A mutation detection strategy for the human keratin 6A gene and novel missense mutations in two cases of pachyonychia congenita type 1

Pachyonychia congenita type 1 (PC-1) is an autosomal dominant ectodermal dy splasia characterized by hypertrophic nail dystrophy, focal non-epidermolyt ic palmoplantar keratoderma and variable features of oral leukokeratosis an d follicular keratosis. Previously, we have shown that this disease can be caused by mutations in type I keratin K16 and one mutation has been reporte d in its type II keratin expression partner, K6a, Mutation analysis for K6a has been hampered by the presence of multiple copies of the K6 gene in the human genome, of which some are expressed and others are pseudogenes. Here , we describe a mutation detection strategy where the entire KRT6A gene, si milar to 7 kb, is specifically amplified by long-range PCR. Using this tech nique, we have detected two novel mutations in the 1A domain of the K6a pol ypeptide, N171K and F174S. Mutations were confirmed in the affected individ uals and were excluded from 50 unaffected unrelated individuals by restrict ion enzyme analysis of KRT6A PCR products. Additionally, mutation N171K was confirmed by RT-PCR in mRNA derived from lesional palmoplantar epidermis o f an affected individual, confirming the specificity of the genomic PCR for the functional K6a gene. This, together with a similar strategy which we h ave developed for the K16 gene, provide a robust system for mutation detect ion and prenatal diagnosis for patients with PC-1.