Carsten Hohoff

Results of a collaborative study of the EDNAP group regarding the reproducibility and robustness of the Y-chromosome STRs DYS19, DYS389 I and II, DYS390 and DYS393 in a PCR pentaplex format

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in the frame work of the STADNAP program, i.e. standardization of DNA profiling in Europe, in order to evaluate the performance of a Y-chromosome STR pentaplex, which includes the loci DYS19, DYS389 I and II, DYS390 and DYS393 and to determine whether uniformity of results could be achieved among different European laboratories. Laboratories were asked to analyze the five Y-STRs using singleplex and multiplex conditions in three bloodstains and one mixed stain (95% female and 5% male). All the laboratories reported the same results even for the mixed stain included in the exercise. This demonstrates the reproducibility and robustness of Y-chromosome STR typing even with multiplex formats and proves the usefulness of Y-STR systems for analyzing mixed stains with a male component.A total of 930 male samples from 10 different populations from Europe were also analysed for all the loci included in the pentaplex. Eight of these ten populations also included haplotype data. As for single gene analysis, haplotype diversity was higher in Germany and Italy and lower in Western European countries and Finland. Pairwise haplotype analysis shows the Finnish departure from the rest of the populations and a relatively homogeneity in the other European populations with F(ST) estimates lower than 0.05.UPGMA analysis shows an association of Western European population (Ireland, UK, Portugal and Galicia) on the one hand and central European populations on the other.

Mitochondrial control region sequences from a Vietnamese population sample.

Entire mitochondrial control region data were generated for 187 individuals from Vietnam. These samples have been previously typed for 16 autosomal short-tandem repeats (STRs) [1].

Allelic drop-out in the STR system ACTBP2 (SE33) as a result of mutations in the primer binding region

In the course of routine genotyping of forensic reference samples by multiplex PCR, an allelic drop-out due to mutations in the primer binding regions of the highly polymorphic STR marker ACTBP2 was observed in 17 samples. The variation rate was estimated to be 0.0014 (95% confidence interval: 0.0006–0.003). The most frequently found mutation was an G to A transition in the reverse primer binding region which was present in 14 out of 17 cases. To overcome the problem we have added a modified reverse primer to different multiplex kits that led to the correct genotype.

Y-chromosomal microsatellite mutation rates in a population sample from northwestern Germany.

To estimate Y-chromosomal short tandem repeat (Y-STR) mutation rates, 15 loci (i.e., DYS19, DYS389 I/II, DYS390, and DYS393; DYS437, DYS438, DYS439, and DYS385; DYS391, DYS392, YCA II, and DXYS156) were analyzed in a sample of 1,029 father/son pairs from Westphalia, northwestern Germany. Among 15,435 meiotic allele transfers, 32 mutations were observed; thus, the mutation rate across all 15 Y-STR loci was 2.1 × 10−3 per locus (95% C.I.: 1.5–3.0 × 10−3). With the exception of a three-repeat mutation at DYS385, all remaining mutations were single repeat mutations. Repeat losses were more frequent than gains (20:12), and the mutation rate appeared to increase with age. The Y haplogroups that were detected in the individuals showing a mutation reflect the haplogroup distribution in the Westphalian population. Additionally, the correlation of surnames and haplotypes was tested: Only 49 surnames occurred more than once, and only two men with the same rare surname shared the same haplotype. All other men with identical surnames carried different haplotypes.

Analysis of 15 short tandem repeats reveals significant differences between the Arabian populations from Morocco and Syria

The short tandem repeat (STR) systems D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA were studied in Arabian population samples from Morocco and Syria. No significant deviation from Hardy-Weinberg equilibrium could be observed in either preparation. Comparing the Moroccan and Syrian populations using the program RxC, no similarity could be observed at all 15 loci. In the Moroccan and Syrian populations the matching probability is 1 in 1.4 x 10(17) and 1 in 2.6 x 10(17), respectively. Thus, the combination of these 15 STR loci is powerful tool for forensic identification in Arabian populations.

The GEDNAP blind trial concept part II. Trends and developments

This article presents a review of the developments in the GEDNAP blind trials over the period covering the past 10 years (1993–2003), demonstrating the changing approach to DNA investigations in the European community as a whole. The results of the trials also identify the most common types of error encountered which can also occur during routine DNA typing and ways of recognising such errors are suggested.

A cautionary note on switching mitochondrial DNA reference sequences in forensic genetics

The first human mitochondrial DNA (mtDNA) sequence was produced in 1981 from an individual of European descent [1]. Since then, this sequence has been known as the Cambridge Reference Sequence (CRS) with a total length of 16,569 base pairs. As is common practice in other fields of genome research, this first mitochondrial genome (mtGenome) served as reference for the scientific community, relative to which other mtDNA haplotypes were reported. Eighteen years later the CRS was re-sequenced and corrected at 10 positions (3423T, 4985A, 9559C, 11335C, 13702C, 14199T, 14272C, 14365C, 14368C, and 14766C) to form the revised Cambridge Reference Sequence (rCRS) [2]. The new analysis also revealed that this mtGenome consists of only 16,568 nucleotides, as a base at position 3107 was mistakenly reported in the CRS. Instead of redefining all nucleotide positions downstream of 3107, this position is indicated in the rCRS as a deletion (unfortunately often indicated as ‘‘N’’, which is reserved for any base in the IUPcode [3]). Thus, the numbering system employed for the CRS and the body of established data can continuously be used with the rCRS. More than 15,500 mtGenomes and well over 150,000 (partial) control region sequences (including databases) have been published to date (http://www.phylotree.org/, Phylotree Build 14 [4]), in which the CRS and the rCRS have been cited 5603 and 968 times, respectively (http://apps.isiknowledge.com/; queried on May 2012). In a recent study, the switch to a new reference sequence, the so-called Reconstructed Sapiens Reference Sequence (RSRS), has been proposed [5]. This ancestral reconstructed sequence represents the deepest root in the known human mtDNA phylogeny at the base of the split of haplogroups L0 and L10203040506 after combining sequence information from all available mtGenomes from Homo neanderthalensis and novel human mtGenomes. The authors believe that the switch would solve misunderstandings and problems associated with the existing nomenclature relative to the rCRS, which belongs to the recently coalescing European haplogroup H2a2a1. In the following we briefly review developments in forensic mitochondrial genetics and discuss possible implications of the proposed switch. Mitochondrial DNA is highly abundant in cells compared to nuclear DNA (nDNA) with increased typing success rates for analysis of highly degraded samples and also hair shafts that often do not harbor detectable amounts of nDNA. To assess the significance of a match between two mtDNA haplotypes (e.g. from the crime scene and from a suspect), mtDNA databases have been developed. Earlier, the difference-coded haplotypes (with respect to the rCRS) were directly compared to the haplotypes in the mtDNA databases with the risk that multiple different alignments of the same sequence led to biased results [6]. The

Characterisation of variant alleles in the STR systems D2S1338, D3S1358 and D19S433

We have observed three hitherto undescribed off-ladder alleles at three widely used STR loci. These were isolated, sequenced and designated as follows: allele 10 (D2S1338, one case), allele 21 (D3S1358, two cases) and allele 6.2 (D19S433, six cases). These sequences are described in comparison to non-variant alleles, and their implications for the semi-automated STR analysis will be discussed.

Elevated germline mutation rate in teenage fathers.

Men age and die, while cells in their germline are programmed to be immortal. To elucidate how germ cells maintain viable DNA despite increasing parental age, we analysed DNA from 24 097 parents and their children, from Europe, the Middle East and Africa. We chose repetitive microsatellite DNA that mutates (unlike point mutations) only as a result of cellular replication, providing us with a natural ‘cell-cycle counter’. We observe, as expected, that the overall mutation rate for fathers is seven times higher than for mothers. Also as expected, mothers have a low and lifelong constant DNA mutation rate. Surprisingly, however, we discover that (i) teenage fathers already set out from a much higher mutation rate than teenage mothers (potentially equivalent to 77–196 male germline cell divisions by puberty); and (ii) ageing men maintain sperm DNA quality similar to that of teenagers, presumably by using fresh batches of stem cells known as ‘A-dark spermatogonia’.

Identification of West Eurasian mitochondrial haplogroups by mtDNA SNP screening: Results of the 2006-2007 EDNAP collaborative exercise

The European DNA Profiling (EDNAP) Group performed a collaborative exercise on a mitochondrial (mt) DNA screening assay that targeted 16 nucleotide positions in the coding region and allowed for the discrimination of major west Eurasian mtDNA haplogroups. The purpose of the exercise was to evaluate the stability and reproducibility of the self-developed multiplex-PCR and multiplex-single base extension kit by blind-testing saliva and hair shaft samples provided by the organizing laboratory. The overall success rate in obtaining useful results was high given that some of the participating laboratories had no previous experience with the technology and/or mtDNA analysis. The results of this collaborative exercise stimulate the expansion of screening methods in forensic laboratories to increase efficiency and performance of mtDNA typing, and thus demonstrates that mtDNA SNP typing is a powerful tool for forensic casework analysis.