Forensic identification of urine samples: a comparison between nuclear and mitochondrial DNA markers.

Urine samples from 20 male volunteers of European Caucasian origin were stored at 4 degrees C over a 4-month period in order to compare the identification potential of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) markers. The amount of nDNA recovered from urines dramatically declined over time. Consequently, nDNA likelihood ratios (LRs) greater than 1,000 were obtained for 100, 70 and 55% of the urines analysed after 6, 60 and 120 days, respectively. For the mtDNA, HVI and HVII sequences were obtained for all samples tested, whatever the period considered. Nevertheless, the highest mtDNA LR of 435 was relatively low compared to its nDNA equivalent. Indeed, LRs obtained with only three nDNA loci could easily exceed this value and are quite easier to obtain. Overall, the joint use of nDNA and mtDNA markers enabled the 20 urine samples to be identified, even after the 4-month period.

Forensic evaluation of the QIAshredder/QIAamp DNA extraction procedure.

The potential to recover genetic profiles from evidence samples has substantially increased since robust and sensitive amplification kits are commercially available. Nevertheless, even the best amplification kits cannot succeed when the extracted DNA is of poor quality. In this study we compared the efficiency of silica (QIAamp DNA Mini Kit), Chelex and Phenol-Chloroform (PC) based protocols to recover DNA from different categories of samples (blood and saliva on cotton swabs, muscles, cigarette butts, saliva on foods and epidermal cells on clothes). The efficiency of the QIAamp system was improved when samples were treated with QIAshredder homogenizing columns. Overall, conventional Chelex or PC protocols allowed to recover conclusive SGM Plus profiles for 61% of the samples considered in this study. Contrastingly, 82% of them were successfully genotyped after being treated with a combination of QIAshredder and QIAamp systems. Our results further suggested that the QIAshredder/QIAamp protocol was particularly helpful to analyze evidence samples with few DNA and/or that were collected on substrates containing PCR inhibitors.

Results of a collaborative study of the EDNAP group regarding mitochondrial DNA heteroplasmy and segregation in hair shafts.

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the distribution of mitochondrial DNA (mtDNA) heteroplasmy amongst the hairs of an individual who displays point heteroplasmy in blood and buccal cells. A second aim of the exercise was to study reproducibility of mtDNA sequencing of hairs between laboratories using differing chemistries, further to the first mtDNA reproducibility study carried out by the EDNAP group. Laboratories were asked to type 2 sections from each of 10 hairs, such that each hair was typed by at least two laboratories. Ten laboratories participated in the study, and a total of 55 hairs were typed. The results showed that the C/T point heteroplasmy observed in blood and buccal cells at position 16234 segregated differentially between hairs, such that some hairs showed only C, others only T and the remainder, C/T heteroplasmy at varying ratios. Additionally, differential segregation of heteroplasmic variants was confirmed in independent extracts at positions 16093 and the poly(C) tract at 302-309, whilst a complete A-G transition was confirmed at position 16129 in one hair. Heteroplasmy was observed at position 16195 on both strands of a single extract from one hair segment, but was not observed in the extracts from any other segment of the same hair. Similarly, heteroplasmy at position 16304 was observed on both strands of a single extract from one hair. Additional variants at positions 73, 249 and the HVII poly(C) region were reported by one laboratory; as these were not confirmed in independent extracts, the possibility of contamination cannot be excluded. Additionally, the electrophoresis and detection equipment used by this laboratory was different to those of the other laboratories, and the discrepancies at position 249 and the HVII poly(C) region appear to be due to reading errors that may be associated with this technology. The results, and their implications for forensic mtDNA typing, are discussed in the light of the biology of hair formation.

Tunisian population data on 10 Y-chromosomal loci.

Alleles and haplotypes frequencies for 10 Y-chromosome STR loci (DYS19, DYS385 I/II, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438 and DYS439), included in the Y-Plex6 and Y-Plex5 kits were determined for a Tunisian population sample of 100 male individuals.

Tunisian population data on 15 PCR-based loci.

Genotype and allele frequencies distribution for 15 PCR-based loci included in the Promega PowerPlex 16 kit were determined for a Tunisian population sample of 196 unrelated individuals.

Is it possible to differentiate mtDNA by means of HVIII in samples that cannot be distinguished by sequencing the HVI and HVII regions?

The mitochondrial control region includes three so-called hypervariable (HV) regions, in which the polymorphic positions show a particularly high frequency. According to a population study of 200 unrelated individuals from Germany, HVI (positions 16,024-16,365, according to Anderson) showed 88 variable positions in a total length of 342 bp (26%) and HVII (positions 73-340) displayed 65 mutable sites in 268 bp (24%). HVIII (positions 438-574) exhibited a slightly lower variability, with 25 polymorphic sites within 137 bp (18%), but contrasted clearly with the background, which showed variability rates of only 7% (positions 16,366-16,569, 1-72) and 3% (positions 341-437), respectively. At present, the displacement (D)-loop database in Magdeburg comprises 904 sequences of the mitochondrial HVI region and HVII region from Germans, Austrians and Swiss. By means of this material, the extent to which the mtDNA sequences that do not differ in the HVI and HVII regions can be differentiated by additionally sequencing HVIII was investigated.

Mitochondrial DNA in the Central European population. Human identification with the help of the forensic mt-DNA D-loop-base database.

Sequencing of mtDNA is an advanced method for the individualisation of traces. Disadvantages of this method are expensive and time-consuming analysis and evaluation procedures as well as the necessary stock of population-genetic data which is still insufficient. Central European institutes of forensic medicine from Germany, Austria, and Switzerland have been working together since the beginning of 1998 to establish a mtDNA database. The aim is to build up a large stock of forensically established data and provide population-genetic data for frequency investigations, which will serve as a basis for expert opinions and scientific research. Good data quality is ensured by using original sequences only. Ring tests, which have been conducted to enhance analytical reliability, revealed a high correspondence rate of the analytical results obtained by the individual member institutes. Today 1410 sequences are available for comparison, of which 1285 sequences in the HV1 and HV2 regions cover the full ranges from 16051 to 16365 and from 73 to 340 (according to Anderson). The major part is formed by Central European sequences comprising 1256 data sets from Germany, Austria, and Switzerland. Today the database contains sequences from a total of 12 European, six African and three Asian countries including 100 sequences from Japan. This paper is aimed at discussing the individualisation potentials of mtDNA as well as the possibilities and limits of ethnic differentiation by means of pairwise sequence differences on the basis of the data stock available.

Forensic evaluation of mtDNA in a population from south west Switzerland.

The polymorphism of the two hypervariable segments (HVI and HVII) of the control region of mtDNA was analyzed in a population of 154 unrelated individuals from south west Switzerland using a fluorescent based capillary electrophoresis sequencing method. In our population data of 154 random individuals, 137 mtDNA types were observed. Of these, 124 sequences were observed only in one individual whereas 10 sequences were observed in 2 individuals, 2 sequences in 3 individuals and 1 sequence in 4 individuals. The probability of two unrelated individuals having the same sequence was 0.84%. The results were compared with four other Caucasian populations. Furthermore, the usefulness of the mtDNA sequencing was tested, for exclusion and inclusion, in 18 forensic cases including 69 evidence samples and 44 reference samples. Despite the fact that 55% of the evidence samples yielded a negative result for the nuclear DNA with the human dot quantitation system, the success rate of the mtDNA sequencing was 71.0%. This validation study proves the great usefulness and sensitivity of the mtDNA sequencing technique using nested PCR and fluorescent capillary electrophoresis.

Forensic validation of the short tandem repeat HUMACTBP2 using capillary electrophoresis.

Experiments were performed to evaluate the forensic identification of the short tandem repeat (STR) HUMACTBP2 (human beta-actin-related pseudogene) using automated fluorescence-based capillary electrophoresis. The HUMACTBP2 is a complex tetranucleotide STR locus with more than 32 alleles in the range of 202-323 bp. The reproducibility of genetic typing using a fluorescent labeled allelic ladder was determined by comparison of the calculated fragment size after consecutive (within-day) and nonconsecutive (day to day) injection. The maximum variation in size (window) observed for any allele was 0.23 bp for the within-day and 0.8 bp for the day-to-day precision. Furthermore, it is possible to achieve a 1 bp resolution, the precision of the reproducibility assays being about 99.95%. Sixty blood samples and twenty stains were typed with both automated fluorescent sequencer ABI 373A and ABI 310. Identical genotypes were obtained with both techniques and the ABI 310 seemed to be more sensitive than the ABI 373A. A population sample of 197 unrelated individuals from southwest Switzerland was analyzed and the genotype frequencies observed were similar to those reported by others. Thirty-one alleles and 126 genotypes were found. The observed heterozygosity was 0.934. Mixtures from two different blood samples varying in their ratio were typed and the minor fraction was detectable to about 1:10. The practical usefulness of the HUMACTBP2 is illustrated by analyzing casework samples. This validation study proves the usefulness of the HUMACTBP2 locus in forensics and the detection efficiency using fluorescent capillary electrophoresis.

PCR-based forensic testing of DNA from stained cytological smears.

Experiments were performed to evaluate the efficiency of PCR-STR (Short Tandem Repeats) and PCR-sequence polymorphisms for the identification of stained pap smears and postcoital slides stained with cytological and forensic techniques. HLA-DQA1, PolyMarker, Amelogenin, HUMTH01, HUMVWFA31, HUMF13B, and HUMFES/FPS were determined. With the exception of the forensic Baecchi stain, all the PCR-systems gave consistent results in comparison with the reference blood from the donors. Cytological stained smears can be important evidence for identification in sexual assault cases and in missing person cases.

F13B and CD4 allele frequencies in south west Switzerland.

Allele frequencies of the two short tandem repeat (STR) systems F13B and CD4 were determined in a population sample from South West Switzerland using PCR analysis. We found five alleles for both STRs in a population sample of 205 unrelated individuals. No significant deviation from Hardy-Weinberg equilibrium was observed.

Evaluation and usefulness of reverse dot blot DNA-PolyMarker typing in forensic case work.

Experiments were performed to evaluate the Amplitype PolyMarker DNA typing system for application to forensic casework. DNA extraction using chelex was compared with phenol-chloroform extraction for various biological materials including postmortem blood, blood samples used for alcohol quantification, fresh urine, envelopes and cigarette butts. Different amounts of genomic DNA were amplified to test the sensitivity of the Amplitype PM. Mixed samples of two different bloods were typed to determine the dilution at which mixtures could be detected. Different storage conditions were evaluated using urine samples. Postmortem blood samples were typed during 4 months to determine the effects of natural degradation. A population sample of 105 unrelated individuals from South-West Switzerland was analyzed and the genotype frequencies were compared with those reported by others. Finally, practical usefulness of the Amplitype PM system is illustrated by analysing casework samples. The results of this validation proved the great usefulness and sensitivity of the Amplitype PM system using the appropriate extraction and typing method. However, mixed samples had to be interpreted with caution owing to the possibility of non-specific alleles with stored material such as urine and postmortem blood.

Isérables: a Bedouin village in Switzerland?

Isérables is an alpine village--about 1000 inhabitants--which remained isolated till these recent years because of its particular geographical situation. The Isérables inhabitants call themselves "Bedjuis" (Bedouin in local dialect) and regard themselves as descendants of the Sarrazins who invaded the Alps during the VIII-X centuries. Our goal, in studying several DNA-VNTR polymorphims, in addition to some blood groups, within the Isérables community, was to see if there was any evidence supporting this popular belief. As a preliminary phase of this project, the allelic frequencies for six VNTR loci analysed for 102 individuals of the village (all descendants of nine original families) are presented. The results are compared with those reported for Swiss and white populations.

Chemiluminescent DNA probes: evaluation and usefulness in forensic cases.

Five phosphatase-labelled oligonucleotide probes were evaluated in respect to their sensitivity, with the help of an optimized chemiluminescent protocol, for DNA-VNTR polymorphism determination. Their usefulness for the identification of biological traces is illustrated with casework examples.

Simultaneous phenotyping of erythrocyte acid phosphatase and esterase D by nonequilibrium agarose isoelectric focusing.

Nonequilibrium agarose isoelectric focusing in a pH 4.5-7 gradient with 3-(N-morpholinopropanesulfonic acid (MOPS) and taurine as chemical separators presents a fast, easy and reliable method for the simultaneous determination of the common erythrocyte acid phosphatase and esterase D phenotypes in hemolysates and dried bloodstains. The application of the method to experimental and casework bloodstains is described.

A, B and H group specific substances in human vitreous humor.

The presence of A, B and H group specific substances in vitreous humor taken from 105 human corpses was determined. Good agreement was obtained between these group substances and the ABO blood group. The relationship with the secretor type is discussed.

A "new" allele of esterase D in a retinoblastoma family.

We describe a new rare allele for esterase D (EsD) occurring in a Portuguese family with retinoblastoma in two generations.

A plasminogen silent allele detected in a Swiss family.

An apparent incompatibility between mother and child in the plasminogen system has been clarified by the demonstration of a silent allele in the family.

Gm(1,2,4,10,21) and Km(1) factors in vitreous humor.

The presence of Gm(1,2,4,10,21) and Km(1) factors in vitreous humor taken from human corpses was investigated. The results revealed a good agreement between the factors detected in this biological material and in blood. Their presence in vitreous humor is independent of the secretor type.