Donor splice site mutation in keratin 5 causes in-frame removal of 22 amino acids of H1 and 1A rod domains in Dowling-Meara epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) arises from mutations within the kerati n 5 and 14 (K5 and K14) genes which alter the integrity of basal keratinocy tes cytoskeleton, The majority of these defects are missense mutations in t he rod domain, whose locations influence the disease severity, We investiga ted a large family dominantly affected with the Dowling-Meara form of EBS ( EBS-DM), Sequencing of amplified and cloned K5 cDNA from cultured keratinoc ytes revealed a 66 nucleotide deletion in one allele corresponding to the l ast 22 amino acid residues encoded by exon 1 (Val164 to Lys185), Sequencing of amplified genomic DNA spanning the mutant region revealed a heterozygou s G-to-A transition at +1 position of the consensus GT donor splice site of intron 1 of K5, This mutation leads to the use of an exonic GT cryptic don or splice site, located 66 nucleotides upstream from the normal donor splic e site of intron 1, The corresponding peptide deletion includes the last fi ve amino acids of the H1 head domain and the first 17 amino acids of the co nserved amino terminal end of the 1A rod domain, including the first two he ptad repeats and the helix initiation peptide, The shortened polypeptide is expressed in cultured keratinocytes at levels which are comparable to the normal K5 protein, This is the first splice site mutation to be reported as a cause of EBS-DM, Owing to the functional importance of the removed regio n, our data strongly suggest that shortened keratin polypeptide can impair keratin filament assembly in a dominant manner and causes EBS-DM.