Thorsten Schwark

Reliable genetic identification of burnt human remains

The identification of severely burnt human remains by genetic fingerprinting is a common task in forensic routine work. In cases of extreme fire impact, only hard tissues (bones, teeth) may be left for DNA analysis. DNA extracted from burnt bone fragments may be highly degraded, making an amplification of genetic markers difficult or even impossible. Furthermore, heavily burnt bones are very prone to contamination with external DNA. We investigated whether authentic DNA profiles can be generated from human bones showing different stages of fire induced destruction (well preserved, semi-burnt, black burnt, blue-grey burnt, blue-grey-white burnt). DNA was extracted from 71 bone fragments derived from 13 individuals. Obtained genetic patterns (STRs and mtDNA sequences) were compared to the genetic pattern of the respective bodies. Our results show that the identification via DNA analysis is reliably and reproducibly possible from well preserved and semi-burnt bones. In black burnt bones the DNA was highly degraded and in some cases no nuclear DNA was left, leaving mitochondrial DNA analysis as an option. Blue-grey burnt bones lead only sporadically to authentic profiles. The investigation of blue-grey-white burnt bones barely led to reliable results.

Vesicular Monoamine Transporter 2 (VMAT2) Expression in Hematopoietic Cells and in Patients with Systemic Mastocytosis

Uptake of monoamines into secretory granules is mediated by the vesicular monoamine transporters VMAT1 and VMAT2. In this study, we analyzed their expression in inflammatory and hematopoietic cells and in patients suffering from systemic mastocytosis (SM) and chronic myelogenous leukemia (CML). Normal human and monkey tissue specimens and tissues from patients suffering from SM and CML were analyzed by means of immunohistochemistry, radioactive in situ hybridization, real time RT-PCR, double fluorescence confocal laser scanning microscopy, and immunoelectron microscopy. In normal tissue specimens, VMAT2, but not VMAT1, was expressed in mast cells, megakaryocytes, thrombocytes, basophil granulocytes, and cutaneous Langerhans cells. Further hematopoietic and lymphoid cells showed no expression of VMATs. VMAT2 was expressed in all types of SM, as indicated by coexpression with the mast cell marker tryptase. In CML, VMAT2 expression was retained in neoplastic megakaryocytes and basophil granulocytes. In conclusion, the identification of VMAT2 in mast cells, megakaryocytes, thrombocytes, basophil granulocytes, and cutaneous Langerhans cells provides evidence that these cells possess molecular mechanisms for monoamine storage and handling. VMAT2 identifies normal and neoplastic mast cells, megakaryocytes, and basophil granulocytes and may therefore become a valuable tool for the diagnosis of mastocytosis and malignant systemic diseases involving megakaryocytes and basophil granulocytes.

Regulation of activin A synthesis in microglial cells: Pathophysiological implications for bacterial meningitis

Previous studies have shown that activin A, a neuroprotective cytokine and dimeric polypeptide composed of two βA subunits, is elevated in the cerebrospinal fluid of patients suffering from bacterial meningitis. In this study, to elucidate further the functional significance and pathophysiological implications of these findings, we demonstrated that microglial cells are not only the source but also the target cells of activin A in the central nervous system: immunohistochemistry and RT‐PCR revealed expression of activin subunit βA mRNA as well as activin receptor type I and type II mRNA in rat microglia in vitro. Further studies showed that activin enhances microglial proliferation and decreases the γ‐interferon‐induced synthesis of nitric oxide, one of several microglial mediators involved in the inflammatory response in microglia activation. Furthermore, quantitative RT‐PCR, Western blotting, and ELISA showed an inhibitory effect of activin on inducible nitric oxide synthase, tumor necrosis factor‐α, interleukin‐6, and interleukin‐1β gene and protein levels after lipopolysaccharide treatment. We suggest that the increased synthesis of activin A is directly involved, via influence on microglia cell functions, in the modulation of the inflammatory response in bacterial meningitis.

A new multiplex-PCR comprising autosomal and y-specific STRs and mitochondrial DNA to analyze highly degraded material

The analysis of short tandem repeats is one of the most powerful tools in forensic genetics. Forensic practice sometimes requires the individualization of samples that may contain only highly degraded nuclear DNA, mitochondrial DNA or PCR inhibitors that hamper DNA amplification. We designed a new multiplex PCR with reduced size amplicons (<200 bp), providing a double sex determination (amelogenin plus two Y-STRs), the detection of two autosomal markers and the amplification of mitochondrial specific fragments from the hypervariable region I (HVI). Additionally, a quality sensor was developed to check for the presence of any PCR inhibitors. The new multiplex PCR shows a reproducible detection threshold down to 25 pg and gives signals even out of highly degraded materials. All signals are reproducible and reliable as it could be shown in comparison to results from commercially available STR multiplex-PCRs. In no case DNA fragments were detectable using any other assay when the quality sensor was not detectable. There was a good correlation between detection of mitochondrial specific fragments in the multiplex-PCR and success of subsequent sequencing of HVI region. The same could be shown for STR analysis: Most samples successfully analyzed in our PCR yielded at least a partial STR profile using a commercial STR kit. We present an assay that allows an easy, reliable, and cost efficient evaluation of DNA sample quality combined with a first rough sample individualization and sex determination suitable for forensic purposes. This assay may help the forensic lab personnel to decide on further sample processing.

Low level of the mtDNA4977 deletion in blood of exceptionally old individuals

The common 4977-bp deletion in mitochondrial DNA (dmtDNA(4977)) occurs frequently in tissues of high oxygen demand and low mitotic activity, e.g. brain, heart and skeletal muscle, where it appears to show an age-related accumulation. Although dmtDNA(4977) can also be detected in very low amounts in fast replicating tissues such as blood, it is still unclear whether an age-dependent distribution of dmtDNA(4977) occurs in blood. In view of these uncertainties, we investigated the presence of the mutation and changes in the dmtDNA(4977) level in whole blood samples from 473 individuals who belong to two different age groups, i.e. elderly (aged 61-75 years) and long-lived individuals (LLI, aged 95-109 years). We applied a highly sensitive and reliable duplex-PCR method that allowed relative quantification of dmtDNA(4977). For validation, we additionally performed absolute quantification on a subset of samples using real time-PCR. Our results showed that the proportion of dmtDNA(4977) carriers was very similar in both groups, but that the individual mutational load was on average much lower in the nonagenarians and centenarians than in the elderly. The finding was independent of smoking habits, gender or variation in APOE and FOXO3A but could be caused by other environmental and/or genetic factors.

A special family history

Y-STR analysis is a common tool in forensic case work. Lately it has also been popular in genealogical research to determine the male lineage. We present the investigation of members of a family with a pedigree dating back to the 14th century. The aim of the study was to genetically confirm the kinship established genealogically, a method that is mainly based on the fact that individuals of the male lineage carry the same family name. We investigated fifteen male individuals who were connected to approximately forty different branches of the family tree using the Powerplex Y kit (Promega) that allows the detection of eleven Y-STRs. Our study shows that there can be differences between genealogical and genetic pedigrees indicating the need for additional genetic analysis.

Genetic determination of sibship and twin zygosity in a case of an alleged double infant homicide

A 32 year-old woman was hospitalised with severe septic symptoms. The examination of the patient revealed placenta residues in the womans uterus as cause of the infection. The police was called and investigated her apartment in search of the missing baby. Finally, the bodies of two newborn babies were found in the freezer. According to the mother, the babies were stillborn twins. The autopsy revealed that the smaller, moderately putrefied child was stillborn, while the larger infant showed typical signs of drowning and only slight putrefaction. The placenta was judged by gynaecologists to be that of a single pregnancy. Thus the prosecution suspected that the woman had had two independent pregnancies and ordered an additional genetic analysis. A genetic analysis clearly revealed that the babies were monozygotic twins, supporting the mothers statement of a twin pregnancy.