Cornelia Schmitt

Evaluation of Y-chromosomal STRs: a multicenter study

Abstract A multicenter study has been carried out to characterize 13 polymorphic short tandem repeat (STR) systems located on the male specific part of the human Y chromosome (DYS19, DYS288, DYS385, DYS388, DYS389I/II, DYS390, DYS391, DYS392, DYS393, YCAI, YCAII, YCAIII, DXYS156Y). Amplification parameters and electrophoresis protocols including multiplex approaches were compiled. The typing of non-recombining Y loci with uniparental inheritance requires special attention to population substructuring due to prevalent male lineages. To assess the extent of these subheterogeneities up to 3825 unrelated males were typed in up to 48 population samples for the respective loci. A consistent repeat based nomenclature for most of the loci has been introduced. Moreover we have estimated the average mutation rate for DYS19 in 626 confirmed father-son pairs as 3.2 × 10–3 (95% confidence interval limits of 0.00041–0.00677), a value which can also be expected for other Y-STR loci with similar repeat structure. Recommendations are given for the forensic application of a basic set of 7 STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393) for standard Y-haplotyping in forensic and paternity casework. We recommend further the inclusion of the highly polymorphic bilocal Y-STRs DYS385, YCAII, YCAIII for a nearly complete individualisation of almost any given unrelated male individual. Together, these results suggest that Y-STR loci are useful markers to identify males and male lineages in forensic practice.

Chromosome Y microsatellites : population genetic and evolutionary aspects

Abstract By means of a multicenter study, a large number of males have been characterized for Y-chromosome specific short tandem repeats (STRs) or microsatellites. A complete summary of the allele frequency distributions for these Y-STRs is presented in the Appendix. This manuscript describes in more detail some of the population genetic and evolutionary aspects for a restricted set of seven chromosome Y STRs in a selected number of population samples. For all the chromosome Y STRs markedly different region-specific allele frequency distributions were observed, also when closely related populations were compared. Haplotype analyses using AMOVA showed that when four different European male groups (Germans, Dutch, Swiss, Italians) were compared, less than 10% of the total genetic variability was due to differences between these populations. Nevertheless, these pairwise comparisons revealed significant differences between most population pairs. Assuming a step-wise mutation model and a mutation frequency of 0.21%, it was estimated that chromosome Y STR-based evolutionary lines of descent can be reliably inferred over a time-span of only 1950 generations (or about 49000 years). This reduces the reliability of the inference of population affinities to a historical, rather than evolutionary time scale. This is best illustrated by the construction of a human evolutionary tree based on chromosome Y STRs in which most of the branches connect in a markedly different way compared with trees based on classical protein polymorphisms and/or mtDNA sequence variation. Thus, the chromosome Y STRs seem to be very useful in comparing closely related populations which cannot probably be separated by e.g. autosomal STRs. However, in order to be used in an evolutionary context they need to be combined with more stable Y-polymorphisms e.g. base-substitutions.

Asian online Y-STR Haplotype Reference Database

For several years Y-chromosomal microsatellites (short tandem repeats, STRs) have been well established in forensic practice. In this context, the genetic characteristics of the Y chromosome (i.e. its paternal inheritance and lack of recombination) render STRs particularly powerful. However, genetic differences between male populations appear to be larger for Y-STRs than for autosomal STRs, a fact that is most likely due to the higher sensitivity of Y-chromosomal lineages to genetic drift (Forensic Sci Int 118 (2001) 153). The assessment of probabilities for matches between haplotyped male persons or traces/persons requires the typing of a large number of haplotypes in the appropriate reference populations. The haplotype data of a large number of European as well as South and North American populations have been collected and are continuously published online (Y-STR Haplotype Reference Database--YHRD; http://www.ystr.org). The most recent multicentric effort has led to the establishment of an Asian YHRD (http://www.ystr.org/asia) which has been available since January 2002. All databases are maintained and curated at the Institute of Legal Medicine, Humboldt-University, Berlin and will soon be fused to a global repository including populations from all continents.

Haplotype frequencies and population data of nine Y-chromosomal STR polymorphisms in a German and a Chinese population

Y-chromosomal STR loci are of increasing interest in paternity testing, forensic casework, anthropological and evolutionary studies. We participate in a cooperation to establish an international reference database of at least nine Y-chromosomal STR loci to be used for biostatistic calculations. We present frequency distributions of nine Y-chromosome specific STR polymorphisms and frequencies of compound haplotypes in two populations. We chose the loci DYS393, DYS19, DYS392, DYS385I, DYS385II, DYS390, DYS391 and DYS389I and II. Blood samples were taken from 136 unrelated male individuals from Cologne (Germany) and of 63 unrelated males from Chengdu (Sichuan Province, PR China). DNA was extracted by a salting out procedure or chelex extraction. PCR was carried out in two multiplex reactions. Fragment analysis was conducted on an ALF- or ALF-express sequencer. Frequency profiles of the German men showed no significant differences compared to most European populations. Mean exclusion chances were between 0.44 for DYS393 and 0.94 for DYS385. Haplotype diversity for the complete haplotype was 86.66% in Germans and 98% in Chinese. The Chinese men showed for all analysed loci except for DYS389I and DYS390 remarkably different allele distributions.

High sensitive DNA typing approaches for the analysis of forensic evidence: comparison of nested variable number of tandem repeats (VNTR) amplification and a short tandem repeats (STR) polymorphism

The approach of using nested primers for the APO B variable number of tandem repeats (VNTR) increases the sensitivity of the polymerase chain reaction (PCR) to single cell level. Different experiments and a comparison to the short tandem repeats (STR) system VWA were carried out, to determine the applicability of this method to forensic samples. Nested amplification of the Apo B VNTR was affected by a strong tendency towards preferential amplification of the shorter alleles. This phenomenon was observed for DNA quantities as low as 100 pg and impaired, depending on the allele length, the results for mixed samples. As expected, VWA polymorphism showed less preferential amplification. The high sensitivity of both PCR systems is accompanied by an increased susceptibility to contamination. Using artificially contaminated bloodstains, the bloodstain genotype, the contamination or both genotypes could be found on one piece of evidence. Here a single analysis can lead to an incorrect result. Therefore a strategy for obtaining reliable results should consist of multiple stain extractions and the amplification of different stepped dilutions of the DNA solution.

Analysis of the co-amplified STR loci D1S1656, D12S391 and D18S51: population data and validation study for a highly discriminating triplex-PCR

A multiplex-PCR composed of the three highly variable STR loci D1S1656, D12S391 and D18S51 has been established. The non-overlapping fragment sizes allow allele detection using a monochrome automated laser fluorescent sequencer (A.L.F. express, Pharmacia Biotech). The typing results of the triplex-PCR showed no difference to those of singleplex-PCR. Allele frequencies were determined in a Western German population of 228 individuals from Cologne. The heterozygosities and exclusion chances (D1S1656, 0.982; D12S391, 0.979; D18S51, 0.97) are very high compared to other short tandem repeats used for forensic applications. No deviations from the Hardy-Weinberg equilibrium were found. Successful typing of DNA amounts down to 50-100 pg is possible. Mixtures of up to 1:10 can be identified. In conclusion, the high combined exclusion chance due to the well-balanced allelic distribution and its high sensitivity make this triplex-PCR a valuable tool for forensic casework.

Population Genetics of Three STR Polymorphisms in a Chinese Population

Short tandem repeats (STRs) belong to a type of DNA polymorphisms with core sequences of a few base pairs in length. They are analysed using the polymerase chain reaction (PCR) and show variable allele lengths from 100 to 500 bp. The PCR products have to be separated by polyacrylamide gel electrophoresis with a resolution of one base pair (1). STRs offer three obvious advantages for forensic purposes. Firstly, the allele classifications can be based upon the number of tandem repeats relative to an allele ladder, so that correct typing is permitted. Secondly, samples containing very small amounts of DNA can be analysed. Thirdly, the amplification of alleles is also possible with highly degraded DNA. Therefore, we analysed 3 STR systems (VWA, HUMTH01 and FES)(2–4) to obtain preliminary allele frequency data for a Chinese population.

Y-chromosomal short tandem repeat haplotypes at the loci DYS393, DYS19, DYS392, and DYS385-I/II, DYS390, DYS389-I/II, and DYS391 in a Filipino population sample.

Whole blood samples were obtained from 106 unrelated male individuals living in Metro Manila, Philippines, through the Department of Health, Manila. DNA was extracted by isopropanol fractionation-sodium iodide precipitation (4) and quantified by spectrophotometry. Nine Y-chromosomal short tandem repeats (STRs) were analyzed from a population sample of 106 unrelated males by means of a quadruplex PCR (DYS393, DYS19, DYS392, DYS385-I/II) and a triplex PCR (DYS390, DYS389-I/II, and DYS391).

Zwei Beispiele zur Anwendbarkeit Y-chromosomaler DNA-Polymorphismen in der forensischen Spurenanalytik

Abstract Two cases where DNA typing has been carried out for stain analysis are described. In both cases Y chromosomal STR systems have been used in combination with autosomal STR systems. In the first case the DNA from the fingernails of a female victim with skin particles of the male murderer was typed. The male DNA in this mixture of male and female DNA could be typed. The excess of female DNA in the samples did not distrub the PCR reaction of Y-chromosomal systems. In the second case several body parts could be identified to belong to a single male person. The typing of the suspect’s DNA did not give any information because of a family relationship. Different kinds of stains have been analysed with DYS 389 I/II, DYS 390, DYS 391, DYS 392 DYS 393 and DYS19. Two multiplex PCR reactions were carried out to save time and stain material. Although the Y-chromosomal systems are not as polymorphic as autosomal STR systems the analysis of combined haplotypes can give additional information in forensic casework.Zusammenfassung Vorgestellt werden zwei Kasuistiken, bei denen DNA-Typisierungen verschiedener Spurenmaterialien durchgeführt wurden. Dabei wird besonders Gewicht auf die Anwendung der Y-chromosomalen STR-Polymorphismen, DYS 389 I/II, DYS 390, DYS 391, DYS 392, DYS 393 and DYS 19, gelegt. Als besonders zeit- und kostensparend hat sich dabei die Multiplex-PCR mehrerer Systeme erwiesen. Im ersten Fall handelte es sich um eine DNA-Mischung aus weiblicher Opfer- und männlicher Tatverdächtigen-DNA. Die Darstellung der Y-chromosomalen Polymorphismen gelang hier vor dem Hintergrund weiblicher DNA. Im zweiten Fall konnten Leichenteile und Gewebespuren einer Person zugeordnet werden. Die Typisierung der DNA der Tatverdächtigen ergab aufgrund der Verwandtschaftsverhältnisse keine weiteren Informationen. In vielen Fällen ist die Analyse Y-chromsomaler STR-Systeme in Verbindung mit autosomalen Polymorphismen eine zusätzliche Informationsquelle, die sich in der Spurenanalytik anbietet, wenn Mischungen von DNAs vorliegen oder zahlreiche Spuren männlichen Personen zugeordnet werden sollen.

Genetic variation of the amplified VNTR polymorphism COL2A1 in chinese and german populations

The amplifiable VNTR polymorphic system COL2A1 has been investigated in Chinese and German population samples. In order to accurately identify COL2A1 alleles, a number of human allele ladders were prepared. A total of 14 different alleles in 23 genotypes was observed in the Chinese population, while in the German population there were 11 alleles in 24 genotypes. Four new alleles were observed. All of them were found in the Chinese and one was also observed in the German population. There was no statistical difference in the allele distribution between two German populations from Münster and Köln, while the allele frequency distribution in the Chinese population differed significantly from the German.