DNA methylation-based age estimation for adults and minors: considering sex-specific differences and non-linear correlations.

DNA methylation patterns change during human lifetime; thus, they can be used to estimate an individual's age. It is known, however, that correlation between DNA methylation and aging might not be linear and that the sex might influence the methylation status. In this study, we conducted a comparative evaluation of linear and several non-linear regressions, as well as sex-specific versus unisex models. Buccal swab samples from 230 donors aged 1 to 88 years were analyzed using a minisequencing multiplex array. Samples were divided into a training set (n = 161) and a validation set (n = 69). The training set was used for a sequential replacement regression and a simultaneous 10-fold cross-validation. The resulting model was improved by including a cut-off of 20 years, dividing the younger individuals with non-linear from the older individuals with linear dependence between age and methylation status. Sex-specific models were developed and improved prediction accuracy in females but not in males, which might be explained by a small sample set. We finally established a non-linear, unisex model combining the markers EDARADD, KLF14, ELOVL2, FHL2, C1orf132, and TRIM59. While age- and sex-adjustments did not generally improve the performance of our model, we discuss how other models and large cohorts might benefit from such adjustments. Our model showed a cross-validated MAD and RMSE of 4.680 and 6.436 years in the training set and of 4.695 and 6.602 years in the validation set, respectively. We briefly explain how to apply the model for age prediction.

Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research.

The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1: Expansion of hiPSCs Basic Protocol 2: Cell banking of hiPSCs Support Protocol 1: Pluripotency assessment by flow cytometry Support Protocol 2: Thawing control: Viability and sterility Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT-PCR Support Protocol 4: Identity: Short tandem repeat analysis.

Mitochondrial DNA variation in Sub-Saharan Africa: Forensic data from a mixed West African sample, Côte d'Ivoire (Ivory Coast), and Rwanda.

This study provides 398 novel complete mitochondrial control region sequences that augment the still underrepresented data from Africa by three datasets: a mixed West African sample set deriving from 12 countries (n = 145) and datasets from Côte d'Ivoire (Ivory Coast) (n = 100) as well as Rwanda (n = 153). The analysis of mtDNA variation and genetic comparisons with published data revealed low random match probabilities in all three datasets and typical West African and East African diversity, respectively. Genetic parameters indicate that the presented mixed West African dataset may serve as first forensic mtDNA control region database for West Africa in general. In addition, a strategy for responsible forensic application of precious mtDNA population samples potentially containing close maternal relatives is outlined. The datasets will be uploaded to the forensic mtDNA database EMPOP (https://empop.online) upon publication.

The use of liquid latex for detecting traces of blood following thermal exposure.

In cases of crimes involving blood, the perpetrators often attempt to remove the traces they have left behind. Setting fire to the crime scene, aside from cleaning measures, seems to achieve this goal and presents a major challenge for crime scene investigators. There is only very little published information available on the effect of fire and extreme heat on blood and the detection thereof. After exposure to high temperatures of or exceeding 1.000 °C, blood is deemed to be undetectable. This study exposed 11 different potentially crime-relevant objects using a standardized and controlled procedure to temperatures of 300 °C, 700 °C, and 1.000 °C documenting the influence of heat on bloodstains and the detection of blood. The results of the forensic collection of blood traces with and without liquid latex confirmed the advantage of using the latex method. Almost all objects showed a clear luminescence-caused visualization of traces of blood after removing the soot with a latex lift. There were also fewer false positive results than in tests not using latex.

Cementum as a source of DNA in challenging forensic cases.

Each forensic case is characterized by its own uniqueness. Deficient forensic cases require additional sources of human identifiers to assure the identity. We report on two different cases illustrating the role of teeth in answering challenging forensic questions. The first case involves identification of an adipocere male found in a car submersed in water for approximately 2 years. The second scenario, which involves paternity DNA testing of an exhumed body, was performed approximately 2.8 years post-mortem. The difficulty in anticipating the degradation of the DNA is one of the main obstacles. DNA profiling of dental tissues, DNA quantification by using real-time PCR (PowerQuant™ System/Promega) and a histological dental examination have been performed to address the encountered impediments of adverse post-mortem changes. Our results demonstrate that despite the adverse environmental conditions, a successful STR profile of DNA isolated from the root of teeth can be generated with respect to tooth type and apportion. We conclude that cementocytes are a fruitful source of DNA. Cementum resists DNA degradation in comparison to other tissues with respect to the intra- and inter-individual variation of histological and anatomical structures.

[Reconstructive investigations and identification measures in unknown soldiers of the Second World War]. / Rekonstruktive Untersuchungen und Identifikationsmassnahmen bei unbekannten Soldaten aus dem Zweiten Weltkrieg.

The article reports on the exhumation and identification of unknown soldiers from the Second World War. With the help of medicolegal investigation and reconstruction methods an American pilot presumably murdered by a shot to the head (lynch law) and an interned Italian soldier could be identified after about 70 years and brought back home.

The assessment of adipocere to estimate the post-mortem interval - a skeleton from the tidelands.

SUMMARY: This paper discusses the discovery of a skeletonized water corpse with hollow bones filled with adipocere found in the tidelands of the river Elbe close to Otterndorf (Wesermarsch, Cuxhaven). Through macroscopic and microscopic methods, the existing adipocere was described. The post-mortem interval was assessed by a comparison of the radiocarbon data and the indications about the preservation of adipocere from the literature. The investigation has shown that the knowledge of post-mortem changes in adipocere within bone structures is still incomplete, especially for the assessment of water corpses with long post-mortem intervals.

Diagnostic value of PSA and AP tests for the detection of spermatozoa in postmortem swabs from the genital and anal region in males.

The aim of this study was to clarify whether positive results for prostate-specific antigen (PSA) and acid phosphatase (AP) occur in postmortem swabs from the genito-anal region in males (n = 80; 4 regions) and females (n = 20; 3 regions) and to calculate the positive predictive value (PPV) concerning the presence of spermatozoa. In male subjects, the highest incidence of positive test results was found in urethral swabs (PSA 76%, AP 71%) and the lowest frequencies appeared in perianal and rectal swabs (15-20%). Microscopic evaluation for spermatozoa was positive between 39% in urethral swabs and 1% in rectal swabs. PPV regarding positive identification of spermatozoa was 33.3% for PSA and 31.5% for AP. The combination of both tests yielded a PPV of 38.2%. In female cases, no spermatozoa were identified, and one case was PSA- and AP-positive in perianal swabs. Our findings indicate that PSA and AP tests are of limited value for the postmortem detection of spermatozoa in male subjects.

[Criminology and victimology of rape in context with war-like conflicts using the example of the former Yugoslavia and Rwanda]. / Kriminologie und Viktimologie von Vergewaltigungen im Rahmen kriegerischer Auseinandersetzungen am Beispiel des ehemaligen Jugoslawiens und Ruandas.

The topic of this article is sexual violence in context with war-like conflicts in the former Yugoslavia and Rwanda. The fundamental categories of sexual violence in war-like conflicts are described. The authors discuss the types of sexual violence as defined in the report of the UN Commission of Experts on the war-like conflicts in the former Yugoslavia. Four criminal trials were evaluated: three held before the International Criminal Tribunal for the Former Yugoslavia (ICTY) in The Hague/Netherlands and one before the International Criminal Tribunal for Rwanda (ICTR) in Arusha/Tansania. The defendants were found guilty of torture, crime against humanity and genocide. Potential procedures with respect to similar crimes in current or prospective conflicts are discussed. An alternative may be the assignment of medical personnel (for example of the German Federal Armed Forces). Finally, the post-war cooperation between the Institute of Legal Medicine at the University Medical Centre of Hamburg-Eppendorf as well as the medical and government institutions in Rwanda is presented, which has been going on since 2005.

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.

Evaluation of seven X-chromosomal short tandem repeat loci located within the Xq26 region.

In this study a set of 29 X-chromosomal short tandem repeats (STRs) located within the Xq26 region was evaluated. These STRs were found within the 133.14-133.45Mb region around the HPRTB locus. Evaluation of the microsatellites was performed with regard to polymorphism, reliable amplification, and low stutter artefacts. DXS10101, DXS10102, and DXS10103 were identified as those X-STRs with highest diversity; i.e. PIC values of 0.7174-0.8933. The locus DXS10101 was the optimal candidate for the integration in the commercial available test system Mentype Argus X-8 PCR amplification kit.

X chromosomal recombination--a family study analysing 39 STR markers in German three-generation pedigrees.

Typing of polymorphisms on the human chromosome X (ChrX) has become a standard technique in forensic genetics, and a growing number of short tandem repeats (STRs) has been established. Knowledge of marker recombination is of great significance especially when ChrX typing is used in forensic kinship testing. It is known that meiotic recombination is not a simple function of physical distance but crossing over events tend to be clustered. Information on genetic distances between markers can be gathered by family studies and by interpolation of gene bank data such as the Rutgers map. We typed DNA samples of pedigrees consisting of mothers with several sons and grandfather-mother-son constellations and report here the recombination characteristics of 39 ChrX STRs in up to 135 meioses.

Validation of six closely linked STRs located in the chromosome X centromere region.

We propose that clusters of closely linked markers, which segregate as stable haplotypes, provide a high potential to solve complex kinship cases. It is known that the X-chromosomal centromere region shows an extremely low degree of recombination. Hence, we focused our interest on the region between 56 and 64 Mb distant from the Xp telomere and considered 6 STRs which are now registered in the Genome Data Base as DXS10161, DXS10159, DXS10162, DXS10163, DXS10164, and DXS10165. All of these markers show a tetranucleotide or pentanucleotide structure and exhibit high or medium polymorphic information content. As a peculiarity, DXS10163 is a combination of a pentanucleotide STR and an 18 bp INDEL polymorphism. We report here the primer sequences, the repeat structures, the allele distributions and parameters of forensic interest for a German population sample.

Characterisation of the STR markers DXS10146, DXS10134 and DXS10147 located within a 79.1 kb region at Xq28.

Three polymorphic X-chromosomal STR markers within a 79 kb region at Xq28 were studied and registered in the GDB as DXS10146, DXS10134 and DXS10147. These markers were molecular characterised and evaluated for their forensic usage. As a result DXS10134 was recently integrated in the commercial available test kit Mentype Argus X-8. At locus DXS10146 we found 23 alleles with PIC and HET values of 0.878 and 0.887. Locus DXS10134 showed 17 alleles with PIC and HET values of 0.844 and 0.858. At locus DXS10147 only 5 alleles with some lower PIC and HET values of 0.636 and 0.692 were found. Additionally, the already known and closely linked STR DXS7423 was included into the haplotyping and recombination studies. Testing this cluster a German population of 404 males revealed the presence of 311 haplotypes. Recombination analysis was performed in 109 father-daughter-grandson trios in which two crossing over events were observed located in the 65.8 kb region between DXS10146 and DXS10134. By using this STR complex for haplotyping in kinship testing further genetic analyses are required to establish an exact recombination rate.

Population genetic evaluation of eight X-chromosomal short tandem repeat loci using Mentype Argus X-8 PCR amplification kit.

The evaluation of four pairs of X-chromosomal short tandem repeats (STRs), i.e. DXS10135-DXS8378, DXS7132-DXS10074, HPRTB-DXS10101 and DXS7423-DXS10134 was carried out using the Argus X-8 Multiplex amplification kit. These eight STRs are distributed as four closely linked pairs over the entire X-chromosome (ChrX), and for practical reasons they are assigned to four linkage groups 1-4. The genetic distance within the STR pairs is assumed to be <1cM, whereas the pair to pair space is about 50 cM or more. Here, we present single STR allele frequencies, haplotype frequencies of the respective STR pairs and further population genetic parameters of forensic interest. Most data refer to a German population, however small samples from Ghana and Japan were also investigated. Furthermore, sequencing of all STR loci displayed the presence of microvariant alleles and variations in the repeat flanking region. A total of 350 meioses investigated here revealed only one sperm DXS7132 mutation. For analysis of linkages within the STR pairs a study involving 104 female meiosis with respect to recombination events was performed. The STR panel presented here provides a powerful tool for solving complex kinship in the case that X-chromosomal lineages can be taken under investigation.

The STR cluster DXS10148-DXS8378-DXS10135 provides a powerful tool for X-chromosomal haplotyping at Xp22.

The evaluation of four pairs of tightly linked chromosome X (ChrX) short tandem repeat (STR)s at Xp22, Xq12, Xq26 and Xq28 led to the creation of the Argus X 8 multiplex amplification kit. These eight STRs are distributed as four closely linked pairs over the entire X-chromosome, and for practical reasons, they are assigned to four linkage groups 1-4. To achieve a further considerable enhancement in discrimination power, we suggest to include additional markers. A recent paper referred to the earlier evaluation of STR clusters at Xq12, Xq26 and Xq28, and here we present the pending data of linkage group 1 at Xp22. The newly established STR updates the Xp22 STR cluster which now presents three polymorphic markers: DXS10148 (PIC = 0.8556), DXS10135 (PIC = 0.9093) and DXS 8378 (PIC = 0.6454). Typing of 398 X-chromosomes provided 278 different and 200 unique haplotypes. All the other haplotypes observed appeared with frequencies in the range between 0.005 and 0.015. Considering this STR triple in the context with the three further triple clusters Xq12, Xq26 and Xq28 published earlier, we announced the development of a next generation of a ChrX STR cluster typing kit.

Dual examinations for identification of urine as being of human origin and for DNA-typing from small stains of human urine.

Concurrent methods for identification of urine as being of human origin, and for DNA-typing from small stains of human urine were examined. A urine stain was extracted with phosphate-buffered saline (PBS), and the extract was filtered using a Centricon-100 device. The filtrate was subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) for identification of human urine and a DNA-typing sample was obtained by dialfiltration of the residue using a DNA purification kit. After the purified residue was treated with an AmpflSTR Profiler PCR amplification kit, the DNA-types were analyzed by capillary electrophoresis using a Genetic Analyzer. It was possible to identify a urine stain as being of human origin, and complete DNA profiles could be successfully obtained from a urine stain which had been created by 50 microL of female urine. Serial analyses of urine stains found at a crime scene provide effective information for forensic investigation. This method is recommended for stain identification and for DNA-typing from a urine stain.

An illicit love affair during the Third Reich: who is my grandfather?

More than 60 years after an illicit love affair had occurred between Erika H, wife of a Wehrmacht soldier, and a Polish slave worker during World War II, we could clarify the blood relationships of her daughter Uta. When Erika H had become pregnant both of the men could have fathered the child. Erika H was found guilty of fraternization and imprisoned at Ravensbrück concentration camp. She gave birth to Uta and died there in 1944. Uta survived the war as did Erika's husband Gustav, who accepted Uta as his child. Blood samples from family members were taken and DNA extracted. A panel of 16 short tandem repeat (STR) loci were amplified and separated by capillary electrophoresis and the likelihoods calculated using the MLINK software. The combined genotypes yielded a cumulative likelihood ratio of over 200,000 against paternity of Gustav H. This case serves to illustrate the utility of STR profiles for complex deficiency kinship analysis.

Complex variability of intron 40 of the von Willebrand factor (vWF) gene.

Intron 40 of the von Willebrand factor (vWF) gene exhibits a highly variable region of about 0.65 kb, which contains 5 juxtaposed STRs. We sequenced 0.65 kb amplicons from 68 chromosomes and found 2 frequent indel polymorphisms and 5 SNPs. The 68 chromosomes investigated here presented a total of 47 different haplotypes. Regarding the SNP allele distribution in our sample, we arranged our results of the vWF intron 40 into a system of 3 haplotypes, i.e. haplotypes a, b and c. Our review may be valuable in further optimising vWF typing in forensic applications and in avoiding pitfalls. Further attempts to develop sophisticated techniques may soon enable haplotyping using autosomale STR clusters.

DXS10079, DXS10074 and DXS10075 are STRs located within a 280-kb region of Xq12 and provide stable haplotypes useful for complex kinship cases.

The evaluation of the short tandem repeat (STR) markers DXS10079, DXS10074 and DXS10075 was amended to establish a STR cluster spanning a genetic distance<1 cM. These three STRs are located within a 280-kb region at Xq12 and provide stable haplotypes useful for solving complex kinship cases. Theoretically, this cluster could give rise to 2,548 different haplotypes in the German population and the genotyping of 781 men revealed the presence of 172 haplotypes. Since the three STRs were shown to be in strong linkage disequilibrium (LD), haplotype frequencies cannot be computed on the basis of a single locus allele frequency alone but have to be estimated directly. Here, we present data on linkage, haplotype frequencies and LD in a German population. Further clusters from other regions of the X chromosome will be published in the future to cover the chromosome with a well-structured network of highly informative sites.