In this study, we identified homozygous or compound-heterozygous PNKP mutations in eight of the nine Portuguese families we studied (with a total of 11 affected individuals), suggesting that, in Portugal, mutations in PNKP are the most frequent cause of AOA. However, additional studies are needed to determine whether this is also the case in other populations. Our results also extend the phenotype associated with mutations in this gene. Future studies clarifying PNKP regulation and interaction with other repair enzymes might help explain this phenotypic variability.