Forty-two novel COL7A1 mutations and the role of a frequent single nucleotide polymorphism in the MMP1 promoter in modulation of disease severity in a large European dystrophic epidermolysis bullosa cohort.

BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is a severe genetic skin blistering disorder caused by mutations in the gene COL7A1, encoding collagen VII. Recently, the MMP1 promoter single nucleotide polymorphism (SNP) rs1799750, designated as 1G 2G, was shown to be involved in modulation of disease severity in patients with recessive DEB (RDEB), and was proposed as a genetic modifier. OBJECTIVES: To identify the molecular basis of DEB in 103 individuals and to replicate the results of the MMP1 promoter SNP analysis in an independent patient group, as verification is necessary in such a rare and heterogeneous disorder. METHODS: To determine the molecular basis of the disease, we performed COL7A1 mutation screening, reverse transcription-polymerase chain reaction (PCR) and real-time quantitative PCR. The status of the MMP1 SNP was analysed by PCR and restriction enzyme digestion and verified by sequencing. RESULTS: We disclosed 42 novel COL7A1 mutations, including the first large genomic deletion of 4 kb affecting only the COL7A1 gene, and three apparently silent mutations affecting splicing. Even though the frequency of the high-risk allele was increased in patients with RDEB, no statistically significant correlation between disease severity and genotype could be made. Also, no correlation was observed with development of squamous cell carcinoma, a severe complication of DEB. CONCLUSIONS: Taken together, the results suggest that the MMP1 SNP is not the sole disease modifier in different forms of DEB, and other genetic and environmental factors contribute to the clinical phenotype.

Functional properties of a novel neutrophil chemotactic factor derived from human monocytes.

Neutrophilic granulocytes (PMN) are attracted to sites of inflammation by chemotactic factors, the most potent of which are the complement split product C5a, the leukotriene B4 and the bacterial chemotactic factor-related tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP). In addition to inducing directed migration, these agents increase the adherence of PMN to synthetic surfaces and endothelial cells; some stimulate an oxidative burst and the production of reactive oxygen derivatives, and they may be involved in the release of granule constituents. Here, we describe studies on the activities stimulated by a novel monocyte-derived chemotaxin (MOC). Human MOC attracted human PMN, but not monocytes or eosinophils. Like all chemotactic agents, it increased the adherence of PMN on nylon fibers. In contrast to other chemotactic factors it did not stimulate the release of superoxide anion regardless whether the cells were in suspension or adherent on nylon fibers. There was no release of the primary granule enzyme glucosaminidase or the secondary granule component vitamin B12-binding protein in the absence or presence of cytochalasin B. The results suggest that MOC is a unique chemotactic agent with properties different from the most potent chemotactic factors C5a, LTB4 and FMLP. The delayed release from macrophages suggests its involvement in protracted and chronic inflammatory reactions.

Experimental production of cholesteatoma in rabbits by using non-irritants (skin tolerants).

The application of various skin tolerant substances to the postero-superior and antero-inferior aspects of the external auditory canal of the rabbit, with an intact ear drum and middle ear, produced cholesteatomas in the skin of the auditory canal and in the tympanic membrane, particularly in the pars flaccida. At the antero-inferior insertion of the tympanic membrane, however, cholesteatoma growth could not be induced in these animal experiments. In severe diffuse otitis externa, secondary to the operative closure of the external auditory canal, cholesteatomas also develop preferentially in the pars flaccida. The relatively thick, loose intermediate layer of connective tissue allows a rapid expansion of epithelial ridges, thus favouring the formation of cholesteatoma in this region.

Interference with the fluorometric histamine assay by biogenic amines and amino acids.

Naturally occurring low molecular weight biogenic amines and amino acids were analyzed for interference in the fluorometric histamine assay. Three mechanisms of interference were detected, mimicking of histamine, suppression of histamine fluorescence and generation of increasing histamine-like fluorescence during excitation or preirradiation of the o-phthaldialdehyde condensation product with daylight or UVA light. The latter effect was seen only with amino acids. Rat peritoneal lavage fluid contained no fluorescence-suppressing substances, but there were considerable amounts of histamine-mimicking compounds which could not be digested with a diamine oxidase, and of substances generating increasing histamine-like fluorescence when exposed to UV light after condensation with phthaldialdehyde. Although butanol extraction was highly effective in selecting histamine over interfering compounds, it was not sufficient to avoid interference. In contrast, Dowex 50 W-X8 ion exchange chromatography added little to separating histamine and interfering substances. Precautions are recommended to avoid the different types of interference when biological samples are analyzed for histamine content with the fluorometric assay.