Entwicklung einer dualen Strategie zur DNA-Analyse von Mischspuren zur Aufklärung von Straftaten gegen die sexuelle Selbstbestimmung

The goal of forensic DNA analysis is to reliably distinguish individuals by their DNA profiles and clearly assign them to a trace. Full autosomal STR profiles are individual, while the evidence of Y-chromosomal STR profiles (haplotype) is still significantly lower. A reason for this is the paternal inheritance of the Y chromosome, meaning the recombination-free transmission of identical copies from generation to generation, which can only be differentiated by spontaneous mutations. However, it is precisely this male-specific property that makes the Y-STRs so valuable for complex mixed biological traces in cases of sexual assault, which is why nowadays Y-chromosomal analyses belong to the standard repertoire of forensic laboratories. With the use of the new Y-STR marker sets, the discrimination capacity of Y-chromosomal haplotypes is improved and thus the evidential value of a Y-chromosomal match in forensic applications is clearly increased. In my thesis, two new high-resolution Y-STR marker sets (23 and 13 Y-STR panel) were examined in population genetics and analyzed and evaluated for molecular genetic and forensic parameters. The 36 Y-STRs showed high gene diversity, in particular the 13 Y-STRs with higher mutation rates (μ > 10-2 [1]). Their application reached (almost) complete differentiation of unrelated males of a population. With the increasing number of Y-STRs used and their selection for mutability and variability, the individualization level of a DNA sample was significantly increased, which means that a proportion of the relatives in the paternal line can now be distinguished. The results for the establishment of a new DNA-based analytical method using autosomal and Y-chromosomal markers (total of 39 STRs) in mixed biological traces and touch stains in sexual assault cases showed a clear gain in information. In 287 cases, 110 informative autosomal (reportable to database) profiles and a total of 133 infor-mative (complete) Y-chromosomal single-source profiles were generated, an increase of ~ 21 % informative profiles compared to stand-alone autosomal STR analysis. With the help of the single-copy Y-STRs, mixtures of several male donors could be detected three times more frequently than with autosomal STRs. It remains to be noted that one-tenth of all cases would have remained inconclusive; the (autosomal) latent male DNA would have remained undetected, if Y-STR analysis had been omitted. The Y-chromosomal analysis with the markers described here thus improves the sensitivity and accuracy of the forensic DNA analysis.Das Ziel der forensischen DNA-Analyse besteht darin, Individuen anhand ihrer DNA-Profile verlässlich voneinander zu unterscheiden und einer Spur eindeutig zuzuordnen. Vollständige autosomale STR-Profile sind individuell, während der Beweiswert Y-chromosomaler STR-Profile (Haplotyp) noch deutlich geringer ist. Ein Grund dafür ist der paternale Erbgang des Y-Chromosoms, d. h. die rekombinationsfreie Weitergabe identischer Kopien von Generation zu Generation, die sich nur durch Spontanmutationen differenzieren lassen. Doch gerade diese mannspezifische Eigenschaft macht die Y-STRs für die komplexen Mischspuren in der Delikt-gruppe der Sexualstraftaten so wertvoll, weshalb Y-chromosomale Analysen heutzutage zum Standardrepertoire forensischer Labore gehören. Mit Hilfe neuer Y-STR Markersets soll die Dif-ferenzierungskraft von Y-chromosomalen Haplotypen verbessert und somit der Beweiswert einer Y-chromosomalen Übereinstimmung bei forensischen Fragestellungen deutlich gesteigert werden. Im Rahmen meiner wissenschaftlichen Arbeit wurden zwei neue hochauflösende Y-STR Markersets (ein 23 und ein 13 Y-STR Panel) populationsgenetisch untersucht und hin-sichtlich molekulargenetischer und forensischer Charakteristika analysiert und bewertet. Die 36 Y-STRs zeigten insgesamt hohe Gendiversitäten, insbesondere die 13 Y-STRs mit überdurch-schnittlich hohen Mutationsraten (μ > 10-2 [1]). Ihre Anwendung erreichte die nahezu vollstän-dige Differenzierung unverwandter Individuen einer Population. Mit zunehmender Anzahl der eingesetzten Y-STRs und ihrer Auswahl nach Mutabilität und Variabilität wurde die Individualisierbarkeit einer DNA-Probe deutlich gesteigert, d. h. das nunmehr auch ein Anteil der Verwandten in väterlicher Linie unterschieden werden kann. Die Ergebnisse zur Etablierung einer neuen DNA-basierten Analysemethode mit autosomalen und Y-chromosomalen Markern (insgesamt 39 STRs) bei Misch- und Kontaktspuren aus Straftaten gegen die sexuelle Selbst-bestimmung zeigten einen deutlichen Informationszugewinn. In 287 Fällen wurden 110 informative autosomale (datenbankfähige) Profile und insgesamt 133 informative (vollstän-dige) Y-chromosomale Einzelprofile generiert, ein Zugewinn an informativen Profilen von ~21 % gegenüber einer ausschließlich autosomalen STR-Analyse. Mit Hilfe der single-copy Y-STRs konnten Mischungen mehrerer männlicher Spurenverursacher dreimal häufiger als mit autosomalen STRs erkannt werden. Es bleibt festzuhalten, dass ein Zehntel aller untersuchten Fälle ohne Y-STR Analyse ergebnislos, die (autosomal) latente männliche DNA also unentdeckt geblieben wäre. Die Y-chromosomale Analyse mit den hier beschriebenen Markern verbessert somit die Empfindlichkeit und die Aussagekraft der forensischen DNA-Analyse

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.Fil: Corach, Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Caputo, Mariela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Servicio de Huellas Digitales Genéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marino, Miguel Eduardo. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Analisis de ADN; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Purps, Josephine. Charité-Universitätsmedizin; AlemaniaFil: Siegert, Sabine. University of Cologne; AlemaniaFil: Willuweit, Sascha. Charité-Universitätsmedizin; AlemaniaFil: Nagy, Marion. Charité-Universitätsmedizin; AlemaniaFil: Alves, Cíntia. Universidad de Porto; PortugalFil: Salazar, Renato. Universidad de Porto; PortugalFil: Angustia, Sheila M. T.. Philippine National Police Crime Laboratory; FilipinasFil: Santos, Lorna H.. Philippine National Police Crime Laboratory; FilipinasFil: Anslinger, Katja. Universitat Genzentrum Der Ludwing-maximilians; AlemaniaFil: Bayer, Birgit. Universitat Genzentrum Der Ludwing-maximilians; AlemaniaFil: Ayub, Qasim. The Wellcome Trust Sanger Institute; Reino UnidoFil: Wei, Wei. The Wellcome Trust Sanger Institute; Reino UnidoFil: Xue, Yali. The Wellcome Trust Sanger Institute; Reino UnidoFil: Tyler Smith, Chris. The Wellcome Trust Sanger Institute; Reino UnidoFil: Baeta Bafalluy, Miriam. Universidad de Zaragoza; EspañaFil: Martínez Jarreta, Begoña. Universidad de Zaragoza; EspañaFil: Egyed, Balazs. Eotvos University, Budapest; ArgentinaFil: Balitzki, Beate. Universidad de Basilea; SuizaFil: Tschumi, Sibylle. Universidad de Basilea; SuizaFil: Ballard, David. King; Reino UnidoFil: Syndercombe Court, Denise. King; Reino UnidoFil: Barrantes, Xinia. Poder Judicial, Forensic Sciences Department; Costa RicaFil: Bäßler, Gerhard. Landeskriminalamt Baden-Württemberg; AlemaniaFil: Berger, Burkhard. Universidad de Innsbruck; AustriaFil: Niederstätter, Haral. Universidad de Innsbruck; AustriaFil: Parson, Walther. Universidad de Innsbruck; Austria. University Park; Estados UnidosFil: Davis, Carey. Department of Molecular and Medical Genetics; Estados Unidos. Institute of Applied Genetics; Estados UnidosFil: Furfuro, Sandra. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Análisis de ADN; ArgentinaFil: Locarno, Laura. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Laboratorio de Análisis de ADN; Argentin

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.Peer reviewe

A Novel MCPH1 Isoform Complements the Defective Chromosome Condensation of Human MCPH1-Deficient Cells

Biallelic mutations in MCPH1 cause primary microcephaly (MCPH) with the cellular phenotype of defective chromosome condensation. MCPH1 encodes a multifunctional protein that notably is involved in brain development, regulation of chromosome condensation, and DNA damage response. In the present studies, we detected that MCPH1 encodes several distinct transcripts, including two major forms: full-length MCPH1 (MCPH1-FL) and a second transcript lacking the six 39 exons (MCPH1De9–14). Both variants show comparable tissue-specific expression patterns, demonstrate nuclear localization that is mediated independently via separate NLS motifs, and are more abundant in certain fetal than adult organs. In addition, the expression of either isoform complements the chromosome condensation defect found in genetically MCPH1-deficient or MCPH1 siRNA-depleted cells, demonstrating a redundancy of both MCPH1 isoforms for the regulation of chromosome condensation. Strikingly however, both transcripts are regulated antagonistically during cell-cycle progression and there are functional differences between the isoforms with regard to the DNA damage response; MCPH1-FL localizes to phosphorylated H2AX repair foci following ionizing irradiation, while MCPH1De9–14 was evenly distributed in the nucleus. In summary, our results demonstrate here that MCPH1 encodes different isoforms that are differentially regulated at the transcript level and have different functions at the protein level

Y-chromosomal analysis identifies the skeletal remains of Swiss national hero Jörg Jenatsch (1596–1639)

Abstract not availableCordula Haas, Natallia Shved, Frank Jakobus Rühli, Christina Papageorgopoulou, Josephine Purps, Maria Geppert, Sascha Willuweit, Lutz Roewer, Michael Krawcza

Refining the Y chromosome phylogeny with southern African sequences

International audienceThe recent availability of large-scale sequence data for the human Y chromosome has revolutionized analyses of and insights gained from this non-recombining, paternally inherited chromosome. However, the studies to date focus on Eurasian variation, and hence the diversity of early-diverging branches found in Africa has not been adequately documented. Here, we analyze over 900 kb of Y chromosome sequence obtained from 547 individuals from southern African Khoisan- and Bantu-speaking populations, identifying 232 new sequences from basal haplogroups A and B. We identify new clades in the phylogeny, an older age for the root, and substantially older ages for some individual haplogroups. Furthermore, while haplogroup B2a is traditionally associated with the spread of Bantu speakers, we find that it probably also existed in Khoisan groups before the arrival of Bantu speakers. Finally, there is pronounced variation in branch length between major haplogroups; in particular, haplogroups associated with Bantu speakers have significantly longer branches. Technical artifacts cannot explain this branch length variation, which instead likely reflects aspects of the demographic history of Bantu speakers, such as recent population expansion and an older average paternal age. The influence of demographic factors on branch length variation has broader implications both for the human Y phylogeny and for similar analyses of other species

Genetic structure and sex-biased gene flow in the history of southern African populations

International audienceObjectives: We investigated the genetic history of southern African populations with a special focus on their paternal history. We reexamined previous claims that the Y-chromosome hap-logroup E1b1b (E-M293) was brought to southern Africa by pastoralists from eastern Africa, and investigated patterns of sex-biased gene flow in southern Africa. Materials and methods: We analyzed previously published complete mtDNA genome sequences and 900 kb of NRY sequences from 23 populations from Namibia, Botswana, and Zambia, as well as haplogroup frequencies from a large sample of southern African populations and 23 newly genotyped Y-linked STR loci for samples assigned to haplogroup E1b1b. Results: Our results support an eastern African origin for Y-chromosome haplogroup E1b1b (E-M293); however, its current distribution in southern Africa is not strongly associated with pasto-ralism, suggesting more complex demographic events and/or changes in subsistence practices in this region. The Bantu expansion in southern Africa had a notable genetic impact and was probably a rapid, male-dominated expansion. Our finding of a significant increase in the intensity of the sex-biased gene flow from north to south may reflect changes in the social dynamics between Khoisan and Bantu groups over time. Conclusions: Our study shows that the population history of southern Africa has been complex, with different immigrating groups mixing to different degrees with the autochthonous populations. The Bantu expansion led to heavily sex-biased admixture as a result of interactions between Khoisan females and Bantu males, with a geographic gradient which may reflect changes in the social dynamics between Khoisan and Bantu groups over time

Genetic structure and sex-biased gene flow in the history of southern African populations

OBJECTIVES: We investigated the genetic history of southern African populations with a special focus on their paternal history. We reexamined previous claims that the Y-chromosome haplogroup E1b1b (E-M293) was brought to southern Africa by pastoralists from eastern Africa, and investigated patterns of sex-biased gene flow in southern Africa. MATERIALS AND METHODS: We analyzed previously published complete mtDNA genome sequences and ∼900 kb of NRY sequences from 23 populations from Namibia, Botswana, and Zambia, as well as haplogroup frequencies from a large sample of southern African populations and 23 newly genotyped Y-linked STR loci for samples assigned to haplogroup E1b1b. RESULTS: Our results support an eastern African origin for Y-chromosome haplogroup E1b1b (E-M293); however, its current distribution in southern Africa is not strongly associated with pastoralism, suggesting more complex demographic events and/or changes in subsistence practices in this region. The Bantu expansion in southern Africa had a notable genetic impact and was probably a rapid, male-dominated expansion. Our finding of a significant increase in the intensity of the sex-biased gene flow from north to south may reflect changes in the social dynamics between Khoisan and Bantu groups over time. CONCLUSIONS: Our study shows that the population history of southern Africa has been complex, with different immigrating groups mixing to different degrees with the autochthonous populations. The Bantu expansion led to heavily sex-biased admixture as a result of interactions between Khoisan females and Bantu males, with a geographic gradient which may reflect changes in the social dynamics between Khoisan and Bantu groups over time

Intracellular distribution of MCPH1 isoforms.

<p>(A) MCPH1-deficient fibroblasts expressing GFP alone or the specified GFP-MCPH1 fusion proteins were fractionated and cytoplasmic (Cyt) and nuclear (Nuc) protein extracts were analyzed using immunoblotting with an antibody against GFP. The nuclear matrix protein p84 and GAPDH were used as index proteins and loading controls. (B) Cells indicated in A stained with an anti-GFP antibody (green), counterstained with DAPI (blue) and analyzed using fluorescence microscopy. Arrows indicate the prophase-like nuclei. Scale bar = 10 µm. All MCPH1 isoforms exhibit unambiguous nuclear localization.</p

Colocalization of MCPH1 and γH2AX in ionizing irradiation-induced nuclear foci.

<p>(A) Non-transduced (NT) MCPH1-deficient 562T cells and 562T stably expressing GFP alone or the specified GFP-MCPH1 fusion proteins were fixed 2 h after irradiation with 10 Gy and co-stained with antibodies against γH2AX (red) and GFP (green). Nuclei were counterstained with DAPI (blue). Rectangles frame areas, which are shown enlarged in the bottom row. MCPH1 focus formation was observed for MCPH1 isoforms containing the C-terminal BRCT tandem. (B) Quantification of cells expressing foci containing γH2AX and/or (C) MCPH1. Error bars indicate the S.D. of three different measurements, counting approximately 300 nuclei. * <i>p</i>≤0.05 vs. NT as calculated using the Student's <i>t</i>-test.</p