Ulrike Schmidt

Online reference database of European Y-chromosomal short tandem repeat (STR) haplotypes

The reference database of highly informative Y-chromosomal short tandem repeat (STR) haplotypes (YHRD), available online at http://ystr.charite.de, represents the largest collection of male-specific genetic profiles currently available for European populations. By September 2000, YHRD contained 4688 9-locus (so-called minimal) haplotypes, 40% of which have been extended further to include two additional loci. Establishment of YHRD has been facilitated by the joint efforts of 31 forensic and anthropological institutions. All contributing laboratories have agreed to standardize their Y-STR haplotyping protocols and to participate in a quality assurance exercise prior to the inclusion of any data. In view of its collaborative character, and in order to put YHRD to its intended use, viz. the support of forensic caseworkers in their routine decision-making process, the database has been made publicly available via the Internet in February 2000. Online searches for complete or partial Y-STR haplotypes from evidentiary or non-probative material can be performed on a non-commercial basis, and yield observed haplotype counts as well as extrapolated population frequency estimates. In addition, the YHRD website provides information about the quality control test, genotyping protocols, haplotype formats and informativity, population genetic analysis, literature references, and a list of contact addresses of the contributing laboratories.

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmonier’s algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.

Successful RNA extraction from various human postmortem tissues

Recently, several authors described the observation that RNA degradation does not correlate with the postmortem interval (PMI), but rather with other parameters like environmental impact and the circumstances of death. Therefore, the question arose if the analysis of gene expression could be a valuable tool in forensic genetics to contribute to the determination of the cause of death. In our study, six human tissues obtained from six individuals with PMI varying between 15 and 118xa0h were used for total RNA extraction. Quantification was performed using a GAPDH real-time assay, and the quality of mRNA was checked by amplification of different fragment lengths of the GAPDH transcript. In our set of samples, nearly all tissues in all PMI revealed satisfactory results, while skeletal muscle, followed by brain and heart, gave the best results. No correlation between PMI and RNA degradation could be detected, as very good results were observed for all tissues from the individual with the longest PMI. The highly promising results obtained in this study raise hopes that in the near future several fields of forensic investigation may profit from additional information about gene expression patterns and their correlation with pathological findings.

Validation of adequate endogenous reference genes for the normalisation of qPCR gene expression data in human post mortem tissue

Gene expression analyses based on messenger RNA (mRNA) profiling require accurate data normalisation. When using endogenous reference genes, these have to be validated carefully. Therefore, we examined the transcript stability of 10 potential reference genes using quantitative real-time polymerase chain reaction: beta actin, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase, TATA box-binding protein, hypoxanthine phosphoribosyl-transferase I, beta-2-microglobulin, hydroxymethylbilane synthase, succinate dehydrogenase complex, subunit A, cyclophilin A and ubiquitin C. The aim of the current study was to assess which reference genes show stable mRNA levels in human post mortem cardiac muscle, skeletal muscle and brain tissue. Considering cardiac muscle tissue, CYCA and TBP were identified as the most stable while in skeletal muscle tissue, SDHA and TBP, and in brain tissue, SDHA and HMBS turned out to be the most stable. Furthermore, we recommend a minimum of four carefully validated endogenous control genes for reliable data normalisation in human post mortem tissue. Parameters influencing the stability of transcript amounts were found to be mainly the post mortem interval in cardiac muscle and skeletal muscle tissue and the donor’s cause of death in skeletal muscle and brain samples. Further parameters like gender, age at death and body mass index were found to influence mRNA quantities in skeletal muscle only. The set of stable control genes identified in this study may be used in further studies if the composition of the samples is similar to the one used here.

Low-volume amplification on chemically structured chips using the PowerPlex16 DNA amplification kit

In forensic DNA analysis, improvement of DNA typing technologies has always been an issue. It has been shown that DNA amplification in low volumes is a suitable way to enhance the sensitivity and efficiency of amplification. In this study, DNA amplification was performed on a flat, chemically structured glass slide in 1-μl reaction volumes from cell line DNA contents between 1,000 and 4xa0pg. On-chip DNA amplification reproducibly yielded full allelic profiles from as little as 32xa0pg of template DNA. Applicability on the simultaneous amplification of 15 short tandem repeats and of a segment of the Amelogenin gene, which are routinely used in forensic DNA analysis, is shown. The results are compared to conventional in-tube amplification carried out in 25-μl reaction volumes.

Sequence polymorphisms within the human mitochondrial genes MTATP6, MTATP8 and MTND4

By sequencing the control region of mitochondrial DNA, the majority of human DNA samples can be differentiated. A further increase in differentiation probability may be possible, e.g. by extending the sequenced region to coding regions of the mitochondrial genome. Restriction to those positions that do not result in a change of the amino acids guarantees that the information thus obtained does not refer to phenotypically relevant information. In the present study the sequence data of the mitochondrial genes MTATP6, MTATP8 and MTND4 were collected from 109 subjects and analyzed in order to define variable positions suitable for identification purposes. There were 32 variable base positions among 850 bases studied from MTATPase genes and 1,200 bases of the MTND4 gene showed 28 variable positions. Hot spots for base exchanges were found in both regions and one position (position 11719 in the MTND4 gene) seems to be suitable for SNP investigation for forensic purposes.

Real-time PCR detection of five different “endogenous control gene” transcripts in forensic autopsy material

Relative quantification of mRNA using quantitative real-time reverse transcription (RT)-PCR is a commonly used method for analysis and comparison of gene expression levels. This method requires a normalisation of data against expression levels of a control gene. In the past, several ubiquitously expressed genes were used as such endogenous controls. When working with human tissue samples obtained during autopsy one has to deal with postmortem intervals of usually more than 10 h. The aim of this study was to investigate whether commonly used endogenous control genes show stability over various postmortem intervals. For this purpose, RNA was extracted from three different human tissues of five postmortem intervals ranging from 15 to 118 h. The Ct values from five commonly used endogenous control genes--beta-actin, B2M, CyPA, TBP, and UBC--were obtained by real-time RT-PCR. Results revealed a relatively high stability of Ct values in skeletal muscle tissue regarding different postmortem intervals. In heart and brain tissues, all endogenous controls were found to be highly variable. B2M appeared to be the least unstable control in this set. Nevertheless, all endogenous controls showed variability in their expression levels regarding both the stability among different tissues and different postmortem intervals. Data obtained in the present study show that postmortem mRNA degradation is a complex process, and that the use of one single endogenous control in gene expression studies of postmortem tissue would lead to erroneous data interpretation. Further studies on this topic should be performed in the future including an increased number of well documented samples.

Diagnosis of a captive-bolt injury in a skull extremely destroyed by fire

The authors report on a combined suicide of a 71-year-old farmer who fired a shot to his forehead with a livestock stunner before burning himself. As the fire was fueled by a pile of firewood it burnt for many hours, thus, causing subtotal incineration of the body. The remaining bones were calcined and reduced to a residual mass of only 3 kg. In spite of the extreme destruction, a circular bone defect corresponding to the site where the captive-bolt had entered the skull could be identified in the frontal squama. The example of this suicide is used to illustrate the problems of distinguishing between mechanical and thermal fractures. As expected, the attempted isolation and amplification of both nuclear and mitochondrial DNA for the purpose of identification was not successful.

Single lymphocytes from two healthy individuals with mitochondrial point heteroplasmy are mainly homoplasmic

The nature of mitochondrial DNA heteroplasmy is still unclear. It could either be caused by two mitochondrial DNA (mtDNA) haplotypes coexisting within a single cell or by an admixture of homoplasmic cells, each of which contains only one type of mtDNA molecule. To address this question, single lymphocytes were separated by flow cytometry assisted cell sorting and analyzed by cycle sequencing or minisequencing. To attain the required PCR sensitivity, the reactions were carried out on the surface of chemically structured glass slides in a reaction volume of 1–2xa0μl. In this study, blood samples from two healthy donors showing mitochondrial point heteroplasmy in direct sequencing (195Y and 234R, respectively) were analyzed. Nearly 96% of single lymphocytes tested were found to be in a homoplasmic state, but heteroplasmic cells were also detected. These results suggest that mitochondrial point heteroplasmy in blood may well be mainly due to the mixture of homoplasmic cells.

Homicides by Sharp Force

Injuries caused by sharp or pointed objects are common. They rarely cause fatal injuries, however, and the fatality rate is estimated to be 3% at most. Most fatalities caused by sharp force are homicides. The ratio of homicide to suicide is estimated at 6:1 to 5:2. When investigating deaths owing to sharp force, the forensic pathologist is expected to give an opinion on the following points: the type of injuries; the number and anatomical distribution of injuries; the shape, size, length, and depth of injuries; the object (weapon) used; the amount of force needed to inflict the injuries; the extent of internal injuries; the cause of death; and the victim’s capability to act. These points are of decisive importance for the reconstruction of the sequence of events and, thus, are essential for distinguishing between self-infliction and involvement of another party. Most homicides by sharp force are committed by males, often under the influence of alcohol. The most common tool used is a knife, but other pointed objects, such as scissors, ice picks, forks, or broken glass, may also be used. The victims are usually family members or acquaintances. Homicides committed by females are comparatively rare. In such cases, the victims are mostly the life partners. The scene of death is most frequently the victim’s home. Fatal stabs are usually located in the precordial or cervical region. The number of stabs does not allow the drawing of conclusions as to the mode of death, the motive, or sex of the perpetrator. When the number of stabs is higher than necessary to kill the victim, this is referred to as “overkill,” and may point to a strong emotional conflict between the perpetrator and the victim.