Towards a comprehensive resource for elucidating the pathogenesis of inherited keratodermas
Keratoderma – pathological hyperkeratosis of palms and soles - is a cause of disability in many clinical situations, including the rare and heterogeneous group of inherited palmoplantar keratodermas (PPKs). The aim of this study was to work towards better understanding of molecular mechanisms active in the pathogenesis of PPK by the creation of a cell and tissue culture resource and its initial application to laboratory studies. My study was based on a diverse group of autosomal dominant disorders, previously ascertained in families from Scotland, in whom the precise genetic aetiology was known. I established a tissue and cell culture resource of inherited keratodermas of known single-gene aetiology from patients with proven keratin 1, 9, 17, loricrin and mitochondrial mutations. An additional pedigree with striate keratoderma with an unknown mutation was recruited, and the causative mutation identified as a novel heterozygous A-to-T transversion in exon 5 (c.430A>T) of the desmoglein 1 gene, converting an arginine residue to a premature termination codon (p.Arg144stop). The keratinocyte culture resource was established from patients with keratin 1, 9, 17 and loricrin mutations, as well as controls. Due to the pain associated with direct infiltration of plantar skin, biopsies were obtained using peripheral nerve block for plantar biopsy. The effectiveness of this approach, which may be useful for future administration of treatment, was made the subject of an open clinical trial. Histological and immunocytochemical studies were carried out on affected plantar skin obtained from PPK patients and compared to control tissue, in an attempt to identify common and distinct pathways resulting in hyperkeratosis. Histological changes, e.g. hypergranulosis, extent of hyperkeratosis, acanthosis or acantholysis, were not uniform across different subtypes of inherited PPK and varied even between individuals within subtypes. Prominent eosin staining of spinous cells was a common feature in inherited PPK due to underlying K1 and K17 mutations. Electron microscopy showed abnormal keratin filaments in PPK with underlying keratin mutations only but was not a uniform finding within subtypes, and other electron microscopic features also varied between individuals. Immunocytochemical study did not demonstrate significant differences in expression of a selection of markers of differentiation (keratins 1, 9, 14 and 17), and cornified envelope protein filaggrin. Abnormal involucrin expression was observed, with premature expression in basal and lower spinous layers in all PPK subtypes raising the possibility of a common underlying mechanism in the development of hyperkeratosis. Prominent loricrin staining was noted in areas of acantholysis in K1 and K9 subtypes, but was uniform across other subtypes. Markers of proliferation and apoptosis demonstrated no overt change in epidermal turnover, although it is possible that only small changes in proliferative index are required to produce plantar hyperkeratosis. Overall, using morphological criteria, plantar hyperkeratosis was not readily distinguishable between inherited PPK of different underlying genetic causes. This raises the possibility that many of the reported structural features of inherited PPK are secondary phenomena as opposed to critical steps in the pathogenesis of hyperkeratosis. Initial attempts at RNA extraction using laser and manual microdissection have to date been unsuccessful in generating RNA of the quality and concentration to run a pilot microarray experiment, using standard RNA extraction kits. Plans for future projects include the further development of a possible microarray experiment in the Pachyonychia Congenita type 2 pedigree with the McLean laboratory in Dundee. The tissue resource has been made available for collaborative study via the GENESKIN project, as well as through the McLean and Lane laboratories, Dundee for both functional studies and immortalisation of cell lines.