Genetic Approaches to Appearance and Ancestry

Traditionally, routine forensic casework is based on comparative grounds. DNA profiles obtained from crime-scenes are compared with those of potential suspects or DNA profiles deposited in forensic DNA databases. The principal limitation of such comparative approach is that trace donors unknown to the investigators with their DNA profiles cannot be identified. The recent advancements in Forensic DNA Phenotyping can provide investigative leads to help find previously unknown individuals by inferring externally visible characteristics and biogeographic ancestry prediction without comparative DNA testing. Some predictive DNA tests for appearance and ancestry were previously developed by our group, but their forensic validation, as prerequisite for casework application, was mostly missing thus far. For other appearance traits the genetic knowledge was previously not advanced enough to develop predictive DNA tests. Moreover, the genotyping technology capable of tolerating quality and quantity issues of trace DNA and at the same time allowing the parallel analysis of large number of DNA markers was previously not available, which limited previously developed forensic ancestry DNA tests. The major aims of this thesis therefore are to: - forensically validate DNA test systems for predicting appearance and ancestry that were recently developed by our group i.e., IrisPlex for eye color prediction, HIrisPlex for hair and eye color prediction, and a set of mtDNA multiplexes for maternal ancestry prediction; - develop and forensically validate a new DNA test system for skin color prediction in combination with eye and hair color prediction from trace DNA; - develop and forensically validate a new DNA test system for complete mitogenome analysis providing maximum-resolution maternal lineage and maternal ancestry inference from trace DNA

Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine

Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylo

Global skin colour prediction from DNA

Human skin colour is highly heritable and externally visible with relevance in medical, forensic, and anthropological genetics. Although eye and hair colour can already be predicted with high accuracies from small sets of carefully selected DNA markers, knowledge about the genetic predictability of skin colour is limited. Here, we investigate the skin colour predictive value of 77 single-nucleotide polymorphisms (SNPs) from 37 genetic loci previously associated with human pigmentation using 2025 individuals from 31 global populations. We identified a minimal set of 36 highly informative skin colour predictive SNPs and developed a statistical prediction model capable of skin colour prediction on a global scale. Average cross-validated prediction accuracies expressed as area under the receiver-operating characteristic curve (AUC) ± standard deviation were 0.97 ± 0.02 for Light, 0.83 ± 0.11 for Dark, and 0.96 ± 0.03 for Dark-Black. When using a 5-category, this resulted in 0.74 ± 0.05 for Very Pale, 0.72 ± 0.03 for Pale, 0.73 ± 0.03 for Intermediate, 0.87±0.1 for Dark, and 0.97 ± 0.03 for Dark-Black. A comparative analysis in 194 independent samples from 17 populations demonstrated that our model outperformed a previously proposed 10-SNP-classifier approach with AUCs rising from 0.79 to 0.82 for White, comparable at the intermediate level of 0.63 and 0.62, respectively, and a large increase from 0.64 to 0.92 for Black. Overall, this study demonstrates that the chosen DNA markers and prediction model, particularly the 5-category level; allow skin colour predictions within and between continental regions for the first time, which will serve as a valuable resource for future applications in forensic and anthropologic genetics

Genetics of skin color variation in Europeans: genome-wide association studies with functional follow-up

In the International Visible Trait Genetics (VisiGen) Consortium, we investigated the genetics of human skin color by combining a series of genome-wide association studies (GWAS) in a total of 17,262 Europeans with functional follow-up of discovered loci. Our GWAS provide the first genome-wide significant evidence for chromosome 20q11.22 harboring the ASIP gene being explicitly associated with skin color in Europeans. In addition, genomic loci at 5p13.2 (SLC45A2), 6p25.3 (IRF4), 15q13.1 (HERC2/OCA2), and 16q24.3 (MC1R) were confirmed to be involved in skin coloration in Europeans. In follow-up gene expression and regulation studies of 22 genes in 20q11.22, we highlighted two novel genes EIF2S2 and GSS, serving as competing functional candidates in this region and providing future research lines. A genetically inferred skin color score obtained from the 9 top-associated SNPs from 9 genes in 940 worldwide samples (HGDP-CEPH) showed a clear gradual pattern in Western Eurasians similar to the distribution of physical skin color, suggesting the used 9 SNPs as suitable markers for DNA prediction of skin color in Europeans and neighboring populations, relevant in future forensic and anthropological investigations

Bringing colour back after 70 years: Predicting eye and hair colour from skeletal remains of World War II victims using the HIrisPlex system

Retrieving information about externally visible characteristics from DNA can provide investigative leads to find unknown perpetrators, and can also help in disaster victim and other missing person identification cases. Aiming for the application to both types of forensic casework, we previously developed and forensically validated the HIrisPlex test system enabling parallel DNA prediction of eye and hair colour. Although a recent proof-of-principle study demonstrated the general suitability of the HIrisPlex system for successfully analysing DNA from bones and teeth of various storage times and conditions, practical case applications to human remains are scarce. In this study, we applied the HIrisPlex system to 49 DNA samples obtained from bones or teeth of World War II victims excavated at six sites, mostly mass graves, in Slovenia. PCR-based DNA quantification ranged from 4 pg/μl to 313 pg/μl and on an average was 41 pg/μl across all samples. All 49 samples generated complete HIrisPlex profiles with the exception of one MC1R DNA marker (N29insA) missing in 83.7% of the samples. In 44 of the 49 samples (89.8%) complete 15-loci autosomal STR (plus amelogenin) profiles were obtained. Of 5 pairs of skeletal remains for which STR profiling suggested an origin in the same individuals, respectively, 4 showed the same HIrisPlex profiles and predicted eye and hair colours, respectively, while discrepancies in one pair (sample 26 and 43) are likely to be explained by DNA quantity and quality issues observed in sample 43. Sample 43 had the lowest DNA concentration of only 4 pg/μl, producing least reliable STR results and could be misleading in concluding that samples 43 and 26 originate from the same individual. The HIrisPlex-predicted eye and hair colours from two skeletal samples, suggested to derive from two brothers via STR profiling together with a living sister, were confirmed by the living sister's report. Overall, we demonstrate that after more than 70 years, HIrisPlex-based eye and hair colour prediction from skeletal remains is feasible with high success rate. Our results further encourage the use of the HIrisPlex system in missing person/disaster victim identification to aid the identification process in cases where ante-mortem samples or putative relatives are not directly available, and DNA predicted eye and hair colour information provides leads for locating them, allowing STRbased individual identification

Developmental validation of mitochondrial DNA genotyping assays for adept matrilineal inference of biogeographic ancestry at a continental level

Mitochondrial DNA (mtDNA) can be used for matrilineal biogeographic ancestry prediction and can thus provide investigative leads towards identifying unknown suspects, when conventional autosomal short tandem repeat (STR) profiling fails to provide a match. Recently, six multiplex genotyping assays targeting 62 ancestry-informative mitochondrial single nucleotide polymorphisms (mt-SNPs) were developed. This hierarchical system of assays allows detection of the major haplogroups present in Africa, America, Western Eurasia, Eastern Eurasia, Australia and Oceania, thus revealing the broad geographic region of matrilineal origin of a DNA donor. Here, we provide a forensic developmental validation study of five multiplex assays targeting all the 62 ancestry-informative mt-SNPs following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. We demonstrate that the assays are highly sensitive; being able to produce full profiles at input DNA amounts of as little as 1 pg. The assays were shown to be highly robust and efficient in providing information from degraded samples and from simulated casework samples of different substrates such as blood, semen, hair, saliva and trace DNA samples. Reproducible results were successfully achieved from concordance testing across three independent laboratories depicting the ease and reliability of these assays. Overall, our results demonstrate the suitability of these five mt-SNP assays for application to forensic casework and other purposes aiming to establish an individual's matrilineal genetic ancestry. With this validated tool, it is now possible to determine the matrilineal biogeographic origin of unknown individuals on the level of continental resolution from forensic DNA samples to provide investigative leads in criminal and missing person cases where autosomal STR profiling is uninformative

High-quality mtDNA control region sequences from 680 individuals sampled across the Netherlands to establish a national forensic mtDNA reference database

The use of mitochondrial DNA (mtDNA) for maternal lineage identification often marks the last resort when investigating forensic and missing-person cases involving highly degraded biological materials. As with all comparative DNA testing, a match between evidence and reference sample requires a statistical interpretation, for which high-quality mtDNA population frequency data are crucial. Here, we determined, under high quality standards, the complete mtDNA control-region sequences of 680 individuals from across the Netherlands sampled at 54 sites, covering the entire country with 10 geographic sub-regions. The complete mtDNA control region (nucleotide positions 16,024-16,569 and 1-576) was amplified with two PCR primers and sequenced with ten different sequencing primers using the EMPOP protocol. Haplotype diversity of the entire sample set was very high at 99.63% and, accordingly, the random-match probability was 0.37%. No population substructure within the Netherlands was detected with our dataset. Phylogenetic analyses were performed to determine mtDNA haplogroups. Inclusion of these high-quality data in the EMPOP database (accession number: EMP00666) will improve its overall data content and geographic coverage in the interest of all EMPOP users worldwide. Moreover, this dataset will serve as (the start of) a national reference database for mtDNA applications in forensic and missing person casework in the Netherlands

Erratum to: Global skin colour prediction from DNA

Unfortunately, during the typesetting the Table 2 column head has been incorrectly aligned. The corrected Table 2 is given below: We apologize for any inconveniences caused

A collaborative EDNAP exercise on SNaPshot™-based mtDNA control region typing

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation

Collaborative EDNAP exercise on the IrisPlex system for DNA-based prediction of human eye colour

The IrisPlex system is a DNA-based test system for the prediction of human eye colour from biological samples and consists of a single forensically validated multiplex genotyping assay together with a statistical prediction model that is based on genotypes and phenotypes from thousands of individuals. IrisPlex predicts blue and brown human eye colour with, on average, >94% precision accuracy using six of the currently most eye colour informative single nucleotide polymorphisms (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 (MATP) rs16891982, TYR rs1393350, and IRF4 rs12203592) according to a previous study, while the accuracy in predicting non-blue and non-brown eye colours is considerably lower. In an effort to vigorously assess the IrisPlex system at the international level, testing was performed by 21 laboratories in the context of a collaborative exercise divided into three tasks and organised by the European DNA Profiling (EDNAP) Group of the International Society of Forensic Genetics (ISFG). Task 1 involved the assessment of 10 blood and saliva samples provided on F