[The genetics of inherited bullous diseases of the skin. Future aspects in perinatal and prenatal diagnosis].
Explore the source record for details and available documents.
Cloning, Molecular↗
Biomedical subjects
J Uitto
Publications and source records attributed to J Uitto.
Glycine substitutions in the triple-helical region of type VII collagen result in a spectrum of dystrophic epidermolysis bullosa phenotypes and patterns of inheritance.
The dystrophic forms of epidermolysis bullosa (DEB) are characterized by fragility of the skin and mucous membranes. DEB can be inherited in either an autosomal dominant or autosomal recessive pattern, and the spectrum of clinical severity is highly variable. The unifying diagnostic hallmark of DEB is abnormalities in the anchoring fibrils, which consist of type VII collagen, and, recently, mutations in the corresponding gene, COL7A1, have been disclosed in a number of families. In this study, we report six families with glycine substitution mutations in the triple-helical region of type VII collagen. Among the six families, two demonstrated a mild phenotype, and the inheritance of the mutation was consistent with the dominantly inherited form of DEB. In the four other families, the mutation was silent in the heterozygous state but, when present in the homozygous state, or combined with a second mutation, resulted in a recessively inherited DEB phenotype. Type VII collagen is, therefore, unique among the collagen genes, in that different glycine substitutions can be either silent in heterozygous individuals or result in a dominantly inherited DEB. Inspection of the locations of the glycine substitutions along the COL7A1 polypeptide suggests that the consequences of these mutations, in terms of phenotype and pattern of inheritance, are position independent.
Adult↗
Compound heterozygosity for COL7A1 mutations in twins with dystrophic epidermolysis bullosa: a recessive paternal deletion/insertion mutation and a dominant negative maternal glycine substitution result in a severe phenotype.
We have previously demonstrated genetic linkage between the type VII collagen gene (COL7A1) and the dominant (DDEB) and recessive (RDEB) forms of dystrophic epidermolysis bullosa (DEB) and have subsequently identified pathogenetic mutations in several families. Mutations in DDEB identified thus far are glycine substitutions in the collagenous domain of COL7A1, while the most severe forms of RDEB result from premature termination codon (PTC) mutations on both alleles. In this study, we performed mutation analysis in the COL7A1 gene in twins who displayed a severe DEB phenotype. Mutational analysis revealed a paternal 2-bp deletion/1-bp insertion in exon 56, designated 5103CC-->G, which results in a frameshift and downstream PTC. Analysis of the maternal COL7A1 allele revealed a glycine-to-arginine substitution in exon 91 (G2351R). Careful questioning of the mother revealed that she and her father had a history of shedding of toenails and occasional poorly healing erosions, consistent with a mild form of DDEB. Immunoprecipitation of type VII collagen from fibroblasts of the twins revealed a marked reduction in intracellular protein production, consistent with the drastic reduction in mRNA transcript from the paternal mutant allele, while the majority of polypeptides bearing the glycine substitution appeared to be degraded intracellularly. Thus, the severe RDEB phenotype in the probands results from compound heterozygosity for one glycine substitution and one PTC mutation in COL7A1.
Base Sequence↗
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↗
Molecular diagnosis of inherited skin diseases: the paradigm of dystrophic epidermolysis bullosa.
Explore the source record for details and available documents.
Diagnosis, Differential↗
Prenatal diagnosis for recessive dystrophic epidermolysis bullosa in 10 families by mutation and haplotype analysis in the type VII collagen gene (COL7A1).
BACKGROUND: Epidermolysis bullosa (EB) is a group of heritable diseases that manifest as blistering and erosions of the skin and mucous membranes. In the dystrophic forms of EB (DEB), the diagnostic hallmark is abnormalities in the anchoring fibrils, attachment structures beneath the cutaneous basement membrane zone. The major component of anchoring fibrils is type VII collagen, and DEB has been linked to the type VII collagen gene (COL7A1) at 3p21, with no evidence for locus heterogeneity. Due to life-threatening complications and significant long-term morbidity associated with the severe, mutilating form of recessive dystrophic EB (RDEB), there has been a demand for prenatal diagnosis from families with affected offspring. MATERIALS AND METHODS: Intragenic polymorphisms in COL7A1 and flanking microsatellite markers on chromosome 3p21, as well as detection of pathogenetic mutations in families, were used to perform PCR-based prenatal diagnosis from DNA obtained by chorionic villus sampling at 10-15 weeks or amniocentesis at 12-15 weeks gestation in 10 families at risk for recurrence of RDEB. RESULTS: In nine cases, the fetus was predicted to be normal or a clinically unaffected carrier of a mutation in one allele. These predictions have been validated in nine cases by the birth of a healthy child. In one case, an affected fetus was predicted, and the diagnosis was confirmed by fetal skin biopsy. CONCLUSIONS: DNA-based prenatal diagnosis of RDEB offers an early, expedient method of testing which will largely replace the previously available invasive fetal skin biopsy at 18-20 weeks gestation.
Amniocentesis↗
Cloning of the laminin alpha 3 chain gene (LAMA3) and identification of a homozygous deletion in a patient with Herlitz junctional epidermolysis bullosa.
Laminin 5 and laminin 6 are basement membrane proteins synthesized by the basal cells of stratifying squamous epithelia. Altered expression of laminin 5 has been associated with Herlitz junctional epidermolysis bullosa (H-JEB), a severe epidermal blistering disorder inherited as an autosomal recessive disease. We have isolated cDNA clones encoding the alpha 3 chain of laminin 5 and searched for mutations in the LAMA3 gene in H-JEB patients. In one H-JEB family, an affected individual exhibited drastically reduced immunoreactivity to antibodies directed against the alpha 3 chain of laminin 5 and an impaired expression of the corresponding mRNA transcripts. RT-PCR analysis of mRNA extracted from the proband's keratinocytes identified a homozygous single basepair deletion in the transcripts encoding the laminin alpha 3A and alpha 3B isoforms. The mutation causes a frameshift and premature termination codon in both alleles of the LAMA3 gene. Inheritance of the clinical H-JEB phenotype was consistent with the segregation of the mutated allele in the family. We also report the identity of the alpha chains of laminin 5 and epiligrin and provide evidence that LAMA3 transcripts are distinct from the laminin 6 alpha chain mRNA.
3T3 Cells↗
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↗
Differential cytokine modulation of the genes LAMA3, LAMB3, and LAMC2, encoding the constitutive polypeptides, alpha 3, beta 3, and gamma 2, of human laminin 5 in epidermal keratinocytes.
Laminin 5, an anchoring filament protein previously known as nicein/kalinin/epiligrin, consists of three polypeptide chains, alpha 3, beta 3, and gamma 2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. The expression of laminin 5 was detected by Northern hybridization with specific cDNA probes in various epidermal keratinocyte cultures, whereas no expression of any of the three genes could be detected in foreskin fibroblast cultures. Transforming growth factor-beta (TGF-beta) enhanced LAMA3, LAMB3, and LAMC2 gene expression in human epidermal keratinocytes, as well as in HaCaT and Balb/K cells in culture, although the extent of enhancement was greater for LAMA3 and LAMC2 genes than for LAMB3. Interestingly, tumor necrosis factor-alpha, (TNF-alpha) alone did not alter the expression of LAMB3 and LAMC2 genes in human epidermal keratinocytes, whereas it inhibited the expression of LAMA3. These results suggest that the expression of the three genes encoding the laminin 5 subunits is not coordinately regulated by the cytokines tested.
Animals↗
Transcriptional regulation of decorin gene expression. Induction by quiescence and repression by tumor necrosis factor-alpha.
Decorin, a leucine-rich proteoglycan with ubiquitous tissue distribution, may play essential biological roles during inflammation and cancer growth through its ability to bind extracellular matrix constituents and growth factors. In this study, we demonstrate that decorin gene expression is greatly enhanced after normal diploid fibroblasts reach confluency and cease to proliferate. Elevation of decorin mRNA steady state levels was maintained for up to 16 days postconfluency. In vitro transcription analyses indicated enhanced transcriptional activity in quiescent fibroblasts when compared to cells harvested in their logarithmic phase of growth. This phenotypic trait was reversed by the exogenous addition of tumor necrosis factor-alpha (TNF-alpha). Furthermore, transforming growth factor-beta (TGF-beta) down-regulated decorin gene expression in an additive manner with TNF-alpha. Transient cell transfection assays using plasmid constructs harboring the decorin promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated a dose-dependent transcriptional repression by TNF-alpha. These findings were further corroborated by in vitro transcription experiments using nuclear extracts from control and TNF-alpha-treated quiescent fibroblasts. In contrast, the decorin promoter constructs failed to respond to TGF-beta, thus suggesting either post-transcriptional regulation by this growth factor or lack of TGF-beta-responsive elements. Further experiments with 5' deletion constructs showed two TNF-alpha response elements, one residing within the 5'-untranslated region (exon Ib), the other one between residues -188 and -140 of the decorin promoter. Collectively, our results indicate that TNF-alpha, through its ability to transcriptionally inhibit decorin gene expression in growth-arrested cells, may be a key modulator of the biological functions of this proteoglycan.
Adult↗
Suppression of elastin gene expression in dermal fibroblasts by protein phosphatase inhibitor okadaic acid.
Treatment of human skin fibroblasts with okadaic acid, an inhibitor of serine/threonine specific protein phosphatases 1 and 2A, resulted in a marked reduction in elastin mRNA levels, with maximal inhibition of 80%. The inhibitory effect of okadaic acid on elastin mRNA levels was efficiently prevented by retinoic acid and cycloheximide and was further enhanced by phorbol ester treatment. Upregulation of elastin mRNA levels by transforming growth factor-beta 2 was abrogated by simultaneous treatment of cells with okadaic acid. Okadaic acid had no effect on the expression of human elastin promoter/CAT chimeric gene in fibroblasts cultured from skin of transgenic mice. These results provide evidence that protein phosphatases 1 and 2A play an important role as positive regulators of elastin gene expression in dermal fibroblasts.
Adult↗
Tissue-specific expression of the 230-kDa bullous pemphigoid antigen gene (BPAG1). Identification of a novel keratinocyte regulatory cis-element KRE3.
The 230-kDa bullous pemphigoid antigen gene (BPAG1) is expressed exclusively in basal keratinocytes of epidermis. In this study, we have identified a novel cis-element, keratinocyte responsive element 3 (KRE3), at position -216 to -197 of the human BPAG1 gene. A promoter-CAT construct containing this element had approximately 50-fold higher expression than a similar construct devoid of this sequence when tested in transient transfections of cultured human keratinocytes. However, there was no effect on the low base-line level of expression in cultured skin fibroblasts. KRE3 contains a palindromic sequence 5'-CAAATATTTG-3', and mutations in this sequence significantly reduced the promoter activity. Gel mobility shift assays with an oligomer containing KRE3 sequence demonstrated binding activity with nuclear proteins isolated from keratinocytes. One of the DNA/protein complexes was clearly specific, since competition with > 12.5-fold excess of the unlabeled oligomer resulted in disappearance of this band. No specific binding activity was noted with nuclear proteins extracted from fibroblasts. Thus, KRE3 appears to serve as the binding site for keratinocyte-specific trans-activating factor(s), and KRE3 may thus confer the tissue-specific expression to the BPAG1 gene.
Autoantigens↗
Interferon-gamma-mediated inactivation of transcription of the 230-kDa bullous pemphigoid antigen gene (BPAG1) provides novel insight into keratinocyte differentiation.
Interferon-gamma (IFN-gamma) has been shown to regulate epidermal keratinocyte growth and differentiation. In this study, we examined the effects of recombinant human IFN-gamma on the expression of the gene encoding the 230-kDa bullous pemphigoid antigen (BPAG1), a marker of the mitotic basal cell phenotype in the epidermis. Northern analysis revealed a dose- and time-dependent suppression of BPAG1 expression by IFN-gamma in cultured human keratinocytes from several different donors, and incubation of the cells with IFN-gamma in the presence of cycloheximide demonstrated that this effect required ongoing protein synthesis. The inhibition of BPAG1 gene expression was also demonstrated at the protein level by indirect immunofluorescence using a monoclonal antibody recognizing the human 230-kDa bullous pemphigoid antigen. Transient transfections of cultured keratinocytes with BPAG1 promoter-chloramphenicol acetyltransferase reporter gene plasmids indicated marked suppression of the promoter activity by IFN-gamma, and deletion constructs were able to identify a defined region containing the responsive element (IFN-gamma inhibitory element). Reduced transcription of the BPAG1 gene by IFN-gamma was also demonstrated by in vitro nuclear run-on assays. These data, which indicate inactivation of transcription of a basal keratinocyte-specific gene of transcription of a basal keratinocyte-specific gene (BPAG1) by IFN-gamma, provide novel insight into the mechanisms of IFN-gamma-mediated keratinocyte gene regulation and epidermal differentiation in inflammatory diseases.
Autoantigens↗
Cloning of the beta 3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa.
Laminin 5 consists of three polypeptides, alpha 3, beta 3, and gamma 2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. In this study, we have elucidated the exon-intron organization of the human LAMB3 gene. Characterization of five overlapping lambda phage DNA clones revealed that the gene was approximately 29 kb in size. Subsequent sequence data revealed that the gene consisted of 23 exons that varied from 64 to 379 bp in size, accounting for the full-length cDNA with an open reading frame of 3516 bp encoding 1172 amino acids. Comparison of the LAMB3 gene structure with the previously characterized LAMB1 gene revealed that LAMB3 was considerably more compact. Knowledge of the exon-intron organization of the LAMB3 gene will facilitate elucidation of mutations in patients with the junctional forms of epidermolysis bullosa, some of which have been associated with mutations in the laminin 5 genes.
Base Sequence↗
Skin elastic fibres: regulation of human elastin promoter activity in transgenic mice.
Elastic fibres form an extracellular network which provides elasticity and resilience to tissues such as the skin. To study the regulation of human elastin gene expression, we have developed a line of transgenic mice which harbour 5.2 kb of human elastin gene promoter region in their genome. This promoter is linked to the chloramphenicol acetyltransferase (CAT) reporter gene which allows determination of the expression of human elastin promoter in different tissues. The highest CAT activity was found in the lungs and aorta, tissues rich in elastin, while lower levels were detected in a variety of other tissues, including skin. Assay of CAT activity in the lungs of fetal and newborn animals revealed high activity which progressively declined during the postnatal period up to six months. Thus, there was evidence of tissue-specific and developmentally regulated expression of the human elastin promoter activity in these mice. These animals were then used to examine the expression of the elastin gene by a variety of factors which have previously shown to alter elastin gene expression, as determined at the mRNA or protein levels. First, injection of transforming growth factor beta 1 (100 ng) subcutaneously into the transgenic animals resulted in a time-dependent elevation of the promoter activity up to 10-fold after a single injection. Secondly, enhancement of the human elastin promoter activity by interleukin 1 beta injected subcutaneously resulted in an approximately 10-fold elevation of the CAT activity. Finally, subcutaneous injection of these animals with triamcinolone acetonide or dexamethasone, two glucocorticosteroids in clinical use, resulted in marked enhancement of human elastin promoter activity. Similar changes were noted in fibroblast cultures established from the transgenic animals. These data indicate that the 5.2 kb upstream segment of the human elastin gene contains cis-elements which allow tissue-specific and developmentally regulated expression of the human elastin promoter. Furthermore, this segment of the gene contains responsive elements to a variety of cytokines and pharmacological agents. Collectively, these data indicate that elastin gene expression in the skin in vivo can be regulated at the transcriptional level.
Animals↗
Detection of sequence variants in the gene encoding the beta 3 chain of laminin 5 (LAMB3).
Laminin 5, a candidate gene/protein system for mutations in the junctional forms of epidermolysis bullosa (JEB), consists of three polypeptides encoded by the LAMA3, LAMB3, and LAMC2 genes. In this study, primer pairs for the amplification of the complete cDNA as well as 22 exons of the LAMB3 gene encoding the entire beta 3 chain of laminin 5, were established. The primers for amplification of individual exons from genomic DNA were placed at least 50 bp away from the exon-intron borders in the flanking intronic sequences. For amplification of cDNA generated by RT-PCR, eight primer pairs covering overlapping segments of mRNA were used. The amplified sequences were used to study sequence variations of the LAMB3 gene in patients with JEB and unrelated individuals using heteroduplex analysis. Nine out of 13 JEB patients examined showed heteroduplexes in at least one of the PCR products, indicating the existence of two variable alleles in their DNA. Sequence analyses revealed putative pathogenetic mutations in seven of the JEB patients, while four of the heteroduplexes resulted from polymorphisms, reflecting a single basepair substitution. The results demonstrate that this method is useful in the detection of JEB mutations, as well as polymorphisms in the LAMB3 gene.
Base Sequence↗
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↗
Fibrillin immunofluorescence in pseudoxanthoma elasticum.
BACKGROUND: Pseudoxanthoma elasticum (PXE) is a rare heritable connective tissue disorder manifested by skin, ocular, and cardiovascular anomalies. The basic defect is unknown; however, the microscopic findings are indicative of defects in elastic fibers. Among the components of the elastic fibers are elastin and elastin-associated microfibrils. OBJECTIVE: We assessed the fidelity of this fibrillar system in PXE with the use of antibodies to fibrillin, a major component of elastin-associated microfibrils. METHODS: Using a well-established immunofluorescence assay, we studied fibrillin deposition in dermal fibroblast cultures from 16 patients with PXE. RESULTS: Six of the 16 patients (37%) showed some abnormality of fibrillin deposition in fibroblasts derived from lesional skin. Fibroblasts from nonlesional skin displayed normal fibrillin immunofluorescence. The only sibship studied, however, was discordant for fibrillin immunostaining. CONCLUSION: Unlike the findings in Marfan syndrome, these data are not suggestive of causal fibrillin defects in PXE.
Actin Cytoskeleton↗