Development of two age estimation models for buccal swab samples based on 3 CpG sites analyzed with pyrosequencing and minisequencing.

The analysis of DNA methylation levels of specific CpG sites is one of the most promising molecular techniques to estimate an individual's age. Numerous studies were published recently presenting age estimation models based on DNA methylation patterns from blood samples, with only a few using saliva or buccal swabs. The aim of this study was to identify age-dependent methylation of 88 CpG sites in eight different marker regions (PDE4C, ELOVL2, ITGA2B, ASPA, EDARADD, SST, KLF14 and SLC12A5) in buccal swab samples. A total of 141 buccal swabs from individuals with age ranging from 21 to 69 years were split into a training set (n = 95) and a validation set (n = 46). Samples of the training set were analyzed by pyrosequencing and markers with best age correlation were identified. Stepwise linear regression analysis was performed resulting in an age estimation model including three of the examined CpG sites and showing a mean absolute deviation of estimated from chronological age of 5.11 years. To allow easy implementation into forensic laboratories without the need for pyrosequencing equipment, a multiplex minisequencing reaction was developed, including the same CpG sites previously identified by pyrosequencing. An adjusted age estimation model was evaluated with a mean absolute deviation of estimated from chronological age of 5.16 years. The independent validation set of 46 buccal swab samples was used to test model performances. Mean absolute deviation of estimated from chronological age was 5.33 years and 6.44 years for the pyrosequencing model and the minisequencing model, respectively. Comparison of the two methods showed a high concordance of results, both, qualitatively and quantitatively. In conclusion, buccal swabs offer a suitable alternative to blood samples for molecular age estimation with the additional advantage of being collected non-invasively. Furthermore we showed that minisequencing offers a cost-effective and easy-to-integrate alternative to pyrosequencing for the analysis of methylation status of individual CpG sites.

Evaluation of the Ion AmpliSeq™ PhenoTrivium Panel: MPS-Based Assay for Ancestry and Phenotype Predictions Challenged by Casework Samples.

As the field of forensic DNA analysis has started to transition from genetics to genomics, new methods to aid in crime scene investigations have arisen. The development of informative single nucleotide polymorphism (SNP) markers has led the forensic community to question if DNA can be a reliable "eye-witness" and whether the data it provides can shed light on unknown perpetrators. We have developed an assay called the Ion AmpliSeq™ PhenoTrivium Panel, which combines three groups of markers: 41 phenotype- and 163 ancestry-informative autosomal SNPs together with 120 lineage-specific Y-SNPs. Here, we report the results of testing the assay's sensitivity and the predictions obtained for known reference samples. Moreover, we present the outcome of a blind study performed on real casework samples in order to understand the value and reliability of the information that would be provided to police investigators. Furthermore, we evaluated the accuracy of admixture prediction in Converge™ Software. The results show the panel to be a robust and sensitive assay which can be used to analyze casework samples. We conclude that the combination of the obtained predictions of phenotype, biogeographical ancestry, and male lineage can serve as a potential lead in challenging police investigations such as cold cases or cases with no suspect.

Marker evaluation for differentiation of blood and menstrual fluid by methylation-sensitive SNaPshot analysis.

The differentiation of blood and menstrual fluid is especially important in cases of alleged sexual assault. While the identification of blood is relatively straightforward, the identification of menstrual fluid in trace evidence has been shown to be more challenging. This may be due to the complex nature of menstrual fluid that leads to intra- and inter-individual differences in composition. Nevertheless, recent advances in DNA methylation profiling have revealed promising markers for the differentiation of the two body fluids and furthermore, markers to distinguish menstrual fluid from vaginal fluid. A literature study was performed and in total, 11 markers were evaluated in this study of which seven could be validated for menstrual fluid and blood identification purposes. Marker "BLU2" (chr16:29757334) was identified as most suitable for differentiation of blood and menstrual fluid.

Forensic differentiation between peripheral and menstrual blood in cases of alleged sexual assault-validating an immunochromatographic multiplex assay for simultaneous detection of human hemoglobin and D-dimer.

Sexual assault is a serious offense and identification of body fluids originating from sexual activity has been a crucial aspect of forensic investigations for a long time. While reliable tests for the detection of semen and saliva have been successfully implemented into forensic laboratories, the detection of other body fluids, such as vaginal or menstrual fluid, is more challenging. Especially, the discrimination between peripheral and menstrual blood can be highly relevant for police investigations because it provides potential evidence regarding the issue of consent. We report the forensic validation of an immunochromatographic test that allows for such discrimination in forensic stains, the SERATEC PMB test, and its performance on real casework samples. The PMB test is a duplex test combining human hemoglobin and D-dimer detection and was developed for the identification of blood and menstrual fluid, both at the crime scene and in the laboratory. The results of this study showed that the duplex D-dimer/hemoglobin assay reliably detects the presence of human hemoglobin and identifies samples containing menstrual fluid by detecting the presence of D-dimers. The method distinguished between menstrual and peripheral blood in a swab from a historical artifact and in real casework samples of alleged sexual assaults. Results show that the development of the new duplex test is a substantial progress towards analyzing and interpreting evidence from sexual assault cases.

Improving body fluid identification in forensic trace evidence-construction of an immunochromatographic test array to rapidly detect up to five body fluids simultaneously.

Body fluid identification is a substantial part of forensic trace analyses. The correct determination of the origin of a biological stain may give valuable information regarding the circumstances of a crime. A simple way to detect a body fluid in a stain is the use of immunochromatographic strip tests. They are easy to use, user-independent, quick, and cheap. Currently, however, it is only possible to analyze one body fluid at a time, requiring the analyst to make previous, possibly subjective, assumptions on the body fluid at hand. Also, identification of mixed body fluids requires the use of several tests, which results in additional sample and time consumption. To combine a simple approach with the possibility to simultaneously detect several body fluids, we constructed a combined immunochromatographic strip test array based on commercially available tests. The array rapidly detects up to five body fluids with a single analysis, and allowing for subsequent DNA extraction from the same material. With this test it was possible to identify the components of a mixture, the test was easily incorporated into standard laboratory work, and its sensitivity and specificity were shown to be comparable to those of conventional strip tests.

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.

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.