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UNLABELLED: Human age estimation from trace samples may give important leads early in a police investigation by contributing to the description of the perpetrator. Several molecular biomarkers are available for the estimation of chronological age, and currently, DNA methylation patterns are the most promising. In this study, a QIAGEN age protocol for age estimation was tested by five forensic genetic laboratories. The assay comprised bisulfite treatment of the extracted DNA, amplification of five CpG loci (in the genes of ELOVL2, C1orf132, TRIM59, KLF14, and FHL2), and sequencing of the amplicons using the PyroMark Q48 platform. Blood samples from 49 individuals with ages ranging from 18 to 64 years as well as negative and methylation controls were analyzed. An existing age estimation model was applied to display a mean absolute deviation of 3.62 years within the reference data set.KEY POINTS: Age determination as an intelligence tool during investigations can be a powerful tool in forensic genetics.In this study, five laboratories ran 49 samples and obtained a mean absolute deviation of 3.62 years.Five markers were analyzed on a PyroMark Q48 platform.

DNA profiling is central in the field of forensic genetics and allows for the forensic identification of individuals. Biological trace samples consisting of very few cells, however, are a challenge. In Denmark, biological trace samples containing less than 100 pg DNA are termed low template DNA (ltDNA) samples and are not subjected to DNA profiling. In the case of such ltDNA samples, RNA may be used instead of DNA. Each cell only contains two copies of the autosomal DNA, while abundant RNA species may be present and expressed in high copy numbers. As such, much of the genomic information, including sequence variation, should be replicated in multiple copies of messenger RNA (mRNA). Single nucleotide polymorphisms (SNPs) may be used for human identification, and the coding region SNPs in RNA represent a direct link between the DNA and RNA. This thesis describes the development of a prototype mRNA SNP profile for the forensic identification of individuals. Di-allelic SNP markers with high allele frequency were identified in blood stains left to degrade over a two-week time period. Promising markers were retained and further screened in a small subset of the Danish population (41 individuals). Markers in linkage disequilibrium or with skewed allele balances were excluded. The prototype profile of 39 SNPs gives a global mean match probability of 1.88 · 10−12 as well as a mean match probability of 1.86 · 10−10 in the European (non-Finnish) population. Further, the mRNA SNP profile also has the potential for simultaneous identification of the body fluid of a stain and its donor due to the presence of blood-specific mRNA markers. Furthermore, this thesis examined the potential for using RNA markers identified through RNAseq to determine the time since deposition (TSD) of a stain. An accurate method to esti- mate the TSD of forensic trace samples would help investigators evaluate the true re

Estimating an individual’s age can be relevant in several areas primarily related to the clinical and forensic fields. In the latter, estimation of an individual’s chronological age from biological material left by the perpetrator at a crime scene may provide helpful information for police investigation. Estimation of age is also beneficial in immigration cases, where age can affect the person’s protection status under the law, or in disaster victim identification to narrow the list of potential missing persons. In the last decade, research has focused on establishing new approaches for age prediction in the forensic field. From the first forensic age estimations based on morphological inspections of macroscopic changes in bone and teeth, the focus has shifted to molecular methods for age estimation. These methods allow the use of samples from human biological material that does not contain morphological age features and can, in theory, be investigated in traces containing only small amounts of biological material. Molecular methods involving DNA analyses are the primary choice and estimation of DNA methylation levels at specific sites in the genome is the most promising tool. This review aims to provide an overview of the status of forensic age prediction using molecular methods, with particular focus in DNA methylation. The frequent challenges that impact forensic age prediction model development will be addressed, together with the importance of validation efforts within the forensic community.

This report was prepared by Marie-Louise Kampmann, MSc, PhD and Niels Morling, MD, DMSc, Department of Forensic Medicine, University of Copenhagen, Denmark, commissioned by Magistrate Don Mario Carroza Espinosa and Magistrate Ms. Paola Plaza to investigate the death of Pablo Neruda. The objective was to secure the identity of the human remains examined and determine the state of human and bacterial DNA preservation and the presence of contamination and possible pathogens in the remains of Pablo Neruda. This was accomplished with the use of advanced forensic genetic examinations, DNA extraction, sequencing technology, and in-depth bioinformatic analysis.

Sodium hypochlorite is an efficient reagent for removal of unwanted DNA from laboratory surfaces. Here, we tested two different chlorine wipes and compared their performance to a 0.9–1.8% hypochlorite solution. WipeClean Chlorine Disinfection wipes contain > 0.1 g sodium hypochlorite/kg, whereas WetWipe Chlorine Desinfection wipes contain > 1000 ppm active chlorine. Clean surfaces were contaminated with 10 µL 0.5 ng/µL of massively parallel sequencing libraries. The DNA was dried and left for 45 min before any treatment. The surfaces were cleaned using either 1) a 0.9–1.8% hypochlorite solution and clean wipes, 2) a WipeClean wipe, 3) a WetWipe, or 4) the surface was not cleaned. All experiments were repeated three times. Subsequently, the surfaces were swabbed using cotton swabs. DNA was extracted from the swabs and the DNA concentrations were determined in quadruplicates by real-time PCR. This protocol was repeated after the soft plastic wrapping around the wipes were left open or closed for several weeks. The results showed that the WipeClean wipes efficiently removed DNA for up to four weeks after the box with the wipes were opened, whereas the WetWipe wipes dried faster and gradually lost their cleaning effect.

Background: No biomarkers have been identified that can classify subtypes of hand eczema (HE). Although skin biopsies represent the gold standard for investigations of the skin, the invasive technique is not favorable when investigating skin from sensitive areas. Recent advances in the use of skin-tape strips for molecular investigations enable noninvasive investigations of HE. Objective: By using whole transcriptome sequencing (WTS), the molecular profile of HE according to different localizations on the hands, etiologies, and clinical/morphological subtypes was investigated. Methods: Thirty adult, Danish HE patients, 12 with and 18 without concurrent atopic dermatitis (AD), as well as 16 controls were included. Tape strip samples were collected from lesional, nonlesional, and healthy skin. Total RNA was extracted and WTS was performed. Results: The largest molecular difference of HE patients with and without AD was found in nonlesional skin areas and included a downregulation of CXCL8 for HE patients without AD. Differences between allergic and irritant contact dermatitis included promising epidermal biomarkers such as EPHA1. Conclusion: Skin tape strip samples could be used to assess the gene expression profile of HE on different localizations of the hands. The skin tape strip method identified new molecular markers that showed promising result for the identification of HE subtypes.

Cerebral palsy (CP) is the most common cause of movement disorders in children. Next generation sequencing (NGS) studies have previously shown that expression levels are fundamentally different in children with CP compared to typically developing (TD). However, given that children are in full development, we might expect gene expression levels to change once maturity is reached. Therefore, the main purpose of this study was to investigate gene expression levels of 93 target genes in adults with CP using NGS on muscle biopsies of the gastrocnemius, taken from 22 participants (n = 12 adults with CP; n = 10 TD adults). Subsequently, we carried out NGS of the mitochondrial genome to identify mtDNA variants, and additionally we studied the mitochondrial content using transmission electron microscopy images of the gastrocnemius muscle. Finally, we compared systemic ion levels between TD adults and adults with CP. Differential gene expression levels were found in genes involved in muscle contraction (MYH1 and MYBPC2), mitochondrial function kATP5J, CYCS and NDUFB6), calcium handling (CAMK2B and ATP2A), metabolism (LPL), muscle signaling (MYC, CREB1, ACVR2B, LMNA and TRIM54), and ECM (TNC). There was no statistical significant difference between CP and TD for mtDNA variant frequencies and mitochondrial content. The ion levels of Ca2+, Na+ and K+ were statistically significantly reduced while the Cl− levels were significant increased in adults with CP compared to TD adults. These results highlight that most transcriptional differences are related to muscle function in adults with CP and that mitochondrial function might be altered but not mitochondrial content.