Contamination incidents in the pre-analytical phase of forensic DNA analysis in Austria-Statistics of 17 years.

Forensic DNA analyses have become more and more sensitive in the past years. With the ability to generate DNA profiles even from minute amounts of cellular material also the possibility to detect DNA on trace material that originates from persons not linked to the crime event, such as crime scene investigators, increases. The contamination of crime scene samples can lead to false positive results and misinterpretation that can cause deceptive investigations. In this work we continue a study of 2010 that compared the number of detected contamination incidents that were caused in the pre-analytical phase of forensic DNA analysis with the number of crime scene samples analyzed by our laboratory. Within the past 17 years we were able to detect a total of 347 contamination incidents caused by police officers in approximately 46,000 trace samples to their origin (0.75%). Additionally we demonstrate the usefulness of reference profile databases that contain DNA profiles of police officers to detect contamination incidents of trace material.

Discordant growth pattern and ovarian function in monozygotic twins with 45,X/46,XX mosaicism.

BACKGROUND: We report on phenotypically discordant female monozygotic twins with 45X/46,XX mosaicism in both lymphocytes and fibroblasts. RESULTS: At 11.5 years, twin A was prepubertal, her height was 126.8 cm (-3.15 SD), bone age (BA) 9.7 years (TW2), FSH 47 IU/l and IGF-I 280 ng/ml (-0.89 SD), but twin B was pubertal (P2, B3), her height was 143.4 cm (-0.92 SD), BA 13.6 years (TW2), FSH 3.4 IU/l and IGF-I 380 ng/ml (-0.21 SD). One year later, twin A had grown 11.1 cm due to growth hormone therapy and had IGF-I 1,400 ng/ml (+5.91 SD), whereas the growth velocity of twin B (no therapy) was 5.9 cm, IGF-I 540 ng/ml (+0.57 SD) and she started regular menstruation at 12.1 years. CONCLUSION: To our knowledge, this is the first report on monozygotic twins with Turner mosaicism in both lymphocytes and fibroblasts who developed a discordant phenotype probably due to an unequal distribution of the two cell lines in distinct tissues.

Evaluation of an alkaline lysis method for the extraction of DNA from whole blood and forensic stains for STR analysis.

A modified alkaline lysis protocol for extracting DNA from forensically relevant specimens is evaluated and compared with the chelex 100 method. For whole blood, bloodstains and sperm stains, both methods yielded comparable results after amplification for a pentameric STR locus (HumCD4). The main advantages of the new method are: only approximately ten minutes and two pipetting steps are necessary and the expenses for the extraction are extremely low as only NaOH, TrisHCl buffer and a single microcentrifuge tube are required. Alkaline lysis also proved to yield DNA suitable for typing longer STRs by using dye-labeled primers and capillary electrophoresis. These advantages should render this protocol especially interesting for high-throughput laboratories in combination with multiplex PCR and fluorescent dye technology, although the storability of the extracts proved to be problematic.

Nine STR markers plus amelogenin (AmpFlSTR Profiler Plus): a forensic study in an Austrian population.

Genetic efficiency data of nine short tandem repeat (STR) loci were determined by multiplex PCR using fluorescently labeled primers and subsequent analysis by capillary electrophoresis (ABI 310). For each locus 7-14 alleles were detected. The combined matching probability is about 1 x 10(-11). No deviations from Hardy-Weinberg equilibrium were observed.

Study on the STR TPOX in an Italian and an Austrian population using two different primer pairs and three different electrophoretic methods.

The short tandem repeat TPOX was studied using two different pairs of primers and three different electrophoretic methods with the aim of optimizing and standardizing the typing conditions for this locus. A genetic population study was subsequently conducted on two population samples from Central Italy (151 individuals) and from Austria (153 individuals) and compared using an R x C contingency table. With the aim of using this system for forensic samples, differences in sensitivity between the methods utilized were studied and several parameters of forensic interest for the two populations (PD, MEC, MEP, pM, PIC) were calculated. A new multiplex system for the loci CSF1PO, TPOX and CD4 is also presented.

Mutation rate in human microsatellites: influence of the structure and length of the tandem repeat.

In 10,844 parent/child allelic transfers at nine short-tandem-repeat (STR) loci, 23 isolated STR mismatches were observed. The parenthood in each of these cases was highly validated (probability >99.97%). The event was always repeat related, owing to either a single-step mutation (n=22) or a double-step mutation (n=1). The mutation rate was between 0 and 7 x 10(-3) per locus per gamete per generation. No mutations were observed in three of the nine loci. Mutation events in the male germ line were five to six times more frequent than in the female germ line. A positive exponential correlation between the geometric mean of the number of uninterrupted repeats and the mutation rate was observed. Our data demonstrate that mutation rates of different loci can differ by several orders of magnitude and that different alleles at one locus exhibit different mutation rates.

A collaborative genetic study on the STR system FGA in two Austrian population samples.

Population genetic data of the short tandem repeat system FGA were determined by PCR analysis in two Austrian population samples, one population north of the Alps and one population south of the Alps. A total of 15 different alleles could be observed in 500 unrelated individuals. No significant differences were found between the phenotype frequencies in the two populations, as determined by R x C contingency test, so the populations could be pooled for further analysis. Both the single populations and the pooled population are in accordance with Hardy-Weinberg equilibrium. FGA proves to be very efficient for both stain analysis and paternity testing. The presented allele and genotype data allow the statistical interpretation of this system for Austrians.

A study on the short tandem repeat system ACTBP2 (SE33) in an Austrian population sample.

Population genetic studies were carried out on 932 caucasians from Austria using the short tandem repeat system ACTBP2 (SEE33). A sequenced allelic ladder was used for typing (Möller et al. 1995). After native polyacrylamide gel electrophoresis all 26 alleles of the ladder were found as well as 194 alleles which migrated differently from those in the ladder. Forensically relevant parameters were calculated (discrimination power: 0.989, mean exclusion chance: 0.854, observed heterozygosity 0.946). An allele consisting of 9 repeats which is not part of the allelic ladder was also found. In 692 meioses 5 mutations were found (mutation rate 0.72%).

Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco.

To study the influence of genomic context on transgene expression, we have determined the T-DNA structure, flanking DNA sequences, and chromosomal location of four independent transgene loci in tobacco. Two of these loci were stably expressed in the homozygous condition over many generations, whereas the other two loci became unstable after several generations of homozygosity. The stably expressed loci comprised relatively simple T-DNA arrangements that were flanked on at least one side by plant DNA containing AT-rich regions that bind to nuclear matrices in vitro. Of the unstably expressed loci, one consisted of multiple incomplete T-DNA copies, and the second contained a single intact T-DNA; in both cases, however, binary vector sequences were directly contiguous to a right T-DNA border. Fluorescence in situ hybridization demonstrated that the two stably expressed inserts were present in the vicinity of telomeres. The two unstably expressed inserts occupied intercalary and paracentromeric locations, respectively. Results on the stability of transgene expression in F1 progeny obtained by intercrossing the four lines and the sensitivity of the four transgene loci to inactivation in the presence of an unlinked "trans-silencing" locus are also presented. The findings are discussed in the context of repetitive DNA sequences and the allotetraploid nature of the tobacco genome.

A genetic study of the short tandem repeat systems VWA and TH01 in an Austrian population.

Allele- and genotype frequencies of the two short tandem repeat (STR) systems VWA and TH01 were determined in an Austrian population sample by polymerase chain reaction (PCR) analysis. A total of 9 alleles for VWA and 6 alleles for TH01 could be observed in a population of 278 (VWA) and 276 (TH01) individuals. Both systems are in accordance with Hardy-Weinberg equilibrium. They are highly informative for individualization from stain analysis and, in addition, they are very useful for paternity testing. The data presented here allow the statistical interpretation of PCR results for an Austrian population.

[Detection of saliva traces on perpetrator masks and their attribution to a particular criminal]. / Zum Nachweis von Speichelspuren auf Tätermasken und deren Zuordnung zu einem Verursacher.

Face masks are commonly used in robberies and sometimes found nearby crime scenes. Usually saliva traces can be found on such masks, which contain cellular material useful for PCR typing. The method described in this study makes it possible to localize such regions on face mask, thereby allowing a much more directed investigation of the objects to analyse. This means a simplification of the DNA extraction procedure and a minimization of loss of material during DNA isolation. The general suitability of this method is demonstrated by a casework example.

Population genetic study of the AmpFLP system APO B in an Austrian population sample.

Allele frequencies of the AmpFLP system APO B were determined in an Austrian population sample consisting of 210 unrelated Caucasian individuals living in the Salzburg region. A total of 25 different alleles could be observed. The allele distributions were in accordance with Hardy-Weinberg equilibrium. No new mutations were found in 184 meioses and seven "interalleles" and four alleles < 29 could be detected.

Analysis of STR-PCR products with high-resolution denaturing PAGE.

The Pharmacia Multiphor II horizontal electrophoresis chamber is a widespread tool for analysis of PCR products in forensic casework. Up to date, however, there is no protocol for successfully running high-resolution denaturing PAGE (poly-acrylamide gel electrophoresis) on a horizontal electrophoresis chamber. We modified the electrophoresis conditions to make this possible.

F13B and CD4 allele frequencies in an Austrian population sample.

Allele frequencies of the two short tandem repeat (STR) systems F13B and CD4 were determined in an Austrian population sample by PCR analysis. A total of 6 alleles for F13B and 8 alleles for CD4 could be observed in a population of 216 (F13B) and 198 (CD4) unrelated individuals. No significant deviations from Hardy-Weinberg equilibrium were observed.

[Clarification of a forced entry theft based on a beverage bottle left at the scene and PCR DNA typing of superficial saliva traces]. / Klärung eines Einbruchdiebstahls anhand einer am Tatort zurückgelassenen Getränkeflasche und PCR-DNA-Typisierung von daran befindlichen Speichelspuren.

This paper describes the first case in Austria where a suspect was been convicted and condemned because of salivary traces found on an empty bottle which were successfully typed by PCR-DNA-analysis. This was the only evidence in that case. In contrast to cigarette butts, which are already relatively routine for PCR-DNA-typing, bottles found on crime scenes usually contain much less material which can be investigated. As the result of permanent refinement of the methods used, it is demonstrated that it should be possible to solve other similar cases in the future.

Inheritance and expression of a transgene insert in an aneuploid tobacco line.

A T-DNA locus comprising nptII, uidA and nos genes--all under the control of the nos promoter (this locus was designated K because it encodes resistance to Kanamycin)--was found to be inherited erratically in a transgenic tobacco line. This anomalous behavior was partially explained following a karyotype analysis of plants representing several generations: these plants were aneuploids, presumably for the K-containing chromosome. During four generations of sexual propagation, transgenic plants that were either trisomic or tetrasomic for the K-containing chromosome (i.e. 2n = 49 or 2n = 50, respectively) were obtained. The trisomic plants (2n = 48 + 1) were virtually indistinguishable phenotypically from normal euploids (2n = 4x = 48), whereas the tetrasomic plants (2n = 48 + 2) were smaller, had somewhat misshapen leaves and exhibited reduced fertility. Although the amount of NPTII protein in different trisomic (K--, KK-, KKK) and tetrasomic (KK--, KKK-) plants was generally consistent with a K dosage effect, the genetic behavior of each trisomic--with respect to segregation of KanR and marker gene activity in progeny--was unique and not completely explicable by invoking aneuploidy. Specifically, unexpected gains or losses of K could occur, suggesting the formation of double reductional gametes and/or frequent gene conversion at this locus. The susceptibility of K locus marker genes to trans-inactivation in the trisomic and tetrasomic lines was tested by crossing in partially homologous silencing loci. In all transgenotypes tested, the three K marker genes were sensitive to trans-silencing, which was accompanied by methylation in all copies of the nos promotor. In addition to this directed inactivation/methylation, the K locus could also undergo infrequent, spontaneous partial methylation, which produced stable epialleles. In most plants, however, the multiple copies of the nos promoter at this locus remained unmethylated and active through four generations in all transgenotypes examined. The significance of these results for irregular inheritance patterns, aneuploid syndromes and homology-dependent gene silencing is discussed.

Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes.

Previous work has shown that two homologous, unlinked transgene loci can interact in plant nuclei, leading to non-reciprocal trans-inactivation and methylation of genes at one locus. Here, we report the structure and methylation of different transgene loci that contain the same construct but are variably able to inactivate and methylate a partially homologous, unlinked target locus. Silencing loci comprised multiple, methylated copies of the transgene construct, whereas a non-silencing locus contained a single, unmethylated copy. The correspondence between strength of silencing activity and copy number/degree of methylation was further demonstrated by producing novel alleles of a strong silencing locus: reducing the transgene copy number and methylation within this silencing locus decreased its ability to inactivate the target locus. The strong silencing locus, which was located close to a telomere, trans-inactivated various structural variants of the original target construct, regardless of their location in the genome. This suggests that the silencing locus can scan the entire genome for homologous regions, a process possibly aided by its telomeric location. Our data support the idea that epistatic trans-inactivation of unlinked, homologous transgenes in plants results from a pre-existing epigenetic difference between transgene loci, which is subsequently equalized by "epigene conversion" involving DNA-DNA pairing.

Susceptibility of transgene loci to homology-dependent gene silencing.

Previous work has shown that two unlinked, partially homologous transgene loci can interact in plant nuclei, leading to reversible methylation and inactivation of one transgene locus in the presence of the second. To study whether the chromosomal location of a transgene influences its susceptibility to trans-inactivation, we retransformed four transgenic lines, which contained the same construct (H) integrated in different chromosomal locations, with a second, partially homologous construct (K). At least 50 double transformants (DTs) were regenerated from each single transformants (ST) and screened for inactivation of markers [chloramphenicol acetyltransferase (CAT); hygromycin resistance (HYGR)] at the resident H locus. For two STs, H locus markers were inactivated in less than 1% of the DTs, suggesting that, at these integration sites, H was relatively resistant to trans-inactivation. In contrast, the other two STs appeared to be more sensitive to trans-inactivation: 4-10% of the DTs were CAT- and/or Hygs. Inactivation of H locus markers could be attributed to two distinct phenomena: 1. Regeneration from cells containing different epigenetic states of H, in which either both, one or none of the H alleles was active. This instability in the expression of the H locus, which was independent of K, was more pronounced in the homozygous state, and was associated with cellular mosaicism of expression and methylation. 2. The presence of an unlinked K locus could weaken the HygR phenotype by transcriptional inactivation and increased methylation of the hph gene at the H locus. These results indicated that a susceptible transgene locus is inherently unstable and partially methylated, and that these characteristics are exacerbated when the locus is homozygous for the transgene and/or when an unlinked homologous transgene is present.

A variety of epistatic interactions can occur between partially homologous transgene loci brought together by sexual crossing.

Epistatic interactions between unlinked transgene loci in tobacco plants were studied following sexual crosses between different transgenic lines. Three potential "modifier" transgene loci, which were structurally similar but integrated at different chromosomal locations, were tested for their ability to influence the expression of a partially homologous "target" transgene locus. After introduction of an individual modifier locus, the target locus could be either unaffected, completely inactivated and methylated or differentially sensitive, showing more complete inactivation and methylation when homozygous than when hemizygous. The implications of these results for inbreeding depression in plants are discussed.