Rapidly Mutating Y-STRs Research Articles

Improving the differentiation of closely related males by RMplex analysis of 30 Y-STRs with high mutation rates

The discovery of rapidly mutating (RM) Y-STRs started to move the field of forensic Y-STR analysis from male lineage identification towards male individual identification. Previously, the forensic value of RM Y-STRs for differentiating male relatives was limited due to the modest number of 13 identified RM Y-STRs. Recently, new RM Y-STRs were discovered, with strong expectations for significantly improving male relative differentiation; however, empirical evidence is missing yet. More recently, the genotyping method RMplex for efficiently analyzing 30 Y-STRs with high mutation rates, including all 26 currently known RM Y-STRs, was introduced. Here, we applied RMplex as well as the current state-of-the-art commercial Y-STR kit: Yfiler™ Plus PCR Amplification kit, to several hundreds of DNA-confirmed father-son pairs. Newly established estimates confirmed the high mutation rates of novel and previous RM Y-STRs. By combining current with previous data, we provide updated consensus estimates of mutation rates for all 49 Y-STRs targeted with both methods. Based on RMplex, 42% of 499 father-son pairs were differentiated, while 14% of 530 pairs based on Yfiler™ Plus, and 48% of 499 pairs based on both methods combined. Regarding brothers, RMplex also clearly outperformed Yfiler™ Plus, with differentiation rates of 62% and 33%, respectively. By combining both methods 72.9% of the brothers showed at least one mutation. For unrelated males, both methods achieved a discrimination capacity of 99.8% and a haplotype diversity of 0.999991, since all males had different haplotypes, except for two, perhaps indicating a hidden paternal relationship. Overall, this study underlines the value of RM Y-STRs in general and RMplex in particular for differentiating male relatives highly relevant in forensic genetics. It provides the first empirical evidence on the high value of RMplex for differentiating close male relatives, which for father-son pairs was almost 60% higher than with the initial set of 13 RM Y-STRs and three times higher than with Yfiler™ Plus. Based on our results from closely related males, we expect RMplex to also improve the differentiation of more distantly related males significantly, which needs empirical demonstration in future studies. We encourage the forensic community to apply RMplex in all forensic cases where a match with a commercial Y-STR kit was obtained between the male suspect and the evidence material, or to solely use RMplex in such cases, aiming to find out if the male suspect or any of his male paternal relatives left the evidence material at the crime scene.

RETRACTED: Developmental validation of the VersaPlex™ 27PY system

The VersaPlex™ 27PY System is an STR multiplex from Promega offering co-amplification of 27 loci. This six-dye multiplex was designed for casework samples and includes loci that meet database requirements for the US (CODIS), Europe (ESS) and China (Chinese National Database). The 23 autosomal loci include the D6S1043 locus because of its high level of discrimination power, especially in Asian populations. The multiplex also includes two rapidly-mutating Y-STRs that can provide useful genetic information for forensic samples. In this article we describe the developmental validation experiments performed on the VersaPlex™ 27PY System that were designed to meet SWGDAM requirements. Changes in the performance of the multiplex are documented in response to reaction variables including PCR inhibitors, reaction volume, cycle number, annealing temperature, and magnesium concentration. The sensitivity, specificity, precision, and reproducibility of the VersaPlex™ 27PY System were also evaluated. The results of the experiments demonstrate the capabilities of the VersaPlex™ 27PY System as a tool for forensic laboratories.

Forensic Genetic Analysis of 11 Rapidly Mutating Y-STRs (RM-YSTRs) in Sindhi Population of Pakistan

Forensic Science is robustly associated with DNA that has a complex genetic blue print information intended to resolve the queries related to civil cases, judiciary, investigatory purposes and crime scenes. The study was carried out to contribute the data set of RMYSTRs (Rapidly Mutating YSTRs) globally. The study focused on the significance of using RM-YSTRs as compared to the set of 9-17 YSTRs (mutation rate 1 × 10-2), as the later one is not being able to attain a high magnitude of male relatives differentiation. In the present work, 100 Sindh DNA male samples were collected, by genotyping the amplified samples, using RM-YSTRs, having a mutation rate of 1 × 10-3. Forensic efficiency parameters such as, PIC (Polymorphism Information Content) was observed in the range of 0.7745 at locus DYS576 to 0.9313 at locus DYF399S1b, maximum gene diversity (0.9350) was found at locus DYF399S1b, minimum gene diversity (0.7978) was seen at locus DYS576. Highest value of allele frequency was found as 0.350 with the allele no 18 at locus DYS576. The results had shown that haplotype frequency and haplotype diversity value were 0.010 and 0.99297, as no haplotype was repeated, which highlighted on the fact that nearly complete male individualization can be obtainedusing RM YSTRs, thus helpful in avoiding the adventitious matches or exclusion or inclusion of male relatives in forensics cases work. It might be an initiative towards establishing RM YSTR database of Pakistani Sindhi population.

Identification and characterization of novel rapidly mutating Y-chromosomal short tandem repeat markers.

Short tandem repeat polymorphisms on the male-specific part of the human Y-chromosome (Y-STRs) are valuable tools in many areas of human genetics. Although their paternal inheritance and moderate mutation rate (~10-3 mutations per marker per meiosis) allow detecting paternal relationships, they typically fail to separate male relatives. Previously, we identified 13 Y-STR markers with untypically high mutation rates (>10-2 ), termed rapidly mutating (RM) Y-STRs, and showed that they improved male relative differentiation over standard Y-STRs. By applying a newly developed in silico search approach to the Y-chromosome reference sequence, we identified 27 novel RM Y-STR candidates. Genotyping them in 1,616 DNA-confirmed father-son pairs for mutation rate estimation empirically highlighted 12 novel RM Y-STRs. Their capacity to differentiate males related by 1, 2, and 3 meioses was 27%, 47%, and 61%, respectively, while for all 25 currently known RM Y-STRs, it was 44%, 69%, and 83%. Of the 647 Y-STR mutations observed in total, almost all were single repeat changes, repeat gains, and losses were well balanced; allele length and fathers' age were positively correlated with mutation rate. We expect these new RM Y-STRs, together with the previously known ones, to significantly improving male relative differentiation in future human genetic applications.

Investigation on rapidly mutating Y-STRs multiplex in Indian population: A pilot study

Importance of rapidly mutating (RM) Y- STRs) is already established and is evident with the addition of seven RM Y-STR markers in new generation Yfiler Plus (YFP) multiplex system. Performance of the 7 RM Y-STRs included in YFP is evaluated in central Indian population. We also report here a pilot study on genetic polymorphism using 13 Rapidly Mutating Y STRs (RM Y-STR) on Indian population. Forensic parameters including allele frequencies, gene diversities, discrimination capacity and Haplotype diversity will be calculated for both the amplification assays. Forensic application and kinship analysis will be highlighted in thisstudy.

Evaluation of rapidly mutating Y-STRs in Pakistani population

Y-chromosomal Short Tandem Repeats have been widely used in forensic investigations, identification of males for criminal justice purpose and population genetics. Commercially available Y-STRs kits allow the identification of male pedigrees and has a limited application in forensic genetics because of its limitation in differentiating closely related male individuals. Recent research with the Rapidly Mutating Y-STRs (RM Y-STRs) have revealed that these loci deliver significantly higher discrimination capacity and haplotype diversity in worldwide populations when compared with the conventional Y-STRs. Although a number of RM Y-STRs have found their way in most updated commercial kits, there are still some loci that are not yet used in such kits. The aim of this study is to develop RM Y-STR haplotypes frequency database for the Pakistani population, in order to appraise the resolution power of these loci. A total of 212 unrelated males from the Pakistani population were typed with 13 RM Y-STRs which comprise DYF399S1, DYF387S1, DYS570, DYS576, DYS518, DYS526a + b, DYS626, DYS627, DYF403S1a + b, DYF404S1, DYS449, DYS547 and DYS612. 211 unique haplotypes were identified, out of which 1 haplotype was shared between two individuals, accounting for 0.9952 discrimination capacity (DC). Haplotype diversity was found to be 0.999925. Gene diversity (GD) values of all the loci were higher than 0.5, where the highest GD values were observed at DYF399S1, DYF403S1a and DYF404S1; with values of 0.99419, 0.98252 and 0.93061 respectively. Results of our study revealed that these 13 RM Y-STRs produced significantly stronger discriminatory power in Pakistani populations.

COULD RAPIDLY MUTATING Y-STRS BE A POTENTIAL FORENSIC TOOL IN DISCRIMINATING LEBANESE MONOZYGOTIC TWINS?

Despite the high power of discrimination that characterizes the well identified 16 to 24 autosomal short tandem repeat markers, monozygotic twins differentiation is generally limited. Arising from a single fertilized egg, monozygotic twins share the same genotype therefore the same DNA profile. This situation imposes a challenge in forensics especially considering that lineage markers are in general less informative than autosomal ones. Although in some cases Y haplotype is considered a powerful investigative tool, it cannot distinguish males belonging to the same paternal lineage. The use of rapidly mutating Y-STRs with mutation rate above 1x10-2 that were recently included in forensic casework is presumed helpful. Since science is always dynamic and each population has its own characteristics, we aim in this study to distinguish between Lebanese monozygotic male twins using rapidly mutating Y-STRs. For this purpose, fourteen unrelated pairs of male monozygotic twins were recruited. Participants filled in a well-designed questionnaire and signed an informed consent. DNA was extracted using PureLink Genomic DNA Mini kit, genotyped using the Identifiler Plus kit, and separated on 3500 Genetic Analyzer to confirm the monozygosity status. DNA samples underwent a second amplification using the Y Filer Plus kit. According to our results, all the Y Filer Plus DNA profiles showed complete match for each twin pair. By consequence, the use of rapidly mutating Y-STRs in this study did not improve discrimination.

Genetic characterisation of 13 rapidly mutating Y-STR loci in 100 father and son pairs from South and East Turkey

Background: Rapidly mutating (RM) Y-STRs recently emerged as a useful genotyping tool that can counteract problems normally associated with traditional Y-STRs. For instance, RM Y-STRs have the potential to differentiate Y-chromosomes from both close and distant paternal relatives.Aim: Characterisation of 13 RM Y-STR loci in a new sample pool from Turkey in terms of population genetic data and mutation rates.Subjects and methods: One hundred father–son pairs from South and East Turkey were genotyped. Based on the 99 father haplotypes unique to the current study, statistical parameters of forensic interest were computed. Nei’s DA distances among 112 global population datasets were estimated and visualised by phylogenetic and multidimensional scaling (MDS) analyses.Results: Fifteen father–son pairs were found to differ at a single locus and four at two loci, resulting in a differentiation rate of 19%. Mutations were observed at 10 out of 13 loci, with rates ranging from 1 × 10−2 to 6 × 10−2.Conclusion: Mutation rates and differentiation rates between the father–son pairs were similar to those from the literature. In contrast to previous work, novel phylogenetic tree construction results based on Nei’s DA distances suggested a close correlation between the geographic and genetic distances observed, except for known cases of past mass migration events.

Rapidly mutating Y-STRs in rapidly expanding populations: Discrimination power of the Yfiler Plus multiplex in northern Africa

The male-specific northern African genetic pool is characterised by a high frequency of the E-M81 haplogroup, which expanded in very recent times (2–3 kiloyears ago). As a consequence of their recent coalescence, E-M81 chromosomes often cannot be completely distinguished on the basis of their Y-STR profiles, unless rapidly-mutating Y-STRs (RM Y-STRs) are analysed. In this study, we used the Yfiler® Plus kit, which includes 7 RM Y-STRs and 20 standard Y-STR, to analyse 477 unrelated males coming from 11 northern African populations sampled from Morocco, Algeria, Libya and Egypt. The Y chromosomes were assigned to monophyletic lineages after the analysis of 72 stable biallelic polymorphisms and, as expected, we found a high proportion of E-M81 subjects (about 46%), with frequencies decreasing from west to east. We found low intra-population diversity indexes, in particular in the populations that experienced long-term isolation. The AMOVA analysis showed significant differences between the countries and between most of the 11 populations, with a rough differentiation between northwestern Africa and northeastern Africa, where the Egyptians Berbers from Siwa represented an outlier population. The comparison between the Yfiler® and the Yfiler® Plus network of the E-M81 Y chromosomes confirmed the high power of discrimination of the latter kit, thanks to higher variability of the RM Y-STRs: indeed, the number of chromosomes sharing the same haplotype was drastically reduced from 201 to 81 and limited, in the latter case, to subjects from the same population.

Mutation rates of 13 RM Y-STRs in a Han population from Shandong province, China

13 rapidly mutating (RM) Y-STRs in Shandong Han population were investigated to verify the mutation characteristic in order to assess the power of the STR loci to differentiate Chinese Han individuals. Using a novel multiplex PCR system, we identified 2880 alleles transferring in 180 father-son pairs at the 13 RM Y-STR loci, differentiating 19.44% of the 180 father-son pairs. Inconsistencies were observed in only four father-son pairs at two loci. 39 mutation events were observed at the 13 loci, with an average mutation rate of 0.0135 (95% CI, 0.0096–0.0185). The most rapidly mutating loci among the 13 STR loci are DYF399S1abc, DYF403S1b2 and DYS570. All mutations discovered in this study are one-step mutations. The frequency of loss to gain of repeats equals to 1.17:1.

Comprehensive mutation analysis of 53 Y-STR markers in father-son pairs

The knowledge of mutation rate at Y-STR markers is essential in forensic casework, especially kinship genetic studies. In the present study, one hundred father–son pairs from Chinese Han population were typed with 53 Y STR markers (38 conventional markers and 15 rapidly mutating markers). A total of 28 mutations were detected in 6201 allele transfers. All were one-step mutation. The average estimated mutation rate of 15 rapidly mutating (RM) Y-STRs was 1.05×10−2 (95% CI 5.45×10−3–1.39×10−2), while the average mutation rate of 38 conventional Y-STRs was 1.46×10−3 (95% CI 3.07×10−4–2.59×10−3). In summary, the average mutation rate of 15 RM Y-STRs seemed to be seven times higher than that of 38 conventional Y-STRs.

Investigation of extended Y chromosome STR haplotypes in Sardinia

Y-chromosomal variation of selected single nucleotide polymorphisms (SNPs) and 32 short tandem repeat (STR) loci was evaluated in Sardinia in three open population groups (Northern Sardinia, n=40; Central Sardinia, n=56; Southern Sardinia, n=91) and three isolates (Desulo, n=34; Benetutti, n=45, Carloforte, n=42). The tested Y-STRs consisted of Yfiler® Plus markers and the seven rapidly mutating (RM) loci not included in the YFiler® Plus kit (DYF399S1, DYF403S1ab, DYF404S1, DYS526ab, DYS547, DYS612, and DYS626).As expected, inclusion of additional Y-STR loci increased haplotype diversity (h), though complete differentiation of male lineages was impossible even by means of RM Y-STRs (h=0.99997).Analysis of molecular variance indicated that the three open populations were fairly homogeneous, whereas signs of genetic heterogeneity could be detected when the three isolates were also included in the analysis.Multidimensional scaling analysis showed that, even for extended haplotypes including RM Y-STR markers, Sardinians were clearly differentiated from populations of the Italian peninsula and Sicily. The only exception was represented by the Carloforte sample that, in accordance with its peculiar population history, clustered with Northern/Central Italian populations.The introduction of extended forensic Y-STR panels, including highly variable RM Y-STR markers, is expected to reduce the impact of population structure on haplotype frequency estimations. However, our results show that the availability of geographically detailed reference databases is still important for the assessment of the evidential value of a Y-haplotype match.

Mutation Rates and Discriminating Power for 13 Rapidly-Mutating Y-STRs between Related and Unrelated Individuals.

Rapidly Mutating Y-STRs (RM Y-STRs) were recently introduced in forensics in order to increase the differentiation of Y-chromosomal profiles even in case of close relatives. We estimate RM Y-STRs mutation rates and their power to discriminate between related individuals by using samples extracted from a wide set of paternal pedigrees and by comparing RM Y-STRs results with those obtained from the Y-filer set. In addition, we tested the ability of RM Y-STRs to discriminate between unrelated individuals carrying the same Y-filer haplotype, using the haplogroup R-M269 (reportedly characterised by a strong resemblance in Y-STR profiles) as a case study. Our results, despite confirming the high mutability of RM Y-STRs, show significantly lower mutation rates than reference germline ones. Consequently, their power to discriminate between related individuals, despite being higher than the one of Y-filer, does not seem to improve significantly the performance of the latter. On the contrary, when considering R-M269 unrelated individuals, RM Y-STRs reveal significant discriminatory power and retain some phylogenetic signal, allowing the correct classification of individuals for some R-M269-derived sub-lineages. These results have important implications not only for forensics, but also for molecular anthropology, suggesting that RM Y-STRs are useful tools for exploring subtle genetic variability within Y-chromosomal haplogroups.

Differences in urbanization degree and consequences on the diversity of conventional vs. rapidly mutating Y-STRs in five municipalities from a small region of the Tyrolean Alps in Austria

In this study we set out to test at a micro-geographic scale for the potential effects of differences in urbanization degree on Y-chromosomal diversity and the paternal lineage differentiation of “conventional” and rapidly-mutating (RM) Y-STR markers. To avoid systematic underrepresentation of common lineages, 551 male samples were collected under a sampling regime allowing for the inclusion of paternal relatives. All participants came from a small, topographically highly structured, yet culturally homogeneous settlement area in the Tyrolean Alps of Austria, a region that is characterized by a longstanding coexistence of communities differing considerably in size and connection. The study participants reported provenance in one of the three rural villages Alpbach, Brandenberg, and Wildschönau – all being separated by topographical barriers from each other – or in one of the two more urban-like and better connected municipalities Kitzbühel and St. Johann in Tirol. When compared with the sample pools from the two larger communities, the three small villages showed distinctly higher rates of self-reported patrilocality since the paternal grandfather (85–95% vs. ∼42%), and featured evidence for a considerably higher proportion of close and cryptic paternal relationships among the study participants. We observed marked differences in the Y-SNP haplogroup frequency spectra and statistically significant Y-STR-based FST distances among the municipality samples, suggesting population sub-structuring along municipality borders. While for the two larger settlements a widely used “core” set of 17 conventional Y-STRs (Yfiler) provided reasonably high lineage resolution (Ĥ: 0.99515±0.00256, 0.99739±0.00224), a markedly reduced haplotype diversity was seen in samples from the rural villages (Ĥ: 0.96126±0.00701–0.98515±0.00278). This difference largely diminished when instead using a set of 13 RM Y-STRs (Ĥ: 0.99180±0.00380–0.99922±0.00187, for all groups). Most notably, in the Alpbach sample the number of different haplotypes rose from 42 (Yfiler) to 99 (RM Y-STRs) and the proportion of matching haplotypes dropped from nearly 4% (Yfiler) to about 0.4% (RM Y-STRs) of the pairwise comparisons. Consistent results were obtained with a reduced version of the dataset, being devoid of inferred close male relatives up to the degree of first cousins. Finally, consequences potentially arising from a gain in lineage-resolution for population reference-sample size requirements will be addressed briefly.

Analysis of Rapidly Mutating Y Chromosome Short Tandem Repeats (RM Y-STRs).

Rapidly mutating (RM) Y-STRs were recently identified and can help to differentiate between paternally related males in paternity and forensic casework. Normal Y-STRs are often used in casework due to their paternal inheritance, which can help to resolve kinship cases, and identify male components in male/female mixtures; however, more discriminating profiles are obtained if rapidly mutating Y-STRs are used. Previously two or three multiplex PCRs have been used to amplify 13 RM Y-STRs; here, an assay amplifying these 13 markers in a single multiplex PCR is described. Commercially available male control DNA samples have been genotyped during the validation of this assay, thus providing a tool for calibrating genotyping results. It is expected that the assay will provide a niche tool for genotyping casework samples.

Development and validation of an allelic frequency database for Qatari population using 13 rapidly mutating Y-STRs multiplex assay

Differentiating male lineages using non-recombining Y-chromosomal genetic markers is highly informative for tracing human migration and for forensic studies. Recently, it has been shown that the level of male lineage resolution can be enhanced by analysing rapidly mutating (RM) Y-STRs. The aim of this study was to develop an allelic frequency database for Qatari population to evaluate the resolution power of 13 RM Y-STRs. The overall haplotype diversity (HD) was 100%. It was found that the markers which contributed the most toward high HD were DYF399S1 and DYF403S1a/b. Together with their value for paternal male relative differentiation, these RM Y-STRs will be a valuable asset for forensic casework. AMOVA test was performed between Qatari population in comparison to Gulf countries, Middle East, and several worldwide population data sets. FST values were also calculated. Geography was found to account considerably for the pattern of population sub structuring. The RM Y-STR markers showed remarkable haplotype resolution power in the Qatari population, high gene diversity and sufficient robustness for a diverse range of applications.