidesmosome-anchoring filament components. Because hemidesmosomes are involved not only in keratinocyte-extracellular matrix adherence, but also in normal anchorage of keratin intermediate filaments to the basal keratinocyte membrane, we questioned whether this intracellular function of hemidesmosomes was also perturbed in junctional epidermolysis bullosa. We used quantitative electron microscopic methods to assess certain morphologic features of hemidesmosome-keratin intermediate filaments interactions in skin from normal subjects (n = 11) and from patients with different forms of junctional epidermolysis bullosa (n = 13). In addition, skin from patients with autosomal recessive epidermolysis bullosa simplex with plectin defects (n = 3) or with autosomal recessive dystrophic epidermolysis bullosa (n = 4) were included as controls. Values were expressed as a percentage of the total number of hemidesmosomes counted. In normal skin 83.3% +/- 3.3 (SEM) hemidesmosomes were associated with keratin intermediate filaments and 90.1% +/- 1.9 had inner plaques. In Herlitz junctional epidermolysis bullosa (laminin 5 abnormalities, n = 4) these values were reduced to 45.3% +/- 11.5 (p < 0.001; analysis of variance) and 50.3% +/- 12.8 (p < 0.001), respectively. In junctional epidermolysis bullosa with pyloric atresia (alpha6beta4 abnormalities, n = 3) the values were also reduced [41.8% +/- 7.0 (p < 0.001) and 44.5% +/- 5.7 (p < 0.001), respectively]. In the non-Herlitz group (laminin 5 mutations, n = 3) the counts were 66.7% +/- 7.1 (p > 0.05) and 70.5% +/- 8.5 (p < 0.05), and in skin from patients with bullous pemphigoid antigen 2 mutations (n = 3) the counts were 54.3% +/- 13.8 (p < 0.01) and 57.1% +/- 13.9 (p < 0.01). In epidermolysis bullosa simplex associated with plectin mutations the values were 31.9% +/- 8.9 (p < 0.001) for keratin intermediate filaments association and 39.9% +/- 7.1 (p < 0.001) for inner plaques. Findings in recessive dystrophic epidermolysis bullosa patients' skin were indistinguishable from normal control skin with inner plaques (90.5% +/- 2.5) and keratin intermediate filaments attachment (86.3% +/- 2.1). These findings suggest that the molecular abnormalities underlying different forms of junctional epidermolysis bullosa appear to affect certain critical intracellular functions of hemidesmosomes, such as the normal connections with keratin intermediate filaments. This may have important implications for the maintenance of basal keratinocyte integrity and resilience in junctional epidermolysis bullosa.

Adolescent↗

PubMed1998

Severe palmo-plantar hyperkeratosis in Dowling-Meara epidermolysis bullosa simplex caused by a mutation in the keratin 14 gene (KRT14).

Mutant keratins 5 or 14 are implicated in the etiology of epidermolysis bullosa simplex (EBS). The catalog of mutations has established certain patterns of mutation clusters from which it may be possible, along with associated biochemical data, to predict phenotypic severity. It is becoming apparent that some of these assumptions may now require modification. We report a mutation in the gene encoding keratin 14 (KRT14) that changes the predicted amino acid at position 119, at the start of the helix initiation motif, from methionine to threonine (K14 M119T) in a patient with an EBS Dowling-Meara phenotype with severe palmo-plantar hyperkeratosis. This demonstrates that the three major types of EBS can arise from missense mutations in the same codon. The findings suggest that the specific nature of the missense mutation, in the context of the protein sequence, can contribute far more to the clinical severity than previously thought. The different EBS subtypes should be viewed as gradations of clinical severity rather than distinct genetic diseases.

Adult↗

PubMed1998

Molecular basis of blistering skin diseases.

Structural molecules in the skin may serve as candidate proteins or target antigens in both inherited and acquired blistering skin diseases. Molecular characterization of the proteins and genes that encode them has led to an improved understanding of the mechanisms of blistering in the skin, and has also allowed the development of new approaches to diagnosis and treatment.

Autoantibodies↗

PubMed1997

Heparan sulphate proteoglycan and wound healing in skin.

Normal wound healing in skin embraces several reparative processes, many of which directly involve components of the extracellular matrix and the cutaneous basement membrane zone. Proteoglycans are a group of extracellular matrix macromolecules that have both structural and regulatory properties. In wound healing, certain proteoglycans fulfill a mechanical function of absorbing water and preventing tissue compression. However, proteoglycans may also have other roles in wound healing including a direct influence on inflammation, cell attachment and migration, and growth factor binding. Furthermore, proteoglycans may help to determine other aspects of the long-term quality of wound healing in skin through regulation of basement membrane permeability, epidermal hyperproliferation, and dermal fibrosis.

Basement Membrane↗

PubMed1997

The role of immunohistochemistry in the diagnosis of the non-lethal forms of junctional epidermolysis bullosa.

Skin biopsies from four patients with similar clinical phenotypes of non-lethal junctional epidermolysis bullosa (JEB) were investigated by electron microscopy and immunohistochemistry. Electron microscopy revealed cleavage within the lamina lucida in each case, but in addition, there was considerable variability in the number and ultrastructure of hemidesmosome-anchoring filament complexes. Immunohistochemical staining of the dermal-epidermal junction, using monoclonal antibodies to laminin 5 and the 180-kDa bullous pemphigoid antigen, showed reduced laminin 5 staining with normal 180-kDa bullous pemphigoid antigen labelling in two cases, and reduced or absent 180-kDa bullous pemphigoid antigen staining with normal laminin 5 labelling in the other two cases. Whereas electron microscopy is invaluable for the diagnosis of JEB as a group of diseases, immunohistochemistry is able to provide insight into the molecular pathology of non-lethal JEB and reveal abnormalities in candidate genes and proteins underlying this group of genetic blistering disorders.

Adolescent↗

PubMed1997

Mutations in the plakophilin 1 gene result in ectodermal dysplasia/skin fragility syndrome.

Members of the armadillo protein gene family, which includes plakoglobin and beta-catenin, have important functions in cytoskeleton/cell membrane interactions. These proteins may act as linker molecules at adherens junctions and desmosomes at the plasma membrane; in addition, they may have pivotal roles in signal transduction pathways and significant effects on cell behaviour during development. Here, we describe the first human mutations in one of these dual function proteins, plakophilin 1 (band-6 protein; refs 8-10). The affected individual has a complete absence of immunostaining for plakophilin 1 in the skin and is a compound heterozygote for autosomal-recessively inherited premature termination codons of translation on both alleles of the plakophilin 1 gene (PKP1). Clinically, there are features of both cutaneous fragility and congenital ectodermal dysplasia affecting skin, hair and nails. There is no evidence of significant abnormalities in other epithelia or tissues. Desmosomes in the skin are small and poorly formed with widening of keratinocyte intercellular spaces and perturbed desmosome/keratin intermediate filament interactions. The molecular findings and clinical observations in this patient attest to the dual importance of plakophilin 1 in both cutaneous cell-call adhesion and epidermal morphogenesis.

Base Sequence↗

PubMed1997

Bullous pemphigoid and cicatricial pemphigoid autoantibodies react with ultrastructurally separable epitopes on the BP180 ectodomain: evidence that BP180 spans the lamina lucida.

The BP180 antigen is a hemidesmosomal glycoprotein that is recognized by autoantibodies associated with three autoimmune disorders, bullous pemphigoid (BP), herpes gestationis (HG), and cicatricial pemphigoid (CP). BP and HG sera have been shown to recognize a common extracellular site located near the membrane-spanning domain of this protein, whereas CP sera react predominantly with a distinct site near the C terminus. In the current study, the main immunogenic sites on the BP180 ectodomain were ultrastructurally localized using six BP sera, four CP sera, and two rabbit antisera. The immunolocalization pattern of BP sera was largely restricted to the upper lamina lucida region immediately subjacent to the epidermal hemidesmosome and closely resembled that of a rabbit antiserum directed against the NC16A (membrane-proximal) domain of BP180. CP sera, on the other hand, exhibited a lower lamina lucida/lamina densa labeling pattern that was strikingly similar to that of rabbit antibodies to the BP180 C-terminal region. Finally, antibodies to the BP180 C-terminal region co-localized with an anti-laminin-5 antibody in the anchoring filament zone. These findings strongly suggest that the BP180 extracellular domain exists in an extended conformation, with the C terminus of this protein projecting into the lamina densa. These data support the hypothesis that BP180 contributes to the structure and function of the anchoring filaments. Differences in the ultrastructural mapping of BP and CP autoantibodies appear to correlate with epitope mapping data, which, together, may help to explain the clinical heterogeneity observed in this group of bullous disorders.

Actin Cytoskeleton↗

PubMed1997

Recurrent mutations in the type VII collagen gene (COL7A1) in patients with recessive dystrophic epidermolysis bullosa.

Mutations in the type VII collagen gene (COL7A1) are known to underlie different forms of the inherited blistering skin disease dystrophic epidermolysis bullosa (DEB). Most COL7A1 mutations are unique to individual families, and therefore it is usually necessary to screen all 118 exons of the gene to determine the molecular pathology in a patient with DEB. This study aimed to identify any recurrent mutations in COL7A1 that might be applicable to mutation-detection strategies in these patients. Mutational analysis was undertaken in 23 British patients with autosomal recessive DEB using PCR amplification of genomic DNA followed by heteroduplex analysis, nucleotide sequencing, and restriction site analysis. Two recurrent mutations were identified: R578X (6 of 46 alleles) and 7786delG (7 of 46 alleles). Haplotype analysis revealed that the mutations existed on similar allelic backgrounds in different patients, consistent with propagation of common British ancestral haplotypes, although R578X and 7786delG also have been described in DEB patients from other ethnic backgrounds. Given the high relative frequency of these two COL7A1 mutations, British patients with recessive DEB should be screened initially for these nucleotide changes by PCR amplification of genomic DNA and restriction analysis before more exhaustive screening of COL7A1.

Alleles↗

PubMed1997

Loricrin mutation in Vohwinkel's keratoderma is unique to the variant with ichthyosis.

A mutation in the glycine-rich cornified envelope protein loricrin has recently been reported in Vohwinkel's keratoderma (honeycomb keratoderma with pseudoainhum), in a pedigree amongst whom ichthyosis was also a feature. We have studied two further families with Vohwinkel's keratoderma for evidence ofloricrin mutations. Our first family (VK1) also had ichthyosis but not deafness. In lesional and nonlesional skin, granular and transitional cell layers were increased. In immunoelectron-microscopic studies cornified envelopes were abnormally thin and were labeled densely by anti-involucrin antibodies, but only sparsely by antiloricrin antibodies; however, abnormal intranuclear granules seen in granular and cornified layer cells were labeled by antibodies to both C- and N-terminal loricrin. Microsatellite markers in VK1 supported linkage to the loricrin locus in the epidermal differentiation complex at 1q21 (Zmax = 2.48). The loricrin gene was sequenced, identifying a heterozygous mutation as previously reported: a G insertion producing a frameshift after codon 231 and an abnormal C-terminal peptide lacking residues necessary for cross-linking. In our second family (VK2), affected members had sensorineural deafness but not ichthyosis. Immunoelectron-microscopic studies showed normal loricrin distribution, and assuming complete penetrance, linkage to 1q21 was excluded. Vohwinkel's keratoderma is thus clinically and genetically heterogeneous. Only the variant with ichthyosis appears to be due to loricrin mutation. As the arginine-rich domain in C-terminal loricrin caused by the frameshift contains several potential bipartite nuclear localization signals, we suggest that the intranuclear accumulation of loricrin in VK1 is due to these motifs, and may be unique to insertional mutation.

Adolescent↗

PubMed1997

The prevalence of epidermolysis bullosa in Scotland.

The prevalence of epidermolysis bullosa (EB) in Britain and most other countries is unknown. Patients suffering from the inherited forms of EB and living in Scotland have been traced. Two hundred and fifty-nine affected people from 76 families have been identified, of whom 211 were clinically assessed. One-third of these Scottish EB sufferers had never been seen by a dermatologist. In Lothian, where there appears to be a relatively high prevalence of EB, 75% of patients were unknown to their general practitioners. The point prevalence of all forms of EB at the outset of the study was 49.0 per million, comprising EB simplex 28.6 per million and dystrophic EB 20.4 per million. Extrapolation of accurate data available for the Lothians suggests that the point prevalence of all forms of EB in Scotland is in excess of these figures.

Adolescent↗

PubMed1997

A recurrent laminin 5 mutation in British patients with lethal (Herlitz) junctional epidermolysis bullosa: evidence for a mutational hotspot rather than propagation of an ancestral allele.

The three genes (LAMA3, LAB3 and LAMC2) that encode the anchoring filament protein, laminin 5, may all harbour pathogenetic mutations in the autosomal recessive blistering skin disorder, junctional epidermolysis bullosa (JEB). Recently, one particular mutation, R635X in the LAMB3 gene, has been found to account for approximately 40% of all JEB laminin 5 mutations (Kivirikko et al., Hum Mol Genet 1996; 5: 231-7). In this study, we assessed the frequency of this mutation in 12 British patients with lethal (Herlitz) JEB using PCR amplification of genomic DNA and restriction endonuclease digestion. The mutation R635X was fond in seven of 24 (29%) mutant alleles, confirming its relative frequency within the British gene pool. In addition, haplotype analysis using intragenic polymorphisms showed that the mutation arose on at least four different haplotype backgrounds, suggesting it represents a mutational hotspot rather than propagation of a common British ancestral allele. These findings support the hypermutable nature of this CpG dinucleotide and have implications in screening for laminin 5 gene mutations in British and other patients with JEB.

Alleles↗

PubMed1997

Immunohistochemical analysis of the skin in junctional epidermolysis bullosa using laminin 5 chain specific antibodies is of limited value in predicting the underlying gene mutation.

The anchoring filament protein laminin 5 is composed of three polypeptide chains (alpha 3, beta 3 and gamma 2) each encoded by separate genes (LAMA3, LAMB3 and LAMC2, respectively). Mutations in any of these three genes may give rise to the autosomal recessive blistering skin disease, junctional epidermolysis bullosa. At present, there is no easy way of predicting which of these three genes might harbour the pathogenetic laminin 5 mutations in a case of junctional epidermolysis bullosa. In this study, we assessed whether immunohistochemistry might be helpful in this regard. We performed immunohistochemical labelling of the dermal-epidermal junction using alpha 3, beta 3 and gamma 2 chain-specific antibodies in 11 patients with junctional epidermolysis bullosa, in whom the laminin 5 mutations had been previously delineated. Although, labelling for the laminin 5 chain bearing the mutations was attenuated or undetectable in all cases, a complete absence of labelling or a reduction in the staining intensity for the other two chains was also seen in all cases. The results showed that immunohistochemical labelling of the dermal-epidermal junction using alpha 3, beta 3 and gamma 2 chain-specific antibodies is not a specific indicator for which of the laminin 5 chain genes contains the pathogenetic mutations, and is therefore unreliable in screening for individual laminin 5 gene mutations in cases of junctional epidermolysis bullosa.

Adolescent↗