Multiplex analysis of genetic polymorphisms within UGT1A9, a gene involved in phase II of Δ9-THC metabolism.

We present a novel multiplex assay for the simultaneous detection of 12 polymorphisms within the UGT1A9 sequence, which codes for enzymes involved in phase II biotransformation. The assay combines a multiplexed amplification step with single-base extension sequencing. The method described here is fast, cost-effective, and easy-to-use, combining the relevant features of screening methods for research and diagnostics in pharmacogenetics. To validate the assay, we tested reproducibility and sensitivity and analysed allele frequencies of 110 Caucasian individuals. Furthermore, we describe combining genetic information of individuals consuming Cannabis sativa products with respective plasma concentrations of a metabolite.

Sudden infant death syndrome: exposure to cigarette smoke leads to hypomethylation upstream of the growth factor independent 1 (GFI1) gene promoter.

PURPOSE: Smoking during pregnancy has long been known as an important risk factor for sudden infant death syndrome (SIDS). However, the precise relationship between the smoking behavior of the mother and SIDS still remains unclear. In this study, the influence of prenatal smoking exposure on the childrens' DNA methylation state of a CpG island located upstream of the promoter of the growth factor independent 1 (GFI1) gene was analyzed. METHODS: Blood samples of well-defined SIDS cases with non-smoking mothers (n = 11), SIDS cases with smoking mothers during pregnancy (n = 11), and non-SIDS cases (n = 6) were obtained from a previous study and methylation states were determined by bisulfite sequencing. RESULTS: Significant hypomethylation was observed in this CpG island in SIDS cases with cigarette smoke exposure compared to non-exposed cases. The strongest effect in this CpG island was observed for 49 CpG sites located within a transcription factor binding site. Coding for a transcriptional repressor, GFI1 plays an important role in various developmental processes. Alterations in the GFI1 expression might be linked to various conditions that are known to be associated with SIDS, such as dysregulated hematopoiesis and excessive inflammatory response. CONCLUSION: Data obtained in this study show that analysis of methylation states in cases of sudden infant death syndrome might provide a further important piece of knowledge toward understanding SIDS, and should be investigated in further studies.

Elevated germline mutation rate in teenage fathers.

Men age and die, while cells in their germline are programmed to be immortal. To elucidate how germ cells maintain viable DNA despite increasing parental age, we analysed DNA from 24 097 parents and their children, from Europe, the Middle East and Africa. We chose repetitive microsatellite DNA that mutates (unlike point mutations) only as a result of cellular replication, providing us with a natural 'cell-cycle counter'. We observe, as expected, that the overall mutation rate for fathers is seven times higher than for mothers. Also as expected, mothers have a low and lifelong constant DNA mutation rate. Surprisingly, however, we discover that (i) teenage fathers already set out from a much higher mutation rate than teenage mothers (potentially equivalent to 77-196 male germline cell divisions by puberty); and (ii) ageing men maintain sperm DNA quality similar to that of teenagers, presumably by using fresh batches of stem cells known as 'A-dark spermatogonia'.

Validation of an immunochromatographic D-dimer test to presumptively identify menstrual fluid in forensic exhibits.

Identifying the biological source of a crime scene stain can be crucial for police investigations in many scenarios. Blood is one of the most common fluids found, and accurate differentiation between peripheral blood and menstrual fluid could provide valuable information regarding the issue of consent in sexual assault cases. For the detection of menstrual fluid, no easy-to-use presumptive test is available to date. Therefore, this study aimed to validate a simple immunochromatographic test for the indication of menstrual fluid, focusing on a D-dimer assay. The Clearview® rapid D-dimer test provides a diagnostic assay for the detection of fibrin degradation products. We validated the sensitivity and robustness of the assay using fresh and dried menstrual fluid samples, body fluid mixtures, diluted samples, and casework swabs. Cross reactivity was tested for saliva, semen, vaginal fluid, and blood. No false positive results were obtained; it was possible to successfully analyze mixtures, highly diluted samples, and casework swabs. The results of this study indicate that the D-dimer assay reliably detects menstrual fluid in forensic exhibits and is easy to implement into the current workflow of body fluid identification.

Meiosis study in a population sample from Nigeria: allele frequencies and mutation rates of 16 STR loci.

Allele frequencies for the 16 short tandem repeat (STR) loci D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, ACTBP2, CSF1PO, FGA, TH01, TPOX and VWA were determined for 337 immigrants from Nigeria. All loci were in Hardy-Weinberg equilibrium. More than 6,000 meiotic transfers were investigated and ten mutations were observed. Single mutations were observed in the STR systems D2S1338, D3S1358, D7S820, D8S1179, D16S539 and FGA, whereas two mutations were observed in the systems D21S11 and VWA.

Secondary DNA transfer by working gloves.

With the development of highly sensitive STR profiling methods, combined with sound statistical tools, DNA analysis on the (sub-)source level is hardly ever seriously questioned in court. More often, the exact mode of DNA transfer to the crime scene is questioned. In burglary cases, in particular when gloves are worn, secondary DNA transfer is often discussed as explanation for finding a DNA profile matching the accused because it is well known that gloves can act as a potential vector for indirect DNA transfer. In this study we investigated the shedder status as a possible factor influencing the extent of secondary DNA transfer to a crime scene, with the person committing the crime wearing working gloves. Firstly, the shedder status for 40 participants (20 male, 20 female) was determined, following a previously published procedure. Good shedders (n = 12) were found to deposit a higher amount and quality of DNA onto objects, compared to bad shedders (n = 25). Secondly, participants were paired into four groups (good with good; good with bad; bad with good; bad with bad), each group consisting of five pairs. The first participant (P1) of each pair used working gloves to pack and carry a box to simulate a house move. Two days later, the second participant (P2) of the pair wore the same pair of gloves to simulate a burglary, using a screwdriver as a break-in tool. After taking swabs of the outside and inside of a glove (primary DNA transfer) and the handle of the screwdriver (secondary DNA transfer), full DNA analysis was performed. Our experiments show that good shedders, overall, deposit more DNA than bad shedders, both onto the outside and the inside of the glove, regardless of being P1 or P2. When conducting the experiments with two participants sharing the same shedder status, no significant differences occurred in the number of deposited alleles. In six out of 19 cases a DNA profile matching P1 was found (binary LR>106) on the screwdriver and in all six cases P1 was a good shedder. Our results indicate that the shedder status of an individual affects the extent of DNA transfer. They further confirm the possibility of an innocent person's DNA profile being found on an object they never handled.

Independent validation of body fluid-specific CpG markers and construction of a robust multiplex assay.

Potential forensic use of tissue-specific DNA methylation markers has recently been discussed for the identification of the biological source of a stain. In this study 13 promising markers were evaluated to identify suitable candidate markers for the development of a robust and reliable multiplex assay. The results of this study suggest that a combination of only four highly informative markers will be enough for clear body fluid identification. A multiplex assay was developed for the identification of menstrual blood, saliva, semen, and venous blood. This assay was successfully applied to the identification of these body fluids in mixtures and crime scene stains. The multiplex assay aids in the identification of not only single source body fluids but also of body fluid mixtures. The main advantage of using DNA methylation assays over alternative tests is that it can be applied at a later time point in the investigative process since testing is possible even after DNA analysis.

Meiosis study in a population sample from Afghanistan: allele frequencies and mutation rates of 16 STR loci.

The 16 short tandem repeat systems D3S1358, VWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820, ACTBP2, D2S1338, D16S539, D19S433, D21S11, D18S51 and D8S1179 were amplified in a population sample composed of 333 immigrants from Afghanistan. The 16 loci met Hardy-Weinberg expectations and possess a combined matching probability of 1 in 3.6 x 10(14) and a combined mean exclusion chance greater than 0.9996 in this Afghan population. Approximately 12,000 meiotic transfers were investigated and 19 mutations were observed in the repeat units of FGA (n=6), ACTBP2 (n=5), D3S1358 (n=2), D5S818 (n=2), D7S820 (n=2), VWA (n=1) and D8S1179 (n=1).

The GEDNAP (German DNA profiling group) blind trial concept.

This paper presents a review of the organisation and background of blind trial systems in general and in particular the system developed in Münster originally for the German Society of Forensic Medicine. This system, known as GEDNAP (German DNA profiling group), has now evolved into a multinational DNA blind trial open to all laboratories involved in paternity and forensic DNA testing.