TGFß level in healthy and children with Marfan syndrome-effective reduction under sartan therapy.

Introduction: Transforming growth factor ß (TGFß) metabolism plays an important role in the pathogenesis of Marfan syndrome (MFS). Accordingly, drug therapy uses TGFß receptor blockade to slow down the cardiovascular manifestations, above all aortic root dilatation. Angiotensin II type 1 receptor blockers (ARBs) have been shown to reduce TGFß levels in adults. Data on childhood are lacking and are now being investigated in the TiGer For Kids study presented here. Methods: We examined 125 children without chronic disease and 31 pediatric Marfan patients with a proven FBN1 variant with regard to TGFß levels. In addition, we measured TGFß levels during the initiation of ARB therapy in pediatric Marfan patients. Results: In children without chronic disease, TGFß levels were found to decrease from childhood to adolescence (p < 0.0125). We could not measure a relevantly increased TGFß level in pediatric Marfan patients. However, we showed a significant suppression of the TGFß level after treatment with ARBs (p < 0.0125) and a renewed increase shortly before the next dose. Discussion: The TGFß level in childhood changes in an age-dependent manner and decreases with age. The TGFß level drops significantly after taking ARBs. Based on our experience and data, a TGFß receptor blockade in childhood seems reasonable. So far, TGFß level cannot be used as an MFS screening biomarker.

Comparison of massively parallel sequencing to capillary electrophoresis for short tandem repeat genotyping of trace DNA.

In forensic genetics, massively parallel sequencing (MPS) offers several advantages over the current golden standard, capillary electrophoresis (CE): additional sequence information, shorter amplicon lengths, and the simultaneous analysis of many markers. These benefits result in a reduced number of reactions necessary while improving the amount of data obtained, thereby conserving valuable sample extracts. This proves particularly advantageous for the analysis of trace DNA. This study assessed the suitability of MPS for short tandem repeat (STR) typing of low template samples compared with results obtained through CE. The MPS genotypes showed higher concordance to reference genotypes, with donor alleles being more frequently assigned to be the major contributor, meeting the requirements for database entry. However, the MPS workflow is more time-consuming and associated with higher costs.

A compact gas attenuator for the SwissFEL ATHOS beamline realized using additive manufacturing.

Gas attenuators are important devices providing accurate variation of photon intensity for soft X-ray beamlines. In the SwissFEL ATHOS beamline front-end the space is very limited and an innovative approach has been taken to provide attenuation of three orders of magnitude up to an energy of 1200 eV. Additive manufacturing of a differential pumping system vacuum manifold allowed a triple pumping stage to be realized in a space of less than half a meter. Measurements have shown that the response of the device is as expected from theoretical calculations.

Whole-genome sequencing of artificial single-nucleotide variants induced by DNA degradation in biological crime scene traces.

The aim of this study was to identify artificial single-nucleotide variants (SNVs) in degraded trace DNA samples. In a preliminary study, blood samples were stored for up to 120 days and whole-genome sequencing was performed using the Snakemake workflow dna-seq-gatk-variant-calling to identify positions that vary between the time point 0 sample and the aged samples. In a follow-up study on blood and saliva samples stored under humid and dry conditions, potential marker candidates for the estimation of the age of a blood stain (= time since deposition) were identified. Both studies show that a general decrease in the mean fragment size of the libraries over time was observed, presumably due to the formation of abasic sites during DNA degradation which are more susceptible to strand breaks by mechanical shearing of DNA. Unsurprisingly, an increase in the number of failed genotype calls (no coverage) was detected over time. Both studies indicated the presence of artificial SNVs with the majority of changes happening at guanine and cytosine positions. This confirms previous studies and can be explained by depurination through hydrolytic attacks which more likely deplete guanine while deamination leads to cytosine to thymine variants. Even complete genotype switches from homozygote 0/0 genotypes to the opposite 1/1 genotypes were observed. While positions with such drastic changes might provide suitable candidate markers for estimating short-term time since deposition (TsD), 11 markers were identified which show a slower gradual change of the relative abundance of the artificial variant in both blood and saliva samples, irrespective of storage conditions.

Detecting DNA damage in stored blood samples.

Several commercially available quantitative real-time PCR (qPCR) systems enable highly sensitive detection of human DNA and provide a degradation index (DI) to assess DNA quality. From routine casework in forensic genetics, it was observed that DNA degradation in forensic samples such as blood samples stored under sub-optimal conditions leads to visible effects in multiplex analyses of short tandem repeat markers (STRs) due to decreased amplification efficiencies in longer amplicons. It was further noticed that degradation indices often remain below the value that is considered to be critical. Thus, the aim of this work was to systematically analyze this effect and to compare conventional qPCR assays with a modified qPCR approach using uracil DNA glycosylase (UNG) and DNA quality assessment methods based on electrophoresis. Blood samples were stored at three different storage temperatures for up to 316 days. Significantly increased DNA recovery was observed from samples stored at high temperatures (37 °C) compared samples stored at room temperature and 4 °C. We observed typical effects of degradation in STR analyses but no correlation between DI and storage time in any of the storage conditions. Adding UNG slightly increased the sensitivity of detecting DNA degradation in one of the qPCR kits used in this study. This observation was not confirmed when using a second qPCR system. Electrophoretic systems did also not reveal significant correlations between integrity values and time. Methods for detecting DNA degradation are usually limited to the detection of DNA fragmentation, and we conclude that degradation affecting forensic STR typing is more complex.

Development of a multiplex assay for detection of autosomal and Y-chromosomal STRs, assessment of the degradation state of mitochondrial DNA and presence of mitochondrial length heteroplasmies.

The current focus in most routine forensic casework is detection of autosomal or gonosomal Short Tandem Repeats (STRs). With increasing degradation, STR analysis tends to be less successful up to complete failure. For challenging samples such as telogen hair roots and shafts, touch DNA samples or skeletal remains, mitochondrial DNA (mtDNA) analysis provides a powerful tool. Determination of DNA quantity is an important part in the casework workflow. Several ready-to-use kits are commercially available for nuclear DNA targets. However, quantification of mtDNA targets requires the establishment of an in-house method. Some assays even contain assessment of degradation, which alleviates the choice of target enrichment for sequencing through medium or small amplicons. As Sanger-type Sequencing (STS) still remains the golden standard in many laboratories, identification of heteroplasmies in C-tract regions prior to the sequencing reaction is advantageous. Firstly, primer selection can be expanded with primers binding near the C-tract and secondly, determination of the dominant variant is straightforward. All those quantity (nuclear and mtDNA) and quality (degradation and length heteroplasmies) evaluations usually require at least two separate reactions. Therefore, the aim of this project was the combination of all these targets in one multiplex assay using capillary electrophoresis to spare valuable sample extract. Amplification of representative autosomal and Y-chromosomal STRs allows estimate of success of (Y-)STR analysis. Simultaneously, five length heteroplasmies in the mitochondrial control region are targeted as well as three conservative regions of differing fragment lengths for assessment of the mitochondrial degradation state. Based on the outcome of this assay, forensic examiners can decide if STR analysis may be suitable. In case of absent STR peaks, appropriate proceeding of mtDNA sequencing can be determined.

Best of both: A simultaneous analysis of mRNA and miRNA markers for body fluid identification.

The body fluid identification of traces found at crime scenes is crucial in relation of the circumstance of crime. For this reason, the body fluid identification (BFI) by molecular biological methods has been increasingly investigated in recent decades. Especially the use of messenger RNA (mRNA) has been established and validated by various studies. mRNAs can resist degradation for several decades under dry and dark environmental conditions, but degradation increases greatly e.g., in humid environments and UV radiation. In contrast, the shorter and protein-protected micro RNAs (miRNAs) are less susceptible to degradation, but not all potential markers are tissue-specific. The aim of this study was to develop a simultaneous mRNA/miRNA multiplex assay to take advantage of both types of RNA. The final assay was tested for various body fluids, dilutions, and mixtures. To demonstrate the advantage of a combined mRNA/miRNA assay, older and mostly degraded samples were examined and compared to an established mRNA assay. Initial results from degraded samples show that tissue-specific miRNAs expected could be detected for 93% of the degraded samples compared to mRNA markers with 25% of the mRNA assay. The result is a simultaneous mRNA/miRNA multiplex assay on capillary electrophoresis (CE) for the first time.

Tricuspid valve prolapse as an early predictor for severe phenotype in children with Marfan syndrome.

AIM: In Marfan syndrome, various cardiovascular pathologies, such as aortic dilatation and mitral valve pathologies, already occur in childhood and determine course of the disease. This study aimed to establish additional cardiovascular risk markers for severe Marfan phenotypes. We investigated tricuspid valve prolapse (TVP) and its predictive value for outcome of paediatric Marfan disease. METHODS: In this retrospective, observational cohort study, we identified 130 paediatric Marfan patients (10.7 ± 4.8 years) with FBN1 variants. We divided patients into two groups based on TVP presence and performed a cross-sectional analysis to investigate the association of TVP with other cardiovascular, ocular and systemic pathologies, at first and last visit. A longitudinal analysis was performed with follow-up data. RESULTS: At baseline, patients with TVP had higher incidence of aortic root dilatation (p = 0.013), mitral valve prolapse (p = 0.0001) and systemic manifestations (p = 0.025) than patients without TVP. At follow-up, previous presence of TVP predicted higher probability of aortic root dilatation (p = 0.002), mitral valve prolapse (p = 0.0001) and systemic manifestations (p = 0.002). CONCLUSION: This shows that TVP is linked to both cardiac and extracardiac Marfan manifestations and TVP is an important marker for a disease severity in these children. Therefore, TVP should be assessed routinely using echocardiography in paediatric Marfan patients.

Persistence of blood (DNA/RNA) on shoe soles under varying casework related conditions.

Blunt force traumas by footwear can result in severe and even fatal head and upper body injuries. Oftentimes, footwear impressions are only partially available and evidential value is limited. DNA evidence on shoe soles could provide crucial evidence helping to solve crimes by linking target DNA to the activity of interest. Little is known about the persistence and detectability of biological material post such offenses and the interplay of factors affecting the analytical success. In this study, we assessed the persistence of blood on shoe soles under varying parameters such as blood location, different sneakers, weather condition, gait, amount of blood, underground and step count. We applied an optimized DNA/RNA workflow adapted to micro-traces without constraints for the primary DNA pipeline. There is a high probability to link donor DNA to the shoe sole for up to 300-400 steps, regardless of the underground, blood location, and amount of blood. Depending on the sole material and the degree of abrasion of the sole, a longer blood persistence can be observed. Considering blood, 98.2% of the initial DNA amount (1 µl initial blood volume) was lost after 100 steps walked on sole areas that are in constant contact with the ground. Proportion of foreign DNA was marginal (avg. 4.4 alleles), minimizing the probability of unintentional DNA transfer in this context. RNA typing showed high specificity but lower sensitivity than presumptive tests used for body fluid identification (BFI). Luminol is essential for targeted sampling on shoe soles, as latent blood traces (>100-200 steps) provided sufficient biological material for DNA/RNA typing. The generated data help to address the activity of interest and evaluate probabilities about prevalence of target DNA important for casework implications and assessments on activity level.

Casework-related DNA transfer on footwear in consideration of the shedder status.

DNA evidence on shoes can play an important role in solving a variety of crimes. We investigated the transfer, persistence, prevalence and recovery of DNA (DNAtppr) on shoes (sneakers) and their soles in realistic handling scenarios taking into account the shedder status. This study aims to increase the understanding of the expected composition of DNA profiles and their probative value, providing a basis for activity level assessments. Samples were analyzed using a direct lysis method, suggesting its versatility and increasing the DNA typing success compared to previous studies on footwear. The data showed surface-dependent background DNA (bDNA) levels on shoe soles and prevalence of bDNA on the upper parts of the shoe. The owner of the shoe was allocatable to the mixture for almost every shoe and sampling location. Alternating scenarios of shoe handling were simulated through different pairs of shedders to distinguish shoe owner and subsequent user. Secondary users were attributable to DNA mixtures regardless of shedder status after wearing shoes a single time. The influence of the shedder status follows specific trends in this context. However, particularly intermediate shedders show inconsistent results. The prevalence of bDNA appears to have a greater effect on the impact of the shedder status on DNA profile composition than previously reported. The data help researchers to better resolve suspect statements and determine if a person of interest wore the shoes relevant to the investigation.

Statistefix 4.0: A novel probabilistic software tool.

Latest innovations indicate that continuous tools are promising DNA trace assessment methods. In this study, we present the continuous software solution Statistefix 4.0. The software supports DNA experts in deducing DNA profiles for database queries and can help to preselect DNA samples suitable for further processing using advanced probabilistic search engines. The novel tool weights genotype contributions and deduces major contributors from high- and low-quality DNA traces. Peak height, degradation, stutter as well as allelic drop-in/-out events are incorporated in the statistical model. We analyzed reference and casework samples as well as artificially generated mixture samples for software evaluation. The tool offers the completely automated assessment of reference and mixture samples. Deconvolution outcomes of mixtures are compared with EuroForMix, GenoProof Mixture 3 and STRmix™. Data show that Statistefix 4.0 is as successful as analogously tested and implemented software. Deduced DNA profiles from casework samples highlight the potential benefit in routine casework. Statistefix 4.0 is freely available, works with replicates of different autosomal kits and enables bulk sample processing. This inter-laboratory study includes a variety of sample types and indicates a timesaving, robust and easily implemented software that supports DNA analysts in evaluating DNA traces.

Development and validation of an mRNA-based multiplex body fluid identification workflow and a rectal mucosa marker pilot study.

Molecular identification of body fluids and tissues is crucial in order to understand the circumstances of crimes. For that reason, molecular investigations used to identify body fluids/tissues have increasingly been examined recently. Various studies have proved that messenger RNA (mRNA) profiling is a sensitive and robust method for body fluid/tissue identification. The forensically relevant body fluids/tissues blood, semen, saliva, vaginal secretion, menstrual blood and skin have all been detected successfully by applying suitable mRNA assay. However, rectal mucosa, which can be found as evidence in sexual assault cases, has been neglected in forensic investigations. So far there is no mRNA marker to detect rectal mucosa, although anal penetration occurs in a large number of sexual assaults (23.2% of female victims and 50% of male victims). In this study, specific and sensitive mRNA markers for forensically relevant body fluids were adapted and validated in an mRNA multiplex assay for routine casework. This included the implementation of a DNA/RNA re-extraction method for automated extraction that can be integrated into casework without loss of DNA. This re-extraction method and the mRNA multiplex assay were tested using casework samples. PCR-primers were designed for the identification of rectal mucosa and the more effective marker MUC12 was integrated into an extended multiplex assay. The result of our study is a highly specific and sensitive mRNA multiplex assay plus an automated DNA/RNA re-extraction method, that can be integrated into casework and identify rectal mucosa for the first time.

The diversity of shedder tests and a novel factor that affects DNA transfer.

Since the first shedder test was formulated almost 20 years ago, a plethora of different test strategies has emerged. The amount of data generated so far is considerable. However, because of the limited reproducibility of its results, the reliability of the shedder concept is frequently questioned. This study provides a literature overview of applied shedder tests that capture the diversity of the concept. It is pointed out to what extent different classification criteria, workflows, and trace evaluation can impair the classification outcome. The robustness of shedder status was assessed by applying a promising approach established by Fonneløp et al. (Forensic Sci Int Genet 29:48-60, 21). Data provide similar results to those in recent studies but also ambiguous shedder classifications. The applied shedder test was adapted based on our own as well as the reviewed data. With novel classification parameters, promising results were achieved. This study reveals uncertainties and inconsistencies of the shedder concept. Recommendations for harmonization and transparency are proposed. Implementation of the recommendations may result in an increased impact on casework and transfer studies, including activity-level assessments. Furthermore, this study shows that moisturizers affect participants' shedder status as well as DNA transfer. The impact appears to remain relevant even 60 min post ointment application but depends greatly on the type of moisturizer applied.

mRNA profiling of mock casework samples: Results of a FoRNAP collaborative exercise.

In recent years, forensic mRNA profiling has increasingly been used to identify the origin of human body fluids. By now, several laboratories have implemented mRNA profiling and also use it in criminal casework. In 2018 the FoRNAP (Forensic RNA Profiling) group was established among a number of these laboratories with the aim of sharing experiences, discussing optimization potential, identifying challenges and suggesting solutions with regards to mRNA profiling and casework. To compare mRNA profiling methods and results a collaborative exercise was organized within the FoRNAP group. Seven laboratories from four countries received 16 stains, comprising six pure body fluid / tissue stains and ten mock casework samples. The laboratories were asked to analyze the provided stains with their in-house method (PCR/CE or MPS) and markers of choice. Five laboratories used a DNA/RNA co-extraction strategy. Overall, up to 11 mRNA markers per body fluid were analyzed. We found that mRNA profiling using different extraction and analysis methods as well as different multiplexes can be applied to casework-like samples. In general, high input samples were typed with high accuracy by all laboratories, regardless of the method used. Irrespective of the analysis strategy, samples of low input or mixed stains were more challenging to analyze and interpret since, alike to DNA profiling, a higher number of markers dropped out and/or additional unexpected markers not consistent with the cell type in question were detected. It could be shown that a plethora of different but valid analysis and interpretation strategies exist and are successfully applied in the Forensic Genetics community. Nevertheless, efforts aiming at optimizing and harmonizing interpretation approaches in order to achieve a higher consistency between laboratories might be desirable in the future. The simultaneous extraction of DNA alongside RNA showed to be an effective approach to identify not only the body fluid present but also to identify the donor(s) of the stain. This allows investigators to gain valuable information about the origin of crime scene samples and the course of events in a crime case.

U-DCS: characterization of the first permanent human dendritic sarcoma cell line.

A dendritic cell sarcoma cell line, U-DCS, was established from a dendritic cell sarcoma in a 53-year-old Caucasian male patient. Since its establishment, U-DCS has maintained stable phenotypic characteristics in vitro and has a doubling time of approximately 2 days under standard culture conditions. U-DCS is growing with typical dendritic cell morphology in tissue and expresses the dendritic cell sarcoma immunophenotypic markers S100 protein, MHCI, MHCII, and vimentin. Expression analysis revealed transcripts for the toll-like receptors TLR3, -4, -9 and DDX58 (RIG-I), but not for TLR2. U-DCS shows functional features of dendritic cells with the ability of phagocytosis and antigen-specific T cell stimulation. Karyotype-, CGH-, and mFISH analysis point to a chromosomal instability and a hypotetraploid karyotype with approximately 130 chromosomes. U-DCS is the first immortalized human dendritic cell sarcoma cell line and has some morphological and functional features of dendritic cells without dependency on growth factors.

Genotype-Phenotype Correlation in Children: The Impact of FBN1 Variants on Pediatric Marfan Care.

Currently, no reliable genotype-phenotype correlation is available for pediatric Marfan patients in everyday clinical practice. We investigated correlations of FBN1 variants with the prevalence and age of onset of Marfan manifestations in childhood and differentiated three groups: missense/in-frame, splice, and nonsense/frameshift variants. In addition, we differentiated missense variants destroying or generating a cysteine (cys-missense) and alterations not affecting cysteine. We categorized 105 FBN1-positive pediatric patients. Patients with cys-missense more frequently developed aortic dilatation (p = 0.03) requiring medication (p = 0.003), tricuspid valve prolapse (p = 0.03), and earlier onset of myopia (p = 0.02) than those with other missense variants. Missense variants correlated with a higher prevalence of ectopia lentis (p = 0.002) and earlier onset of pulmonary artery dilatation (p = 0.03) than nonsense/frameshift, and dural ectasia was more common in the latter (p = 0.005). Pectus excavatum (p = 0.007) appeared more often in patients with splice compared with missense/in-frame variants, while hernia (p = 0.04) appeared earlier in the latter. Findings on genotype-phenotype correlations in Marfan-affected children can improve interdisciplinary therapy. In patients with cys-missense variants, early medical treatment of aortic dilatation seems reasonable and early regular ophthalmologic follow-up essential. Patients with nonsense/frameshift and splice variants require early involvement of orthopedic specialists to support the growing child.

Developmental validation of the monSTR identity panel, a forensic STR multiplex assay for massively parallel sequencing.

The 21-plex STR panel monSTR was designed for high-fidelity forensic genotyping on the Illumina MiSeq platform. In this study, the panel's performance was validated according to the recommended validation guidelines of the Scientific Working Group for DNA Analysis Methods (SWGDAM). Concordance, repeatability and reproducibility, sensitivity of detection, mixture analysis, species-specificity, and the ability to analyze mock samples were assessed. Sequence data was analyzed using the genotyping software toaSTR. The assay performance was evaluated by measuring the read on-target ratio, the genotype accuracy, the inter-locus balance, the heterozygosity balance, and the signal-to-noise ratio. Results showed that profiles of NIST reference DNA samples as well as GEDNAP proficiency samples were fully concordant with CE-based methods. In addition, inter-run and intra-run variation experiments indicated high precision. Furthermore, full profiles could be obtained using 62.5 pg of DNA input amount with proper inter-locus balance and read on-target ratio; 76.4% of alleles were correctly called with 7.8 pg DNA input amount. It was demonstrated that 94.4% of minor contributor alleles were resolved accurately in a 1:49 mixture. Results suggested that the minor contribution could be precisely calculated based on the minor component allele frequency. Validation results described here demonstrate that the monSTR forensic identity panel is a valid tool for forensic STR genotyping using massively parallel sequencing.

Looking for the pinpoint: Optimizing identification, recovery and DNA extraction of micro traces in forensic casework.

Many challenges are encountered in the analysis of micro traces such as touch DNA or telogen hair samples. Although DNA typing methods have become immensely more sensitive over the last years, recovery of the minute amounts of biological material in micro traces requires further enhancement. For example felony cases, where an offender oftentimes only contributes minor amounts of touch DNA, separation of victim and offender DNA poses difficulties. Whereas complete sampling of evidence generates admixed profiles, single particle collection is labor- and time-intensive. Besides optimization of identification and collection of bio particles during this study, a novel sampling strategy for enhanced DNA yield as well as success rate but simultaneous avoidance of mixture creation is proposed for tapings in forensic casework. Improvement of another crucial step in micro trace analysis, DNA extraction, involved evaluation of efficiency and DNA recovery of different extraction methods, namely magnetic bead based Maxwell extraction, Chelex, Casework Direct Kit and Investigator Casework GO! kit. Direct lysis approaches seemed to be most suitable for low template traces. Recently developed commercial kits even allow the presence of inhibitors. Improvement of embraced aspects successfully facilitates processing of micro traces in terms of time and labor.

"The acid test"-validation of the ParaDNA® Body Fluid ID Test for routine forensic casework.

The identification of the cellular origin and composition of crime scene-related traces can provide crucial insight into a crime scene reconstruction. In the last decade, especially mRNA-based body fluid and tissue identification (BFI) has been vigorously examined. Besides capillary electrophoretic (CE) and real-time quantitative PCR (RT-qPCR)-based approaches for mRNA detection, melt curve analysis bears potential as a simple-to-use method for BFI. The ParaDNA® Body Fluid ID Test relies on HyBeacon® probes and was developed as a rapid test for mRNA-based BFI of six different body fluids: vaginal fluid, seminal fluid, sperm cells, saliva, menstrual, and peripheral blood. The herein presented work was performed as an "acid test" of the system and should clarify whether the approach matches the requirements of forensic routine casework in German police departments. Tested samples consisted of single source as well as of mixed samples.