Characterization of 137 Genomic DNA Reference Materials for 28 Pharmacogenetic Genes: A GeT-RM Collaborative Project.

Pharmacogenetic testing is increasingly available from clinical laboratories. However, only a limited number of quality control and other reference materials are currently available to support clinical testing. To address this need, the Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, has characterized 137 genomic DNA samples for 28 genes commonly genotyped by pharmacogenetic testing assays (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4F2, DPYD, GSTM1, GSTP1, GSTT1, NAT1, NAT2, SLC15A2, SLC22A2, SLCO1B1, SLCO2B1, TPMT, UGT1A1, UGT2B7, UGT2B15, UGT2B17, and VKORC1). One hundred thirty-seven Coriell cell lines were selected based on ethnic diversity and partial genotype characterization from earlier testing. DNA samples were coded and distributed to volunteer testing laboratories for targeted genotyping using a number of commercially available and laboratory developed tests. Through consensus verification, we confirmed the presence of at least 108 variant pharmacogenetic alleles. These samples are also being characterized by other pharmacogenetic assays, including next-generation sequencing, which will be reported separately. Genotyping results were consistent among laboratories, with most differences in allele assignments attributed to assay design and variability in reported allele nomenclature, particularly for CYP2D6, UGT1A1, and VKORC1. These publicly available samples will help ensure the accuracy of pharmacogenetic testing.

Development of a genomic DNA reference material panel for Rett syndrome (MECP2-related disorders) genetic testing.

Rett syndrome is a dominant X-linked disorder caused by point mutations (approximately 80%) or by deletions or insertions (approximately 15% to 18%) in the MECP2 gene. It is most common in females but lethal in males, with a distinctly different phenotype. Rett syndrome patients have severe neurological and behavioral problems. Clinical genetic testing laboratories commonly use characterized genomic DNA reference materials to assure the quality of the testing process; however, none are commercially available for MECP2 genetic testing. The Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with the genetic testing community and the Coriell Cell Repositories, established 27 new cell lines and characterized the MECP2 mutations in these and in 8 previously available cell lines. DNA samples from the 35 cell lines were tested by eight clinical genetic testing laboratories using DNA sequence analysis and methods to assess copy number (multiplex ligation-dependent probe amplification, semiquantitative PCR, or array-based comparative genomic hybridization). The eight common point mutations known to cause approximately 60% of Rett syndrome cases were identified, as were other MECP2 variants, including deletions, duplications, and frame shift and splice-site mutations. Two of the 35 samples were from males with MECP2 duplications. These MECP2 and other characterized genomic DNA samples are publicly available from the NIGMS Repository at the Coriell Cell Repositories.

p53 prevents the accumulation of double-strand DNA breaks at stalled-replication forks induced by UV in human cells.

To investigate the mechanism by which UV irradiation causes S-phase-dependent chromosome aberrations and thereby genomic instability, we have developed an assay to study the DNA structure of replication forks (RFs) in UV-irradiated mammalian cells, using pulse-field gel electrophoresis for the DNA analysis. We demonstrate that replication stalling at UV-induced pyrimidine dimers results in the formation of single-strand DNA (ssDNA) regions and incomplete RF structures. In normal and in nucleotide-excision-repair (NER)-defective xeroderma pimentosum (XP) cells, stalling at dimers is rapid and prolonged and recovery depends on dimer repair or bypass. By contrast, XP variant (XPV) cells, defective in replication of a UV-damaged template due to mutation of bypass-polymerase epsilon, fail to arrest at dimers, resulting in a much higher frequency of ssDNA regions in the stalled RFs. We show that the stability of UV-arrested RFs depends directly on functional p53, and indirectly on NER and pol eta. In p53-deficient cells, the stalled sites give rise to double-strand DNA breaks (DSBs), at a frequency inversely correlated with repair capacity of the cell. In normal cells only a fraction of the stalled sites give rise to DSBs, while in XPASV, XPDSV and also XPVSV, all the sites do. XPVSV cells, although repair proficient, accumulate almost double the number of DSBs, suggesting that a high frequency of ssDNA regions in UV-arrested forks cause RF instability. These replication-associated DSBs do not accumulate in p53-proficient human cells. We propose that a major mechanism by which p53 maintains genome stability is the prevention of DSB accumulation at long-lived ssDNA regions in stalled-replication forks.