Are rapidly mutating Y-short tandem repeats useful to resolve a lineage? Expanding mutability data on distant male relationships.

BACKGROUND: Y-chromosomal short tandem repeats (Y-STRs) are essential to relate male lineages in forensic and evolutionary studies. Although large panels of Y-STR markers are now available, none possess sufficient discrimination power to distinguish close male relatives. This limit may be overcome by the use of rapidly mutating Y-STRs (RM Y-STRs), characterized by mutation rates higher than common Y-STRs. Recently, multicenter studies evaluated the ability of RM Y-STRs to differentiate father-son pairs; however, more extensive data on distantly related males are needed. STUDY DESIGN AND METHOD: A total of 157 male relative pairs separated by two to seven meiotic events, originating from Italy, were analyzed by 13 RM Y-STRs and 23 Y-STRs. RESULTS: Overall, 154 mutational events were observed at RM Y-STR loci and the estimated mutation rate was of 2.59 × 10(-2) (95% confidence interval, 2.16 × 10(-2) -2.97 × 10(-2) ). A total of 105 male relative pairs showed at least one mutation in at least one locus and differentiation rates increased from 52.8% to 88.9% from the second to the fourth generation, while 23 Y-STRs provided much lower values, spanning from 10.1% to 29.6%. CONCLUSIONS: These findings confirmed the higher capability of RM Y-STRs than conventional Y-STRs to resolve male lineages, thus suggesting a possible future use for forensic male individual identification.

Kinship analysis: assessment of related vs unrelated based on defined pedigrees.

The study aimed at evaluating whether the adoption of enlarged batteries of STR markers in kinship analysis may provide LR values suitable for discrimination of relatives from non-relatives, in comparison to conventionally used STR panels. The presence of LD among some loci and its effects on LR values were also assessed. Three hundred pairs of related and unrelated individuals, each separated from 1-3 generations and residing in North Italy were genotyped with the Investigator HDplex STR kit (Qiagen), AmpFlSTR Identifiler (Applied Biosystems), and PowerPlex Fusion System (Promega). Loci and alleles shared between each pair and within groups of relatives were compared. Also, combined LR values with and without loci in LD, sensitivity and specificity were calculated for each commercial kit and their combinations. Full siblings displayed the largest number of shared loci and alleles, with a proportion of LR ≥ 10 results significantly higher than other degrees of relatedness and, consequently, with the lowest percentage of inconclusive and false negative results. Only minor differences were detected in the combined LR distributions, after including or omitting loci in LD. However, these became only appreciable when analyzing more distant relative pairs.The implementation of additional STRs into the LR calculation allowed a complete and robust discrimination between relatives and non-relatives only for full siblings, by removing the typical uncertainty of the "grey zone", while this was not achieved among other degrees of relatedness. Furthermore, the presence of loci in LD seems to not significantly affect LR distributions within each generation.

Haplotype data and mutation rates for the 23 Y-STR loci of PowerPlex® Y 23 System in a Northeast Italian population sample.

The PowerPlex® Y 23 System (Promega) is a short tandem repeat (STR) multiplex that allows co-amplification of 23 gonosomal Y-STRs, combining 17 loci commonly included in commercially available kits (DYS389I, DYS448, DYS389II, DYS19, DYS391, DYS438, DYS437, DYS635, DYS390, DYS439, DYS392, DYS393, DYS458, DYS385a/b, DYS456, and Y-GATA-H4) and six new loci (DYS481, DYS549, DYS533, DYS643, DYS576, and DYS570) with the last two being rapidly mutating Y-STRs (RM Y-STRs). In order to assess the possible gain in forensic efficiency provided by the six additional markers, a population sample of 410 unrelated healthy males originating from Northeast Italy (Veneto, Trentino Alto Adige, Lombardia, and Friuli Venezia Giulia regions) was typed. The data (335 of the 410 samples) are available in the Y chromosome haplotype reference database under accession number YA003327. Overall, 410 unique haplotypes were found corresponding to a global haplotype diversity (HD) of 0.999994 with a discriminatory capacity (DC) of 100%. Allelic microvariants, null alleles, and duplications were detected. Pairwise genetic distances (R(ST)) calculated among neighboring European reference populations revealed no significant differences. Furthermore, for studying Y-STR mutation rates, 90 father-son pairs, in which the fathers were already included in the full dataset, were tested. On a total of 2,070 meioses considered, eight single-step mutational events were observed, two of which within the same father-son pair and the average mutation rate was 3.38 × 10(-3) per locus per generation (95% confidence interval, 1.36 × 10(-3)-6.95 × 10(-3)).

Investigator HDplex markers: allele frequencies and mutational events in a North Italian population.

Autosomal short tandem repeats (STRs) analysis represents the method of election in forensic genetics and up to now, 23 STRs are available for these purposes. However, in particular circumstances such as human identification or complex kinship cases, examination of additional STRs may be required in order to obtain reliable conclusions. For this purpose, a new multiplex STR system, namely Investigator® HDplex kit (QIAGEN) that coamplifies a set of 12 autosomal loci, 9 of which, represents novel supplementary STRs, was recently developed. A population sample of 359 unrelated healthy subjects residing in North Italy was typed to determine allele frequencies, forensic parameters and genetic distances among European populations. Furthermore, to evaluate the suitability of the HDplex kit as an auxiliary tool for paternity testing, mutation rates were estimated on 84 confirmed family trios. The 12 loci resulted highly informative with a combined power of discrimination of 0.999998 and no departures from Hardy-Weinberg equilibrium were observed with the sole exception of locus D4S2366. From the comparison of our population sample and European reference populations, a single significant difference was revealed with the Poland population at D4S2366 locus. With regard to the mutation rate study, on a total of 2,016 meioses considered, six single-step mutational events were observed and the average mutation rate calculated was of 2.94 × 10(-3) per locus per generation (95% confidence interval, 1.08 × 10(-3)-6.39 × 10(-3)).

Deletion and duplication at DYS448 and DYS626 loci: unexpected patterns within the AZFc region of the Y-chromosome.

Increasing the knowledge of multiple and microstructural events within the Y-chromosome may prove useful to better characterize abnormal short tandem repeats patterns complicating DNA profile interpretation. On the long arm of the human Y-chromosome, such structural rearrangements were observed in azoospermia factor regions (AZFa, AZFb, AZFc) that play an important role in male fertility and also host Y-STRs commonly used in forensic genetics. Here, we describe two cases, involving two males formerly included in an Italian population study, where DYS448 and DYS626 loci, located within the AZFc region, simultaneously displayed a double deletion in one case and a double duplication in the other. With the aim of better defining the size of both events, low and high-resolution mapping by means of 16 sequence-tagged sites was performed, and unexpected discontinued patterns within the palindromic segments b1/b3 of the AZFc were identified. Extending the analysis to their respective male relatives revealed unaltered transmission of the patterns along the two pedigrees. Reviewing literature data describing DYS448-DYS626 deletion and duplication suggested no close correlation between the occurrence of multiple/microstructural events and geographical origin.

Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

Evaluation of genetic parameters of 22 autosomal STR loci (PowerPlex® Fusion System) in a population sample from Northern Italy.

The PowerPlex® Fusion System (Promega, Madison, WI) is a short tandem repeat (STR) multiplex that allows co-amplification of 22 autosomal STRs, including the CODIS core and the European Standard Set loci, plus amelogenin for gender determination and DYS391 male specific marker included in order to avoid errors in gender assignment when null Y-alleles or deletions of the Y-chromosome short arm involve the amelogenin locus. Allele frequencies and forensic efficiency parameters were estimated in a population sample of 303 unrelated healthy individuals living in Northern Italy. No significant deviations from Hardy-Weinberg expectations were observed after applying Bonferroni's correction for multiple testing. The combined power of discrimination was 0.999999999999 and the combined power of exclusion was 0.9999956. A rare 28 allele at locus D12S391 was observed, while one tri-allelic pattern at Penta E locus was detected. Population differentiation test revealed significant genetic diversity between our population sample and other European populations considered. The results showed that the PowerPlex® Fusion System is one of the most informative kit available in forensic genetics and may prove useful in both human identification and kinship analysis.

Allele and haplotype diversity of X-chromosomal STRs in Ivory Coast.

Twenty-one X-chromosomal short tandem repeat (STR) loci, including the six clusters of linked markers DXS10148-DXS10135-DXS8378 (Xp22), DXS7132-DXS10079-DXS10074 (Xq12), DXS6801-DXS6809-DXS6789 (Xq21), DXS7424-DXS101 (Xq22), DXS10103-HPRTB-DXS10101 (Xq26), DXS8377-DXS10146-DXS10134-DXS7423 (Xq28) and the loci DXS6800, GATA172D05 and DXS10011 were typed in a population sample from Ivory Coast (n=125; 51 men and 74 women). Allele and haplotype frequencies as well as linkage disequilibrium data for kinship calculations are provided. On the whole, no significant differences in the genetic variability of X-STR markers were observed between Ivorians and other sub-Saharan African populations belonging to the Niger-Kordofanian linguistic group.

Analysis of 11 tetrameric STRs in wild boars for forensic purposes.

STR profiling of animal species has a wide range of applications, including forensic identification, wildlife preservation, veterinary public health protection and food safety. We tested the efficacy of a multiplex PCR-based assay including 11 porcine-specific tetrameric STRs in a population sample of wild boars (n=142) originating from Piedmont (North West Italy). Multiple deviations from Hardy-Weinberg expectations were observed, mostly due to a reduction in observed heterozygosity indicative of a high degree of inbreeding. A value of θ of 0.046 and an inbreeding coefficient of 0.089 were estimated. Combined power of discrimination and probability of exclusion values for the STR panel were 0.9999999999996 and 0.99989. In order to test the suitability of the method for meat traceability purposes, a domestic pig reference sample (n=412), consisting of commercial lines commonly used in the meat production process, was also typed. A Bayesian cluster analysis carried out using the observed genotypes, showed a percentage of correct subspecies assignment of individual samples of 0.974 for wild boars and 0.991 for pigs, thus demonstrating the usefulness of the multiplex STR-typing system for discrimination purposes.