Seven novel COL7A1 mutations identified in patients with recessive dystrophic epidermolysis bullosa from Mexico.

Recessive dystrophic epidermolysis bullosa (RDEB; OMIM #226600) is one of the most devastating subtypes of epidermolysis bullosa, a group of skin and mucous membrane blistering disorders often associated with extracutaneous manifestations. RDEB is caused by mutations in COL7A1, the gene encoding type VII collagen (C7), and to date over 700 different mutations in the 8835 nucleotides constituting the open reading frame or adjacent exon-intron boundaries of COL7A1 have been described. We used targeted next-generation sequencing to identify seven previously unreported mutations in a cohort of 17 Mexican patients who were diagnosed with RDEB based on clinical presentation and immunoepitope mapping. Our study expands the spectrum of mutations identified in this cohort, including those suitable for emerging therapies reliant on precise genotyping.

DNA-based prenatal diagnosis of heritable skin diseases.

BACKGROUND: Recent success in identifying candidate genes and demonstrating mutations in such genes has set the stage for DNA-based prenatal diagnosis of genodermatoses. An example of such conditions is epidermolysis bullosa in which discrete mutations have been identified in different forms of the disease. The severity of the clinical phenotype in some forms of epidermolysis bullosa appears to justify prenatal diagnosis in families at risk for recurrence of the disease. OBSERVATIONS: DNA-based prenatal diagnosis can be performed on chorionic villi, which can be obtained as early as the eighth week of gestation. Thus, the approaches that use sensitive and specific molecular probes will allow identification of a fetus at risk relatively early during the pregnancy. Such prenatal diagnosis has successfully been performed in families with recessive dystrophic epidermolysis bullosa using type VII collagen-specific markers. CONCLUSIONS: Combination of informative intragenic and flanking DNA markers, and eventual identification of specific mutations in additional families with recessive dystrophic epidermolysis bullosa, is expected to allow early prenatal diagnosis in most families at risk for this devastating skin disease. An emerging technology in the field of prenatal genetics involves blastomere analysis prior to implantation. These DNA-based technologies will undoubtedly replace invasive skin biopsy in cases where candidate genes or specific mutations have been identified. Finally, identification of specific mutations will provide the foundation for potential gene replacement therapy in individuals affected with severe skin diseases.

Leukoregulin down-regulates type I collagen mRNA levels and promoter activity in human dermal fibroblasts, and counteracts the up-regulation elicited by transforming growth factor-beta.

Leukoregulin (LR), a T-cell-derived growth factor, modulates fibroblast functions in vitro [Mauviel, Rédini, Hartmann, Loyau & Pujol (1991) J. Cell Biol. 113, 1455-1462]. In the present study, incubation of human dermal fibroblasts with LR (0.1-2 units/ml) resulted in decreases in the mRNA steady-state levels for alpha 1(I), alpha 2(I) and alpha 1(III), but not alpha 2(V), collagen genes. LR also down-regulated alpha 2(I) collagen promoter activity in transient cell transfections of control cells as well as those incubated with transforming growth factor-beta, a potent up-regulator of collagen type I gene expression. Thus LR is a strong inhibitor of type I collagen gene expression, acting at the level of transcription.

Reflex sympathetic dystrophy. Occurrence of inflammatory skin lesions in patients with stages II and III disease.

Reflex sympathetic dystrophy is a poorly understood syndrome of posttraumatic pain and sympathetic nervous aberration. We have observed previously unreported cutaneous manifestations of reflex sympathetic dystrophy. Seven patients with reflex sympathetic dystrophy were referred to our institution because of skin disorders. Three had recurrent ulcerating papules, and two had reticulate hyperpigmentation. Xerosis was common, and cutaneous atrophy was infrequent. Cutaneous ulceration and reticulate hyperpigmentation are previously unappreciated aspects of reflex sympathetic dystrophy. Further investigation regarding neural influences on the skin is warranted.

Human type VII collagen: genetic linkage of the gene (COL7A1) on chromosome 3 to dominant dystrophic epidermolysis bullosa.

Epidermolysis bullosa (EB) is a heterogeneous group of heritable blistering disorders affecting the skin and the mucous membranes. Previous ultrastructural studies on the dystrophic (scarring) forms of EB have demonstrated abnormalities in the anchoring fibrils, morphologically distinct structures below the basal lamina at the dermal/epidermal basement membrane zone. Type VII collagen is the major collagenous component of the anchoring fibrils, and it is therefore a candidate gene for mutations in some families with dystrophic forms of EB. In this study, we performed genetic linkage analyses in a large kindred with dominant dystrophic EB. A 1.9-kb type VII collagen cDNA clone was used to identify a PvuII RFLP to follow the inheritance of the gene. This RFLP cosegregated with the EB phenotype in this family, strongly supporting genetic linkage (Z = 5.37; theta = .0). In addition, we assigned the type VII collagen gene (COL7A1) to chromosome 3 by hybridization to a panel of human x rodent somatic cell hybrids. These data demonstrate very close genetic linkage between the clinical phenotype in this family and the polymorphism in the type VII collagen gene mapped to chromosome 3. The absence of recombination between EB and the type VII collagen gene locus, as well as the observed abnormalities in the anchoring fibrils, strongly suggest that this collagen gene is the mutant locus in this kindred.

Eosinophilic fasciitis. Increased collagen production and type I procollagen messenger RNA levels in fibroblasts cultured from involved skin.

Two patients with eosinophilic fasciitis were studied to elucidate the activation of collagen production in this disorder. Histologic examination of biopsy specimens from the involved area of skin revealed the presence of inflammatory cell infiltrates and various degrees of collagen accumulation in the dermis, subcutis, fascia, and underlying muscle. Fibroblast cultures initiated from the involved skin exhibited 2.0- to 3.7-fold increased collagen production when compared with control fibroblast cultures established from the uninvolved skin of the same patients. Eosinophilic fasciitis fibroblasts also displayed 2.4- to 6.2-fold higher steady-state levels of type I procollagen messenger RNA than did the control cells, indicating pretranslational activation of type I procollagen gene expression. In addition, cellular fibronectin messenger RNA steady-state levels were elevated 1.9- to 3.3-fold in eosinophilic fasciitis fibroblasts. These results suggest that fibroblasts in the involved skin of patients with eosinophilic fasciitis exhibit an activated phenotype, similar to that of scleroderma fibroblasts, leading to accumulation of collagen in the skin and the underlying structures.

Genetic linkage analysis of hereditary arthro-ophthalmopathy (Stickler syndrome) and the type II procollagen gene.

Hereditary arthro-ophthalmopathy (AO), or Stickler syndrome, is a dominantly inherited disorder characterized by vitreo-retinal degeneration and frequently accompanied by epiphyseal dysplasia and premature degenerative joint disease. Three large families with AO were analyzed for clinical manifestations of the disease and for coinheritance of the genetic defect with RFLPs in the type II procollagen gene (COL2A1). Genetic linkage between AO and COL2A1 was demonstrated in the largest family, with a maximum LOD score of 3.52 at a recombination distance of zero. Data from a second family also supported linkage of AO and COL2A1, with a LOD score of 1.20 at a recombination distance of zero. These results are consistent with the conclusion that mutations in the COL2A1 gene are responsible for AO in these two families. In a third AO family, however, recombination between AO and COL2A1 occurred in at least one meiosis, and the data were inconclusive with respect to linkage.

Isolation and characterization of human elastin cDNAs, and age-associated variation in elastin gene expression in cultured skin fibroblasts.

In attempts to isolate human elastin cDNAs, a human placental lambda gt11 cDNA library was screened with a 1.3 kilobase sheep genomic DNA subclone, corresponding to the 3'-end of the elastin mRNA. The four largest clones, the largest being approximately 3 kilobase, were characterized by Northern transfer analyses, restriction endonuclease digestions and dideoxy nucleotide sequencing. Northern transfer analyses of poly(A)+RNA revealed hybridization to mRNA transcripts in the region of 3.5 kilobase. Restriction endonuclease mapping and nucleotide sequencing demonstrated distinct domains characteristic of elastin, and identified areas of variability which apparently reflects alternative splicing of the primary elastin transcripts. To demonstrate the utilization of these cDNAs for studies on elastin gene expression in human cells, elastin mRNA was examined in fibroblast cultures established from the skin of several individuals of varying ages. Northern transfer analyses and slot blot hybridizations demonstrated that elastin gene expression is initiated early during fetal development, and continues at a relatively constant level through several decades. The lowest abundance of elastin mRNA was noted in the cell cultures established for the oldest individual studied (61-year-old female). Demonstration of elastin gene expression in cultured fibroblasts provides a system to study diseases affecting the elastic fibers.

Cellular effects of the pulsed tunable dye laser at 577 nanometers on human endothelial cells, fibroblasts, and erythrocytes: an in vitro study.

The 577-nm flashlamp-pumped tunable dye laser pulsed at 450 microseconds is rapidly becoming the treatment of choice for removal of portwine stains and other vascular ectasias. In this study, we examined the mechanisms of vessel destruction by determining the effects of laser irradiation on three types of primary target cells--erythrocytes, endothelial cells, and fibroblasts. Human endothelial cells and fibroblasts in microwell plates were irradiated at various energy densities with the laser, after which several aspects of cellular biology were determined, including 1) viability of cells by trypan blue exclusion test; 2) cell proliferation by [3H]thymidine incorporation; and 3) rate of protein synthesis using [3H]leucine incorporation as a marker. In endothelial cell cultures, both [3H]thymidine and [3H]leucine incorporations were inhibited at energy levels of 5-12 J/cm2 (P less than 0.01). In fibroblast cultures, cell proliferation was similarly inhibited, while supratherapeutic energy density (greater than or equal to 12 J/cm2) was required for inhibition of protein synthesis. The laser energy in the range of 5-8.5 J/cm2 had no effect on cell viability. Erythrocytes as target cells for laser energy demonstrated rapid, dose-dependent lysis, as determined by release of free hemoglobin into culture medium. Addition of erythrocytes into a coculture with endothelial cells abolished the direct inhibitory effect noted in cultures when endothelial cells were present alone. The results of the latter experiment imply that erythrocytes are the primary target cell absorbing the laser energy at 577 nm. However, direct laser effects on endothelial cells may also contribute to the mechanisms of ablation of the vascular ectasias by the tunable dye laser at 577 nm.

Association of hemolytic anemia and early-onset pulmonary emphysema in three siblings.

Three of four siblings born to nonconsanguineous parents of Italian origin were affected with severe congenital hemolytic anemia of unknown cause, and early-onset pulmonary emphysema. Two of the three affected siblings died of septic shock after splenectomy, at the ages of 7 and 3 1/2 years, respectively. The remaining affected sibling was shown to have cutis laxa and severe pulmonary emphysema at 15 years of age. Assay of serum components indicated that alpha 1-antitrypsin and alpha 2-macroglobulin levels were normal or slightly elevated. However, there was markedly elevated activity of an elastase-like serum enzyme. The relation of the hemolytic anemia to the pulmonary findings in this family is not clear; pedigree analysis suggests a recessively inherited defect.