Peter Wiegand

Forensic validation of the STR systems SE 33 and TC 11

SummaryPopulation studies on Caucasians from northwest Germany were carried out using the short tandem repeat (STR) systems SE 33 (Locus: ACTBP2) and TC 11 (Locus: 11p15.5). After electrophoresis in PAG 26 alleles could be identified for SE 33 in a sample size of 180 unrelated individuals and 6 alleles were found for TC 11 in 110 individuals. The combined mean exclusion chance for both systems was 0.96 and the discrimination index 0.999. No significant deviations from Hardy-Weinberg equilibrium could be demonstrated. In a small sample of families (SE 33 −n = 21; TC 11 −n = 30) no new mutations could be found. Positive and reproducible results for both STRs could be obtained from 50 pg template DNA.ZusammenfassungPopulationsstichproben nordwestdeutscher Kaukasier wurden mit den beiden Short tandem repeat (STR)-Systemen SE 33 (Locus: ACTBP2) and TC11 (Locus: 11p15.5) untersucht. Nach elektrophoretischer Auftrennung in PAG konnten 26 Allele für SE 33 in einer Bevölkerungsstichprobe von 180 nicht verwandten Personen und 6 Allele für TC 11 in einer Stichprobe von 110 Individuen differenziert werden. Der resultierende kombinierte AVACH-Wert für beide Systeme lag bei 0.96, der entsprechende Diskriminationsindex bei 0.999. Eine signifikante Abweichung vom Hardy-Weinberg-Gleichgewicht wurde nicht festgestellt. Erste Familienstudien (SE 33 −n = 21; TC 11 −n = 30) gaben keinen Hinweis auf Neumutation. Die Nachweissensitivität beider STRs lag im Bereich von 50 pg template DNA.

DNA typing of debris from fingernails

SummaryIn 3 series paired volunteers were asked to gently scratch each other with the fingernails to produce superficial abrasions only of the stratum corneum. In a 4th series scratch marks were produced in the skin of cadavers but additionally including the deeper epidermal layers. Debris was removed using a thorough technique in series 1 and 2 and a careful technique in series 3. After DNA extraction, the debris was typed using the STR systems HUMACTBP2 (SE33), HUMTH01 (TC11) and HUMVWFA31 (VWA). In the material obtained from series 1 (i.e. scratching with no prior cleaning of the nails) and series 2 (i.e. cleaning of the nails prior to the experiment) the debris was removed with a sharp instrument and only the DNA pattern of the person who carried out the scratching could be detected. In the 3rd series extraneous material was removed very carefully from under the fingernails to avoid contamination with DNA from the nails. In 71% of these cases DNA patterns of the person who had been scratched or mixed DNA patterns of both persons could be detected. In the experiments with postmortem skin the DNA pattern of the cadaver could be detected in all cases. These results show that in crime cases where the perpetrator has been scratched by the victim, sufficient material can be obtained from under the fingernails for DNA typing if removal of the particles is carried out with sufficient care.ZusammeufassungIn 3 Versuchsreihen erzeugten freiwillige Probanden jeweils gegenseitig oberflächliche Kratzverletzungen, die ausschließlich auf das Stratum corneum der Epidermis beschrdnkt waren. In einer vierten Versuchsreihe wurden Kratzverletzungen auch der tieferen Epidermisschichten an Leichenhaut hervorgerufen. Die Epithelschüppchen unter den Fingerndgeln wurden mit verschiedenen Techniken asserviert and nach DNA-Extraktion unter Anwendung der STR-Systeme HUMACTBP2 (SE33), HUMTH01 (TC11) und HUMVWFA31 (VWA) individualisiert. In den beiden ersten Versuchsreihen (1. Kratzen mit zuvor ungereinigten Fingernägeln, 2. Asservieren der Epithelschüppchen mit scharfem Instrument und intensiver Reinigung vor und nach dem Kratzen) konnten lediglich Merkmale des Kratzenden festgestellt werden. Da bei der dritten Versuchsreihe besonderer Wert auf eine vorsichtige Asservierung der Epithelpartikel gelegt wurde, ohne dabei gleichzeitig Schüppchen der Fingernägel abzukratzen, gelang es in 71% der Fälle, die DNA des Gekratzten oder ein Mischmuster des Kratzenden und Gekratzten darzustellen. Die Versuche an Leichenhaut ergaben in allen Fällen das Muster des Verstorbenen. Die Ergebnisse belegen, daß bei entsprechenden Spurenfällen die Untersuchung eines sorgfältig präparierten Fingernagelschmutzes in einem hohen Prozentsatz erfolgreich sein kann und somit zur Identifizierung eines Tatverdächtigen geeignet ist.

Population genetics and forensic efficiency data of 4 AMPFLP's

SummaryFamily studies were carried out in a population sample from north west Germany using 4 amplifiable VNTR polymorphic systems D1S80 (MCT118), ApoB, D17S30 (YNZ22) and COL2A1. Separation was carried out in polyacrylamide gels and visualised using silver staining. In family studies (n = 30) no evidence of new mutations was found. The population study of unrelated individuals (mothers and putative fathers) showed that all 4 systems were highly polymorphic and similar to other population studies. The combined exclusion chance was calculated to be approximately 99% and the combined discrimination index 1.5 · 10−4. The HardyWeinberg equilibrium was checked by forming groups of alleles and no significant deviations could be found in all systems.ZusammenfassungUntersucht wurden Familien einer nordwestdeutschen Bevölkerungsstichprobe mit 4 amplifizierbaren VNTR-Polymorphismen (MCT118, ApoB, YNZ22, COL2A1). Die Darstellung der Systeme erfolgte in Polyacrylamidgelen durch Silberfärbung. Es fand sich kein Hinweis auf Neumutationen. Anhand der typisierten Unverwandten (Mütter und Putativväter) konnten für jedes System Allelfrequenzen ermittelt werden, die im Vergleich zu anderen Bevölkerungsstichproben relativ gute Übereinstimmungen aufwiesen. Die Berechnung der kombinierten Ausschlußchance für die 4 Systeme führte zu einem Wert von etwa 99%. Der dazu korrespondierende Diskriminationsindex betrug 1,5 · 10−4. Zur Uberprüfung des Hardy-Weinberg-Gleichgewichts wurden Allelgruppen gebildet. Für alle 4 Systeme konnte das Hardy-Weinberg-Gleichgewicht nachgewiesen werden.

Population and family data of RFLP's using selected single- and multi-locus systems

The multi-locus systems (MLS) 33.15 and 33.6 (Jeffreys et al 1985a) and the single-locus systems (SLS) MS 1, MS 8, MS 31 and MS 43 (Wong et al. 1987) were investigated. The number of bands and the rate of band sharing were determined for both multi-locus systems and compared to the results of an English survey. Additionally 73 families were investigated using the multi-locus probes and the results compared to those obtained for the traditional blood grouping systems. The results were in full agreement so that no evidence of new mutations could be found. The fragment distribution was calculated for each of the 4 single-locus systems and compared to the values reported in an English survey (Smith et al. 1990b). Distinct discrepancies were seen in the systems MS 1 and MS 8. Family analyses were carried out for the 4 single-locus systems and compared to English data (Jeffreys et al. 1988) to look for any indications of possible new mutations. Only one isolated exclusion could be demonstrated with MS 1.

Phenotype differences of STRs in 7 human populations.

A maximum of 6 STR systems (TH01, VWA, ACTBP2, FES, F13B, D21S11) was investigated in 7 human populations (Germans, Turks, Moroccans, Japanese, Chinese, Papuans, Ovambos). In each population no deviations from Hardy-Weinberg equilibrium were observed. Out of each population the phenotypes of 50 individuals (comprising 3 to 6 STRs) were randomly selected. Based on the phenotype frequencies interpopulation comparisons were carried out using the frequencies of each other population. Within major ethnic groups only minor differences in phenotype frequencies were found. Between major ethnic groups differences of up to several orders of magnitude could be observed. The most discriminative STRs for interpopulation comparisons were TH01, FES and F13B.

Collaborative genetic mapping of 12 forensic short tandem repeat (STR) loci on the human X chromosome

A large number of short tandem repeat (STR) markers spanning the entire human X chromosome have been described and established for use in forensic genetic testing. Due to their particular mode of inheritance, X-STRs often allow easy and informative haplotyping in kinship analyses. Moreover, some X-STRs are known to be tightly linked so that, in combination, they constitute even more complex genetic markers than each STR taken individually. As a consequence, X-STRs have proven particularly powerful in solving complex cases of disputed blood relatedness. However, valid quantification of the evidence provided by X-STR genotypes in the form of likelihood ratios requires that the recombination rates between markers are exactly known. In a collaborative family study, we used X-STR genotype data from 401 two- and three-generation families to derive valid estimates of the recombination rates between 12 forensic markers widely used in forensic testing, namely DXS10148, DXS10135, DXS8378 (together constituting linkage group I), DXS7132, DXS10079, DXS10074 (linkage group II), DXS10103, HPRTB, DXS10101 (linkage group III), DXS10146, DXS10134 and DXS7423 (linkage group IV). Our study is the first to simultaneously allow for mutation and recombination in the underlying likelihood calculations, thereby obviating the bias-prone practice of excluding ambiguous transmission events from further consideration. The statistical analysis confirms that linkage groups I and II are transmitted independently from one another whereas linkage groups II, III and IV are characterised by inter-group recombination fractions that are notably smaller than 50%. Evidence was also found for recombination within all four linkage groups, with recombination fraction estimates ranging as high as 2% in the case of DXS10146 and DXS10134.

Population genetic comparisons of three X-chromosomal STRs

The X-chromosomal short tandem repeats (STRs) DXS6800, DXS101 and DXS8377 were analysed in male and female population samples from Germany and Austria using a PCR multiplex approach. We investigated 135 family trios from Innsbruck (Austria) and surrounding areas and 50 families and further male and female samples from Ulm (Germany) and surrounding areas. The comparisons of the allele frequencies gave similar distributions for Innsbruck and Ulm although minor variations were found for some alleles. Additionally, some differences were found when comparing the allele frequencies of the male and female samples independently. The forensic efficiency values demonstrate that especially DXS101 and DXS8377 are highly informative markers for kinship analysis and deficiency cases. Based on the investigated meiotic events no new mutations were detected.

Application of a quasi-median network analysis for the visualization of character conflicts to a population sample of mitochondrial DNA control region sequences from southern Germany (Ulm).

Entire mtDNA control region sequences from 100 individuals in a west Eurasian population sample from southern Germany (around the city of Ulm) were generated and analyzed. The control region was amplified in one piece and sequenced with ten different sequencing primers. Sequence evaluation was performed independently. Phylogenetic analyses were used for quality assurance purposes and for the determination of the haplogroup affiliation of the samples. The sequences were scrutinized performing a quasi-median network analysis. To visualize character conflicts, frequent mutations were filtered, and the reduced data were represented by the torso of their quasi-median network. Character incompatibilities were found to be based on real biological patterns of homoplasy. The population data will be incorporated in the EMPOP database (http://www.empop.org).

Structure of new mutations in 2 STR systems.

Isolated father/child mismatches in cases with a high probability of paternity (W > 99.9%) have been investigated using short tandem repeat (STR) systems. According to the high probability of paternity new mutations could be assumed in these cases. A new mutation could be observed in 3 cases using the STR system HumACTBP2. Two of these cases showed a deletion and 1 case an insertion of 1 repeat (AAAG-motif) which could be verified by sequencing. In another paternity case a new mutation - 1 - repeat insertion (TCTA-motif) - in the HumVWA system was detected and verified by sequencing. These findings led to a new mutation rate of 0.7% (n = 453 meioses) for HumACTBP2 and 0.2% for HumVWA (n = 484 meioses).ZusammenfassungUntersucht wurden isolierte Vater/ Kind-Ausschlüsse mit Short Tandem Repeat (STR)-Systemen in Paternitätsfällen mit sehr hoher Vaterschaftswahr-scheinlichkeit (W > 99.9%). Aufgrund der hohen Vaterschaftswahrscheinlichkeit war von Neumutationen auszugehen. Mit dem STR-System HumACTBP2 wurde eine Neumutation in 3 Paternitätsfällen beobachtet. In 2 Fällen wurde eine Deletion und in einem Fall eine Insertion um jeweils 1 Repeat (AAAG-Motiv) durch Sequenzierung nachgewiesen. Im HumVWA-System konnte in einem weiteren Paternitätsfall eine 1-Repeat-Insertion (TCTA-Motiv) durch Sequenzierung bestätigt werden. Die 3 beobachteten Neumutationsfälle ergaben für HumACTBP2 eine Mutationsrate von 0.7% (n = 453 Meiosen). Für Hum-VWA lag die Mutationsrate bei 0.2% (n = 484 Meiosen).

Somatic mutations at STR loci—a reason for three-allele pattern and mosaicism

Two families are analysed in which one of the parents exhibited a three-allele pattern at the ACTBP2 locus. Since the alleles were obviously segregated independently to the children, a generalised mosaicism must be assumed involving at least two tissues in one of them and at least four tissues in the other one. The intensity of the PCR amplified alleles in both three-allele individuals indicate an occurrence in a very early embryonic stage. Occurrence was most probably due to a single step mutation in both cases. Forensic implications would include paternity testing as well as stain analysis.