Mutations or exclusion: an unusual case in paternity testing

In an immigration case with the scope of family reunification, the DNA extracted from the saliva samples of the male child, the alleged mother and the putative father was typed with 22 autosomal short tandem repeat (STR) systems. In seven STR systems, the alleged mother could be excluded from maternity, and the case then had to be regarded as a deficiency case. Taking this fact into consideration, only two exclusions were found for the putative father, and the question arose whether there was an exclusion of the putative father or the existence of two mutations. Autosomal STR typing could not clarify the case, but the application of eight Y-chromosomal markers showed that the alleged father could be excluded from paternity.     

Population genetics of Y-chromosome STRs in a population of Northern Greeks

Seventeen Y STR loci were typed in a population sample of 191 unrelated male individuals from Northern Greece. Haplotypes are presented for the following loci: DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438 and DYS448. The overall haplotype diversity was 0.9992. This database study provides significant additional information for the application of Y-chromosomal STRs to forensic identification efforts in Greece by nearly doubling both the number of individuals and the number of Y-loci typed from Greek populations. These samples have been previously typed for autosomal STRs [L. Kovatsi, T.J. Parsons, R.S. Just, J.A. Irwin, Genetic variation for 15 autosomal STR loci (PowerPlex 16) in a population sample from northern Greece, Forensic Sci. Int. 159 (2006) 61–63] and the mitochondrial DNA control region [J. Irwin, J. Saunier, K. Strouss, C. Paintner, T. Diegoli, K. Sturk, L. Kovatsi, A. Brandstatter, M.A. Cariolou, W. Parson, T.J. Parsons, Mitochondrial control region sequences from northern Greece and Greek Cypriots, Int. J. Legal Med. 122 (2008) 87–89].     

Analyse biologischer Spuren

The analysis of mitochondrial DNA and of Y-chromosomal STR markers to investigate biological stains is widely used today. Especially for stain material that does not contain sufficient nuclear DNA, such as telogen or rootless hair, bones, teeth, or severely degraded samples, sequencing of mtDNA has become the method of choice. The maternal segregation, the high sensitivity, and the presence of heteroplasmic sites makes mtDNA investigations different from nuclear DNA testing. This article highlights the various aspects of the biology and genetics of mtDNA and forensic stain investigations using mtDNA. Y-chromosomal STR are especially valuable for the investigation of mixed stains. In cases of mixed stain samples with an excess of female DNA, it is possible to determine a profile for the male contributor to the stain. Therefore, Y-STRs are a useful complement to the investigation of autosomal STR markers.     

Comparative analysis of short tandem repeats and single nucleotide polymorphisms on the Y-chromosome in Germans,Chinese and Thais

We have typed genomic DNA samples from 95 individuals from Western Germany, 78 individuals from Bangkok/Thailand and 56 individuals from Chengdu/China for 11 Y-chromosomal diallelic polymorphisms and eight short tandem repeat (STR) systems. For single nucleotide polymorphism (SNP) analysis, a rapid method was applied using the single base extension technology (minisequencing) in combination with capillary electrophoresis. PCR products for SRY-8299, Tat, SRY2627, 92R7, SRY1532, M9, M13, M17/M19 and M20 were pooled and used as templates for the commercially available SNaPshot kit. In addition to these ten SNPs we also tested the Y-chromosomal diallelic Alu repeat insertion DYS287 (YAP) by agarose gel electrophoresis as well as the Y-chromosomal STR systems DYS19, DYS389I+II, DYS390, DYS391, DYS392, DYS393 and DYS385 by fluorescent multiplex fragment analysis. Among the 11 diallelic SNP/Alu systems, only six were found to be polymorphic in the three population samples. From these a total number of seven different haplogroups could be identified in the three populations. Of these, five haplogroups were present in Germans, five in Thais, and only two in Chinese. These haplogroup trees clearly represent population-specific structures. Haplogroup 26 is represented at a high frequency in the Thai and Chinese populations whereas it is absent in Germans. The Y-STR data confirm a haplogroup-specific distribution of Y-STR haplotypes. Only a few cases of identical STR haplotypes in the same SNP haplogroups were detected in each of the three populations studied.     

First Polish DNA "manhunt"--an application of Y-chromosome STRs

This study presents the application of Y-chromosomal STR polymorphisms to male identification in the case of a serial rapist and woman murderer in Poland. Since August 1996 a rapist from Swinoujscie (northwest Poland) committed at least 14 rapes. In the year 2000 he brutally raped 8 young girls and murdered a 22-year-old girl. DNA profiles obtained from semen stains left at the scenes of crime gave information that one and the same man had committed all the rapes. The Y-chromosome haplotype (9 loci) obtained was used for the elimination process of 421 suspects. One man was found who had an identical DNA profile in all Y-chromosome STR loci analysed and possessed common alleles in 9 out of 10 autosomal loci, strongly suggesting that the real rapist and the typed man were closely related males. Analysis of reference DNA obtained from the man's brother revealed an identical DNA STR profile to that identified at the crime scenes. To the best of our knowledge this is the first case in Poland and probably in Eastern Europe where DNA typing of a large population was used to identify the offender.     

Y-chromosomal STR haplotypes in a population sample from Bavaria

The seven Y-chromosomal STRs DYS19, DYS385 I/II, DYS389 I/II, DYS390, DYS391, DYS392 and DYS393 were amplified using two multiplex PCRs. The optimization of the PCR conditions led to reliable and sensitive systems. Co-amplification of the amelogenin locus was possible in both multiplex systems. In a population sample of 151 Bavarian males, a gene diversity of 0.99 was observed. Sensitivity studies revealed a detection limit of 50 pg DNA per 25 μl reaction volume. PCR experiments with combinations of male/male and male/female DNA showed that in male/male mixtures, the minor component could be detected up to a ratio of 1:15, whereas in male/female mixtures the male component could be found in a higher ratio up to 1: 60. Received: 19 March 1999 / Received in revised form: 16 June 1999     

Validation of a combined autosomal/Y-chromosomal STR approach for analyzing typical biological stains in sexual-assault cases

DNA testing is an established part of the investigation and prosecution of sexual assault. The primary purpose of DNA evidence is to identify a suspect and/or to demonstrate sexual contact. However, due to highly uneven proportions of female and male DNA in typical stains, routine autosomal analysis often fails to detect the DNA of the assailant. To evaluate the forensic efficiency of the combined application of autosomal and Y-chromosomal short tandem repeat (STR) markers, we present a large retrospective casework study of probative evidence collected in sexual-assault cases. We investigated up to 39 STR markers by testing combinations of the 16-locus NGMSElect kit with both the 23-locus PowerPlex Y23 and the 17-locus Yfiler kit. Using this dual approach we analyzed DNA extracts from 2077 biological stains collected in 287 cases over 30 months. To assess the outcome of the combined approach in comparison to stand-alone autosomal analysis we evaluated informative DNA profiles. Our investigation revealed that Y-STR analysis added up to 21% additional, highly informative (complete, single-source) profiles to the set of reportable autosomal STR profiles for typical stains collected in sexual-assault cases. Detection of multiple male contributors was approximately three times more likely with Y-chromosomal profiling than with autosomal STR profiling. In summary, 1/10 cases would have remained inconclusive (and could have been dismissed) if Y-STR analysis had been omitted from DNA profiling in sexual-assault cases.     

DYS19 and amelogenin in artificial blood stains with defined amounts of male and female cells

The sensitivity of the DYS19 and the amelogenin STR systems for amplifying Y-specific fragments was assayed using artificial bloodstains with varying amounts of male and female (non-template) DNA in different ratios. The study confirmed the high sensitivity of both systems in detecting male-specific PCR fragments in stains containing 10–25 template molecules even in the presence of large amounts of female DNA in the mixture by silver-stain detection. However, blood mixtures which contain less than 10% male cells could be reliably typed only when at least 100 template molecules were present in the artificial bloodstain, due to increasing amounts of hemoglobin from the female blood which is a PCR inhibitor. Received: 27 October 1997 / Received in revised form: 29 January 1998     

Haplotype frequencies of eight Y-chromosome STR loci in Barcelona (North-East Spain)

Haplotype frequencies for eight Y-chromosomal short tandem repeat (STR) loci were determined in ¶a population sample from Barcelona (NE Spain). After PCR amplification and denaturing PAGE electrophoresis, DYS19, DYS388, DYS389 I/II, DYS390, DYS391, DYS392 and DYS393 loci were typed. Complete eight ¶Y-chromosomal STRs haplotypes could be formed for 223 subjects, among which 137 different haplotypes were observed. The most common haplotype was shared by 13% of the sample, while 108 haplotypes were unique. The discrimination capacity was 61.5% and the gene diversity was 0.978. From the forensic point of view the combined polymorphisms provide a high diagnostic efficiency.     

Y-Chromosome multiplexes and their potential for the DNA profiling of Koreans

We have developed four multiplex genotyping systems (GeneKin Y-STR multiplexes) using silver staining with allelic ladders for ten Y-chromosome STR markers (DYS19, DYS385, DYS388, DYS389I/II, DYS390, DYS391, DYS392, DYS393 and DXYS156Y), with a view towards the application of rapid and simple genotyping assay methods for DNA profiling. The GeneKin Y-STR multiplexes developed have followed the published nomenclature and ISFG guidelines for STR analysis. Allele and haplotype frequencies at these Y-STRs loci were analysed by PCR amplification using the GeneKin Y-STR multiplexes, followed by denaturing polyacrylamide gel electrophoresis in 316 unrelated males in the Korean population. A total of 295 different haplotypes were found, 279 of them being unique. Gene diversity ranged from 0.4026 at DYS391 to 0.9606 at DYS385. The haplotype diversity value (which is the same as the discrimination index) calculated from all ten loci combined was 0.9995, which is informative. Our results revealed that a set of ten Y-STRs can discriminate between most of the male individuals in the Korean population (discrimination capacity: 93.35%). The Y-STR multiplexes thus provide useful information for forensic analysis and paternity tests and can also be of great benefit for providing information not normally available from autosomal DNA systems. Received: 12 March 2001 / Accepted: 26 June 2001     

Polymorphisms and microvariant sequences in the Japanese population for 25 Y-STR markers and their relationships to Y-chromosome haplogroups

Y-chromosomal short tandem repeat (Y-STR) markers have been used for forensic purposes such as kinship analysis of male-linage and detection of a male DNA component in a mixture of male and female DNA. Recently, rapidly mutating Y-STR (RM Y-STR) markers were reported that are expected to help distinguish close male relatives. This study provides data of Y-chromosomal haplotypes for 25 Y-STR markers, including six RM Y-STR markers (DYS576, DYS627, DYS518, DYS570, DYS449 and DYF387S1) typed with the Yfiler™ Plus kit in 1299 males of the Japanese population. Discrimination capacity increased from 87.2% for 16 Y-STR markers with the Yfiler™ kit to 99.6% for 25 Y-STR markers with the Yfiler™ Plus kit. We characterized sequences of observed microvariant alleles of eight Y-STR markers and a low-amplified allele of DYS390 by Sanger sequencing. DYF387S1, a multi-locus Y-STR marker that is located at two positions on the human Y-chromosome, was observed in tri-allelic patterns in 51 of 1299 samples (3.9%) and we found an extremely high frequency of the tri-allelic pattern of DYF387S1 in haplogroup C-M131. We also analyzed Y-STR gene diversity in each haplogroup and its relevance to mutation rates.     

DNA analysis of family members with deletion in Yp11.2 region containing amelogenin locus

For personal identification of two male bodies discovered at the scene of a fire, autosomal and Y chromosomal STR of the two cadavers and of two living male relatives were genotyped. The four males were incorrectly typed as female due to the lack of the amelogenin Y homolog, whereas all loci of Y-STR except for DYS458 were successfully genotyped. Because PCR of Y-specific amelogenin (AMELY) and DYS458 loci failed to amplify target products when using additional primer sets, it was concluded that deletion in the Yp11.2 region containing the loci of AMELY and of DYS458 on the Y chromosome, rather than mutation in the annealing region of the primer sets, had occurred. Investigation using Y-specific markers showed the deletion extending approximately 2.56 Mb in the Yp11.2 region. The variety of deletion sizes and Y-STR haplotypes among AMELY negative males presented to date suggests that the mutation of the Yp11.2 region occurs independently in different ethnic groups. A study on the frequency of the AMELY deletion in the Japanese population would be helpful for future criminal investigation.     

Genetic polymorphisms for 17 Y-chromosomal STR haplotypes in Jammu and Kashmir Saraswat Brahmin population

In this study 17 Y-chromosomal STRs (including DYS19, DYS389I, DS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and Y GATA H4) were analysed using blood samples of 122 unrelated male individuals belonging to Saraswat Brahmin community from Jammu (ID YP000599) and Kashmir (ID YP000600) region of J&K state of India. The allelic frequency distribution and haplotype diversity of 17 Y-chromosomal STR for both the populations were calculated. In the Kashmiri Saraswat group, a total of 109 haplotypes were identified in 122 individuals, of these haplotypes, 101 were found only once. The gene diversity values of STR loci ranged from 0.4813 (DYS391) to 0.8645 (DYS385a/b) for Jammu & Kashmiri Saraswat Brahmins.     

Developmental validation of the Yfiler® Plus PCR Amplification Kit: An enhanced Y-STR multiplex for casework and database applications

Y-chromosomal loci have proven useful in solving investigations where low levels of male DNA are present in a high female DNA background. An intrinsic limitation of Y-STRs compared with autosomal STRs is a reduced power of discrimination due to a lack of recombination throughout most of the Y-chromosome. Thus, in an effort to increase the power of discrimination we have developed a new 6-dye, 27-plex Y-STR system that includes the 17 loci from the Yfiler^® and Yfiler^® Direct kits (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 (Y GATA C4), and Y GATA H4) plus three highly polymorphic Y-STR loci (DYS460, DYS481, and DYS533), and seven rapidly mutating Y-STR loci (DYF387S1a/b, DYS449, DYS518, DYS570, DYS576, DYS627) which allow for improved discrimination of related individuals. The Yfiler^® Plus PCR Amplification Kit is a dual application assay designed to amplify DNA from extracted casework and database samples from storage cards and swab lysates via direct amplification. Compared to the Yfiler PCR Amplification Kit, the new multiplex shows increased discrimination of male lineages and also improved performance in inhibited samples, improved balance in male DNA samples mixed with female DNA at ratios >1:1000, and faster time to results. The Yfiler Plus Kit shows very high concordance to the Yfiler Kit but discordance with the PowerPlex^® Y23 Kit at the DYS481 locus was observed in 2 out of 30 samples tested. This developmental validation work follows the SWGDAM guidelines and demonstrates that the assay is robust and suitable for use on forensic casework and database samples.     

Genetic polymorphisms for 17 Y-chromosomal STRs haplotypes in Chinese Hui population

We have co-amplified 17 Y-chromosomal STRs (including DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a,b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y GATA H4) for samples of 143 unrelated male individuals of Chinese Hui Ethnic Minority Group. We obtained allelic frequency distribution and haplotype diversity of 17 Y-chromosomal STR for Hui population. A total of 136 different haplotypes were identified in 143 individuals, among which 129 were found only once, and seven haplotypes were found twice. The gene diversity values of STR loci ranged from 0.4161 (DYS391) to 0.9571 (DYS385a,b). The overall haplotype diversity for the 17 Y-STR loci was 0.9933, and the discrimination capacity was 0.9511.     

Ninhydrin-dyed latent fingerprints as a DNA source in a murder case

In this case report, we describe the possibility of using ninhydrin-dyed fingerprints as a DNA source for STR typing. Preliminary tests prove that ninhydrin-dyed material still can be useful for STR typing. The case material consisted of seven ninhydrin-labeled latent fingerprints found at a murder crime site, which could not be typed in a classical manner. We were able to swap DNA from the ninhydrin-treated areas and successfully use it for STR typing.     

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.     

Point mutations in the flanking regions of the Y-chromosome specific STRs DYS391, DYS437 and DYS438

Sequence analysis of the DNA fragments amplified with the DYS391, DYS437 and DYS438 primers allowed the detection of biallelic polymorphisms in the flanking region of these STR loci. In this work, we describe a methodology where both the STR alleles and the SNPs at these loci are typed. Sequencing of chimpanzee (Pan troglodytes) homologous loci was performed and the ancestral state of the SNPs was determined. The allele distribution of these biallelic markers was analysed within different haplogroups. For DYS391, allele 1 was found in all samples from haplogroups E3a and E* (xE3a). DYS437 allele 1 was present in all haplogroup E3a samples and absent in the haplogroup E* (xE3a). The presence of allele 1 of DYS438 was restricted to haplogroup J. The SNP typing can be helpful in distinguishing STR haplotype identity by descent from identity by state, thus proving to be very informative in forensic investigations.     

Evaluation of Y-chromosomal STRs: a multicenter study

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. Received: 30 December 1996 / Received in revised form: 26 February 1997     

Highly informative Y-chromosomal haplotypes by the addition of three new STRs DYS437, DYS438 and DYS439

The Y chromosome STRs DYS437, DYS438 and DYS439 were selected from publicly available genome databases and used to analyse an Italian population sample. A tetraplex PCR reaction including the highly informative DYS385 locus, was set up and used for the analysis of 131 male samples to determine allele frequencies and STR diversity values. The number of different haplotypes and the haplotype diversity value found from the analysis of the STRs included in the tetraplex reaction were very similar to those found from the analysis of the basic set of 7 Y-STRs (DYS19, DYS389I/II, DYS390, DYS391, DYS392 and DYS393) previously carried out on the same population sample. By combining the allelic states of the 11 Y-chromosomal STRs we could construct highly informative haplotypes that allowed the discrimination of 93.8% (120 out of 128) of the samples tested. This approach represents a very powerful tool for individual identification and paternity testing in forensic medicine. Received: 29 November 1999 / Accepted: 17 March 2000