Biomedical subjects

Harald Niederstätter

Publications and source records attributed to Harald Niederstätter.

At least 19 recordsLinked to original sources

PubMed2006

Profiling 627 mitochondrial nucleotides via the analysis of a 23-plex polymerase chain reaction by liquid chromatography-electrospray ionization time-of-flight mass spectrometry.

We present a rapid and informative mitochondrial DNA profiling system, which has high forensic impact. The assay is based on the analysis of a 23-plex PCR by ion-pair reversed-phase high-performance liquid chromatography online hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS). In a single 25-min run, an overall number of 627 nucleotide positions were screened. The vast majority of observed sequence variations were explainable by alterations of the allelic states of the 23 target SNPs, which were selected on their ability to increase forensic discrimination within West Eurasian populations. Within an Austrian population sample comprising 90 unrelated men, 14 different, nontarget SNP-related sequence variations--13 base substitutions and 1 deletion--were detected by ICEMS and confirmed by sequencing. All amplified sequences were located outside of the routinely sequenced hypervariable segments (HVS-I and HVS-II) of the noncoding control region. Accordingly, the genetic information obtained by the 23-plex PCR-ICEMS assay could be combined with HVS-I/HVS-II sequencing results to one highly discriminating mtDNA profile, which covered approximately 7.5% of the total mtDNA genome. With the 23-plex PCR-ICEMS assay, DNA mixtures were detected and the allelic ratios were accurately quantified. The observed robustness and sensitivity underlined the practical applicability of the assay in forensic science, which was proven by typing eight representative casework samples.

Alleles↗

PubMed2006

Liquid chromatography-electrospray ionization mass spectrometry for simultaneous detection of mtDNA length and nucleotide polymorphisms.

We demonstrate the applicability of ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry (ICEMS) for the simultaneous characterization of length and nucleotide polymorphisms. Two sections within the first (HVS-I) and second (HVS-II) hypervariable segments of the mitochondrial (mt)DNA control region were selected as targets, both containing poly-cytosine (C) tracts, which display length heteroplasmy at a substantial frequency in the population. The two mtDNA sections were simultaneously amplified and analyzed by ICEMS in 90 maternally unrelated mother-offspring pairs from Austria. The findings were confirmed by direct sequencing of the polymerase chain reaction products. For the detailed characterization of present-length heteroplasmic variants, the results retrieved through ICEMS were more informative compared with those derived from direct sequencing. Hence, ICEMS represents an interesting option for successful application in forensic science.

Austria↗

PubMed2006

Generating population data for the EMPOP database - an overview of the mtDNA sequencing and data evaluation processes considering 273 Austrian control region sequences as example.

The European DNA profiling group (EDNAP) mtDNA population database (EMPOP) is an international collaborative project between DNA laboratories performing mtDNA analysis and the DNA laboratory of the Institute of Legal Medicine (GMI) in Innsbruck, Austria. The goal is to set up a directly accessible mtDNA population database, which can be used in routine forensic casework for frequency investigations. Here we describe a safe laboratory scheme involving electronical data handling and computer-aided data transfer, which help to minimize errors originating from potential sample mix-up, data misinterpretation and incorrect transcription. The procedure is demonstrated by example of an mtDNA control region population study on 273 unrelated individuals from Austria. Our population sample was compared with five other European populations via an analysis of molecular variance (AMOVA). The inclusion of regions outside HVS-I and HVS-II increased the amount of information on the haplogroup diagnostic sites in the control region. Most of the haplotypes in Austrians fell into haplogroups H, J, K, T, and U. The random match probability in Austrians was 1:125; the average number of nucleotide differences between individuals in the Austrian database was 9.32.

Austria↗

PubMed2006

Direct molecular haplotyping of multiple polymorphisms within exon 4 of the human catechol-O-methyltransferase gene by liquid chromatography-electrospray ionization time-of-flight mass spectrometry.

The applicability of ion-pair reversed-phase high-performance liquid chromatography hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS) for the haplotyping of five SNPs (rs769223, rs4818, rs4986871, rs8192488, rs4680) located within exon 4 of the human catechol-O-methyltransferase (COMT, EC 2.1.1.6) gene is demonstrated. Two differently sized products of polymerase chain reaction--a 71-bp amplicon partially covering the sequence of a 124-bp amplicon--were used to determine unequivocally the allelic states of the single nucleotide polymorphisms linked on both chromosomes. The two amplicons were co-loaded onto the chromatographic column and simultaneously analyzed within a single gradient run. Using the described strategy, 101 individuals representing an Austrian population sample were typed. The obtained haplotype frequencies will serve as reference values in future association studies to examine the impact of the COMT gene on neuropsychiatric disorders. Additionally, two newly discovered polymorphic sites within the sequence of the COMT gene are described (a synonymous C>T mutation at the third position of the amino acid codon 99 in the soluble COMT protein or 149 in the membrane-bound COMT protein; a non-synonymous G>A substitution at the second position of the amino acid codon 95 in the soluble COMT protein or 145 in the membrane-bound-COMT protein).

Base Sequence↗

PubMed2006

Unravelling the mystery of Nanga Parbat.

Since the disappearance of Günther Messner, brother of the world-famous mountaineer Reinhold Messner, on an expedition to Nanga Parbat in 1970, the circumstances of his death have given rise to controversy. Reinhold Messner claimed that he and Günther descended the Diamir face together when an avalanche killed his brother, while other expedition members argued that Günther was abandoned by Reinhold to descend the Rupal face. Now, 35 years after the event, Günther's remains have been found at the Diamir face and have been identified by forensic DNA fingerprinting. The location of the remains supports Messner's version, thus putting to rest one of the climbing communities' most publicised controversies.

Amelogenin↗

PubMed2006

Colour-assortative mating among populations of Tropheus moorii, a cichlid fish from Lake Tanganyika, East Africa.

The species flocks of cichlid fishes in the East African Lakes Tanganyika, Malawi and Victoria are prime examples of adaptive radiation and explosive speciation. Several hundreds of endemic species have evolved in each of the lakes over the past several thousands to a few millions years. Sexual selection via colour-assortative mating has often been proposed as a probable causal factor for initiating and maintaining reproductive isolation. Here, we report the consequences of human-mediated admixis among differentially coloured populations of the endemic cichlid fish Tropheus moorii from several localities that have accidentally been put in sympatry in a small harbour bay in the very south of Lake Tanganyika. We analysed the phenotypes (coloration) and genotypes (mitochondrial control region and five microsatellite loci) of almost 500 individuals, sampled over 3 consecutive years. Maximum-likelihood-based parenthood analyses and Bayesian inference of population structure revealed that significantly more juveniles are the product of within-colour-morph matings than could be expected under the assumption of random mating. Our results clearly indicate a marked degree of assortative mating with respect to the different colour morphs. Therefore, we postulate that sexual selection based on social interactions and female mate choice has played an important role in the formation and maintenance of the different colour morphs in Tropheus, and is probably common in other maternally mouthbrooding cichlids as well.

Alleles↗

PubMed2006

Accurate determination of allelic frequencies in mitochondrial DNA mixtures by electrospray ionization time-of-flight mass spectrometry.

The mitochondrial locus 16519T/C was used as a model for the evaluation of the benefits of ion-pair reversed-phase high-performance liquid chromatography on-line hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS assay) for the determination of allelic frequencies of single nucleotide polymorphisms. This marker has gained interest in forensic science owing to its ability to increase the discrimination power of mitochondrial DNA testing as a consequence of its high variability across various populations. In a first set of experiments, artificial mitochondrial DNA mixtures prepared from all four theoretically possible 16519 alleles served as samples. Any allele occurring at a frequency of as low as 1-5% was unequivocally detectable irrespective of the kind of allelic mixture. Measured and expected allelic frequencies correlated well following correction of observed experimental bias, which was most probably attributable to differential PCR amplification and/or preferential ionization. For thirteen different T/C mixtures with C contents in the range 1.0-99.0%, an average error of 1.2% and a maximum error of 2.2% were observed. Furthermore, ICEMS was applied to the quantitative genotyping of eight selected individuals of which four were heteroplasmic with C contents in the range 1.9-34.1%. To check the reliability of these results, allelic proportions were additionally determined by a cloning assay. The results of the two assays correlated well (R (2)=0.9971). In all cases, deviations were obtained that were smaller than 5.4%. The overall observed assay performance suggests that the described mass spectrometric technique represents one of the most powerful assays for the determination of allelic frequencies available today.

DNA, Mitochondrial↗

PubMed2006

Some guidelines for the analysis of genomic DNA by PCR-LC-ESI-MS.

Ion-pair reversed-phase high-performance liquid chromatography online hyphenated to electrospray ionization mass spectrometry (ICEMS) represents an efficient method for the characterization of nucleic acids amplified by polymerase chain reaction (PCR). Since sample preparation is limited to PCR, the optimization of its solution conditions is of utmost importance for efficient mass spectrometric detection. The compatibility of a number of different commercially available PCR components including DNA polymerases, deoxynucleotide triphosphates, bovine serum albumin, enhancer, and ionic buffers was evaluated. These experiments revealed that higher concentration of enhancer and detergents such as Tween-20 or Nonidet P-40 impairs the mass spectrometric detection of nucleic acids and should be avoided within the PCR mixture. The optimized analytical platform was applied to the characterization of PCR products covering parts of the first hypervariable region of the noncoding mitochondrial control region. Truncated amplicons were detected attributable to the use of low quality primers. Furthermore, due to the proofreading activity of the applied polymerase system, mismatches between the primer and the target sequence located at the last or the second last base at the 3'-end of primers were corrected and detected within the corresponding amplicons.

Chromatography, High Pressure Liquid↗

PubMed2006

Dissection of mitochondrial superhaplogroup H using coding region SNPs.

Haplogroup H (hg H) includes about 40-50% of the West Eurasian mitochondrial DNA (mtDNA) samples investigated so far. In order to enhance discrimination within this haplogroup we selected 45 coding region SNPs that allow to ascribe samples to the main phylogenetic branches of super hg HV (that embraces hg H) and, in particular, to H sublineages with a much finer resolution than previous studies. SNP selection was carried out using the most up-to-date available literature on population and forensic genetics and extended by means of phylogenetic analysis of complete or coding region genomes (<430) and control region sequences. A meticulous inspection of the H phylogeny led us to the observation of various but uncharacterized subclades of hg H. The selected SNPs were amplified in two PCR-multiplex reactions and subsequently targeted in three single-base extension multiplex reactions. A total of 2214 West Eurasian samples were screened for hg H specific loci 2706 and 7028, of which 859 fell in hg H and were further subjected to subhaplogroup typing. We observed 35 different subhaplogroups in total, 33 of which were found at frequencies below 5%. This assay can be used as a prescreening tool in forensic casework for rapid discrimination between divergent lineages (very effective for high-volume crime cases) or as discriminatory assay, when identical hg H haplotypes were obtained by control region sequencing.

Adolescent↗

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

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

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

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

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↗