[Purpura pigmentosa progressiva in type III cryoglobulinemia and tartrazine intolerance. A follow-up over 20 years]. / Purpura pigmentosa progressiva bei Kryoglobulinämie Typ III und Tartrazinintoleranz. Ein Verlauf über 20 Jahre.

A 58 year old patient with hepatitis virus C (HCV) infection had a secondary polyclonal IgG-IgM cryoglobulinemia with a benign 20 year course. Clinically the patient suffered from progressive pigmented purpura (PPP). Histologic evaluation revealed a lymphocytic vasculitis. Food containing tartrazine triggered flares of the PPP, as demonstrated with controlled oral provocation testing. In most of the previously described cases of HCV and type III cryoglobulinemia, the typical cutaneous finding was palpable purpura with leukocytoclastic vasculitis.

A combination of a common splice site mutation and a frameshift mutation in the COL7A1 gene: absence of functional collagen VII in keratinocytes and skin.

We describe a patient with severe generalized dystrophic epidermolysis bullosa (EBD) and a novel combination of compound heterozygous mutations in the COL7A1 gene. The maternal mutation was an A-to-G transition (425-A --> G) at position -2 of the donor splice site within exon 3 that causes aberrant splicing of two abnormal transcripts. One includes intron 3, and one excludes both exon 3 and intron 3. Both splice variants contained a premature termination of the translation. The paternal mutation is a 25-bp deletion in exon 20 (2638de125) that leads to a frameshift and a premature termination codon 133 bp downstream from the site of deletion. This combination of mutations allowed expression of collagen VII mRNA. Immunofluorescence staining of the patient's skin and cultured keratinocytes with domain-specific collagen VII antibodies, however, demonstrated markedly reduced levels of alpha1(VII) polypeptides, and no stable collagen VII protein could be extracted from the patient's cells. Electron microscopy showed severely hypoplastic fibrils below the lamina densa, without evidence of normal anchoring fibrils. The clinically unaffected parents were heterozygous for the mutations, suggesting that both COL7A1 gene defects were recessively inherited disease-causing mutations that are "silent" in heterozygous carriers but in combination can severely interfere with the dermal-epidermal adhesion and lead to severe EBD.

Immunohistochemical and mutation analyses demonstrate that procollagen VII is processed to collagen VII through removal of the NC-2 domain.

Collagen VII is the major structural constituent of anchoring fibrils in the skin. It is synthesized as a procollagen that is larger than the collagen deposited in the tissue. In this study, we investigated the conversion of procollagen VII to collagen VII in human skin and in cutaneous cells in vitro and identified the propeptide using domain-specific antibodies. For this purpose, two bacterial fusion proteins containing unique sequences of the carboxy-terminal globular NC-2 domain of procollagen VII were prepared, and polyclonal antibodies raised against them. Immunoblotting showed that the anti-NC2 antibodies reacted with procollagen VII isolated from cultured keratinocytes, but not with collagen VII extracted from the skin. Immunohistochemical experiments with the NC-2 antibodies revealed a strong reaction in cultured keratinocytes, but the basement membrane zone of normal skin remained negative. The staining could not be rendered positive by chemical or enzymatic unmasking of potential hidden epitopes in the skin, indicating that most of the NC-2 domain is absent from normal skin. In contrast, a positive staining with NC-2 antibodies was observed in the skin of a patient with NC-2 antibodies was observed in the skin of a patient with dystrophic epidermolysis bullosa, who carried a 14-bp deletion at one of the intro-exon junctions of the collagen VII gene. This aberration led to an in-frame skipping of exon 115 from the mRNA and eliminated 29 amino acids from the NC-2 domain which include the putative cleavage site for the physiological processing enzyme, procollagen C-proteinase. The results indicate that in normal human skin, the removal of the NC-2 domain from procollagen VII precedes its deposition at the dermal-epidermal junction. Furthermore, they suggest that an aberration in the procollagen VII cleavage interferes with the normal fibrillogenesis of the anchoring fibrils.

Collagen VII in severe recessive dystrophic epidermolysis bullosa: expression of mRNA but lack of intact protein product in skin and cutaneous cells in vitro.

The collagen VII gene, COL7A1, is the candidate gene for both the recessive and dominant forms of dystrophic epidermolysis bullosa (EBD). Collagen VII is a structural protein of the anchoring fibrils, which are rudimentary or altered in several subtypes of EBD. In severe recessive mutilating EBD, anchoring fibrils and collagen VII are not detectable in skin of most patients. To elucidate the underlying pathogenetic mechanisms, we analyzed collagen VII expression in cutaneous cells of six patients with this severe EBD subtype. Neither keratinocytes nor fibroblasts synthesized detectable amounts of collagen VII protein; however, Northern blot analysis revealed small amounts of normal-size collagen VII mRNA in both EBD and control fibroblasts. When the mRNA was amplified using reverse transcription-polymerase chain reaction, correct amplimers were present in all specimens. The results demonstrate that transcription of the COL7A1 gene occurs in these patients with severe mutilating EBD and suggest that post-transcriptional or post-translational events lead to absence of collagen VII protein from skin.