Klaas Heeres

Revertant Mosaicism in Epidermolysis Bullosa Caused by Mitotic Gene Conversion

Mitotic gene conversion acting as reverse mutation has not been previously demonstrated in human. We report here that the revertant mosaicism of a compound heterozygous proband with an autosomal recessive genodermatosis, generalized atrophic benign epidermolysis bullosa, is caused by mitotic gene conversion of one of the two mutated COL17A1 alleles. Specifically, the maternal allele surrounding the mutation site on COL17A1 (1706delA) showed reversion of the mutation and loss of heterozygosity along a tract of at least 381 bp in revertant keratinocytes derived from clinically unaffected skin patches; the paternal mutation (R1226X) remained present in all cell samples. Revertant mosaicism represents a way of natural gene therapy.

180-kD bullous pemphigoid antigen (BP180) is deficient in generalized atrophic benign epidermolysis bullosa.

Generalized atrophic benign epidermolysis bullosa (GABEB) is a form of nonlethal junctional epidermolysis bullosa characterized by universal alopecia and atrophy of the skin. We report a deficiency of the 180-kD bullous pemphigoid antigen in three patients with GABEB from unrelated families. We screened specimens of clinically normal skin from nine junctional epidermolysis bullosa patients (3 GABEB, 4 lethal, 1 cicatricial, 1 pretibial) by immunofluorescence using monoclonal antibodies to the 180-kD and 230-kD bullous pemphigoid antigens (BP180 and BP230). In the skin of the three GABEB patients there was no reactivity with antibodies to BP180, whereas staining for BP230 was normal. In the skin of the other six, non-GABEB patients, included in this study the expression of BP180 and BP230 was normal. Immunoblot analysis of cultured keratinocytes from one of the GABEB patients also failed to detect BP180 antigen, whereas BP230 was present in normal amounts. The deficient expression of BP180 is reflected in the RNA message, as in Northern blot analysis a reduced amount of BP180 transcripts, although of normal length, were detected. Interestingly, in another GABEB patient there were not-involved areas of skin, in which blistering could not be induced by rubbing. Biopsy material from these areas showed interrupted staining for BP180. There was no staining for BP180 in areas of clinically normal but involved skin of this patient. In conclusion, this study reveals that the BP180 antigen is deficient and the BP180 mRNA is reduced in generalized atrophic benign epidermolysis bullosa.

Bullous pemphigoid: serum antibody titre and antigen specificity

Abstract 2 antigens have been identified as possible targets for autoantibody depositions in bullous pemphigoid: a 230‐kD protein (BP230) and a 180‐kD protein (BP180). We studied the relationship of these 2 antigens with the immunofluorescence determined serum antibody titre. 2 groups of bullous pemphigoid patients were selected on the basis of immunoblot‐determined antibody specificity. One group (13 patients) had antibody specificity for BP230 and not for BP180, while the other group (9 patients) had antibody specificity for BP180 and not for BP230. The immunofluorescence titres of the circulating antibodies determined on monkey oesophagus substrate displayed, for the BP230‐specific group, a mean of 1:1102. The maximal observed titre was 1:5120. The mean titre in the BP180‐specific group was only 1:29, with a highest titre of only 1:160. This result suggests that in routine indirect immunofluorescence of bullous pemphigoid sera, the contribution of the BP180‐specific antibodies to the total anti‐epidermal basement membrane zone antibody titre is relatively much lower than that of the BP230‐specilic antibodies. Thus, at high dilutions, only the BP230‐speeific antibodies contribute to the overall indirect immunfluorescence titre.

Fluorescence overlay antigen mapping of the epidermal basement membrane zone: III. Topographic staining and effective resolution.

In this third study on the fluorescence overlay antigen mapping (FOAM) technique, we have addressed the question of which differences of antigen distributions close to the resolving power of the light microscope can be distinguished. An answer to this question should provide clues to future applications of the technique aiming at the topographic differentiation of IgG deposits displayed at the epidermal basement membrane zone (EBMZ) in certain bullous skin disorders. For the present purpose we have developed a topographic staining model in human skin, using structural EBMZ antigens as topographic reference markers. The distribution of these markers relative to one another is visualized in FOAM images obtained by selective double immunofluorescence tracing and videomicroscopic overlay imaging. The theoretical resolution limit of the technique is discussed and suggests an effective lower limit of some 60-65 nm. Although this limit is not reached under present conditions, our results show that it is possible to distinguish topographic differences of antigen distributions with an upper resolution limit of 200 +/- 50 nm. Furthermore, our findings indicate that collagen Type VII and beta 4 integrin are the most suitable molecules to serve as topographic reference markers in future applications of the technique aiming at the differentiation of bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). Preliminary results on this topic are most promising indeed.

Fluorescence overlay antigen mapping of the epidermal basement membrane zone: I. Geometric errors.

To identify in tissue sections the relative positions of antigen distributions close to the resolving power of the microscope, we have developed the fluorescence overlay antigen mapping (FOAM) procedure. As this technique makes high demands on the geometric fidelity of the overlay image, it is essential to recognize geometric errors resulting from optical imperfections. This applies in particular to the image shift difference (ISD) that may routinely occur during fluorescence overlay. We describe here procedures for assessment and mechanical correction of the ISD in tissue sections. Furthermore, we describe an alignment verification test to assess the accuracy of the ISD correction procedure, using collagen Type VII as the geometric verification marker. These procedures should enable reliable evaluation of relative antigen distributions in tissue sections using photomicrographic multicolor fluorescence overlay. Further details of the FOAM technique, such as color fidelity and its utility for diagnostic and research purposes, will be published separately.

Fluorescence overlay antigen mapping of the epidermal basement membrane zone: II. Color fidelity.

In this second report on the fluorescence overlay antigen mapping (FOAM) technique, we highlight some of the errors that may influence faithful color rendition of slide preparations using triple antigen immunofluorescence staining. Reliable interpretation of multicolor fluorescence images requires that the observer can unambiguously assign each color in these images to the presence of a specific combination of the labeled antigens. This is possible only when the image fidelity meets certain standards. The present study concentrates on color fidelity which is easily undermined by spectral matching errors, image contrast errors, and exposure time errors. Evaluation of these errors, using the photomicrographic overlay variant of FOAM, showed the potential unreliability of the simultaneous use of multiple fluorophores for immunofluorescence microscopy. The procedures described here may serve as a solid starting point in formulating technical conditions that allow reliable color rendition in multicolor immunofluorescence microscopy. Furthermore, these procedures can be adapted to studies other than the analysis of basement membrane zone antigens, to which they have been first applied.