11 Study of Polynucleotide Kinase/Phosphatase (PNKP) Mutations Found in a Patient with Microcephaly, Seizures, and Developmental Delay (MCSZ) and Glioblastoma

Abstract

The enzyme polynucleotide kinase/phosphatase (PNKP) plays a key role in DNA repair by resolving the chemistry at DNA strand breaks. Mutations in PNKP (chromosome 19q13.4) are known to cause MCSZ, a serious neurodevelopmental disorder, but to date there has been no link to cancer initiation or progression. However, a child with MCSZ recently presented at Seattle Children's Hospital with a 3-cm glioblastoma. The child was shown to have two germline mutations in PNKP. To study the effects of the PNKP mutations found in this patient, we generated mutant PNKP cDNAs carrying either the individual mutations or the double mutation using site directed mutagenesis. These cDNAs were incorporated into bacterial and mammalian expression vectors. The bacterially expressed mutant proteins as well as the wild type have been purified and are undergoing testing for PNKP DNA kinase and phosphatase activity. The PNKP cDNAs, fused to GFP, were expressed in Hela and HCT116 human cancer cell lines. High-content analysis and micro-irradiation techniques are being used to determine PNKP localization within the cells and recruitment to damaged DNA. Our preliminary results indicate that the mutations alter the ratio of nuclear to cytoplasmic PNKP compared to the wild-type protein.