Publications by authors named "Peter de Knijff"

114 Publications

Application of a probabilistic genotyping software to MPS mixture STR data is supported by similar trends in LRs compared with CE data.

Forensic Sci Int Genet 2021 05 26;52:102489. Epub 2021 Feb 26.

Division of Biological Traces, Netherlands Forensic Institute, The Hague, The Netherlands; University of Amsterdam, Swammerdam Institute for Life Sciences, Amsterdam, The Netherlands. Electronic address:

The interpretation of short tandem repeat (STR) profiles can be challenging when, for example, alleles are masked due to allele sharing among contributors and/or when they are subject to drop-out, for instance from sample degradation. Mixture interpretation can be improved by increasing the number of STRs and/or loci with a higher discriminatory power. Both capillary electrophoresis (CE, 6-dye) and massively parallel sequencing (MPS) provide a platform for analysing relatively large numbers of autosomal STRs. In addition, MPS enables distinguishing between sequence variants, resulting in enlarged discriminatory power. Also, MPS allows for small amplicon sizes for all loci as spacing is not an issue, which is beneficial with degraded DNA. Altogether, MPS has the potential to increase the weights of evidence for true contributors to (complex) DNA profiles. In this study, likelihood ratio (LR) calculations were performed using STR profiles obtained with two different MPS systems and analysed using different settings: 1) MPS PowerSeq™ Auto System profiles analysed using FDSTools equipped with optimized settings such as noise correction, 2) ForenSeq™ DNA Signature Prep Kit profiles analysed using the default settings in the Universal Analysis Software (UAS), and 3) ForenSeq™ DNA Signature Prep Kit profiles analysed using FDSTools empirically adapted to cope with one-directional reads and provisional, basic settings. The LR calculations used genotyping data for two- to four-person mixtures varying for mixture proportion, level of drop-out and allele sharing and were generated with the continuous model EuroForMix. The LR results for the over 2000 sets of propositions were affected by the variation for the number of markers and analysis settings used in the three approaches. Nevertheless, trends for true and non-contributors, effects of replicates, assigned number of contributors, and model validation results were comparable for the three MPS approaches and alike the trends known for CE data. Based on this analogy, we regard the probabilistic interpretation of MPS STR data fit for forensic DNA casework. In addition, guidelines were derived on when to apply LR calculations to MPS autosomal STR data and report the corresponding results.
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http://dx.doi.org/10.1016/j.fsigen.2021.102489DOI Listing
May 2021

Biallelic loss-of-function variants in PLD1 cause congenital right-sided cardiac valve defects and neonatal cardiomyopathy.

J Clin Invest 2021 03;131(5)

Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.
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http://dx.doi.org/10.1172/JCI142148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919725PMC
March 2021

Subdividing Y-chromosome haplogroup R1a1 reveals Norse Viking dispersal lineages in Britain.

Eur J Hum Genet 2021 03 2;29(3):512-523. Epub 2020 Nov 2.

Department of Genetics & Genome Biology, University of Leicester, Leicester, UK.

The influence of Viking-Age migrants to the British Isles is obvious in archaeological and place-names evidence, but their demographic impact has been unclear. Autosomal genetic analyses support Norse Viking contributions to parts of Britain, but show no signal corresponding to the Danelaw, the region under Scandinavian administrative control from the ninth to eleventh centuries. Y-chromosome haplogroup R1a1 has been considered as a possible marker for Viking migrations because of its high frequency in peninsular Scandinavia (Norway and Sweden). Here we select ten Y-SNPs to discriminate informatively among hg R1a1 sub-haplogroups in Europe, analyse these in 619 hg R1a1 Y chromosomes including 163 from the British Isles, and also type 23 short-tandem repeats (Y-STRs) to assess internal diversity. We find three specifically Western-European sub-haplogroups, two of which predominate in Norway and Sweden, and are also found in Britain; star-like features in the STR networks of these lineages indicate histories of expansion. We ask whether geographical distributions of hg R1a1 overall, and of the two sub-lineages in particular, correlate with regions of Scandinavian influence within Britain. Neither shows any frequency difference between regions that have higher (≥10%) or lower autosomal contributions from Norway and Sweden, but both are significantly overrepresented in the region corresponding to the Danelaw. These differences between autosomal and Y-chromosomal histories suggest either male-specific contribution, or the influence of patrilocality. Comparison of modern DNA with recently available ancient DNA data supports the interpretation that two sub-lineages of hg R1a1 spread with the Vikings from peninsular Scandinavia.
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http://dx.doi.org/10.1038/s41431-020-00747-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940619PMC
March 2021

DNA commission of the International Society of Forensic Genetics (ISFG): Recommendations on the interpretation of Y-STR results in forensic analysis.

Forensic Sci Int Genet 2020 09 4;48:102308. Epub 2020 Jun 4.

Institute of Legal Medicine and Forensic Sciences, Dept. Forensic Genetics, Charité - Universitätsmedizin Berlin, Germany.

Forensic genetic laboratories perform a large amount of STR analyses of the Y chromosome, in particular to analyze the male part of complex DNA mixtures. However, the statistical interpretation of evidence retrieved from Y-STR haplotypes is challenging. Due to the uni-parental inheritance mode, Y-STR loci are connected to each other and thus haplotypes show patterns of relationship on the familial and population level. This precludes the treatment of Y-STR loci as independently inherited variables and the application of the product rule. Instead, the dependency structure of Y-STRs needs to be included in the haplotype frequency estimation process affecting also the current paradigm of a random match probability that is in the autosomal case approximated by the population frequency assuming unrelatedness of sampled individuals. Information on the degree of paternal relatedness in the suspect population as well as on the familial network is however needed to interpret Y-chromosomal results in the best possible way. The previous recommendations of the DNA commission of the ISFG on the use of Y-STRs in forensic analysis published more than a decade ago [1] cover the interpretation issue only marginally. The current recommendations address a number of topics (frequency estimators, databases, metapopulations, LR formulation, triage, rapidly mutating Y-STRs) with relevance for the Y-STR statistics and recommend a decision-based procedure, which takes into account legal requirements as well as availability of population data and statistical methods.
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http://dx.doi.org/10.1016/j.fsigen.2020.102308DOI Listing
September 2020

Generation and genetic repair of 2 iPSC clones from a patient bearing a heterozygous c.1120del18 mutation in the ACVRL1 gene leading to Hereditary Hemorrhagic Telangiectasia (HHT) type 2.

Stem Cell Res 2020 07 28;46:101786. Epub 2020 May 28.

LUMC hiPSC Hotel, Leiden University Medical Center, Leiden, 2333 ZC, The Netherlands; Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, 2333 ZC, The Netherlands.

Fibroblasts from a patient carrying a heterozygous 18bp deletion in exon 8 of the ACVRL1 gene (c.1120del18) were reprogrammed using episomal vectors. The in-frame deletion in ACVRL1 causes the loss of 6 amino acids of the protein, which is associated with Hereditary Hemorrhagic Telangiectasia (HHT) type 2 (Letteboer et al., 2005). CRISPR-Cas9 editing was used to genetically correct the mutation in the induced pluripotent stem cells (iPSCs). The top5-predicted off-target sites were not altered. Patient and isogenic iPSCs showed high pluripotent marker expression, in vitro differentiation capacity into all three germ layers and displayed a normal karyotype. The obtained isogenic pairs will enable proper in vitro disease modelling of HHT (Roman and Hinck, 2017).
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http://dx.doi.org/10.1016/j.scr.2020.101786DOI Listing
July 2020

Comprehensive diagnostics of acute myeloid leukemia by whole transcriptome RNA sequencing.

Leukemia 2021 01 3;35(1):47-61. Epub 2020 Mar 3.

Department of Hematology, Leiden University Medical Center, 2300RC, Leiden, The Netherlands.

Acute myeloid leukemia (AML) is caused by genetic aberrations that also govern the prognosis of patients and guide risk-adapted and targeted therapy. Genetic aberrations in AML are structurally diverse and currently detected by different diagnostic assays. This study sought to establish whole transcriptome RNA sequencing as single, comprehensive, and flexible platform for AML diagnostics. We developed HAMLET (Human AML Expedited Transcriptomics) as bioinformatics pipeline for simultaneous detection of fusion genes, small variants, tandem duplications, and gene expression with all information assembled in an annotated, user-friendly output file. Whole transcriptome RNA sequencing was performed on 100 AML cases and HAMLET results were validated by reference assays and targeted resequencing. The data showed that HAMLET accurately detected all fusion genes and overexpression of EVI1 irrespective of 3q26 aberrations. In addition, small variants in 13 genes that are often mutated in AML were called with 99.2% sensitivity and 100% specificity, and tandem duplications in FLT3 and KMT2A were detected by a novel algorithm based on soft-clipped reads with 100% sensitivity and 97.1% specificity. In conclusion, HAMLET has the potential to provide accurate comprehensive diagnostic information relevant for AML classification, risk assessment and targeted therapy on a single technology platform.
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http://dx.doi.org/10.1038/s41375-020-0762-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787979PMC
January 2021

Taxonomic classification and abundance estimation using 16S and WGS-A comparison using controlled reference samples.

Forensic Sci Int Genet 2020 05 5;46:102257. Epub 2020 Feb 5.

Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands; Department of Clinical Genetics, Leiden University Medical Center, Leiden, the Netherlands.

The assessment of microbiome biodiversity is the most common application of metagenomics. While 16S sequencing remains standard procedure for taxonomic profiling of metagenomic data, a growing number of studies have clearly demonstrated biases associated with this method. By using Whole Genome Shotgun sequencing (WGS) metagenomics, most of the known restrictions associated with 16S data are alleviated. However, due to the computationally intensive data analyses and higher sequencing costs, WGS based metagenomics remains a less popular option. Selecting the experiment type that provides a comprehensive, yet manageable amount of information is a challenge encountered in many metagenomics studies. In this work, we created a series of artificial bacterial mixes, each with a different distribution of skin-associated microbial species. These mixes were used to estimate the resolution of two different metagenomic experiments - 16S and WGS - and to evaluate several different bioinformatics approaches for taxonomic read classification. In all test cases, WGS approaches provide much more accurate results, in terms of taxa prediction and abundance estimation, in comparison to those of 16S. Furthermore, we demonstrate that a 16S dataset, analysed using different state of the art techniques and reference databases, can produce widely different results. In light of the fact that most forensic metagenomic analysis are still performed using 16S data, our results are especially important.
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http://dx.doi.org/10.1016/j.fsigen.2020.102257DOI Listing
May 2020

Deciduous Teeth as an Alternative DNA Source for Postmortem Genetic Testing.

Circ Genom Precis Med 2020 04 31;13(2):e002674. Epub 2020 Jan 31.

Department of Genetics, University Medical Center Groningen, University of Groningen, the Netherlands (W.P.t.R., H.H.L., S.Z.J., Y.M.H.).

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http://dx.doi.org/10.1161/CIRCGEN.119.002674DOI Listing
April 2020

Correction to: The Dutch Y-chromosomal landscape.

Eur J Hum Genet 2020 Mar;28(3):399

Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41431-019-0528-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028942PMC
March 2020

The Dutch Y-chromosomal landscape.

Eur J Hum Genet 2020 03 5;28(3):287-299. Epub 2019 Sep 5.

Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.

Previous studies indicated existing, albeit limited, genetic-geographic population substructure in the Dutch population based on genome-wide data and a lack of this for mitochondrial SNP based data. Despite the aforementioned studies, Y-chromosomal SNP data from the Netherlands remain scarce and do not cover the territory of the Netherlands well enough to allow a reliable investigation of genetic-geographic population substructure. Here we provide the first substantial dataset of detailed spatial Y-chromosomal haplogroup information in 2085 males collected across the Netherlands and supplemented with previously published data from northern Belgium. We found Y-chromosomal evidence for genetic-geographic population substructure, and several Y-haplogroups demonstrating significant clinal frequency distributions in different directions. By means of prediction surface maps we could visualize (complex) distribution patterns of individual Y-haplogroups in detail. These results highlight the value of a micro-geographic approach and are of great use for forensic and epidemiological investigations and our understanding of the Dutch population history. Moreover, the previously noted absence of genetic-geographic population substructure in the Netherlands based on mitochondrial DNA in contrast to our Y-chromosome results, hints at different population histories for women and men in the Netherlands.
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http://dx.doi.org/10.1038/s41431-019-0496-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029002PMC
March 2020

Forensic Y-SNP analysis beyond SNaPshot: High-resolution Y-chromosomal haplogrouping from low quality and quantity DNA using Ion AmpliSeq and targeted massively parallel sequencing.

Forensic Sci Int Genet 2019 07 27;41:93-106. Epub 2019 Apr 27.

Department of Genetic Identification, Erasmus MC University Medical Center Rotterdam, Wytemaweg 80, 3000 CA, Rotterdam, the Netherlands. Electronic address:

Y-chromosomal haplogroups assigned from male-specific Y-chromosomal single nucleotide polymorphisms (Y-SNPs) allow paternal lineage identification and paternal bio-geographic ancestry inference, both being relevant in forensic genetics. However, most previously developed forensic Y-SNP tools did not provide Y haplogroup resolution on the high level needed in forensic applications, because the limited multiplex capacity of the DNA technologies used only allowed the inclusion of a relatively small number of Y-SNPs. In a proof-of-principle study, we recently demonstrated that high-resolution Y haplogrouping is feasible via two AmpliSeq PCR analyses and simultaneous massively parallel sequencing (MPS) of 530 Y-SNPs allowing the inference of 432 Y-haplogroups. With the current study, we present a largely improved Y-SNP MPS lab tool that we specifically designed for the analysis of low quality and quantity DNA often confronted with in forensic DNA analysis. Improvements include i) Y-SNP marker selection based on the "minimal reference phylogeny for the human Y chromosome" (PhyloTree Y), ii) strong increase of the number of targeted Y-SNPs allowing many more Y haplogroups to be inferred, iii) focus on short amplicon length enabling successful analysis of degraded DNA, and iv) combination of all amplicons in a single AmpliSeq PCR and simultaneous sequencing allowing single DNA aliquot use. This new MPS tool simultaneously analyses 859 Y-SNPs and allows inferring 640 Y haplogroups. Preliminary forensic developmental validation testing revealed that this tool performs highly accurate, is sensitive and robust. We also provide a revised software tool for analysing the sequencing data produced by the new MPS lab tool including final Y haplogroup assignment. We envision the tools introduced here for high-resolution Y-chromosomal haplogrouping to determine a man's paternal lineage and/or paternal bio-geographic ancestry to become widely used in forensic Y-chromosome DNA analysis and other applications were Y haplogroup information from low quality / quantity DNA samples is required.
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http://dx.doi.org/10.1016/j.fsigen.2019.04.001DOI Listing
July 2019

Diagnostics of short tandem repeat expansion variants using massively parallel sequencing and componential tools.

Eur J Hum Genet 2019 03 19;27(3):400-407. Epub 2018 Nov 19.

Department of Human Genetics, Leiden University Medical Centre, Nijmegen, The Netherlands.

Short tandem repeats (STRs) are scattered throughout the human genome. Some STRs, like trinucleotide repeat expansion (TRE) variants, cause hereditable disorders. Unambiguous molecular diagnostics of TRE disorders is hampered by current technical limitations imposed by traditional PCR and DNA sequencing methods. Here we report a novel pipeline for TRE variant diagnosis employing the massively parallel sequencing (MPS) combined with an opensource software package (FDSTools), which together are designed to distinguish true STR sequences from STR sequencing artifacts. We show that this approach can improve TRE diagnosis, such as Oculopharyngeal muscular dystrophy (OPMD). OPMD is caused by a trinucleotide expansion in the PABPN1 gene. A short GCN expansion, (GCN[10]), coding for a 10 alanine repeat is not pathogenic, but an alanine expansion is pathogenic. Applying this novel procedure in  a Dutch OPMD patient cohort, we found expansion variants from GCN[11] to GCN[16], with the GCN[16] as the most abundant variant. The repeat expansion length did not correlate with clinical features. However, symptom severity was found to correlate with age and with the initial affected muscles, suggesting that aging and muscle-specific factors can play a role in modulating OPMD.
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http://dx.doi.org/10.1038/s41431-018-0302-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6460572PMC
March 2019

From next generation sequencing to now generation sequencing in forensics.

Authors:
Peter de Knijff

Forensic Sci Int Genet 2019 01 3;38:175-180. Epub 2018 Nov 3.

Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands. Electronic address:

In contrast to genetic diagnostic disciplines such as Oncogenetics and Cinical Genetics, where worldwide, since 2010, tens of thousands of DNA samples are routinely screened annually using either targeted genome sequencing or whole genome sequencing using massively parallel sequencing (MPS), the forensic use of MPS is still far from being a routine diagnostic tool. This perspectives focusses on issues that are essential in order to fully understand (i) why MPS of short tandem repeats (STRs) is very different from the capillary electrophoresis (CE) based genotyping of STRs, (ii) what we, DNA experts, should know before explaining MPS-based evidence in court, and (iii) what information should be present in a forensic investigation report that is MPS-based. Here one has to keep in mind that the forensic use of CE was first introduced in 1992-1993 and that it took some time to fully appreciate all intricacies. Obviously, I might be biased in my opinion, having worked on this topic since 2008, but I sincerely hope that MPS will soon be widely accepted and used because, especially in case of mixed-source DNA samples, MPS is much better in the deconvolution of the individual contributors and invariably reveals genetic information that cannot be inferred otherwise.
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http://dx.doi.org/10.1016/j.fsigen.2018.10.017DOI Listing
January 2019

Short hypervariable microhaplotypes: A novel set of very short high discriminating power loci without stutter artefacts.

Forensic Sci Int Genet 2018 07 22;35:169-175. Epub 2018 May 22.

Department of Human Genetics, Leiden University Medical Center, Einthovenweg 20, 2333, ZC, Leiden, The Netherlands. Electronic address:

Since two decades, short tandem repeats (STRs) are the preferred markers for human identification, routinely analysed by fragment length analysis. Here we present a novel set of short hypervariable autosomal microhaplotypes (MH) that have four or more SNPs in a span of less than 70 nucleotides (nt). These MHs display a discriminating power approaching that of STRs and provide a powerful alternative for the analysis;1;is of forensic samples that are problematic when the STR fragment size range exceeds the integrity range of severely degraded DNA or when multiple donors contribute to an evidentiary stain and STR stutter artefacts complicate profile interpretation. MH typing was developed using the power of massively parallel sequencing (MPS) enabling new powerful, fast and efficient SNP-based approaches. MH candidates were obtained from queries in data of the 1000 Genomes, and Genome of the Netherlands (GoNL) projects. Wet-lab analysis of 276 globally dispersed samples and 97 samples of nine large CEPH families assisted locus selection and corroboration of informative value. We infer that MHs represent an alternative marker type with good discriminating power per locus (allowing the use of a limited number of loci), small amplicon sizes and absence of stutter artefacts that can be especially helpful when unbalanced mixed samples are submitted for human identification.
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http://dx.doi.org/10.1016/j.fsigen.2018.05.008DOI Listing
July 2018

Demographic History and Genetic Adaptation in the Himalayan Region Inferred from Genome-Wide SNP Genotypes of 49 Populations.

Mol Biol Evol 2018 08;35(8):1916-1933

The Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom.

We genotyped 738 individuals belonging to 49 populations from Nepal, Bhutan, North India, or Tibet at over 500,000 SNPs, and analyzed the genotypes in the context of available worldwide population data in order to investigate the demographic history of the region and the genetic adaptations to the harsh environment. The Himalayan populations resembled other South and East Asians, but in addition displayed their own specific ancestral component and showed strong population structure and genetic drift. We also found evidence for multiple admixture events involving Himalayan populations and South/East Asians between 200 and 2,000 years ago. In comparisons with available ancient genomes, the Himalayans, like other East and South Asian populations, showed similar genetic affinity to Eurasian hunter-gatherers (a 24,000-year-old Upper Palaeolithic Siberian), and the related Bronze Age Yamnaya. The high-altitude Himalayan populations all shared a specific ancestral component, suggesting that genetic adaptation to life at high altitude originated only once in this region and subsequently spread. Combining four approaches to identifying specific positively selected loci, we confirmed that the strongest signals of high-altitude adaptation were located near the Endothelial PAS domain-containing protein 1 and Egl-9 Family Hypoxia Inducible Factor 1 loci, and discovered eight additional robust signals of high-altitude adaptation, five of which have strong biological functional links to such adaptation. In conclusion, the demographic history of Himalayan populations is complex, with strong local differentiation, reflecting both genetic and cultural factors; these populations also display evidence of multiple genetic adaptations to high-altitude environments.
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http://dx.doi.org/10.1093/molbev/msy094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063301PMC
August 2018

The HIrisPlex-S system for eye, hair and skin colour prediction from DNA: Introduction and forensic developmental validation.

Forensic Sci Int Genet 2018 07 12;35:123-135. Epub 2018 Apr 12.

Department of Biology, Indiana University Purdue University Indianapolis (IUPUI), IN, USA. Electronic address:

Forensic DNA Phenotyping (FDP), i.e. the prediction of human externally visible traits from DNA, has become a fast growing subfield within forensic genetics due to the intelligence information it can provide from DNA traces. FDP outcomes can help focus police investigations in search of unknown perpetrators, who are generally unidentifiable with standard DNA profiling. Therefore, we previously developed and forensically validated the IrisPlex DNA test system for eye colour prediction and the HIrisPlex system for combined eye and hair colour prediction from DNA traces. Here we introduce and forensically validate the HIrisPlex-S DNA test system (S for skin) for the simultaneous prediction of eye, hair, and skin colour from trace DNA. This FDP system consists of two SNaPshot-based multiplex assays targeting a total of 41 SNPs via a novel multiplex assay for 17 skin colour predictive SNPs and the previous HIrisPlex assay for 24 eye and hair colour predictive SNPs, 19 of which also contribute to skin colour prediction. The HIrisPlex-S system further comprises three statistical prediction models, the previously developed IrisPlex model for eye colour prediction based on 6 SNPs, the previous HIrisPlex model for hair colour prediction based on 22 SNPs, and the recently introduced HIrisPlex-S model for skin colour prediction based on 36 SNPs. In the forensic developmental validation testing, the novel 17-plex assay performed in full agreement with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, as previously shown for the 24-plex assay. Sensitivity testing of the 17-plex assay revealed complete SNP profiles from as little as 63 pg of input DNA, equalling the previously demonstrated sensitivity threshold of the 24-plex HIrisPlex assay. Testing of simulated forensic casework samples such as blood, semen, saliva stains, of inhibited DNA samples, of low quantity touch (trace) DNA samples, and of artificially degraded DNA samples as well as concordance testing, demonstrated the robustness, efficiency, and forensic suitability of the new 17-plex assay, as previously shown for the 24-plex assay. Finally, we provide an update to the publically available HIrisPlex website https://hirisplex.erasmusmc.nl/, now allowing the estimation of individual probabilities for 3 eye, 4 hair, and 5 skin colour categories from HIrisPlex-S input genotypes. The HIrisPlex-S DNA test represents the first forensically validated tool for skin colour prediction, and reflects the first forensically validated tool for simultaneous eye, hair and skin colour prediction from DNA.
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http://dx.doi.org/10.1016/j.fsigen.2018.04.004DOI Listing
July 2018

The Beaker phenomenon and the genomic transformation of northwest Europe.

Nature 2018 03 21;555(7695):190-196. Epub 2018 Feb 21.

Departamento de Prehistoria y Arqueología, Universidad Autónoma de Madrid, Madrid 28049, Spain.

From around 2750 to 2500 bc, Bell Beaker pottery became widespread across western and central Europe, before it disappeared between 2200 and 1800 bc. The forces that propelled its expansion are a matter of long-standing debate, and there is support for both cultural diffusion and migration having a role in this process. Here we present genome-wide data from 400 Neolithic, Copper Age and Bronze Age Europeans, including 226 individuals associated with Beaker-complex artefacts. We detected limited genetic affinity between Beaker-complex-associated individuals from Iberia and central Europe, and thus exclude migration as an important mechanism of spread between these two regions. However, migration had a key role in the further dissemination of the Beaker complex. We document this phenomenon most clearly in Britain, where the spread of the Beaker complex introduced high levels of steppe-related ancestry and was associated with the replacement of approximately 90% of Britain's gene pool within a few hundred years, continuing the east-to-west expansion that had brought steppe-related ancestry into central and northern Europe over the previous centuries.
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http://dx.doi.org/10.1038/nature25738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973796PMC
March 2018

A SNP panel for identification of DNA and RNA specimens.

BMC Genomics 2018 01 25;19(1):90. Epub 2018 Jan 25.

Department of Human Genetics, Leiden University Medical Center, Postzone S4-P, PO Box 9600, 2300 RC, Leiden, The Netherlands.

Background: SNP panels that uniquely identify an individual are useful for genetic and forensic research. Previously recommended SNP panels are based on DNA profiles and mostly contain intragenic SNPs. With the increasing interest in RNA expression profiles, we aimed for establishing a SNP panel for both DNA and RNA-based genotyping.

Results: To determine a small set of SNPs with maximally discriminative power, genotype calls were obtained from DNA and blood-derived RNA sequencing data belonging to healthy, geographically dispersed, Dutch individuals. SNPs were selected based on different criteria like genotype call rate, minor allele frequency, Hardy-Weinberg equilibrium and linkage disequilibrium. A panel of 50 SNPs was sufficient to identify an individual uniquely: the probability of identity was 6.9 × 10 when assuming no family relations and 1.2 × 10 when accounting for the presence of full sibs. The ability of the SNP panel to uniquely identify individuals on DNA and RNA level was validated in an independent population dataset. The panel is applicable to individuals from European descent, with slightly lower power in non-Europeans. Whereas most of the genes containing the 50 SNPs are expressed in various tissues, our SNP panel needs optimization for other tissues than blood.

Conclusions: This first DNA/RNA SNP panel will be useful to identify sample mix-ups in biomedical research and for assigning DNA and RNA stains in crime scenes to unique individuals.
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http://dx.doi.org/10.1186/s12864-018-4482-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785835PMC
January 2018

Effect of APOE ε4 allele on survival and fertility in an adverse environment.

PLoS One 2017 6;12(7):e0179497. Epub 2017 Jul 6.

Department of Gerontology and Geriatrics, Leiden University Medical Center, Leiden, the Netherlands.

Background: The apolipoprotein-ε4 allele (APOE-ε4) is strongly associated with detrimental outcomes in affluent populations including atherosclerotic disease, Alzheimer's disease, and reduced lifespan. Despite these detrimental outcomes, population frequencies of APOE-ε4 are high. We hypothesize that the high frequency of APOE-ε4 was maintained because of beneficial effects during evolution when infectious pathogens were more prevalent and a major cause of mortality. We examined a rural Ghanaian population with a high pathogen exposure for selective advantages of APOE-ε4, to survival and or fertility.

Methods And Findings: This rural Ghanaian population (n = 4311) has high levels of mortality from widespread infectious diseases which are the main cause of death. We examined whether APOE-ε4 was associated with survival (total follow-up time was 30,262 years) and fertility after stratifying by exposure to high or low pathogen levels. Households drawing water from open wells and rivers were classified as exposed to high pathogen levels while low pathogen exposure was classified as those drawing water from borehole wells. We found a non-significant, but positive survival benefit, i.e. the hazard ratio per APOE-ε4 allele was 0.80 (95% confidence interval: 0.69 to 1.05), adjusted for sex, tribe, and socioeconomic status. Among women aged 40 years and older (n = 842), APOE-ε4 was not associated with the lifetime number of children. However, APOE-ε4 was associated with higher fertility in women exposed to high pathogen levels. Compared with women not carrying an APOE-ε4 allele, those carrying one APOE-ε4 allele had on average one more child and those carrying two APOE-ε4 alleles had 3.5 more children (p = 0.018).

Conclusions: Contrary to affluent modern-day populations, APOE-ε4 did not carry a survival disadvantage in this rural Ghanaian population. Moreover, APOE-ε4 promotes fertility in highly infectious environments. Our findings suggest that APOE-ε4 may be considered as evolutionarily adaptive. Its adverse associations in affluent modern populations with later onset diseases of aging further characterize APOE-ε4 as an example of antagonistic pleiotropy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179497PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500260PMC
September 2017

Decay of Sexual Trait Genes in an Asexual Parasitoid Wasp.

Genome Biol Evol 2016 12;8(12):3685-3695

Animal Ecology, Department of Ecological Sciences, VU University Amsterdam, The Netherlands.

Trait loss is a widespread phenomenon with pervasive consequences for a species’ evolutionary potential. The genetic changes underlying trait loss have only been clarified in a small number of cases. None of these studies can identify whether the loss of the trait under study was a result of neutral mutation accumulation or negative selection. This distinction is relatively clear-cut in the loss of sexual traits in asexual organisms. Male-specific sexual traits are not expressed and can only decay through neutral mutations, whereas female-specific traits are expressed and subject to negative selection. We present the genome of an asexual parasitoid wasp and compare it to that of a sexual lineage of the same species. We identify a short-list of 16 genes for which the asexual lineage carries deleterious SNP or indel variants, whereas the sexual lineage does not. Using tissue-specific expression data from other insects, we show that fifteen of these are expressed in male-specific reproductive tissues. Only one deleterious variant was found that is expressed in the female-specific spermathecae, a trait that is heavily degraded and thought to be under negative selection in L. clavipes. Although the phenotypic decay of male-specific sexual traits in asexuals is generally slow compared with the decay of female-specific sexual traits, we show that male-specific traits do indeed accumulate deleterious mutations as expected by theory. Our results provide an excellent starting point for detailed study of the genomics of neutral and selected trait decay.
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http://dx.doi.org/10.1093/gbe/evw273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381511PMC
December 2016

FDSTools: A software package for analysis of massively parallel sequencing data with the ability to recognise and correct STR stutter and other PCR or sequencing noise.

Forensic Sci Int Genet 2017 03 27;27:27-40. Epub 2016 Nov 27.

Department of Human Genetics, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands. Electronic address:

Massively parallel sequencing (MPS) is on the advent of a broad scale application in forensic research and casework. The improved capabilities to analyse evidentiary traces representing unbalanced mixtures is often mentioned as one of the major advantages of this technique. However, most of the available software packages that analyse forensic short tandem repeat (STR) sequencing data are not well suited for high throughput analysis of such mixed traces. The largest challenge is the presence of stutter artefacts in STR amplifications, which are not readily discerned from minor contributions. FDSTools is an open-source software solution developed for this purpose. The level of stutter formation is influenced by various aspects of the sequence, such as the length of the longest uninterrupted stretch occurring in an STR. When MPS is used, STRs are evaluated as sequence variants that each have particular stutter characteristics which can be precisely determined. FDSTools uses a database of reference samples to determine stutter and other systemic PCR or sequencing artefacts for each individual allele. In addition, stutter models are created for each repeating element in order to predict stutter artefacts for alleles that are not included in the reference set. This information is subsequently used to recognise and compensate for the noise in a sequence profile. The result is a better representation of the true composition of a sample. Using Promega Powerseq™ Auto System data from 450 reference samples and 31 two-person mixtures, we show that the FDSTools correction module decreases stutter ratios above 20% to below 3%. Consequently, much lower levels of contributions in the mixed traces are detected. FDSTools contains modules to visualise the data in an interactive format allowing users to filter data with their own preferred thresholds.
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http://dx.doi.org/10.1016/j.fsigen.2016.11.007DOI Listing
March 2017

The Simons Genome Diversity Project: 300 genomes from 142 diverse populations.

Nature 2016 Oct 21;538(7624):201-206. Epub 2016 Sep 21.

Department of Zoology, University of Oxford, Oxford OX1 3PS, UK.

Here we report the Simons Genome Diversity Project data set: high quality genomes from 300 individuals from 142 diverse populations. These genomes include at least 5.8 million base pairs that are not present in the human reference genome. Our analysis reveals key features of the landscape of human genome variation, including that the rate of accumulation of mutations has accelerated by about 5% in non-Africans compared to Africans since divergence. We show that the ancestors of some pairs of present-day human populations were substantially separated by 100,000 years ago, well before the archaeologically attested onset of behavioural modernity. We also demonstrate that indigenous Australians, New Guineans and Andamanese do not derive substantial ancestry from an early dispersal of modern humans; instead, their modern human ancestry is consistent with coming from the same source as that of other non-Africans.
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http://dx.doi.org/10.1038/nature18964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161557PMC
October 2016

Genetic variants determining survival and fertility in an adverse African environment: a population-based large-scale candidate gene association study.

Aging (Albany NY) 2016 07;8(7):1364-83

Section of Gerontology and Geriatrics, Department of Internal Medicine, Leiden University Medical Center, Leiden, the Netherlands.

Human survival probability and fertility decline strongly with age. These life history traits have been shaped by evolution. However, research has failed to uncover a consistent genetic determination of variation in survival and fertility. As an explanation, such genetic determinants have been selected in adverse environments, in which humans have lived during most of their history, but are almost exclusively studied in populations in modern affluent environments. Here, we present a large-scale candidate gene association study in a rural African population living in an adverse environment. In 4387 individuals, we studied 4052 SNPs in 148 genes that have previously been identified as possible determinants of survival or fertility in animals or humans. We studied their associations with survival comparing newborns, middle-age adults, and old individuals. In women, we assessed their associations with reported and observed numbers of children. We found no statistically significant associations of these SNPs with survival between the three age groups nor with women's reported and observed fertility. Population stratification was unlikely to explain these results. Apart from a lack of power, we hypothesise that genetic heterogeneity of complex phenotypes and gene-environment interactions prevent the identification of genetic variants explaining variation in survival and fertility in humans.
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http://dx.doi.org/10.18632/aging.100986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993336PMC
July 2016

Massively parallel sequencing of short tandem repeats-Population data and mixture analysis results for the PowerSeq™ system.

Forensic Sci Int Genet 2016 09 7;24:86-96. Epub 2016 Jun 7.

Forensic Laboratory for DNA Research, Department of Human Genetics, Leiden University Medical Centre, Postzone S 05 P, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Electronic address:

Current forensic DNA analysis predominantly involves identification of human donors by analysis of short tandem repeats (STRs) using Capillary Electrophoresis (CE). Recent developments in Massively Parallel Sequencing (MPS) technologies offer new possibilities in analysis of STRs since they might overcome some of the limitations of CE analysis. In this study 17 STRs and Amelogenin were sequenced in high coverage using a prototype version of the Promega PowerSeq™ system for 297 population samples from the Netherlands, Nepal, Bhutan and Central African Pygmies. In addition, 45 two-person mixtures with different minor contributions down to 1% were analysed to investigate the performance of this system for mixed samples. Regarding fragment length, complete concordance between the MPS and CE-based data was found, marking the reliability of MPS PowerSeq™ system. As expected, MPS presented a broader allele range and higher power of discrimination and exclusion rate. The high coverage sequencing data were used to determine stutter characteristics for all loci and stutter ratios were compared to CE data. The separation of alleles with the same length but exhibiting different stutter ratios lowers the overall variation in stutter ratio and helps in differentiation of stutters from genuine alleles in mixed samples. All alleles of the minor contributors were detected in the sequence reads even for the 1% contributions, but analysis of mixtures below 5% without prior information of the mixture ratio is complicated by PCR and sequencing artefacts.
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http://dx.doi.org/10.1016/j.fsigen.2016.05.016DOI Listing
September 2016

Transmission of human mtDNA heteroplasmy in the Genome of the Netherlands families: support for a variable-size bottleneck.

Genome Res 2016 Apr 25;26(4):417-26. Epub 2016 Feb 25.

Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany;

Although previous studies have documented a bottleneck in the transmission of mtDNA genomes from mothers to offspring, several aspects remain unclear, including the size and nature of the bottleneck. Here, we analyze the dynamics of mtDNA heteroplasmy transmission in the Genomes of the Netherlands (GoNL) data, which consists of complete mtDNA genome sequences from 228 trios, eight dizygotic (DZ) twin quartets, and 10 monozygotic (MZ) twin quartets. Using a minor allele frequency (MAF) threshold of 2%, we identified 189 heteroplasmies in the trio mothers, of which 59% were transmitted to offspring, and 159 heteroplasmies in the trio offspring, of which 70% were inherited from the mothers. MZ twin pairs exhibited greater similarity in MAF at heteroplasmic sites than DZ twin pairs, suggesting that the heteroplasmy MAF in the oocyte is the major determinant of the heteroplasmy MAF in the offspring. We used a likelihood method to estimate the effective number of mtDNA genomes transmitted to offspring under different bottleneck models; a variable bottleneck size model provided the best fit to the data, with an estimated mean of nine individual mtDNA genomes transmitted. We also found evidence for negative selection during transmission against novel heteroplasmies (in which the minor allele has never been observed in polymorphism data). These novel heteroplasmies are enhanced for tRNA and rRNA genes, and mutations associated with mtDNA diseases frequently occur in these genes. Our results thus suggest that the female germ line is able to recognize and select against deleterious heteroplasmies.
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http://dx.doi.org/10.1101/gr.203216.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817766PMC
April 2016

Wide distribution and altitude correlation of an archaic high-altitude-adaptive EPAS1 haplotype in the Himalayas.

Hum Genet 2016 Apr 16;135(4):393-402. Epub 2016 Feb 16.

The Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK.

High-altitude adaptation in Tibetans is influenced by introgression of a 32.7-kb haplotype from the Denisovans, an extinct branch of archaic humans, lying within the endothelial PAS domain protein 1 (EPAS1), and has also been reported in Sherpa. We genotyped 19 variants in this genomic region in 1507 Eurasian individuals, including 1188 from Bhutan and Nepal residing at altitudes between 86 and 4550 m above sea level. Derived alleles for five SNPs characterizing the core Denisovan haplotype (AGGAA) were present at high frequency not only in Tibetans and Sherpa, but also among many populations from the Himalayas, showing a significant correlation with altitude (Spearman's correlation coefficient = 0.75, p value 3.9 × 10(-11)). Seven East- and South-Asian 1000 Genomes Project individuals shared the Denisovan haplotype extending beyond the 32-kb region, enabling us to refine the haplotype structure and identify a candidate regulatory variant (rs370299814) that might be interacting in an additive manner with the derived G allele of rs150877473, the variant previously associated with high-altitude adaptation in Tibetans. Denisovan-derived alleles were also observed at frequencies of 3-14% in the 1000 Genomes Project African samples. The closest African haplotype is, however, separated from the Asian high-altitude haplotype by 22 mutations whereas only three mutations, including rs150877473, separate the Asians from the Denisovan, consistent with distant shared ancestry for African and Asian haplotypes and Denisovan adaptive introgression.
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http://dx.doi.org/10.1007/s00439-016-1641-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4796332PMC
April 2016

Massively parallel sequencing of forensic STRs: Considerations of the DNA commission of the International Society for Forensic Genetics (ISFG) on minimal nomenclature requirements.

Forensic Sci Int Genet 2016 May 21;22:54-63. Epub 2016 Jan 21.

Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Galicia, Spain.

The DNA Commission of the International Society for Forensic Genetics (ISFG) is reviewing factors that need to be considered ahead of the adoption by the forensic community of short tandem repeat (STR) genotyping by massively parallel sequencing (MPS) technologies. MPS produces sequence data that provide a precise description of the repeat allele structure of a STR marker and variants that may reside in the flanking areas of the repeat region. When a STR contains a complex arrangement of repeat motifs, the level of genetic polymorphism revealed by the sequence data can increase substantially. As repeat structures can be complex and include substitutions, insertions, deletions, variable tandem repeat arrangements of multiple nucleotide motifs, and flanking region SNPs, established capillary electrophoresis (CE) allele descriptions must be supplemented by a new system of STR allele nomenclature, which retains backward compatibility with the CE data that currently populate national DNA databases and that will continue to be produced for the coming years. Thus, there is a pressing need to produce a standardized framework for describing complex sequences that enable comparison with currently used repeat allele nomenclature derived from conventional CE systems. It is important to discern three levels of information in hierarchical order (i) the sequence, (ii) the alignment, and (iii) the nomenclature of STR sequence data. We propose a sequence (text) string format the minimal requirement of data storage that laboratories should follow when adopting MPS of STRs. We further discuss the variant annotation and sequence comparison framework necessary to maintain compatibility among established and future data. This system must be easy to use and interpret by the DNA specialist, based on a universally accessible genome assembly, and in place before the uptake of MPS by the general forensic community starts to generate sequence data on a large scale. While the established nomenclature for CE-based STR analysis will remain unchanged in the future, the nomenclature of sequence-based STR genotypes will need to follow updated rules and be generated by expert systems that translate MPS sequences to match CE conventions in order to guarantee compatibility between the different generations of STR data.
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http://dx.doi.org/10.1016/j.fsigen.2016.01.009DOI Listing
May 2016

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

Forensic Sci Int Genet 2016 Mar 10;21:158-67. Epub 2015 Dec 10.

Department of Genetic Identification, Erasmus MC University Medical Center Rotterdam, Rotterdam, The Netherlands. Electronic address:

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.
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http://dx.doi.org/10.1016/j.fsigen.2015.12.002DOI Listing
March 2016
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