Epidermolysis bullosa simplex is a hereditary skin blistering disorder caused by 
mutations in the KRT5 or KRT14 genes. More than 50 different mutations have been 
described so far. These, and reports of other keratin gene mutations, have 
highlighted the existence of mutation "hotspots" in keratin proteins at which 
sequence changes are most likely to be detrimental to protein function. 
Pathogenic mutations that occur outside these hotspots are usually associated 
with less severe disease phenotypes. We describe a novel K5 mutation (V186L) 
that produces a conservative amino acid change (valine to leucine) at position 
18 of the 1A helix. The phenotype of this case is unexpectedly severe for the 
location of the mutation, which lies outside the consensus helix initiation 
motif mutation hotspot, and other mutations at this position have been 
associated in Weber--Cockayne (mild) epidermolysis bullosa simplex only. The 
mutation was confirmed by mismatch-allele-specific polymerase chain reaction and 
the entire KRT5 coding region was sequenced, but no other changes were 
identified. De novo K5/K14 (mutant and wild-type) filament assembly in cultured 
cells was studied to determine the effect of this mutation on filament 
polymerization and stability. A computer model of the 1A region of the K5/K14 
coiled-coil was generated to visualize the structural impact of this mutation 
and to compare it with an analogous mutation causing mild disease. The results 
show a high level of concordance between genetic, cell culture and molecular 
modeling data, suggesting that even a conservative substitution can cause severe 
dysfunction in a structural protein, depending on the size and structure of the 
amino acid involved.