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European Americans are often treated as a homogeneous group, but in fact form a structured population due to historical immigration of diverse source populations. Discerning the ancestry of European Americans genotyped in association studies is important in order to prevent false-positive or false-negative associations due to population stratification and to identify genetic variants whose contribution to disease risk differs across European ancestries. Here, we investigate empirical patterns of population structure in European Americans, analyzing 4,198 samples from four genome-wide association studies to show that components roughly corresponding to northwest European, southeast European, and Ashkenazi Jewish ancestry are the main sources of European American population structure. Building on this insight, we constructed a panel of 300 validated markers that are highly informative for distinguishing these ancestries. We demonstrate that this panel of markers can be used to correct for stratification in association studies that do not generate dense genotype data. © 2008 Price et al.

According to the ISFG guidelines on the use of X-chromosome, the biostatistical evaluation in kinship analysis is based on a likelihood ratio approach, but since in calculation linkage and recombination events should be accounted for, an accurate estimate of mutation and recombination rates of X-markers analyzed is required. Due to the mode of genetic transmission and physical location, sometimes X-chromosome markers can be more informative than autosomal STRs and their analysis could be considered a supplementary tool in DNA testing. The increased demand to forensic laboratories for kinship investigations in complex cases explains the need not only to enlarge the Italian population database for 12 X-STRs routinely used for forensic application, but also to investigate the recombination fractions among markers. A collaborative exercise of the Italian Speaking Working Group Ge.F.I. was organized to evaluate mutation and recombination events in 12 X-STRs included in the Investigator Argus X12 kit. In order to explore the segregation stability, three-generation families (grandpa-mother-son) and two-generation families (mother-sons, father-daughters) for a total of 269 pedigrees were analyzed and calculations to estimate the recombination fractions between pairs of markers and mutation rates were performed. The statistical analysis showed evidence of inter- and intra-cluster recombination events, with almost free recombination for junction markers of linkage groups I and II and reduced recombination for linkage groups III and VI. We observed one- and two-step mutations, with an average value of 2.9 × 10−3.

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 μg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.

Forensic mRNA profiling assays normally include a set of vaginal-specific markers. Although it is known that vagina undergoes characteristic age-related morphological and physiological changes over a lifetime, few studies have evaluated the efficacy of proposed forensic vaginal mRNA markers in women from different age groups. In this collaborative study involving ten GeFI (Italian working group of ISFG) laboratories, a 19-plex mRNA profiling assay including three vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of vaginal swabs obtained from female volunteer donors in their reproductive years (n = 84) and postmenopausa (n = 55). Differential expression of vaginal markers in the two age categories was assessed by means of: a) overall success rate of mRNA profiling (vaginal mucosa “observed” in the tested sample according to scoring protocol) b) average peak height ratios between vaginal-specific markers within mRNA profiling replicates. Other factors potentially influencing mRNA profiling outcomes, like time interval between vaginal swab collection and analysis, concurrence of menstrual cycle and recent sexual activity at the time of sampling were also investigated.

A collaborative exercise on DNA/RNA co-analysis and RNA cell typing involving 15 GEFI (Italian working group of ISFG) laboratories was organized in collaboration with the Netherlands Forensic Institute. Participants received: 1) PCR primers for a 19-plex mRNA profiling assay, with reference purified PCR products for each cell type targeted in the multiplex; 2) detailed protocols for DNA/RNA co-extraction, mRNA profiling, and interpretation of results; 3) a set of 8 mock forensic stains (7 single source, one a mixture of two body fluids). All but one laboratory generated correct DNA typing results. As expected, stochastic effects were seen for low template DNA extracted from a skin stain. As for mRNA profiling, the percentage of laboratories that correctly identified body fluids was ≥60% for blood, saliva, vaginal mucosa, semen, and skin. Success rates were

In the recent years the Y chromosome genealogy has been refined by a number of newly discovered SNPs. The non-random distribution of the Y chromosome lineages worldwide makes fundamental the dissection and characterisation of haplogroups associated with specific geographic areas. In Southern Europe the haplogroup J2, as defined by the M172 marker, can reach frequencies up to 35%, making the dissection of such lineage critical for population studies. Here we present a study on J2 chromosomes from the Italian peninsula. Populations and forensic implications are discussed. A total of 900 individuals were previously genotyped for a number of SNPs, including M172. More than 200 of these have been now genotyped for 7 SNPs within the J2 lineage using a multiplex SNaPshot approach. The different distribution of the various lineages in different geographic areas probably reflects different historical demographic events and points to differential Y chromosome haplotype distribution, with implication for forensic application of this genetic marker. © 2008 Elsevier Ireland Ltd. All rights reserved.

Recently introduced rapidly mutating Y-chromosomal short tandem repeat (RM Y-STR) loci, displaying a multiple-fold higher mutation rate relative to any other Y-STRs, including those conventionally used in forensic casework, have been demonstrated to improve the resolution of male lineage differentiation and to allow male relative separation usually impossible with standard Y-STRs. However, large and geographically-detailed frequency haplotype databases are required to estimate the statistical weight of RM Y-STR haplotype matches if observed in forensic casework. With this in mind, the Italian Working Group (GEFI) of the International Society for Forensic Genetics launched a collaborative exercise aimed at generating an Italian quality controlled forensic RM Y-STR haplotype database. Overall 1509 male individuals from 13 regional populations covering northern, central and southern areas of the Italian peninsula plus Sicily were collected, including both "rural'' and "urban'' samples classified according to population density in the sampling area. A subset of individuals was additionally genotyped for Y-STR loci included in the Yfiler and PowerPlex Y23 (PPY23) systems (75% and 62%, respectively), allowing the comparison of RM and conventional Y-STRs. Considering the whole set of 13 RM Y-STRs, 1501 unique haplotypes were observed among the 1509 sampled Italian men with a haplotype diversity of 0.999996, largely superior to Yfiler and PPY23 with 0.999914 and 0.999950, respectively. AMOVA indicated that 99.996% of the haplotype variation was within populations, confirming that genetic-geographic structure is almost undetected by RM Y-STRs. Haplotype sharing among regional Italian populations was not observed at all with the complete set of 13 RM Y-STRs. Haplotype sharing within Italian populations was very rare (0.27% non-unique haplotypes), and lower in urban (0.22%) than rural (0.29%) areas. Additionally, 422 father-son pairs were investigated, and 20.1% of them could be discriminated by the whole set of 13 RM Y-STRs, which was very close to the theoretically expected estimate of 19.5% given the mutation rates of the markers used. Results obtained from a high-coverage Italian haplotype dataset confirm on the regional scale the exceptional ability of RM Y-STRs to resolve male lineages previously observed globally, and attest the unsurpassed value of RM Y-STRs for male-relative differentiation purposes. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

To investigate the male genetic legacy of the Arab rule in southern Europe during medieval times, we focused on specific Northwest African haplogroups and identified evolutionary close STR-defined haplotypes in Iberia, Sicily and the Italian peninsula. Our results point to a higher recent Northwest African contribution in Iberia and Sicily in agreement with historical data, southern Italian regions known to have experienced long-term Arab presence also show an enrichment of Northwest African types. The forensic and genomic implications of these findings are discussed.

Within an Italian collaborative exercise on the extended haplotype of the Y-chromosome, 1288 subjects were typed by the AmpFlSTR YFiler Amplification Kit (AB Applied Biosystems) and other 526 were typed by the PowerPlex Y ® System (Promega). The sampling scheme included either a "regional" or a "local" recruitment, the first referring to individuals born in the region of the participating lab, the second referring to individuals coming from small villages. Total sample sizes were N =954 and 860, respectively. A significant decrease of haplotype diversity was found in the local samples. The results may be of interest in forensic applications of the Y-chromosome.