Birgit Bayer

Identification of the skeletal remains of Martin Bormann by mtDNA analysis.

Abstract Contrary to statements of an eye-witness who reported that Martin Bormann, the second most powerful man in the Third Reich, died on 2 May 1945 in Berlin, rumours persisted over the years that he had escaped from Germany after World War II. In 1972, skeletal remains were found during construction work, and by investigating the teeth and the bones experts concluded that they were from Bormann. Nevertheless, new rumours arose and in order to end this speculation we were commissioned to identify the skeletal remains by mitochondrial DNA analysis. The comparison of the sequence of HV1 and HV2 from the skeletal remains and a living maternal relative of Martin Bormann revealed no differences and this sequence was not found in 1500 Caucasoid reference sequences. Based on this investigation, we support the hypothesis that the skeletal remains are those of Martin Bormann.

Sex-specific fluorescent labelling of cells for laser microdissection and DNA profiling

Sex-specific isolation of cells from mixtures would greatly facilitate forensic casework. Thus, male and female cell mixtures were marked with a fluorescent X/Y-probe CEP X SpectrumOrange/Y SpectrumGreen DNA probe kit for fluorescence in situ hybridization, and single cells were isolated via laser microdissection (LMD). DNA profiling of LMD isolated, hybridized cells showed usable short tandem repeat profiles for at least 20 cells, which are comparable with results from other studies. To simulate casework samples, the method was also optimized for air-dried samples.

Digoxigenin labelling and laser capture microdissection of male cells

Laser capture microdissection (LMD) is a relatively new technique for the isolation of single cells. The application in forensic investigations has become more and more widespread, especially to select spermatozoa out of mixtures with vaginal cells. In particular in cases with low numbers of sperm it could be profitable to isolate all male cells (e.g. sperm and male epithelial cells) instead of focussing on the sperm only. Therefore, the specific labelling and detection of the male cells in a male/female cell mixture is necessary. In order to label all cells carrying a Y-chromosome we used a digoxigenin labelled chromosome Y hybridisation probe (Q Biogen). The stained cells were isolated with the SL μCut LMD system from Molecular Machines & Industries AG (MMI). At least ten diploid male cells were required to obtain a partial STR profile, with 20 cells, a full profile could be obtained.

Decidual Macrophages Are Significantly Increased in Spontaneous Miscarriages and Over-Express FasL: A Potential Role for Macrophages in Trophoblast Apoptosis

Decidual macrophages (DM) are the second most abundant population in the fetal-maternal interface. Their role has been so far identified as being local immuno-modulators favoring the maternal tolerance to the fetus. Herein we investigated tissue samples from 11 cases of spontaneous miscarriages and from 9 cases of elective terminations of pregnancy. Using immunohistochemistry and dual immunofluorescence we have demonstrated that in spontaneous miscarriages the DM are significantly increased. Additionally, we noted a significant up-regulation of macrophage FasL expression. Our results further support a dual role for DM during pregnancy and miscarriages. We hypothesize that the baseline DM population in normal pregnancy is in line with an M2 phenotype supporting the ongoing gestation. In contrast, during spontaneous miscarriages, the increased FasL-expressing population could be a part of an M1 phenotype participating in Fas/FasL-related apoptosis. Our results highlight a new aspect of macrophage biology in pregnancy physiology and pathophysiology. Further studies with larger samples are needed to verify the current results and evaluate their clinical impact.

Ninhydrin treatment as a screening method for the suitability of swabs taken from contact stains for DNA analysis

More and more swabs containing unknown traces of biological material are submitted for forensic DNA analysis. Most of the samples are swabs taken from handled items such as tools, weapons and handles etc. Therefore, we tried to develop a screening method in order to focus the investigation on samples containing biomolecules, such as amino acids which might be associated with nucleic acids. A total of 285 swabs taken from various items collected during crime scene investigations were treated with ninhydrin which leads to a purple colour for samples containing amino acids. Of the swabs 158 were classified as ninhydrin positive and 76% of these samples yielded DNA profiles that fulfil the criteria for inclusion in the German national DNA database (profile frequency greater than 1 in 100,000) or in DNA mixtures which could at least be compared with suspects. In comparison only 9% of the 127 samples shown to be ninhydrin negative, revealed a usable DNA profile. Consequently, ninhydrin treatment was found to be an effective screening method which resulted in an increase in the rate of successfully typed samples and subsequently in a reduction of the costs due to the lower number of samples that needed to be typed.

Expression of Thyroid Hormone Receptors in Villous Trophoblasts and Decidual Tissue at Protein and mRNA Levels Is Downregulated in Spontaneous and Recurrent Miscarriages

Thyroid hormones are essential for the maintenance of pregnancy, and a deficiency in maternal thyroid hormones has been associated with early pregnancy losses. The expression of THRα1, THRβ1 and THRα2 increases with gestational age. The aim of this study was the investigation of the protein and mRNA-levels of THR isoforms THRα1, THRα2, THRβ1 and THRβ2 in normal, spontaneous and recurrent miscarriages. The identification of THR-expressing cells in the decidua was done with double immunofluorescence. The nuclear expression of THRα1, THRα2, THRβ1 and THRβ2 is downregulated at protein level in spontaneous and recurrent miscarriages in villous trophoblast tissue. In decidual tissue, we found a significant downregulation only for THRα1 in spontaneous miscarriages. For recurrent miscarriages, THRα1 and THRβ1 were both significantly downregulated in decidual tissue. By applying HLA-G as a trophoblast marker, we found a significant co-expression only for THRβ2. The results of our study show that thyroid hormone receptors THRα1, THRα2, THRβ1 and THRβ2 are downregulated in spontaneous and recurrent miscarriages. The majority of cells expressing the thyroid hormone receptors in the decidua are decidual stromal cells.

The expression of thyroid hormone receptors (THR) is regulated by the progesterone receptor system in first trimester placental tissue and in BeWo cells in vitro.

BACKGROUND Thyroid hormones are essential for the maintenance of pregnancy and a deficiency in maternal thyroid hormones has been associated with early pregnancy losses. The aim of this study was a systematic investigation of the influence of mifepristone (RU 486) on the expression of the thyroid hormone receptor (THR) isoforms THRα1, THRα2, THRβ1 and THRβ2 on protein and mRNA-level. METHODS Samples of placental tissue were obtained from patients with mifepristone induced termination of pregnancy (n=13) or mechanical induced termination of normal pregnancy (n=20), each from the 4th to 13th week of pregnancy. Expression of THRα1, THRα2, THRβ1 and THRβ2 was analysed on protein level by immunohistochemistry and on mRNA level by real time RT-PCR (TaqMan). The influence of progesterone on THR gene expression was analysed in the trophoblast tumour cell line BeWo by real time RT-PCR (TaqMan). RESULTS Nuclear expression of THRα1, THRα2 and THRβ1 is downregulated on protein level in mifepristone (RU 486) treated villous trophoblast tissue. In decidual tissue, we found a significant downregulation only for THRα1 in mifepristone treated tissue. On mRNA level, we also found a significantly reduced expression of THRA but no significant downregulation for THRB in placental tissue. The gene THRA encodes the isoform THRα and the gene THRB encodes the isoform THRβ. The majority of cells expressing the thyroid hormone receptors in the decidua are decidual stromal cells. In addition, in vitro experiments with trophoblast tumour cells showed that progesterone significantly induced THRA but not THRB expression. CONCLUSIONS Termination of pregnancy with mifepristone (RU 486) leads to a downregulation of THRα1, THRα2 and THRβ1 in villous trophoblasts and in addition to a decreased expression of THRA in placental tissue. Decreased expression of THRα1 induced by RU486 could also be found in the decidua. Therefore inhibition of the progesterone receptor may be responsible for this downregulation. This assumption is supported by the finding, that stimulation of the progesterone receptor by progesterone itself up-regulated THRA in trophoblast cells in vitro.

Mass spectrometric base composition profiling: Implications for forensic mtDNA databasing

In forensic genetics mitochondrial DNA (mtDNA) is usually analyzed by direct Sanger-type sequencing (STS). This method is known to be laborious and sometimes prone to human error. Alternative methods have been proposed that lead to faster results. Among these are methods that involve mass-spectrometry resulting in base composition profiles that are, by definition, less informative than the full nucleotide sequence. Here, we applied a highly automated electrospray ionization mass spectrometry (ESI-MS) system (PLEX-ID) to an mtDNA population study to compare its performance with respect to throughput and concordance to STS. We found that the loss of information power was relatively low compared to the gain in speed and analytical standardization. The detection of point and length heteroplasmy turned out to be roughly comparable between the technologies with some individual differences related to the processes. We confirm that ESI-MS provides a valuable platform for analyzing mtDNA variation that can also be applied in the forensic context.

Comparative investigation of hair with the genRES® MPX-SP1, genRES MPX-SP2, and genRES MPX-2 kits

In recent years there has been considerable improvement in short-tandem repeat (STR) investigations of hair, which were previously marred by small amounts of nuclear DNA and its degradation. This study examined the suitability of two STR kits with shortened amplicons for the investigation of hairs from routine casework. The overall sucess rate was more than 20%. Furthermore, the usefulness of quantification with real-time ploymerase chain reaction as a screening method was demonstrated.

Comparison of telogen hair analyses: genRES® MPX-2SP kit versus genRES® MPX-SP1 and genRES® MPX-SP2 kits

STR investigations of telogen hair are invariably difficult due to the small amounts of nuclear DNA and its degradation products. However, in recent years there has been a considerable improvement. This study examined the suitability of a new STR kit with shortened amplicons for the investigation of hair in routine casework. This kit allows the simultaneous amplification of the eight STR-loci D3S1358, VWA, FGA, TH01, SE33, D8S1179, D18S51, and D21S11, and the sex-determining amelogenin system. It was tested against the genRES MPX-SP1 and genRES MPX-SP2 kits. The sensitivity of the new genRES MPX-2SP kit was demonstrated to be inferior to that of the genRES MPX-SP1, but almost equal to that of the genRES MPX-SP2 kit.