Ribosomal DNA as target for the assessment of DNA degradation of human and canine DNA.

The assessment of DNA amount and DNA integrity can support forensic DNA analysis, in particular of problematic traces such as single telogen hairs where STR typing success is often hampered by low amounts and strong degradation of nuclear DNA. Common strategies consist of quantitative polymerase chain reaction (qPCR)-based analysis of the abundance of a short versus a long nuclear amplicon, the latter prone to DNA degradation. To increase sensitivity, commercial qPCR solutions rest on amplification of multi-copy DNA sequences. Here we show that ribosomal DNA (rDNA) sequences are well suited for the same purpose. Because rDNA sequences are present in high copy number in most eukaryotic species, qPCR strategies can easily be adapted to non-human species. In this paper, we establish qPCR-based assays for human or dog DNA, respectively, which allow for sensitive analysis of DNA amounts and DNA degradation. We show that the human system can be applied to DNA of single telogen hairs, where STR typing success correlates with measured amounts and integrity of the DNA. By adapting the system to dog rDNA sequences we found that single telogen dog hairs often displayed less DNA degradation than human telogen hairs, in most cases allowing for successful STR typing. Thus, qPCR-based analysis of rDNA represents a cost-effective, highly sensitive strategy to assess DNA amount and integrity that can be adapted to hairs or other traces from various animal species.

The germlines of male monozygotic (MZ) twins: Very similar, but not identical.

In 2012, a thought experiment in this journal suggested that paternity cases involving monozygotic (MZ) twins as putative fathers could be solved by means of whole genome sequencing (WGS). Although arising from a single fertilization event, MZ twins nevertheless continue to acquire somatic mutations during their development, including those that occur in the germline. Provided that paternity had been narrowed down to the twin pair beforehand by classical DNA analysis, one post-zygotic mutation would suffice to assign the paternal compartment of an offspring genome unambiguously to either twin if that mutation is found in the offspring and one twin, but not in the other twin. Since the publication of a proof-of-principle report in 2014, we have worked up five additional cases of MZ twin germline discrimination in real life, four paternity disputes and one criminal case requiring the identification of a sperm trace donor among a pair of MZ twin brothers. In this opinion paper, we report on the experiences made in the course of our work and take a look at possibilities for further development of the approach.

Distinguishing genetically between the germlines of male monozygotic twins.

Identification of the potential donor(s) of human germline-derived cells is an issue in many criminal investigations and in paternity testing. The experimental and statistical methodology necessary to work up such cases is well established but may be more challenging if monozygotic (MZ) twins are involved. Then, elaborate genome-wide searches are required for the detection of early somatic mutations that distinguish the cell sample and its donor from the other twin, usually relying upon reference material other than semen (e.g. saliva). The first such cases, involving either criminal sexual offenses or paternity disputes, have been processed successfully by Eurofins Genomics and Forensics Campus. However, when presenting the experimental results in court, common forensic genetic practice requires that the residual uncertainty about donorship is quantified in the form of a likelihood ratio (LR). Hence, we developed a general mathematical framework for LR calculation, presented herein, which allows quantification of the evidence in favour of the true donor in the respective cases, based upon observed DNA sequencing read counts.

Finding the needle in the haystack: differentiating "identical" twins in paternity testing and forensics by ultra-deep next generation sequencing.

Monozygotic (MZ) twins are considered being genetically identical, therefore they cannot be differentiated using standard forensic DNA testing. Here we describe how identification of extremely rare mutations by ultra-deep next generation sequencing can solve such cases. We sequenced DNA from sperm samples of two twins and from a blood sample of the child of one twin. Bioinformatics analysis revealed five single nucleotide polymorphisms (SNPs) present in the twin father and the child, but not in the twin uncle. The SNPs were confirmed by classical Sanger sequencing. Our results give experimental evidence for the hypothesis that rare mutations will occur early after the human blastocyst has split into two, the origin of twins, and that such mutations will be carried on into somatic tissue and the germline. The method provides a solution to solve paternity and forensic cases involving monozygotic twins as alleged fathers or originators of DNA traces.

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.

Insertion-/deletion polymorphisms close to the repeat region of STR loci can cause discordant genotypes with different STR kits.

It is well known that mutations in the primer binding site of STR loci can cause "null alleles". If a sample is typed with different sets of PCR primers (for example if different kits or kits of different manufacturers are used) discordant results might be observed, thus making the use of different STR kits for deviant genotypes necessary. Here we present another reason for using different kits: Insertion-/deletion polymorphism between the repeat region of the STR locus and the primer binding site can also cause genotypes that vary between different kits/primer sets.

Correlation of inter-individual variations of amitriptyline metabolism examined in hairs with CYP2C19 and CYP2D6 polymorphisms.

The metabolite ratio of amitriptyline (AT), nortriptyline (NT) and its 10-hydroxy metabolites (E10-OHAT, Z10-OHAT, E10-OHNT and Z10-OHNT) was examined by liquid chromatography-mass spectrometry in hair samples of 23 white infants after long-term administration of AT. High inter-individual variation of the metabolite ratios were observed (e.g. NT/AT = 0.8-8.1, E10-OHNT/Z10-OHNT = 1.6-10.3). The significance of these variations was proven by confirmation of the temporary stability of these ratios within a hair fibre. Moreover, an association of the metabolic phenotype with genetic disposition was observed. The genotypes of CYP2C19 (alleles *2, *3 and *4) and of CYP2D6 (*3, *4, and *6) were examined by conventional polymerase chain reaction genotyping experiments. The relative amount of demethylation (NT/AT) is clearly affected by the number of functional alleles of CYP2C19. The demethylation capacity of CYP2C19 poor metabolizers (3 individuals, compared to 15 extensive metabolizers) was 4.3 times depleted. Moreover, the selectivity of hydroxylation (e.g. E10-OHNT/Z10-OHNT) is significantly correlated with CYP2C19.

Proof of a 1-(3-chlorophenyl)piperazine (mCPP) intake: use as adulterant of cocaine resulting in drug-drug interactions?

Since 2005, increasing numbers of seizures of the designer drug of abuse 1-(3-chlorophenyl)piperazine (mCPP) have been reported. This paper describes the unequivocal proof of a mCPP intake. Differentiation from the intake of its precursor drugs trazodone and nefazodone was performed by a systematic toxicological analysis (STA) procedure using full-scan GC-MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation. The found mCPP/hydroxy-mCPP ratio indicated altered metabolism of this cytochrome (CYP) 2D6 catalyzed reaction compared to published studies using the same procedure. Although this might be ascribed to a poor metabolizer (PM) phenotype, genotyping revealed no PM genotype but indications for an intermediate metabolizer genotype. However, a PM phenotype could also be caused by drug-drug interactions with CYP2D6 inhibitors or substrates such as the co-consumed cocaine and diltiazem and/or diltiazem metabolites, respectively. In conclusion, the presented data indicate a possible relevance of CYP2D6 polymorphism and/or drug interactions to mCPP toxicokinetics, which is important for risk assessment of this new designer drug of abuse, in particular if it is used as adulterant of CYP2D6 substrates such as cocaine.

New alleles and mutational events at 14 STR loci from different German populations.

The molecular origin of DNA mutations and the mutation rates were analyzed at 14 short tandem repeat (STR) loci with samples from trio cases derived from 10 different German population samples. STR loci comprised of D2S1360, D3S1744, D4S2366, D5S2500, D6S474, D7S1517, D8S1132, D10S2325, D12S391, D18S51, D19S246, D20S480, D21S226, and D22S689. In a total of 488 meioses, 16 isolated genetic inconsistencies in 8 different STRs were observed, whereas no mutations were found at the other loci. The data of five mutations suggested the presence of silent or null alleles due to sequence variation in primer binding site. This could be confirmed for four suspected cases by the use of alternative primer sets and by DNA sequence analyses. Furthermore, this study revealed nine new allelic variants at five different loci.

Wonder or fake - investigations in the case of the stigmatisation of Therese Neumann von Konnersreuth.

We investigated two compresses used by Therese Neumann (T.N.), a woman who lived from 1898 until 1962 in Konnersreuth, Germany. The compresses were soaked with blood during the appearance of stigmata on T.N.'s body on a Friday. T.N. became very popular among the faithful in Germany at this time. The question was whether this blood was from T.N. herself or from a family relative or an animal. The comparison of the HV1 and HV2 mtDNA sequence obtained from the compresses with the sequences from a reference sample from a maternally related niece of T.N. revealed an identity. Furthermore, we obtained a short tandem repeat (STR) profile from the bloodstains that were identical with the STR profile from a gummed envelope. The envelope contained a letter written by T.N. in the 1930s. Therefore, our investigations gave no indication for any manipulation.

Peripheral dendritic cell chimerism in allogeneic hematopoietic stem cell recipients.

BACKGROUND: Relapse and graft-versus-host disease (GVHD) represent major causes of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Although leukocyte and T-cell chimerism analyses are performed routinely suggesting a predictive value on the patients outcome, little is known about chimerism of dendritic cells (DC) representing strong initiators of immune responses. METHODS: In this prospective study, peripheral DC1 (CD11c+) and DC2 (CD123+) chimerism was determined in hematopoetic stem cell recipients. DCs were isolated from peripheral blood by fluorescence activated cell sorting. Chimerism analyses were performed by fluorescent in situ hybridization or by polymerase chain reaction-based typing of short tandem repeats. RESULTS: At time of engraftment, DC chimerism analyses showed complete chimerism in 76.3% (DC1)/79.5% (DC2), mixed chimerism (MC) in 21.0% (DC1)/17.9% (DC2) and no chimerism in 2.7% (DC1)/2.6% (DC2) of the patients. Peripheral DC chimerism had no significant effect on relapse-free or overall survival. Although acute GVHD was observed more often in patients with MC for DC1/DC2 and chronic GVHD occurred more often in patients with MC for DC2, there was no statistically significant correlation. CONCLUSIONS: Although DCs as antigen presenting cells are supposed to have an impact on the induction of GVHD, there was no significant correlation between incidence of GVHD and DC chimerism after HSCT.

mtDNA investigations after differential lysis.

In order to gain information regarding the ethnic origin of an unknown offender in a murder case it was necessary to sequence the HV1 and 2 regions of the mitochondrial DNA (mtDNA). The only evidentiary material that could be linked to the suspect was DNA, extracted from spermatozoa after differential lysis. The observed mtDNA sequences were identical to the sequences of the victim. Therefore, we had to check if this was a coincidence or the result of a technical limitation of the testing procedure. Subsequently, we performed a systematic study. In cases with complete separation of sperm and female cells it wasn't possible to obtain a mtDNA sequence for the sperm fraction. This phenomena is based on the loss of the sperm's flagellum and mid-piece during the first lysis step and a concomitant loss of the sperm's mitochondria. In our murder case, a minor carry-over of female cells to the sperm fraction was responsible for the sequencing result.

Allele frequencies of the ACTBP2 (=SE33), D18S51, D8S1132, D12S391, D2S1360, D3S1744, D5S2500, D7S1517, D10S2325 and D21S2055 loci in a German population sample.

A population study on 10 tetrameric STR loci (ACTBP2 (=SE33), D18S51, D8S1132, D12S391, D2S1360, D3S1744, D5S2500, D7S1517, D10S2325, D21S2055) was performed with Germans from Bavaria.

Application of the BioRobot EZ1 in a forensic laboratory.

In a forensic laboratory, the routine application of an automated DNA extraction and purification robot has to fulfil several conditions, like producing reproducible DNA's of sufficient quantity and quality from all the different forensic biological stains relevant to various carrier materials. In this study, the suitability of the BioRobot EZ1 system from QIAGEN (Hilden, Germany), which offers fully automated extraction and purification of nucleic acids using magnetic bead technology, was tested. In summary, the DNA's obtained from the BioRobot EZ1 for different forensic relevant biological materials showed a quantity and quality comparable to those of the forensic standard protocols normally used in our laboratory. The system saves time, because there is no need of any further purification or concentration step after the automated DNA extraction. It can also be used as a replacement for time consuming organic extractions. A disadvantage of the system was the unsteady quality of the chemical regencies used by the robot. Nineteen different lots were tested with a self designed test system.

Allele frequencies of 13 short tandem repeat (STR) loci in the Romanian population.

A population study on 13 tetra- and pentameric STR loci (D3S1358, VWA, D8S1179, D21S11, D18S51, D16S539, D2S1338, D19S433, THO1, FGA, ACTBP2 (SE33), Penta D and Penta E) was performed with Romanians from the Bucharest area.

Y-chromosomal STR haplotypes in a Romanian population sample.

A set of seven STR-loci mapping on the male-specific region of the human Y chromosome (DYS19, DYS385, DYS389 I/II, DYS390, DYS391, DYS392, DYS393) were typed by means of two multiplex PCR reactions and capillary electrophoresis in a Romanian population sample of 104 unrelated males. Among the 97 different haplotypes observed, 92 were unique, while 5 occurred more than once. The observed haplotype diversity was 0.989.

Recurrence of lymphangioleiomyomatosis after single lung transplantation: new insights into pathogenesis.

Lymphangioleiomyomatosis (LAM) is a rare disease found primarily in white women of childbearing age. The present study describes a case of recurrent LAM after single lung transplantation. Double-staining nonisotopic in situ hybridization, immunohistochemistry, and short tandem repeat loci analysis demonstrated that the recurrent LAM lesions originated from the recipient. The data strongly support that metastatic spread of LAM cells or migration of progenitor cells plays an important role in the pathogenesis of LAM.

Investigation of oral swabs taken from newborn infants after breastfeeding.

The aim of this study was to investigate the influence of breastfeeding on the DNA-profiles from oral swabs of newborn babies. Five mother and child pairs were asked to provide orals swabs from the mother and from the child before and immediately after nursing. Investigation of the samples revealed no maternal alleles in the saliva samples of the child taken directly after breast feeding. Therefore, we conclude that nursing does not influence the STR-typing of oral swabs taken form newborn infants. A possible explanation could be that the DNA content of mother's milk is significantly lower than the DNA content of saliva.