Biomedical subjects

Walther Parson

Publications and source records attributed to Walther Parson.

At least 37 records · Page 2Linked to original sources

PubMed2005

A collaborative study of the EDNAP group regarding Y-chromosome binary polymorphism analysis.

A collaborative study was carried out by the European DNA Profiling Group (EDNAP) in order to evaluate the performance of Y-chromosome binary polymorphism analysis in different European laboratories. Four blood samples were sent to the laboratories, to be analysed for 11 Y-chromosome single nucleotide polymorphisms (SNPs): SRY-1532, M40, M35, M213, M9, 92R7, M17, P25, M18, M153 and M167. All the labs were also asked to submit a population study including these markers. All participating laboratories reported the same results, indicating the reproducibility and robustness of Y-chromosome SNP typing. A total of 535 samples from six different European populations were also analysed. In Galicia (NW Spain) and Belgium, the most frequent haplogroup was R1b*(xR1b1,R1b3df). Haplogroup F*(xK) is one of the most frequent in Austria and Denmark, while the lowest frequency appear in Belgium. Haplogroup frequencies found in this collaborative study were compared with previously published European Y-chromosome haplogroup data.

Chromosomes, Human, Y↗

PubMed2005

Characterization of mtDNA SNP typing and mixture ratio assessment with simultaneous real-time PCR quantification of both allelic states.

We performed a study on the forensic utility of allele-discriminatory quantitative real-time PCR (rtPCR) using Minor Groove Binder TaqMan probes, targeting the highly variable mitochondrial single nucleotide polymorphism 16519T/C. The apparent single-cycle PCR efficiency was virtually 100% for both 16519 alleles. The allele designations made by rtPCR were concordant with the results obtained in a previous study by sequencing analysis. In heteroplasmic samples, minor allele proportions down to 9% were unambiguously detected and quantified. The variation in allele proportion estimates was essentially the same within and between different rtPCR runs, and the differences between total copy number estimates found for rerun samples were comparable to those found with non-allele-discriminatory quantitative rtPCR assays.

DNA Fingerprinting↗

PubMed2005

Detection of DNA sequence variations in homo- and heterozygous samples via molecular mass measurements by electrospray ionization time-of-flight mass spectrometry.

The potential of ion-pair reversed-phase high-performance liquid chromatography on-line hyphenated to electrospray ionization time-of-flight mass spectrometry for the characterization of polymerase chain reaction (PCR) amplified nucleic acids was evaluated. For that purpose, a "SNP toolbox" was constructed by cloning and PCR-mediated site-directed in vitro mutagenesis at nucleotide position (ntp) 16,519 of a sequence-verified fragment of the human mitochondrial genome (ntps 15,900-599). Confirmatory sequencing demonstrated that within the sequences of the clones one and the same base was mutated to all other bases. Using these clones or equimolar mixtures of these clones as PCR templates, 51-401-bp-long amplicons were generated, which were used to determine the upper size limits of PCR products for the unequivocal detection of sequence variations in homo- and heterozygous samples. Based on the high mass spectrometric performance of the applied time-of-flight mass spectrometer, the unequivocal genotyping of all kinds of single base exchanges in PCR amplicons from heterozygous samples with lengths up to 254 base pairs (bp) was demonstrated. Considering homozygous samples, the successful genotyping of single base substitutions in up to 401-bp-long PCR products was possible. Consequently, the described hyphenated technique represents one of the most powerful mass spectrometric genotyping assays available today.

Alleles↗

PubMed2005

CD25-expressing CD8+ T cells are potent memory cells in old age.

We have recently described an IL-2/IL-4-producing CD8+CD25+ non-regulatory memory T cell population that occurs in a subgroup of healthy elderly persons who characteristically still have a good humoral response after vaccination. The present study addresses this specific T cell subset and investigates its origin, clonal composition, Ag specificity, and replicative history. We demonstrate that CD8+CD25+ memory T cells frequently exhibit a CD4+CD8+ double-positive phenotype. The expression of the CD8 alphabeta molecule and the occurrence of signal-joint TCR rearrangement excision circles suggest a thymic origin of these cells. They also have longer telomeres than their CD8+CD25- memory counterparts, thus indicating a shorter replicative history. CD8+CD25+ memory T cells display a polyclonal TCR repertoire and respond to IL-2 as well as to a panel of different Ags, whereas the CD8+CD25- memory T cell population has a more restricted TCR diversity, responds to fewer Ags, and does not proliferate in response to stimulation with IL-2. Molecular tracking of specific clones with clonotypic primers reveals that the same clones occur in CD8+CD25+ and CD8+CD25- memory T cell populations, demonstrating a lineage relationship between CD25+ and CD25- memory CD8+ T cells. Our results suggest that CD25-expressing memory T cells represent an early stage in the differentiation of CD8+ cells. Accumulation of these cells in elderly persons appears to be a prerequisite of intact immune responsiveness in the absence of naive T cells in old age.

Aged↗

PubMed2005

Estimating the probability of identity in a random dog population using 15 highly polymorphic canine STR markers.

Dog DNA-profiling is becoming an important supplementary technology for the investigation of accident and crime, as dogs are intensely integrated in human social life. We investigated 15 highly polymorphic canine STR markers and two sex-related markers of 131 randomly selected dogs from the area around Innsbruck, Tyrol, Austria, which were co-amplified in three PCR multiplex reactions (ZUBECA6, FH2132, FH2087Ua, ZUBECA4, WILMSTF, PEZ15, PEZ6, FH2611, FH2087Ub, FH2054, PEZ12, PEZ2, FH2010, FH2079 and VWF.X). Linkage testing for our set of marker suggested no evidence for linkage between the loci. Heterozygosity (HET), polymorphism information content (PIC) and the probability of identity (P((ID)theoretical), P((ID)unbiased), P((ID)sib)) were calculated for each marker. The HET((exp))-values of the 15 markers lie between 0.6 (VWF.X) and 0.9 (ZUBECA6), P((ID)sib)-values were found to range between 0.49 (VWF.X) and 0.28 (ZUBECA6). Moreover, the P((ID)sib) was computed for sets of loci by sequentially adding single loci to estimate the information content and the usefulness of the selected marker sets for the identification of dogs. The estimated P((ID)sib) value of all 15 markers amounted to 8.5 x 10(-8). The presented estimations turned out to be a helpful approach for a reasonable choice of markers for the individualisation of dogs.

Animals↗

PubMed2005

Y-STR typing of an Austrian population sample using a 17-loci multiplex PCR assay.

Y-chromosomal STR haplotypes were determined from a sample of 135 unrelated men and 70 sons from Tirol (Austria) using the AmpFlSTR Yfiler PCR amplification kit (Applied Biosystems) that coamplifies 17 Y-STRs. The panel of markers includes the 9-loci European minimal haplotype (minHt) and, in addition, the markers DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 (Y GATA C4) and Y GATA H4. A total of 130 different haplotypes (125 were unique) were identified by the 17 Y-STR markers, an increase of 19 compared with the minHt. The gene diversity of DYS635, DYS456 and DYS458 exceeded 0.75 and only that of the duplicated marker DYS385 (0.86) was higher. Consistently high haplotype diversity values were found in all tested Y-SNP haplogroups. Because the simultaneous analysis of 17 Y-STR systems offers a high power of discrimination at minimum sample consumption, the Yfiler kit is a promising tool for forensic applications.

Austria↗

PubMed2005

Recommendations for animal DNA forensic and identity testing.

Genetic analysis in animals has been used for many applications, such as kinship analysis, for determining the sire of an offspring when a female has been exposed to multiple males, determining parentage when an animal switches offspring with another dam, extended lineage reconstruction, estimating inbreeding, identification in breed registries, and speciation. It now also is being used increasingly to characterize animal materials in forensic cases. As such, it is important to operate under a set of minimum guidelines that assures that all service providers have a template to follow for quality practices. None have been delineated for animal genetic identity testing. Based on the model for human DNA forensic analyses, a basic discussion of the issues and guidelines is provided for animal testing to include analytical practices, data evaluation, nomenclature, allele designation, statistics, validation, proficiency testing, lineage markers, casework files, and reporting. These should provide a basis for professional societies and/or working groups to establish more formalized recommendations.

Animals↗

PubMed2005

Cancer cell line identification by short tandem repeat profiling: power and limitations.

Cancer cell lines are used worldwide in biological research, and data interpretation depends on unambiguous attribution of the respective cell line to its original source. Short-tandem-repeat (STR) profiling (DNA fingerprinting) is the method of choice for this purpose; however, the genetic stability of cell lines under various experimental conditions is not well defined. We tested the effect of long-term culture, subcloning, and generation of drug-resistant subclones on fingerprinting profiles in four widely used leukemia cell lines. The DNA fingerprinting profile remained unaltered in two of them (U937 and K562) throughout 12 months in culture, and the vast majority of subclones derived therefrom by limiting dilution after long-term culture revealed the same profile, indicating a high degree of stability and clonotypic homogeneity. In contrast, two other cell lines (CCRF-CEM and Jurkat) showed marked alterations in DNA fingerprinting profiles during long-term culture. Limiting dilution subcloning revealed extensive clonotypic heterogeneity with subclones differing in up to eight STR loci from the parental culture. Similar heterogeneity was observed in subclones generated by selection culture for drug resistance where DNA fingerprinting proved useful in identifying possible resistance mechanisms. Thus, common tissue culture procedures may dramatically affect the fingerprinting profile of certain cell lines and thus render definition of their origin difficult.

Apoptosis↗

PubMed2005

Phantom mutation hotspots in human mitochondrial DNA.

Phantom mutations are systematic artifacts generated in the course of the sequencing process. Contra common belief these artificial mutations are nearly ubiquitous in sequencing results, albeit at frequencies that may vary dramatically. The amount of artifacts depends not only on the sort of automated sequencer and sequencing chemistry employed, but also on other lab-specific factors. An experimental study executed on four samples under various combinations of sequencing conditions revealed a number of phantom mutations occurring at the same sites of mitochondrial DNA (mtDNA) repeatedly. To confirm these and identify further hotspots for artifacts, > 5000 mtDNA electropherograms were screened for artificial patterns. Further, > 30 000 published hypervariable segment I sequences were compared at potential hotspots for phantom mutations, especially for variation at positions 16085 and 16197. Resequencing of several samples confirmed the artificial nature of these and other polymorphisms in the original publications. Single-strand sequencing, as typically executed in medical and anthropological studies, is thus highly vulnerable to this kind of artifacts. In particular, phantom mutation hotspots could easily lead to misidentification of somatic mutations and to misinterpretations in all kinds of clinical mtDNA studies.

Artifacts↗

PubMed2005

Characterization of synthetic nucleic acids by electrospray ionization quadrupole time-of-flight mass spectrometry.

The potential of electrospray ionization quadrupole-quadrupole-time-of-flight mass spectrometry (ESI-QqTOF-MS) for the characterization of synthetic nucleic acids was evaluated. Oligonucleotides ranging in size from 12 up to 51 nucleotides were analyzed via direct infusion MS as well as via liquid chromatography (LC) online hyphenated to MS. These experiments proved the outstanding mass spectrometric performance of the TOF mass analyzer in regard of accuracy, reproducibility, resolution, and sensitivity. During a 1-min run, the monoisotopic mass of (dT)(24) was measured with a maximum relative mass deviation of 7.64 ppm proving the high mass accuracy of the TOF analyzer. Over a period of 1 h, mean deviations were determined in the range between -3.58 ppm and 3.06 ppm demonstrating the high stability of the applied external calibration. The molecular mass of a 51-mer was measured with a deviation smaller than 3.23 ppm from the theoretical value. The resolution exceeded a value of m/Deltam = 20 000 (m is the measured mass and Deltam the full peak width at half-maximum), which enabled the separation of the isotopic peaks of all investigated oligonucleotides. Because of the outstanding transmission and detection efficiency of the TOF mass analyzer, detection limits in the amol/microl to low fmol/microl range were reached. The usability of LC-ESI-QqTOF-MS for the qualitative and quantitative analysis of synthetic oligonucleotide mixtures was demonstrated.

Acetonitriles↗

PubMed2005

DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR.

Forensic laboratories are increasingly confronted with problematic samples from the scene of crime, containing only minute amounts of deoxyribonucleic acid (DNA), which may include polymerase chain reaction (PCR)-inhibiting substances. Efficient DNA extraction procedures, as well as accurate DNA quantification methods, are critical steps involved in the process of successful DNA analysis of such samples. The phenol-chloroform method is a sensitive method for the extraction of DNA from a wide variety of forensic samples, although it is known to be laborious compared with single-tube extraction methods. The relatively high DNA recovery and the quality of the extracted DNA speak for itself. For reliable and sensitive DNA quantitation, the application of real-time PCR is described. We modified a published real-time PCR assay, which allows for the combined analysis of nuclear and mitochondrial DNA, by introducing 1) improved hybridization probes with the use of minor groove binders; 2) an internal positive control (for both nuclear and mitochondrial DNA) for the detection of PCR inhibitors; and 3) different amplicon lengths for the determination of the degradation state of the DNA. The internal positive controls were constructed by site directed mutagenesis by overlap extension of the wild-type mitochondrial and nuclear DNA target with the advantage that no additional probes, which are cost-intensive, are required. The quantitation system is accomplished as a modular concept, which allows for the combined determination of the above-mentioned features (quantity/inhibition or quantity/degradation) depending on the situation.

Base Sequence↗

PubMed2004

Canine-specific STR typing of saliva traces on dog bite wounds.

Forensic investigations in dog attacks usually involve the examination of bite marks and toothprints, the dog's stomach and pathological methods. For identification of the offending dog we evaluated canine STR typing of saliva traces on dog bite marks. The specificity of 15 canine-specific STRs was tested on human-canine DNA mixtures prior to an applied study in which 52 cases of dog bites were investigated. The first-aid wound bandages as well as swab samples from the surrounding area of the wound were used for DNA analyses. Generally, it was possible to obtain a canine-specific STR profile from the dog's saliva left on the wound area, even when high background of human DNA was present (blood). Interestingly, we found canine STR typing to be more successful when the bandages and swabs showed high amounts of human blood, i.e. when the dog bite was severe. Canine saliva was then sometimes visible as white-coloured secretion on the human blood surface. Less severe bite cases, which did not result in bleeding wounds, showed less success in obtaining useful STR results, probably due to the fact that the surface of the wounds may have been treated before the victims consulted medical aid which therefore removed the canine cells.

Adolescent↗

PubMed2004

Separate analysis of DYS385a and b versus conventional DYS385 typing: is there forensic relevance?

In order to determine to what extent the separate analysis of both copies of DYS385 improves Y-chromosomal short tandem repeat (Y-STR) haplotyping, we followed a recently published protocol for the separate amplification of DYS385a and DYS385b with modifications and compared the results with those obtained by conventional analysis in a population sample comprising 133 unrelated Caucasian males from Austria. Additionally, we typed all markers of the minimal haplotype (minHT) and a set of Y-chromosomal single nucleotide polymorphisms (Y-SNPs) in order to interpret the STR data depending on the Y-SNP haplogroup structure. The separate amplification of DYS385a and b improved the power of discrimination of this marker when compared to the results obtained with the conventional non-locus-discriminating amplification strategy. However, the degree of this improvement varied greatly between different haplogroups and was found to be highest in clade K. In the forensically relevant context of the minHT, the separate analysis of the DYS385 alleles had no effect on the differentiation of paternal lineages in our study. Furthermore, the amplicon lengths of 700-780 base pairs obtained in the course of the locus-discriminating approach restrict the applicability of this amplification strategy to high quality DNA samples.

Austria↗

PubMed2004

Duplications of the Y-chromosome specific loci P25 and 92R7 and forensic implications.

In the present study, we demonstrate that two commonly used Y-chromosome single nucleotide polymorphisms (SNPs), P25 and 92R7, are paralogous sequence variants (PSVs) originating from segmental duplications and that at least one of the sequence variants in each group of loci is polymorphic. Several methodologies were used in order to detect the SNP alleles and the PSVs of the loci. All results obtained with the various typing techniques supported the conclusion. The allele distributions of the binary markers were analysed in more than 600 males with seven different haplogroups. For P25, the ancestral allele C was found in several samples from different haplogroups. The derived allele A was always present with an additional C variant. Haplogroup P was defined by the derived allele A at the 92R7 locus. However, the ancestral allele G was always associated with an A variant due to the duplication.

Chromosomes, Human, Y↗

PubMed2004

STR analysis of artificially degraded DNA-results of a collaborative European exercise.

Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit different properties in this respect, and that it is difficult to systematically assess the performance of routinely used typing systems for the analysis of degraded DNA samples. Using a batch of artificially degraded DNA with an average fragment size of approx. 200 bp a collaborative exercise was carried out among 38 forensic laboratories from 17 European countries. The results were assessed according to correct allele detection, peak height and balance as well as the occurrence of artefacts. A number of common problems were identified based on these results such as strong peak imbalance in heterozygous genotypes for the larger short tandem repeat (STR) fragments after increased PCR cycle numbers, artefact signals and allelic drop-out. Based on the observations, strategies are discussed to overcome these problems. The strategies include careful balancing of the amount of template DNA and the PCR cycle numbers, the reaction volume and the amount of Taq polymerase. Furthermore, a careful evaluation of the results of the fragment analysis and of automated allele calling is necessary to identify the correct alleles and avoid artefacts.

Alleles↗

PubMed2004

The EDNAP mitochondrial DNA population database (EMPOP) collaborative exercises: organisation, results and perspectives.

This paper presents an overview of the organisation and the results of the collaborative exercises (CE) of the European DNA Profiling (EDNAP) Group's mitochondrial DNA population database project (EMPOP). The aim of the collaborative exercises was to determine whether uniformity of mtDNA sequencing results could be achieved among different laboratories. These were asked to sequence either the complete mtDNA control region or the two hypervariable regions HVI (16024-16365) and HVII (73-340) from DNA extracts, buccal swabs or bloodstains, proceeding in accordance with the protocol and strategies used in each individual laboratory. The results of the collaborative exercises were employed to identify possible sources of errors that could arise during the analysis and interpretation of mtDNA profiles. These findings were taken as a basis to tentatively make suitable arrangements for the construction of a high quality mtDNA database. One hundred fifty mtDNA profiles were submitted to the evaluating laboratory, and disaccording profiles were classified into four groups corresponding to the source of error: clerical errors, sample mix-ups, contaminations and discrepancies with respect to the mtDNA nomenclature. Overall, 14 disaccording haplotypes (16 individual errors) were observed. The errors included 10 clerical errors, 3 interpretation problems, 2 cases of sample mix-up and 1 case of point heteroplasmic mixture, where the 2 sequencing reactions brought inconsistent base calls. This corresponds to an error rate of 10.7% in a virtual mtDNA database consisting of the collaborative exercise results. However, this estimate is still conservative compared to conclusions drawn by authors of meanwhile numerous publications critically reviewing published mtDNA population databases. Our results and earlier published concerns strongly emphasize the need for appropriate safety regulations when mtDNA profiles are compiled for database purposes in order to accomplish the high standard required for mtDNA databases that are used in the forensic context.

Clinical Laboratory Techniques↗

PubMed2004

Monitoring the inheritance of heteroplasmy by computer-assisted detection of mixed basecalls in the entire human mitochondrial DNA control region.

The entire mitochondrial DNA control region (approximately 1122 bp) of 270 blood samples (135 mother-child pairs) was determined by direct sequencing. Overall, 135 'generational events' were screened and within these, 20 mother-offspring pairs demonstrated more than 1 mtDNA haplotype. In 13 families, differences in the haplotypes between mother and offspring were detected in the form of heteroplasmic substitutions. Intergenerational comparisons led to the identification of three heteroplasmic point mutations and eight heteroplasmic length mutations affecting the children only. In two cases, a point heteroplasmy of the maternal sequence was resolved to homoplasmy in the corresponding sequence of the child. These discordant maternal-offspring haplotypes suggest that the shift in the mtDNA haplotype was the result of segregation of a limited maternal subpopulation of mtDNA. As technical implement, quality values assigned to basecalls were tested for their application in automated point heteroplasmy detection.

DNA, Mitochondrial↗

PubMed2004

A common nonsense mutation in the repetitive Kringle IV-2 domain of human apolipoprotein(a) results in a truncated protein and low plasma Lp(a).

LPA, the gene coding for apolipoprotein(a) [apo(a)], is the major determinant of lipoprotein(a) [Lp(a)] plasma levels, which are associated with risk for coronary heart disease (CHD) and stroke. It is not completely understood how variation in LPA relates to Lp(a) concentrations. One type of variation related to Lp(a) levels is the number of Kringle (K) IV-2 (g.61C>T; GenBank L14005.1) repeats in LPA, but sequence variation may also contribute. Human apo(a) contains from two to >40 nearly identical K IV-2 repeats of genomic size 5.5 kb, which makes it difficult to detect mutations. To elucidate the genetic variation of the apo(a) K IV-2 domain, we isolated a single "nonexpressing" apo(a) allele with 26 K IV-2 repeats, followed by PCR, cloning and sequencing of 96 clones, resulting in an average coverage of each K IV-2 repeat of approximately four-fold. The previously described K IV types 2A and 2B (K IV-2A and K IV-2B) were detected in 74% of the clones. In addition, a new type designated 2C (K IV-2C) was present. A nonsense mutation in the first exon of K IV-2 (g.61C>T) predicted to result in a truncated protein (p.R21X) was found in nine clones on a K IV-2A background. The presence of this mutation was confirmed by analysis of genomic DNA and was shown to represent the rare allele (frequency 0.02) of a SNP. Immunoblot analysis of apo(a) from plasma confirmed the presence of a truncated apo(a) isoform in the index individual and family members. Our data show that SNPs affecting Lp(a) plasma concentrations also exist in the apo(a) K IV-2 domain.

Apolipoproteins A↗