Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis.

The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.

European Network of Forensic Science Institutes (ENFSI): Evaluation of new commercial STR multiplexes that include the European Standard Set (ESS) of markers.

To support and to underpin the European initiative to increase the European set of standard markers (ESS), by the addition of five new loci, a collaborative project was organised by the European Network of Forensic Science Institutes (ENFSI) DNA working group in order to assess the new multiplex kits available. We have prepared allele frequency databases from 26 EU populations. Concordance studies were carried out to verify that genotyping results were consistent between kits. Population genetics studies were conducted and it was estimated that F(ST)<0.001. The results showed that the kits were comparable to each other in terms of performance and major discrepancy issues were highlighted. We provide details of allele frequencies for each of the populations analysed per laboratory.

Glycosylphosphatidylinositol (GPI)-deficient Jurkat T cells as a model to study functions of GPI-anchored proteins.

Many cell surface proteins attached to the membrane by GPI are involved in cell signalling. However, the role of the GPI membrane anchor itself remains poorly understood. GPI-defective cells from patients with paroxysmal nocturnal haemoglobinuria (PNH) are relatively resistant to apoptosis induction. We developed a Jurkat T cell model for GPI deficiency by isolating a GPI-negative mutant, which is defective in the GPI biosynthetic gene PIG-A. Using retroviral PIG-A gene transfer along with the transfer of a vector control, we obtained two genetically identical cell lines, distinguished only by expression of the PIG-A gene and, thus, their ability to produce GPI. Cell proliferation and survival were not affected by this difference. Apoptotic stimuli such as serum starvation and camptothecin exposure elicited similar responses. In contrast, GPI-defective Jurkat cells were more susceptible to Fas-mediated apoptosis than GPI-positive cells. These results indicate that a deficiency in GPI-anchored proteins, as is found in PNH, does not confer resistance to apoptosis.

Biosynthesis of glycosylphosphatidylinositols in mammals and unicellular microbes.

Membrane anchoring of cell surface proteins via glycosylphosphatidylinositol (GPI) occurs in all eukaryotic organisms. In addition, GPI-related glycophospholipids are important constituents of the glycan coat of certain protozoa. Defects in GPI biosynthesis can retard, if not abolish growth of these organisms. In humans, a defect in GPI biosynthesis can cause paroxysmal nocturnal hemoglobinuria (PNH), a severe acquired bone marrow disorder. Here, we review advances in the characterization of GPI biosynthesis in parasitic protozoa, yeast and mammalian cells. The GPI core structure as well as the major steps in its biosynthesis are conserved throughout evolution. However, there are significant biosynthetic differences between mammals and microbes. First indications are that these differences could be exploited as targets in the design of novel pharmacotherapeutics that selectively inhibit GPI biosynthesis in unicellular microbes.

The maize caffeic acid O-methyltransferase gene promoter is active in transgenic tobacco and maize plant tissues.

The pattern of expression directed by the promoter of the maize caffeic acid O-methyltransferase (COMT) gene was studied by histochemical and fluorometric beta-glucuronidase (GUS) analysis in transgenic maize and tobacco plants. The COMT promoter directs GUS expression to the xylem and the other tissues undergoing lignification, and it responds to wounding and to elicitors. In transgenic maize plants, expression of GUS corresponds to the pattern of expression of the endogenous COMT gene as determined by northern analysis and in situ hybridization. The pattern in transgenic tobacco plants clearly shows that the maize promoter sequence is recognized by tobacco transcriptional factors, in spite of the anatomical differences and the evolutionary distance between these two species. The results suggest that the most significant promoter signals that induce the specific expression of the lignin COMT are conserved in different species.