Processing of voided urine for prostate cancer RNA biomarker analysis.

BACKGROUND: Voided urine samples have been shown to contain cells released from prostate tumors. Could good quality RNA from cells in urine be obtained from every donor for multimarker analysis? In addition, could urine donation be as simple as possible, a practical consideration for a lab test, without involving a prostate massage (as indicated for PCA3 testing), which precludes frequent collection; needing it done at a specific time of day (e.g., first or second urine); and requiring prompt processing of samples in clinics with limited molecular biology capability? METHODS: Collected urine samples were pelleted, and the RNA isolated was processed for cDNA synthesis and in vitro transcription to generate amplified sense aRNA. The resultant aRNA was rigorously analyzed for possible introduced changes. DMSO was used as a cell preservative for frozen storage of urine samples. RESULTS: Good quality aRNA was obtained for over 100 samples collected at two different institutions. The process of RNA amplification removed co-isolated DNA in some samples, which did not affect RNA amplification. Amplification did not amplify genes that were absent and produce other expression alterations. The sense aRNA could be used to generate urinary transcriptomes specific to individual patients. No chaotropic agents for RNA preservation were added to the urine samples so that the supernatant could be used for analysis of secreted protein biomarkers. The time of donation was not important since patients were seen during the entire day. DMSO was an effective cell preservative for freezing urine. CONCLUSIONS: Urinary RNA can be readily isolated and amplified for prostate cancer biomarker analysis. Individual patients had unique set of transcripts derived from their tumor.

Clinical and cellular phenotypes associated with sequestosome 1 (SQSTM1) mutations.

Familial Paget's disease of bone has been shown to be associated with mutations in the ubiquitin-associated (UBA) domain of the sequestosome 1 (SQSTM1) gene. We have clinical findings on five families with diverse racial and ethnic backgrounds who all harbor SQSTM1 UBA domain mutations (P387L, P392L, D391fsX394, P392fsX394). Intrafamilial expressivity was highly variable. The probands in two of the families had early-onset disease involving a large number of bones and highly elevated prediagnostic levels of serum alkaline phosphatase. Affected siblings in these same families had limited bone involvement and were only diagnosed by technetium-99m methylene diphosphonate (MDP) bone scans. Furthermore, there was at least one subject in each family with no evidence of Paget's disease, although they carried one mutated copy of the SQSTM1 gene. A total of 18 such individuals were identified across the five kindreds. Thus, the gene seems to have highly variable expressivity, as well as incomplete penetrance, supporting the role of this gene as a predisposition gene for familial Paget's disease of the bone. Molecular studies of the SQSTM1 protein showed different cellular aggregation phenotypes depending on the nature of the mutation. In general, the point mutations formed larger cytoplasmic aggregates than the wildtype or truncation mutations. This aggregation phenotype was not altered on removal of the N-terminal PB1 dimerization domain, implying that aggregate formation is not wholly mediated by interaction through the PB1 domain. Although there was a genotype/phenotype correlation on the cellular level, this was not apparent on the clinical level. This supports the argument that other nongenetic factors play an important role in the pathogenesis of the disease.

Characterization of an Epstein-Barr virus-related gammaherpesvirus from common marmoset (Callithrix jacchus).

A gammaherpesvirus related to Epstein-Barr virus (EBV; Human herpesvirus 4) infects otherwise healthy common marmosets (Callithrix jacchus). Long-term culture of common marmoset peripheral blood lymphocytes resulted in outgrowth of spontaneously immortalized lymphoblastoid cell lines, primarily of B cell lineage. Electron microscopy of cells and supernatants showed herpesvirus particles. There were high rates of serological cross-reactivity to other herpesviruses (68-86%), but with very low geometric mean antibody titres [1:12 to human herpesvirus 6 and 1:14 to Herpesvirus papio (Cercopithecine herpesvirus 12)]. Sequence analysis of the conserved herpesvirus DNA polymerase gene showed that the virus is a member of the lymphocryptovirus subgroup and is most closely related to a lymphocryptovirus from rhesus macaques and is closely related to EBV and Herpesvirus papio. High seroprevalence (79%, with geometric mean antibody titre of 1:110) among 28 common marmosets from two geographically distinct colonies indicated that the virus is likely present in many common marmosets in captivity. A New World primate harbouring a lymphocryptovirus suggests that this subgroup arose much earlier than previously thought.