Anita Brandstätter

Rapid screening of mtDNA coding region SNPs for the identification of west European Caucasian haplogroups

This work presents a selection of 16 SNPs from the coding region of the human mitochondrial DNA. The selected markers are used for the assignment of individuals to one of the nine major European Caucasian mitochondrial haplogroups. The selected SNPs are targeted in two multiplex systems, via the application of the SNaPshot kit, a multiplex method based on the dideoxy single-base extension of unlabeled oligonucleotide primers. The method is conceived as a rapid screening technique prior to sequencing analysis, in order to eliminate multiple suspects from an inquiry or to discriminate between stains in a high volume casework example. Moreover, the ability to assign an unknown sample to an mtDNA type of known Caucasian origin could be of probative value in some investigations. A database of 277 Austrian Caucasians has been constructed, and the probability of a chance match between two unrelated individuals is calculated as 11.4%. This novel multiplex PCR amplification and typing system for mtDNA coding region SNPs promises to be a convenient and informative new DNA profiling system in the forensic field.

Mitochondrial DNA control region sequences from Nairobi (Kenya): inferring phylogenetic parameters for the establishment of a forensic database

Large forensic mtDNA databases which adhere to strict guidelines for generation and maintenance, are not available for many populations outside of the United States and western Europe. We have established a high quality mtDNA control region sequence database for urban Nairobi as both a reference database for forensic investigations, and as a tool to examine the genetic variation of Kenyan sequences in the context of known African variation. The Nairobi sequences exhibited high variation and a low random match probability, indicating utility for forensic testing. Haplogroup identification and frequencies were compared with those reported from other published studies on African, or African-origin populations from Mozambique, Sierra Leone, and the United States, and suggest significant differences in the mtDNA compositions of the various populations. The quality of the sequence data in our study was investigated and supported using phylogenetic measures. Our data demonstrate the diversity and distinctiveness of African populations, and underline the importance of establishing additional forensic mtDNA databases of indigenous African populations.

Phylogeography of the vairone ( Leuciscus souffia , Risso 1826) in Central Europe

The vairone Leuciscus souffia is a cyprinid fish that inhabits river systems in and around the Alps. The complete mitochondrial DNA control region (945 bp) was sequenced in 295 vairone from 22 populations in Central Europe. A total of 51 haplotypes were identified with a maximum pairwise distance between haplotypes of 5.6%. Phylogenetic analyses revealed two major clades in L. souffia, an ‘Italian’ clade, and an ‘Alpine’ clade. Two hybrid zones exist, in the Mediterranean Alps and in the Soca basin. The position of the sister species of L. souffia, L. turskyi, to the ‘Alpine’ and the ‘Italian’ clade could not be resolved unambiguously. However, a linearized tree analysis indicated that L. turskyi represents a third lineage, that originated at the same time as the ‘Alpine’ and the ‘Italian’ clades of L. souffia. In the ‘Alpine’ clade two groups were resolved, a subclade with haplotypes from the Rhône and Var basins and a cluster with haplotypes from the Danube and Rhine systems. Our data suggest a long history of the vairone in Central Europe, predating Pleistocene glacial cycles. Two main refugia during glaciations must have existed, one in Italy and another one most probably in the Danube system. However, age estimates based on molecular clock calibrations suggest the survival of ‘Alpine’ haplotypes in several drainages during the last glaciation cycles. The Rhine system was only recently colonized.

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 (~1122xa0bp) 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.

Mitochondrial DNA heteroplasmy or artefacts--a matter of the amplification strategy?

We compared two different PCR strategies for the amplification of mtDNA hypervariable region 1 (HV1) with regard to the detection and interpretation of point mutation heteroplasmy in human hair roots. We monitored the level of detected heteroplasmy using direct sequence analysis. PCR amplifications were performed in duplicate on each hair root, using 62 cycles of nested PCR versus 35 cycles of direct PCR. As a previous publication reported different sensitivities of heteroplasmy detection based on the number of PCR cycles used, we were interested in whether and how different PCR amplification strategies would impact sequence quality and the detection of point heteroplasmy. We identified 12 out of 93 hair roots as heteroplasmic (7 out of 31 persons) with direct PCR, whereas 2 of these heteroplasmic events could not be identified with the nested PCR approach. Generally, the quality of the sequence electropherograms in terms of background noise was significantly lower for the nested PCR amplification strategy, leading to ambiguous results in some of the nucleotide positions. Thus, the ability to clearly distinguish a genuine mixture of two nucleotides from background noise at a heteroplasmic position was substantially greater with direct PCR amplification, which generally resulted in higher quality sequence electopherograms.

Improved specificity of Y-STR typing in DNA mixture samples.

Abstract. Y-STR loci are beneficial for the analysis of forensic samples especially in sexual assault cases or other situations where mixtures of male and female cells are present. However, the amplification of Y-chromosomal STRs is also known to result in the formation of artefactual amplification products, mainly due to insufficient PCR specificity. This is a major drawback of the method, as the sensitivity as well as the correct Y-STR interpretation are affected. In our study, the addition of a PCR enhancer to the reaction master-mix resulted in a significant increase of specificity of Y-STR typing. This was clearly demonstrated by a loss of artefactual signal with increasing enhancer concentration, while the peak heights of the Y-STR alleles were not significantly affected by the enhancer. Mixtures of up to 1:500 (200xa0pg male and 100xa0ng female DNA) gave correct Y-STR profiles when the PCR enhancer was added to the reaction, while artefactual amplification succeeded over Y-specific amplification when no PCR enhancer was present.

EMPOP—the EDNAP mtDNA population database concept for a new generation, high-quality mtDNA database

Abstract 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. Most forensic laboratories do not have the capacity to rapidly generate massive amounts of sequence data. Altogether, however, they dispose of a respectable body of data. Apart from a mutual, non-competitive exchange of molecular genetic techniques, every forensic institute can contribute to the EMPOP project with its familiarity with the individual technology and with the polymorphisms typical for the population inhabiting its surroundings. The EMPOP core laboratory in Innsbruck provides the bioinformatic infrastructure with which to analyze, transcribe, store, and share the data.

Evaluation of the forensic usefulness of the separate analysis of DYS385a and DYS385b in an Austrian population sample

Abstract The duplicated Y-STR DYS385 is one of the most informative markers on the Y-chromosome. It consists of two copies—DYS385a and DYS385b—that are located close to the inner borders of palindrome P4. The application of standard PCR protocols for DYS385 results in the simultaneous amplification of both copies. Hence, an explicit assignment of the alleles to their loci is not possible which causes a loss of information. Recently, a PCR strategy was published that allows the separate analysis of DYS385a and b, which we followed with modifications in our study on 133 unrelated Caucasian men from Tirol (Austria). Additionally all markers of the minimal haplotype (including DYS385) were amplified using standard PCR protocols. The non-allele-discriminating amplification strategy for DYS385 allowed the discrimination of 27 different genotypes (GD=0.8616). By the separate analysis of the two DYS385 copies, this number increased to 34 (GD=0.9185). However, the cumulative haplotype diversity of the complete minimal haplotype was 0.9951, regardless of the amplification strategy for DYS385. This indicates that in the context of the minimal haplotype, the separate analysis of DYS385a and b has no or only a marginally positive effect on the differentiation of paternal lineages.

MtDNA coding region SNPs for rapid screening and haplogroup identification of forensic samples

Abstract Analysis of single nucleotide polymorphisms (SNPs) is a promising application in forensic human identification. We selected 16 SNPs from the coding region of the human mitochondrial DNA in order to ascribe samples to 1 of the 9 major West European Caucasian mitochondrial haplogroups. The selected SNPs are targeted in two multiplex-systems, via the application of the SNaPshot™ kit, a multiplex method based on the dideoxy single-base extension of unlabeled oligonucleotide primers. By screening these SNPs prior to sequencing analysis of the hypervariable regions in the control region, we would be able to rapidly differentiate between stains or hairs in high volume case work or to eliminate multiple suspects from an inquiry.

The highly discriminating Y-STR DYS464: a reasonable extension of the minimal Y-STR haplotype?

Abstract DYS464 is a multi-copy STR system with four positions on the Y chromosome (DYS464a, b, c, and d) which was recently identified and characterized. The aims of our study were to perform a population study, to estimate the mutation-rate and an extensive sequence analysis in order to confirm the nomenclature. Fourteen different alleles were found with allele lengths varying from 9 to 19 repeats. All alleles were cloned and sequenced. A total of 54 different genotypes were identified in 135 men corresponding to a gene diversity (GD) of 0.97. This value is much higher than those of other Y-STRs. DYS464 has the same discrimination capacity as the combination of the five Y-STR loci with the lowest gene diversity of the Y-STR core set. The mutation-rate estimate based on the 70 meioses analyzed amounts to 2.86×10 −2 .