Search

Background: Prostate-specific antigen (PSA), a widely used biomarker for prostate cancer (PCa), is encoded by a kallikrein gene (KLK3, kallikrein-related peptidase 3). Serum PSA concentrations vary in the population, with PCa patients generally showing higher PSA concentrations than control individuals, although a small proportion of individuals in the population display very low PSA concentrations. We hypothesized that very low PSA concentrations might reflect gene-inactivating mutations in KLK3 that lead to abnormally reduced gene expression., Methods: We have sequenced all KLK3 exons and the promoter and searched for gross deletions or duplications in KLK3 in the 30 individuals with the lowest observed PSA concentrations in a sample of approximately 85 000 men from the Prostate Testing for Cancer and Treatment (ProtecT) study. The ProtecT study examines a community-based population of men from across the UK with little prior PSA testing., Results: We observed no stop codons or frameshift mutations, but we did find 30 single-base genetic variants, including 3 variants not described previously. These variants included missense variants that could be functionally inactivating and splicing variants. At this stage, however, we cannot confidently conclude whether these variants markedly lower PSA concentration or activity. More importantly, we identified 3 individuals with different large heterozygous deletions that encompass all KLK3 exons. The absence of a functional copy of KLK3 in these individuals is consistent with their reduced serum PSA concentrations., Conclusions: The clinical interpretation of the PSA test for individuals with KLK3 gene inactivation could lead to false-negative PSA findings used for screening, diagnosis, or monitoring of PCa., (© 2012 American Association for Clinical Chemistry)

Background: Apolipoprotein E (APOE) is an important element of lipid metabolism and, hence, cardiovascular disorders. APOE has 3 main allelic variants: epsilon3, epsilon4, and epsilon2. Of these, epsilon3 is the most common, followed by epsilon4 and epsilon2. The associations of these isoforms with cardiovascular disorders and Alzheimer disease have been widely studied in different populations. Most of the genotyping in these studies has been performed with gel-based methods, which have important limitations, particularly for large epidemiologic studies. We therefore developed an integrated "one-tube" liquid-phase assay., Methods: To measure APOE isoforms, we developed an integrated single-label liquid-phase fluorescence assay containing 2 PCR primers, 2 probes, and 2 quencher oligonucleotides. We used a 384-well LightTyper, but the assay would be generically applicable for use with any fluorescence detector with thermal ramp control. We validated this method and applied it in the British Women's Heart and Health Study., Results: There were 4 melting peaks, at 41, 56, 61, and 69 degrees C, which generated 6 distinctive patterns representing genotypic combinations of epsilon3, epsilon4, and epsilon2. The magnitude and direction of the associations found with total cholesterol, HDL-cholesterol, triglycerides, and estimated LDL-cholesterol were consistent with previous reports., Conclusion: The one-tube LightTyper assay presented here enables accurate, convenient, and economical genotyping of APOE and can be used for large epidemiologic studies.