Exploring the potential of genetic analysis in historical blood spots for patients with iodine-deficient goiter and thyroid carcinomas in Switzerland and Germany (1929-1989).

Iodine deficiency-induced goiter continues to be a global public health concern, with varying manifestations based on geography, patient's age, and sex. To gain insights into clinical occurrences, a retrospective study analyzed medical records from patients with iodine deficiency-induced goiter or thyroid cancer who underwent surgery at the Community Hospital in Riehen, Switzerland, between 1929 and 1989. Despite today's adequate iodine supplementation, a significant risk for iodine-independent goiter remains in Switzerland, suggesting that genetic factors, among others, might be involved. Thus, a pilot study exploring the feasibility of genetic analysis of blood spots from these medical records was conducted to investigate and enhance the understanding of goiter development, potentially identify genetic variations, and explore the influence of dietary habits and other environmental stimuli on the disease.Blood prints from goiter patients' enlarged organs were collected per decade from medical records. These prints had been made by pressing, drawing, or tracing (i.e., pressed and drawn) the removed organs onto paper sheets. DNA analysis revealed that its yields varied more between the prints than between years. A considerable proportion of the samples exhibited substantial DNA degradation unrelated to sample collection time and DNA mixtures of different contributors. Thus, each goiter imprint must be individually evaluated and cannot be used to predict the success rate of genetic analysis in general. Collecting a large sample or the entire blood ablation for genetic analysis is recommended to mitigate potential insufficient DNA quantities. Researchers should also consider degradation and external biological compounds' impact on the genetic analysis of interest, with the dominant contributor anticipated to originate from the patient's blood.

Zygotic-splitting after in vitro fertilization and prenatal parenthood testing after suspected embryo mix-up - a case report.

After in vitro fertilization with a single embryo, the parents learned about being pregnant with twins in the 10th week with various indications that an embryonic mix-up could have taken place. The affected couple thus expressed the urgent desire for a clarification of parenthood considering an abortion. However, the prenatal test results would not have been available until the 14/15th week of pregnancy. Legally, then, severe physical or mental distress of the pregnant woman must be claimed by physicians to justify an abortion after the twelfth week. However, a lack of genetic relatedness could lead to serious psychological distress for the parents, making a pregnancy termination possible even after the twelfth week, which is discussed in this case study alongside the interdisciplinary team's ethical, legal, and medical considerations.For the invasive relationship testing, cultivated chorionic villi samples (CVS) from both unborn and saliva samples from the putative parents were genetically analyzed using classical short tandem repeats (STR) analysis. The perfect match of both CVS profiles suggested the occurrence of an unusual late twin shaft, for which, fortunately, parenthood could be confirmed. To our knowledge, this is the first report on a prenatal investigation of a suspected embryo mix-up after assisted reproductive technology (ART), in which parenthood should be fixed. We want to draw attention to this unthinkable scenario, which may increase in the future with ART-induced rising multiple pregnancies.

Targeted recovery of male cells in a male and female same-cell mixture.

DNA mixture deconvolution in the forensic DNA community has been addressed in a variety of ways. "Front-end" methods that separate the cellular components of mixtures can provide a significant benefit over computational methods as there is no need to rely on models with inherent uncertainty to generate conclusions. Historically, cell separation methods have been investigated but have been largely ineffective due to high cost, unreliability, and the lack of proper instrumentation. However, the last decade has given rise to more innovative technology that can target and recover cells more effectively. This study focuses on the development and optimization of a method to selectively label and recover male cells in a mixture of male and female epithelial cells using a Y-chromosome labeling kit with DEPArray™ technology, whereby male cells are labeled and recovered into a single extraction-ready tube. Labeling efficiency was tested using freshly collected and aged buccal swabs where 70%-75% and 38% of male cells were labeled, respectively, with less than 1% false positives. DEPArray™ detection was assessed using single buccal epithelial cells where approximately 80% of labeled cells were identified as male. Mixtures (1:1, 1:10, male to female) yielded profiles that were predominantly single source male or those in which the male component was more easily interpreted. The male-specific labeling method was demonstrated to be both robust and reliable when used on freshly collected cells. While the DEPArray™ meditated detection and recovery had notable limitations, it still improved the interpretation of the male component in same-cell mixtures in more recently collected samples.

DEPArray™ single-cell technology: A validation study for forensic applications.

In forensics investigations, it is common to encounter biological mixtures consisting of homogeneous or heterogeneous components from multiple individuals and with different genetic contributions. One promising mixture deconvolution strategy is the DEPArray™ technology, which enables the separation of cell populations before genetic analysis. While technological advances are fundamental, their reliable validation is crucial for successful implementation and use for casework. Thus, this study aimed to 1) systematically validate the DEPArray™ system concerning specificity, sensitivity, repeatability, and contamination occurrences for blood, epithelial, and sperm cells, and 2) evaluate its potential for single-cell analysis in the field of forensic science. Our findings confirmed the effective identification of different cell types and the correct assignment of successfully genotyped single cells to their respective donor(s). Using the NGM Detect™ Amplification Kit, the average profile completeness for diploid cells was approximately 80%, with ∼ 290 RFUs. In contrast, haploid sperm analysis yielded an average completeness of 51% referring to the haploid reference profile, accompanied by mean peak heights of ∼ 176 RFUs. Although certain alleles of heterozygous loci in diploid cells showed strong imbalances, the overall peak balances yielded acceptable values above ≥ 60% with a mean value of 72% ± 0.21, a median of 77%, but with a maximum imbalance of 9% between heterozygous peaks. Locus dropouts were considered stochastic events, exhibiting variations among donors and cell types, with a notable failure incidence observed for TH01. Within the wet-lab experimentation with >500 single cells for the validation, profiling was performed using the consensus approach, where profiles were selected randomly from all data to better mirror real casework results. Nevertheless, complete profiles could be achieved with as few as three diploid cells, while the average success rate increased to 100% when using profiles of 6-10 cells. For sperms, however, a consensus profile with completeness >90% of the autosomal diploid genotype could be attained using ≥15 cells. In addition, the robustness of the consensus approach was evaluated in the absence of the respective reference profile without severe deterioration. Here, increased stutter peaks (≥ 15%) were found as the main artifact in single-cell profiles, while contamination and drop-ins were ascertained as rare events. Lastly, the technique's potential and limitations are discussed, and practical guidance is provided, particularly valuable for cold cases, multiple perpetrator rapes, and analyses of homogeneous mixed evidence.

A systematic approach to improve downstream single-cell analysis for the DEPArray™ technology.

Most commercially available STR amplification kits have never been fully validated for low template DNA analysis, highlighting the need for testing different PCR kits and conditions for improving single-cell profiling. Here, current strategies rely mainly on adjusting PCR cycle number and analytical threshold settings, with a strong preference for using 30 amplification cycles and thresholds at 30-150 RFU for allele detection. This study aimed to (1) determine appropriate conditions for obtaining informative profiles utilizing a dilution series, and (2) test the outcome on single cells using the DEPArray™ technology. Four routinely applied forensic STR kits were compared by using three different amplification volumes and DNA dilutions down to 3.0 pg, while two well-performing kits were used for single/pooled leucocyte and sperm cell genotyping. Besides reduced costs, the results demonstrate that a 50%-75% PCR volume reduction was beneficial for peak height evaluation. However, this was counteracted by an increased artifact generation in diluted DNA volumes. Regarding profile completeness, the advantage of volume reduction was only prominent in samples processed with Fusion 6C. For single and pooled cells, ESIFast and NGMDetect provided a solid basis for consensus profiling regarding locus failure, although locus dropouts were generally observed as stochastic events. Amplification volume of 12.5 µL was confirmed as appropriate in terms of peak heights and stutter frequencies, with increased stutter peaks being the main artifact in single-cell profiles. Limitations associated with these analyses are discussed, providing a solid foundation for further studies on low template DNA.

Collecting touch DNA from glass surfaces using different sampling solutions and volumes: Immediate and storage effects on genetic STR analysis.

Touch DNA has become increasingly important evidence in todays' forensic casework. However, due to its invisible nature and typically minute amounts of DNA, the collection of biological material from touched objects remains a particular challenge that underscores the importance of the best collection methods for maximum recovery efficiency. So far, swabs moistened with water are often utilized in forensic crime scene investigations for touch DNA sampling, even though an aqueous solution provokes osmosis, endangering the cell's integrity. The aim of the research presented here was to systematically determine whether DNA recovery from touched glass items can be significantly increased by varying swabbing solutions and volumes compared with water-moistened swabs and dry swabbing. A second objective was to investigate the possible effects of storage of swab solutions prior to genetic analysis on DNA yield and profile quality when stored for 3 and 12 months, as is often the case with crime scene samples. Overall, the results indicate that adapting volumes of the sampling solutions had no significant effect on DNA yield, while the detergent-based solutions performed better than water and dry removal, with the SDS reagent yielding statistically significant results. Further, stored samples showed an increase in degradation indices for all solutions tested, but no deterioration in DNA content and profile quality, allowing for unrestricted processing of touch DNA samples stored for at least 12 months. One further finding was a strong intraindividual change in DNA amounts observed over the 23 deposition days which may be related to the donor's menstrual cycle.

The DNA-Buster: The evaluation of an alternative DNA recovery approach.

Touch DNA recovery techniques can have limitations, as their effectiveness depends on the substrate on which the DNA of a person of interest can be found. In this study, an in-house dry-vacuuming device, the DNA-Buster, was compared to traditional methods for its DNA recovery performance from items typically examined in forensic casework. The aim was to evaluate whether this dry-vacuuming approach can recover DNA efficiently, potentially complementing the well-established recovery strategies. For this, the performances of swabbing, taping, wet- (M-Vac®) and dry-vacuuming (DNA-Buster) were investigated quantitatively and qualitatively for touch DNA deposited on carpet, cotton sweater, stone, tile and wood. For the sweater, both vacuuming methods outperformed the other collection tools quantitatively. While the highest DNA amounts for the carpet were yielded by swabbing and taping, dry-vacuuming was equally good in reaching full DNA profiles, whereas less complete profiles were observed for the M-Vac®. For stone and tile, swabbing was optimal, whereas dry-vacuuming clearly underperformed for these substrates. Taping was the best recovery method for wood. Despite applying single donor DNA after thoroughly cleaning the items, undesired DNA mixtures were detected for all recovery techniques and all substrates. The overall research findings show first that the novel dry-vacuuming method is suited for DNA recovery from textiles. Secondly, they indicate that more attention should be paid to the substrate-collection dependency to ensure best practices in recovering genetic material in a precise, confident and targeted manner from the variety of forensic casework material.

Recommendations for the successful identification of altered human remains using standard and emerging technologies: Results of a systematic approach.

Successful DNA-based identification of altered human remains relies on the condition of the corpses and varies between tissue types. Therefore, the aim of this prospective multicenter study was to generate evidence-based recommendations for the successful identification of altered remains. For this, 19 commonly used soft and hard tissues from 102 altered human bodies were investigated. The corpses' condition was categorized into three anatomical regions using a practical scoring system. Besides other data, DNA yields, degradation indices, and short tandem repeat (STR) profile completeness were determined in 949 tissue samples. Additionally, varying degrees of alteration and tissue-specific differences were evaluated using the Next Generation Sequencing (NGS) platform MiSeq FGx™. Selected challenging samples were sequenced in parallel with the Ion S5™ platform to assess platform-specific performances in the prediction of the deceased's phenotype and the biogeographic ancestry. Differences between tissue types and DNA extraction methods were found, revealing, for example, the lowest degradation for vertebral disc samples from corpses with initiating, advanced and high degrees of decomposition. With respect to STR profile completeness, blood samples outperformed all other tissues including even profoundly degraded corpses. NGS results revealed higher profile completeness compared to standard capillary electrophoresis (CE) genotyping. Per sample, material and degradation degree, a probability for its genotyping success, including the "extended" European Standard Set (eESS) loci, was provided for the forensic community. Based on the observations, recommendations for the alteration-specific optimal tissue types were made to improve the first-attempt identification success of altered human remains for forensic casework.

Technical note: Comparison of forensic swabs for intravaginal sampling.

This study compared the currently used swab Prionics ForensiX Evidence Collection Kit with the alternatives Prionics ForensiX Evidence Collection Tube SafeDry and Sarstedt Forensic Swab XL. Volunteers provided intravaginal swabs collected with all swab types at specific time points after unprotected sexual intercourse. Quantifiable DNA, detectability of seminal fluid component (prostate specific antigen, PSA) and spermatozoa were evaluated to find the best-performing swab type. While Sarstedt XL showed significantly higher DNA quantities for sperm cell fractions than ForensiX Kit, the more concise PSA test results clearly favour ForensiX SafeDry. Reassuringly, mostly complete autosomal STR profiles of male components were obtained for sperm cell fractions at all time points and tested swabs. Switching to the higher performing ForensiX SafeDry with improved sampling and processing properties will also benefit victims, medical personnel, and investigators.

Collaborative swab performance comparison and the impact of sampling solution volumes on DNA recovery.

The collection of DNA traces marks the first step determining the success of genetic analysis. This study aimed to identify and validate a suitable alternative to the currently used ForensiX Evidence Collection Kit containing a cardboard box for swab storage. This box has to be folded at the crime scene, which is time-consuming and carries the risk of potential contamination and handling difficulties. A collaboration study involving three police departments and one laboratory for forensic genetics was performed to compare the currently used swab against three challenger swabs: ForensiX SafeDry, Copan 4N6FLOQSwab™ Genetics and Copan 4N6FLOQSwab™ Crime Scene. Mock samples consisted mainly of touch DNA, but also blood, saliva and semen were applied to twelve items with different surfaces. Every organisation contributed with three DNA collectors, whose individual collection efficiencies were investigated. The challenge of preparing homogenous traces, especially touch DNA, was addressed by enhancing hand contact frequency and sampling area. As a further part of the swab comparison study, we describe for the first time the influence of different swabbing solution volumes on the sampling efficiency of the different swabs. The application of touch DNA was also tested for a further swab type, the Sarstedt Forensic Swab, which yielded such low DNA concentrations that it was excluded from the collaboration study. The Copan Genetics and Copan Crime Scene swabs yielded significantly lower DNA concentrations than the currently used ForensiX Evidence Collection Kit and ForensiX SafeDry swab. The inter-individual performance results of the operators revealed significant differences in sampling skills. Comparing different swabbing solution volumes showed higher DNA yields or no significant difference for the ForensiX Evidence Collection Kit and ForensiX SafeDry than the Copan Genetics, depending on the item or trace type swabbed. Our results highlight the importance of validating first-step components that are decisive to the success of DNA typing in the context of specific sampling procedures and laboratory methods. Also, the significance of individuals' securing variations, principally unknown for crime scene investigation and laboratory teams, is emphasised for the first time, offering a practical approach for improving and training DNA collecting activities and ensuring the optimal securing evidence process. These findings increase the knowledge of impacts on DNA collection and, thus, benefit other laboratories and forensic services, particularly when using the same extraction methods.

Validation and beyond: Next generation sequencing of forensic casework samples including challenging tissue samples from altered human corpses using the MiSeq FGx system.

The proceeding developments in next generation sequencing (NGS) technologies enable increasing discrimination power for short tandem repeat (STR) analyses and provide new possibilities for human identification. Therefore, the growing relevance and demand in forensic casework display the need for reliable validation studies and experiences with challenging DNA samples. The presented validation of the MiSeq FGx system and the ForenSeq™ DNA Signature Prep Kit (1) investigated sensitivity, repeatability, reproducibility, concordance, pooling variations, DNA extraction method variances, DNA mixtures, degraded, and casework samples and (2) optimized the sequencing workflow for challenging samples from human corpses by testing additional PCR purification, pooling adjustments, and adapter volume reductions. Overall results indicate the system's reliability in concordance to traditional capillary electrophoresis (CE)-based genotyping and reproducibility of sequencing data. Genotyping success rates of 100% were obtained down to 62.5 pg DNA input concentrations. Autosomal STR (aSTR) profiles of artificially degraded samples revealed significantly lower numbers of locus and allelic dropouts than CE. However, it was observed that the system still exposed drawbacks when sequencing highly degraded and inhibited samples from human remains. Due to the lack of studies evaluating the sequencing success of samples from decomposed or skeletonised corpses, the presented optimisation studies provide valuable recommendations such as an additional PCR purification, an increase in library pooling volumes, and a reduction of adapter volumes for samples with concentrations ≥31.2 pg. Thus, this research highlights the importance of all-encompassing validation studies for implementing novel technologies in forensic casework and presents recommendations for challenging samples.

Which tissue to take? A retrospective study of the identification success of altered human remains.

In forensic medicine, deceased are usually identified by comparing ante- and post-mortem dental or radiological features. However, in severe putrefaction, burning or absent reference data, the remaining tool for identifying human remains is DNA genotyping. But even a DNA-based identification can be challenging when confronted with a high post-mortem interval or heat impacts because it can lead to undesirable degradation of the DNA that varies among tissue types. This retrospective study investigated the identification success in 402 altered human corpses over seven years by comparing the examined tissue types from decomposed, skeletonised and burnt corpses as well as bodies found in water. For each tissue, the STR genotyping results and the number of additional or parallel genetic analyses were evaluated. By comparing the amplification success in samples from altered and unaltered remains, condition-based and tissue-specific differences were observed. With a mean number of 1.6 additional amplifications in cases with well-preserved corpses and 4.5 in altered corpses, the results showed significantly more DNA analyses for altered remains. In 83% of the cases, extra amplifications were performed to identify the corpse. The tissue-specific differences revealed an uncertainty in choosing suitable material from altered corpses for a successful DNA profile. Especially for bone and muscle samples, the genotyping success was the most unpredictable. Furthermore, comparing the retrospective outcome with other research findings, a remarkable variety of recommendations for the "best tissue choice" exists in the forensic community. Thus, our survey highlights the advantages of a broader and systematic approach on hard and soft tissues for successful DNA-based identification of altered human remains at first attempt.

Identification and characterization of novel rapidly mutating Y-chromosomal short tandem repeat markers.

Short tandem repeat polymorphisms on the male-specific part of the human Y-chromosome (Y-STRs) are valuable tools in many areas of human genetics. Although their paternal inheritance and moderate mutation rate (~10-3 mutations per marker per meiosis) allow detecting paternal relationships, they typically fail to separate male relatives. Previously, we identified 13 Y-STR markers with untypically high mutation rates (>10-2 ), termed rapidly mutating (RM) Y-STRs, and showed that they improved male relative differentiation over standard Y-STRs. By applying a newly developed in silico search approach to the Y-chromosome reference sequence, we identified 27 novel RM Y-STR candidates. Genotyping them in 1,616 DNA-confirmed father-son pairs for mutation rate estimation empirically highlighted 12 novel RM Y-STRs. Their capacity to differentiate males related by 1, 2, and 3 meioses was 27%, 47%, and 61%, respectively, while for all 25 currently known RM Y-STRs, it was 44%, 69%, and 83%. Of the 647 Y-STR mutations observed in total, almost all were single repeat changes, repeat gains, and losses were well balanced; allele length and fathers' age were positively correlated with mutation rate. We expect these new RM Y-STRs, together with the previously known ones, to significantly improving male relative differentiation in future human genetic applications.

Examination of postmortem animal interference to human remains using cross-species multiplex PCR.

Postmortem animal interference may be confused at first sight with injuries of vital origin, thus arousing suspicion of external violence preceding death. A reliable classification of the origin of such doubtful injuries is of crucial importance, a fact that is especially true for the investigation of suspected homicide and/or mammade body mutilation after death. In forensic pathology, the identification of injuries as caused by animals postmortem and the classification of a particular species as responsible for a specific injury pattern under question is usually done by forensic pathologists with vast practical experience and special knowledge of the appearance and morphology of tooth marks of carnivores and rodents, respectively. However, a molecular biological investigation of such wounds could provide genetic evidence that an injury pattern present on a corpse was truly caused postmortem by animal interference and thus support the pathologist's expertise. For this purpose, we developed a panel of small species-specific short-tandem repeat systems (<150 bp) for animals typically involved in postmortem scavenging of human remains, such as dogs and cats as well as wild-living rodents (mice and rats) having possible access to death scenes inside apartments or buildings. A specific and sensitive cross-species multiplex polymerase chain reaction was then established including the species-specific animal markers, thus enabling the genetic identification of wounds caused postmortem by different animals on human remains.