Angela M. Christiano

Molecular complexity of the cutaneous basement membrane zone

Abstract Spectacular success has recently been made towards elucidation of the molecular basis of various forms of epidermolysis bullosa (EB), a group of heritable blistering skin diseases. The information derived from these studies has already had a profound impact in terms of precise diagnosis and classification, early prenatal prediction of the phenotype and genetic counseling in families at risk for recurrence. This review highlights recent progress made in defining the molecular basis of junctional and dystrophic forms of EB and the genotype/phenotype relationships established from these studies. Extensive molecular studies, such as the ones captured in this review, form a foundation for the rational design of gene therapies to counteract these conditions in the future.

Strategy for identification of sequence variants in COL7A1 and a novel 2-bp deletion mutation in recessive dystrophic epidermolysis bullosa

The diagnostic hallmark of the dystrophic forms of epidermolysis bullosa (DEB), a group of heritable blistering skin diseases, is abnormalities in the anchoring fibrils at the dermal‐epidermal basement membrane zone. Since type VII collagen is the major, if not the exclusive, component of the anchoring fibrils, the corresponding gene (COL7A1) is the candidate gene in DEB. Recent cloning of the type VII collagen cDNA and elucidation of the exon‐intron organization of the gene have provided the basis for us to develop a novel strategy for identification of sequence variants in COL7A1. Optimization of 72 balanced primer pairs corresponding to flanking intronic sequences allowed PCR amplification of all 118 exons directly from genomic DNA. The PCR products were examined by heteroduplex analysis followed by comparative nucleotide sequencing. More than 100 sequence variants have been identified thus far in COL7A1 using this method, some of which are single base pair polymorphisms and many of which are pathogenetic mutations contributing to the blistering phenotype in DEB. The comprehensive method described is useful for rapid, reliable, and sensitive detection of sequence variants in COL7A1. We demonstrate the utility of this novel strategy in mutation detection and prenatal exclusion of RDEB in a consanguineous family at risk for recurrence. Hum Mutat 10:408–414, 1997.

Molecular basis for the dystrophic forms of epidermolysis bullosa: mutations in the type VII collagen gene

Significant progress has recently been made in understanding the molecular basis of heritable skin diseases, such as epidermolysis bullosa, a group of mechano-bullous genodermatoses. In particular, the dystrophic forms of epidermolysis bullosa have been shown to result from distinct mutations in the gene encoding type VII collagen, the major, if not the exclusive, component of the anchoring fibrils. These mutations result in deficient synthesis and/or altered assembly of the anchoring fibrils, thus compromising the integrity of the cutaneous basement membrane zone. The mutations in the type VII collagen gene have implications for understanding the structure-function relationships of the type VII collagen molecule, and also provide the basis for prenatal DNA-based diagnosis in families at risk for recurrence of the disease. Finally, understanding the genetic basis of dystrophic forms of EB sets the stage for gene therapy approaches for the treatment of these devastating skin diseases.

Cloning of the {beta}3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa

Abstract Laminin 5 consists of three polypeptides, α3, β3, and γ2, 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 λ 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.

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, α3, β3, and γ2, 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‐β (TGF‐β) 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‐α, (TNF‐α) 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.

Molecular biology and pathology of type VII collagen

Abstract Type VII collagen is a genetically distinct member of the collagen family of proteins. Type VII collagen has been shown to be the major component of anchoring fibrils, attachment complexes which secure the cutaneous basement membrance of the skin to the underlying dermis. Understanding of the structure of type VII collagen has been advanced by recent cloning of the corresponding gene. Chromosomal mapping of the gene to the short arm of chromosome 3 and identification of intragenic polymorphic markers have allowed demonstration of strong genetic linkage between the type VII collagen locus and the dystrophic forms of EB (epidermolysis bullosa). This overview summarizes the progress made in the molecular genetics of type VII collagen.

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.

Premature termination codons on both alleles of the type VII collagen gene (COL7A1) in three brothers with recessive dystrophic epidermolysis bullosa.

Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. In the most severe, dystrophic (scarring) forms of EB, blisters form below the cutaneous basement membrane at the level of the anchoring fibrils, which are composed of type VII collagen. Ultrastructural observations of altered anchoring fibrils and genetic linkage to the type VII collagen locus (COL7A1) have implicated COL7A1 as the candidate gene in the dystrophic forms of EB. We have recently cloned the entire cDNA and the gene for human COL7A1. In this study, we describe distinct mutations in both COL7A1 alleles in three brothers with severe, mutilating recessive dystrophic EB (the Hallopeau-Siemens type, HS-RDEB). The patients are compound heterozygotes for two different mutations, both of which result in a premature termination codon in COL7A1, and the parents were shown to be clinically heterozygous carries of the respective mutations. Premature termination codons in both alleles of COL7A1 appear to be the underlying cause of severe, recessive dystrophic EB in this family.

Cloning of the Gene for Human Pemphigus Vulgaris Antigen (Desmoglein 3), a Desmosomal Cadherin CHARACTERIZATION OF THE PROMOTER REGION AND IDENTIFICATION OF A KERATINOCYTE-SPECIFIC cis-ELEMENT

Pemphigus vulgaris antigen is a cadherin-like desmosomal cell adhesion molecule expressed primarily in suprabasal keratinocytes within the epidermis. Previously characterized structural features have defined this molecule as a desmoglein, DSG3. In this study, we have cloned the human DSG3 gene and examined the transcriptional regulation of its expression. The total gene consisted of 15 exons and was estimated to span >23 kilobases. Comparison of exon-intron organization of DSG3 with bovine DSG1 and several classical cadherin genes revealed striking conservation of the structure. Up to 2.8 kilobases of the upstream genomic sequences were sequenced and found to contain several putative cis-regulatory elements. The promoter region was GC-rich and TATA-less, similar to previously characterized mammalian cadherin promoters. The putative promoter region was subcloned into a vector containing chloramphenicol acetyl transferase reporter gene. Transient transfections with a series of deletion clones indicated that the DSG3 promoter demonstrated keratinocyte-specific expression, as compared with dermal fibroblasts examined in parallel, and fine mapping identified a 30-base pair segment at −200 to −170 capable of conferring epidermal specific expression. The results provide evidence for the transcriptional regulation of the pemphigus vulgaris antigen gene, potentially critical for development of the epidermis and physiologic terminal differentiation of keratinocytes.

Nothing but skin and bone

Skin and bone - what comes to mind at hearing this phrase? While certainly a metaphor for disease, it also defines two very different tissues, one a flexible and contiguous outer covering, the other a morphologically diverse hard tissue distributed at over 200 sites in the body. As the accompanying series of Reviews highlights, these tissues are indeed diverse, but there are also surprising similarities. Skin is the interface between the internal organs and the environment, and as such plays a crucial role in the bodys defense mechanism. The skin and its many appendages are responsible for functions as diverse as epidermal barrier and defense, immune surveillance, UV protection, thermoregulation, sweating, lubrication, pigmentation, the sensations of pain and touch, and, importantly, the protection of various stem cell niches in the skin. Bone serves a number of purposes: it provides protection for vital organs, a lever for locomotion, a reservoir for calcium, and the site of adult hematopoiesis. The tissue is composed of osteoblasts, osteoclasts, and their individual precursors plus a complex mixture of mesenchymal, myeloid, and lymphoid cells in the marrow space. Finally, the endothelial microenvironment provides nutrition and is a conduit for the influx and emigration of cells that impact bone biology in several important ways. This Review series guides the reader through these various facets of 2 diverse, yet interdependent, tissues.