Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturer's assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research. Many laboratories are testing for pharmacogenetic (PGx) markers, common genetic variants that are usually considered only when a patient is likely to be exposed to a Accepted for publication June 21, 2010. R.B., A.E.-B., C.S., A.V., and M.Z. are employees of AutoGenomics (manufacturer of several pharmacogenetic assays used in this study); M.B., A.B., and K.M. are employees of Quest Diagnostics Inc.; J.M. is an employee of Idaho Technology (manufacturer of the reagents used to genotype CYP2C9 and VKORC1 loci for this project);

Background Diff-Quik stained fine-needle aspiration (FNA) smears and touch preparations from biopsies represent alternative specimens for molecular testing when cell block or biopsy material is insufficient. We describe the use of these samples for targeted next-generation sequencing (NGS) of primary and metastatic lung adenocarcinoma and report DNA quality and success rates of FNA smears compared with other specimens from one year of clinical use. Methods A validation set of 10 slides from 9 patients with prior clinical EGFR Sanger sequencing and KRAS pyrosequencing (5 KRAS +/EGFR−, 4 KRAS/EGFR−) underwent DNA extraction, quality assessment, and targeted NGS. Subseqently, lung adenocarcinoma specimens submitted for NGS solid tumor mutation panel testing in one calendar year (60 biopsies, 57 resections, 33 FNA cell blocks, 12 FNA smears, 10 body fluid cell blocks) were reviewed for specimen adequacy, sequencing success, and DNA quality. Results All 10 validation samples met the DNA quality threshold (ΔCT threshold <8, range −2.2 to 4.9) and yielded 0.5 to 22 μg of DNA. KRAS and EGFR mutation status from FNA smears by NGS were concordant with previous clinical testing for all 10 samples. In the one year review, FNA smears were 100% successful, suggesting performance equivalent to or better than established specimen types, including FNA cell blocks. DNA quality by ΔCT was significantly better from FNA smears than from biopsies, resections, and FNA cell blocks. Conclusions We conclude that FNA smears of lung adenocarcinomas are a high quality alternative specimen for a targeted NGS panel with a high success rate in clinical practice.

We have isolated a protein from Saccharomyces cerevisiae that binds specifically to a nucleotide sequence associated with the autonomously replicating sequence (ARS) ARS120, located in the telomeric region of a yeast chromosome. "Footprinting" analysis revealed that a 26-basepair DNA sequence, 5'-CAAGTGCCGTGCATAATGATGT-GGGT-3', was protected by this protein from DNase I digestion. A plasmid containing 48 direct tandem repeats of this oligonucleotide was constructed and used to affinity-purify the binding activity. The purified protein, OBF1 (origin binding factor), showed specific binding to ARS120. The 26-base-pair OBF1-protected sequence was sufficient for the recognition and binding of the protein, since the mobility of a DNA fragment containing the synthetic binding site was retarded in agarose gels when incubated with OBF1. By performing competition experiments with a number of different ARSs, we showed that OBF1 binds tightly to some but not all ARSs. Interestingly, OBF1 does not appear to have a discernible affinity for ARS) or the ARSs associated with mating type loci, HMLa and HMRa, which are substrates for a DNA-binding activity reported by others. Since OBF1 appears to bind to DNA associated with a number of ARSs, we suggest that this protein may have a function related to ARS activity, perhaps in the initiation of DNA replication at selected ARSs.

Congenital human cytomegalovirus (HCMV) infection affects 1% of children and is the most common infectious cause of sensorineural hearing loss. Due to the difficulty of diagnosing deafness and other neurological disorders in infants , affected individuals may not be recognized until much later when active infection has resolved and culture is no longer informative. To overcome this problem , congenital HCMV infection was diagnosed retrospectively by testing residual blood samples collected from newborns and dried on perinatal cards as part of the North Carolina Newborn Screening Program. We modified the Qiagen method for purifying DNA from dried blood spots to increase the sample size and recovery of the lysate. A multiplex , real-time TaqMan polymerase chain reaction assay on an ABI 7900 instrument measured a highly conserved segment of the HCMV polymerase gene and the APOB human control gene. HCMV DNA was detected in blood dried on perinatal cards from all seven infants with culture-proven congenital infection , and all 24 negative control cases lacked detectable HCMV DNA. Our findings suggest that it is possible to diagnose congenital HCMV infection using dried blood collected up to 20 months earlier.

Comparative chromosomal mapping studies and investigations of tumor-associated chromosomal abnormalities suggest that the development of hepatic tumors in humans and rats may share a common molecular mechanism that involves inactivation of the same tumor suppressor genes or common genetic loci. We investigated the potential of human chromosomes 2 and 11 to suppress the tumorigenic phenotype of rat liver epithelial tumor cell lines. These tumor cell lines (GN6TF and GP7TB) display elevated saturation densities in culture, efficiently form colonies in soft agar, and produce subcutaneous tumors in 100% of syngeneic rat hosts with short latency periods. Introduction of human chromosome 11 by microcell fusion markedly altered the tumorigenicity and the transformed phenotype of GN6TF cells. In contrast, the tumorigenic potential and phenotype of GP7TB cells was unaffected by the introduction of human chromosome 11, indicating that not all rat liver tumor cell lines can be suppressed by loci carried on this chromosome. Introduction of human chromosome 2 had little or no effect on the tumorigenicity or cellular phenotype of either tumor cell line, suggesting the involvement of chromosome 11-specific loci in the suppression of the GN6TF tumor cell line. The GN6TF-11neo microcell hybrid cell lines displayed significantly reduced saturation densities in monolayer cultures, and their ability to grow in soft agar was completely inhibited. Although GN6TF-11neo cells ultimately formed tumors in 80-100% of syngeneic rat hosts, the latency period for tumor formation was much longer. Molecular characterization of GN6TF-11neo microcell hybrid cell lines indicated that some of the clonal lines had spontaneously lost significant portions of the introduced human chromosome, partially delineating the chromosomal location of the putative tumor suppressor locus to the region between the centromere and 11p12. Molecular examination of microcell hybrid-derived tumor cell lines further defined the minimal portion of human chromosome 11 capable of tumor suppression in this model system to the region 11p11.2-p12.