Sabine Lutz-Bonengel

Natural radioactivity and human mitochondrial DNA mutations

Radioactivity is known to induce tumors, chromosome lesions, and minisatellite length mutations, but its effects on the DNA sequence have not previously been studied. A coastal peninsula in Kerala (India) contains the worlds highest level of natural radioactivity in a densely populated area, offering an opportunity to characterize radiation-associated DNA mutations. We sampled 248 pedigrees (988 individuals) in the high-radiation peninsula and in nearby low-radiation islands as a control population. We sequenced their mtDNA, and found that the pedigrees living in the high-radiation area have significantly (P < 0.01) increased germ-line point mutations between mothers and their offspring. In each mutation case, we confirmed maternity by autosomal profiling. Strikingly, the radioactive conditions accelerate mutations at nucleotide positions that have been evolutionary hot spots for at least 60,000 years.

Artificial recombination in forensic mtDNA population databases

Artificial recombination of two or more mitochondrial DNA fragments from different samples would constitute a serious cause of error in forensic DNA typing, and yet one can demonstrate that such events have happened in the preparation of several published mtDNA databases. Focussed database searches, phylogenetic analysis, and network representations can highlight mosaic patterns and thus pinpoint sample mix-up. Therefore, we suggest that this approach should be applied to data prior to publication in order to uncover such errors in time.

RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays

Messenger RNA (mRNA) profiling in post-mortem human tissue might reveal information about gene expression at the time point of death or close to it. When working with post-mortem human tissue, one is confronted with a natural RNA degradation caused by several parameters which are not yet fully understood. The aims of the present study were to analyse the influence of impaired RNA integrity on the reliability of quantitative gene expression data and to identify ante- and post-mortem parameters that might lead to reduced RNA integrities in post-mortem human brain, cardiac muscle and skeletal muscle tissues. Furthermore, this study determined the impact of several parameters like type of tissue, age at death, gender and body mass index (BMI), as well as duration of agony, cause of death and post-mortem interval on the RNA integrity. The influence of RNA integrity on the reliability of quantitative gene expression data was analysed by generating degradation profiles for three gene transcripts. Based on the deduced cycle of quantification data, this study shows that reverse transcription quantitative polymerase chain reaction (RT-qPCR) performance is affected by impaired RNA integrity. Depending on the transcript and tissue type, a shift in cycle threshold values of up to two cycles was observed. Determining RNA integrity number of 136 post-mortem samples revealed significantly different RNA qualities among the three tissue types with brain revealing significantly lower integrities compared to skeletal and cardiac muscle. The body mass index was found to influence RNA integrity in skeletal muscle tissue (M. iliopsoas). Samples originating from deceased with a BMI > 25 were of significantly lower integrity compared to samples from normal weight donors. Correct data normalisation was found to partly diminish the effects caused by impaired RNA quality. Nevertheless, it can be concluded that in post-mortem tissue with low RNA integrity numbers, the detection of large differences in gene expression activities might still be possible, whereas small expression differences are prone to misinterpretation due to degradation. Thus, when working with post-mortem samples, we recommend generating degradation profiles for all transcripts of interest in order to reveal detection limits of RT-qPCR assays.

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.

Collaborative genetic mapping of 12 forensic short tandem repeat (STR) loci on the human X chromosome

A large number of short tandem repeat (STR) markers spanning the entire human X chromosome have been described and established for use in forensic genetic testing. Due to their particular mode of inheritance, X-STRs often allow easy and informative haplotyping in kinship analyses. Moreover, some X-STRs are known to be tightly linked so that, in combination, they constitute even more complex genetic markers than each STR taken individually. As a consequence, X-STRs have proven particularly powerful in solving complex cases of disputed blood relatedness. However, valid quantification of the evidence provided by X-STR genotypes in the form of likelihood ratios requires that the recombination rates between markers are exactly known. In a collaborative family study, we used X-STR genotype data from 401 two- and three-generation families to derive valid estimates of the recombination rates between 12 forensic markers widely used in forensic testing, namely DXS10148, DXS10135, DXS8378 (together constituting linkage group I), DXS7132, DXS10079, DXS10074 (linkage group II), DXS10103, HPRTB, DXS10101 (linkage group III), DXS10146, DXS10134 and DXS7423 (linkage group IV). Our study is the first to simultaneously allow for mutation and recombination in the underlying likelihood calculations, thereby obviating the bias-prone practice of excluding ambiguous transmission events from further consideration. The statistical analysis confirms that linkage groups I and II are transmitted independently from one another whereas linkage groups II, III and IV are characterised by inter-group recombination fractions that are notably smaller than 50%. Evidence was also found for recombination within all four linkage groups, with recombination fraction estimates ranging as high as 2% in the case of DXS10146 and DXS10134.

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.

A cautionary note on switching mitochondrial DNA reference sequences in forensic genetics

The first human mitochondrial DNA (mtDNA) sequence was produced in 1981 from an individual of European descent [1]. Since then, this sequence has been known as the Cambridge Reference Sequence (CRS) with a total length of 16,569 base pairs. As is common practice in other fields of genome research, this first mitochondrial genome (mtGenome) served as reference for the scientific community, relative to which other mtDNA haplotypes were reported. Eighteen years later the CRS was re-sequenced and corrected at 10 positions (3423T, 4985A, 9559C, 11335C, 13702C, 14199T, 14272C, 14365C, 14368C, and 14766C) to form the revised Cambridge Reference Sequence (rCRS) [2]. The new analysis also revealed that this mtGenome consists of only 16,568 nucleotides, as a base at position 3107 was mistakenly reported in the CRS. Instead of redefining all nucleotide positions downstream of 3107, this position is indicated in the rCRS as a deletion (unfortunately often indicated as ‘‘N’’, which is reserved for any base in the IUPcode [3]). Thus, the numbering system employed for the CRS and the body of established data can continuously be used with the rCRS. More than 15,500 mtGenomes and well over 150,000 (partial) control region sequences (including databases) have been published to date (http://www.phylotree.org/, Phylotree Build 14 [4]), in which the CRS and the rCRS have been cited 5603 and 968 times, respectively (http://apps.isiknowledge.com/; queried on May 2012). In a recent study, the switch to a new reference sequence, the so-called Reconstructed Sapiens Reference Sequence (RSRS), has been proposed [5]. This ancestral reconstructed sequence represents the deepest root in the known human mtDNA phylogeny at the base of the split of haplogroups L0 and L10203040506 after combining sequence information from all available mtGenomes from Homo neanderthalensis and novel human mtGenomes. The authors believe that the switch would solve misunderstandings and problems associated with the existing nomenclature relative to the rCRS, which belongs to the recently coalescing European haplogroup H2a2a1. In the following we briefly review developments in forensic mitochondrial genetics and discuss possible implications of the proposed switch. Mitochondrial DNA is highly abundant in cells compared to nuclear DNA (nDNA) with increased typing success rates for analysis of highly degraded samples and also hair shafts that often do not harbor detectable amounts of nDNA. To assess the significance of a match between two mtDNA haplotypes (e.g. from the crime scene and from a suspect), mtDNA databases have been developed. Earlier, the difference-coded haplotypes (with respect to the rCRS) were directly compared to the haplotypes in the mtDNA databases with the risk that multiple different alignments of the same sequence led to biased results [6]. The

Mitochondrial DNA control region diversity in hairs and body fluids of monozygotic triplets

Length heteroplasmy of the homopolymeric cytosine stretch in the hypervariable region II of the mitochondrial D-loop was investigated in blood, buccal cells and hair shafts of monozygotic triplets. The proportions of length heteroplasmy were determined by cloning and sequencing of multiple independent clones. Blood and buccal cells showed an accumulation of molecules with one and two insertions of cytosine residues in relation to the Cambridge Reference Sequence (CRS). The results did not show statistically significant differences between blood and buccal cells of one and the same individual and also not between the three monozygotic brothers. In the hair samples a loss of cytosine residues was established in all three monozygotic individuals compared to blood and buccal cells, suggesting that this must be a regular process. Furthermore, the hair shaft samples showed significant differences between the frequencies of 7, 8 or 9 Cs in the poly C region comparing the three individuals (p<0.008) and in addition there were highly significant differences (p<0.0001) when comparing the results for six different hairs of each individual separately. From these results it can be assumed that besides a common genetic bottleneck during embryonic development, a post-embryonic bottleneck seems to exist in each hair follicle.

Apparent versus true gene expression changes of three hypoxia-related genes in autopsy derived tissue and the importance of normalisation

The aim of our work was to show how a chosen normal-isation strategy can affect the outcome of quantitative gene expression studies. As an example, we analysed the expression of three genes known to be upregulated under hypoxic conditions: HIF1A, VEGF and SLC2A1 (GLUT1). Raw RT-qPCR data were normalised using two different strategies: a straightforward normalisation against a single reference gene, GAPDH, using the 2−ΔΔCt algorithm and a more complex normalisation against a normalisation factor calculated from the quantitative raw data from four previously validated reference genes. We found that the two different normalisation strategies revealed contradicting results: normalising against a validated set of reference genes revealed an upregulation of the three genes of interest in three post-mortem tissue samples (cardiac muscle, skeletal muscle and brain) under hypoxic conditions. Interestingly, we found a statistically significant difference in the relative transcript abundance of VEGF in cardiac muscle between donors who died of asphyxia versus donors who died from cardiac death. Normalisation against GAPDH alone revealed no upregulation but, in some instances, a downregulation of the genes of interest. To further analyse this discrepancy, the stability of all reference genes used were reassessed and the very low expression stability of GAPDH was found to originate from the co-regulation of this gene under hypoxic conditions. We concluded that GAPDH is not a suitable reference gene for the quantitative analysis of gene expression in hypoxia and that validation of reference genes is a crucial step for generating biologically meaningful data.

Factors affecting the detection and quantification of mitochondrial point heteroplasmy using Sanger sequencing and SNaPshot minisequencing

Mitochondrial DNA analysis plays an important role in forensic science as well as in the diagnosis of mitochondrial diseases. The occurrence of two different nucleotides at the same sequence position can be caused either by heteroplasmy or by a mix of samples. The detection of superimposed positions in forensic samples and their quantification can provide additional information and might also be useful to identify a mixed sample. Therefore, the detection and visualization of heteroplasmy has to be robust and sensitive at the same time to allow for reliable interpretation of results and to avoid a loss of information. In this study, different factors influencing the analysis of mitochondrial heteroplasmy (DNA polymerases, PCR and sequencing primers, nucleotide incorporation, and sequence context) were examined. BigDye Sanger sequencing and the SNaPshot minisequencing were compared as to the accuracy of detection using artificially created mitochondrial DNA mixtures. Both sequencing strategies showed to be robust, and the parameters tested showed to have a variable impact on the display of nucleotide ratios. However, experiments revealed a high correlation between the expected and the measured nucleotide ratios in cell mixtures. Compared to the SNaPshot minisequencing, Sanger sequencing proved to be the more robust and reliable method for quantification of nucleotide ratios but showed a lower detection sensitivity of minor cytosine components.