Search Results (9)

This differential extraction protocol details the steps for isolating DNA from sample pads used in lateral flow immunochromatographic (LFI) tests, particularly for cases involving mixed biological samples such as semen and menstrual blood, or other evidence related to sexual assault. This procedure utilizes a differential extraction technique applied to sample pads from immunochromatographic tests, where the sample pads serve as the substrate. The method involves two sequential lysis steps to effectively separate non-sperm and sperm fractions, enabling the targeted isolation of distinct cell types for downstream DNA analysis. The efficiency of this procedure is demonstrated by the results within this paper, which highlights the successful recovery of both male autosomal and Y-STR profiles, even in mixed samples with a high female presence. Overall, this protocol demonstrates the effective recovery of DNA from sample pads, which is beneficial for forensic practitioners dealing with limited sample quantities, underscoring the value of using these pads in forensic analysis. Full article

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na₂EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell^® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis. Full article

DNA analysis plays a crucial role in forensic investigations, helping in criminal cases, missing persons inquiries, and archaeological research. This study focuses on the DNA concentration in different skeletal elements to improve human identification efforts. Ten cases of unidentified skeletal remains brought to the Institute of Forensic Medicine in Timisoara, Romania, underwent DNA analysis between 2019 and 2023. The results showed that teeth are the best source for DNA extraction as they contain the highest concentration of genetic material, at 3.68 ng/µL, compared to the petrous temporal bone (0.936 ng/µL) and femur bone (0.633 ng/µL). These findings highlight the significance of teeth in forensic contexts due to their abundant genetic material. Combining anthropological examination with DNA analysis enhances the understanding and precision of identifying human skeletal remains, thus advancing forensic science. Selecting specific skeletal elements, such as the cochlea or teeth, emerges as crucial for reliable genetic analyses, emphasizing the importance of careful consideration in forensic identification procedures. Our study concludes that automated DNA extraction protocols without liquid nitrogen represent a significant advancement in DNA extraction technology, providing a faster, more efficient, and less labor-intensive method for extracting high-quality DNA from damaged bone and tooth samples. Full article

In the realm of DNA testing with legal implications, the reliability and precision of genetic markers play a pivotal role in confirming or negating paternity claims. This study aimed to assess the potential utility of human leukocyte antigen (HLA) gene polymorphism through massively parallel sequencing (MPS) technology as robust forensic markers for parentage testing involving genetic deficiencies. It sought to redefine the significance of HLA genes in this context. Data on autosomal short tandem repeat (aSTR) mutational events across 18 paternity cases involving 16 commonly employed microsatellite loci were presented. In instances where traditional aSTR analysis failed to establish statistical certainty, kinship determination was pursued via HLA genotyping, encompassing the amplification of 17 linked HLA loci. Within the framework of this investigation, phase-resolved genotypes for HLA genes were meticulously generated, resulting in the definition of 34 inherited HLA haplotypes. An impressive total of 274 unique HLA alleles, which were classified at either the field 3 or 4 level, were identified, including the discovery of four novel HLA alleles. Likelihood ratio (LR) values, which indicated the likelihood of the observed data under a true biological relationship versus no relationship, were subsequently calculated. The analysis of the LR values demonstrated that the HLA genes significantly enhanced kinship determination compared with the aSTR analysis. Combining LR values from aSTR markers and HLA loci yielded conclusive outcomes in duo paternity cases, showcasing the potential of HLA genes and MPS technology for deeper insights and diversity in genetic testing. Comprehensive reference databases and high-resolution HLA typing across diverse populations are essential. Reintegrating HLA alleles into forensic identification complements existing markers, creating a potent method for future forensic analysis. Full article

This study assessed the usefulness of DNA quantification to predict the success of historical samples when analyzing SNPs, mtDNA, and STR targets. Thirty burials from six historical contexts were utilized, ranging in age from 80 to 800 years postmortem. Samples underwent library preparation and hybridization capture with two bait panels (FORCE and mitogenome), and STR typing (autosomal STR and Y-STR). All 30 samples generated small (~80 bp) autosomal DNA target qPCR results, despite mean mappable fragments ranging from 55–125 bp. The qPCR results were positively correlated with DNA profiling success. Samples with human DNA inputs as low as 100 pg resulted in ≥80% FORCE SNPs at 10X coverage. All 30 samples resulted in mitogenome coverage ≥100X despite low human DNA input (as low as 1 pg). With PowerPlex Fusion, ≥30 pg human DNA input resulted in >40% of auSTR loci. At least 59% of Y-STR loci were recovered with Y-target qPCR-based inputs of ≥24 pg. The results also indicate that human DNA quantity is a better predictor of success than the ratio of human to exogenous DNA. Accurate quantification with qPCR is feasible for historical bone samples, allowing for the screening of extracts to predict the success of DNA profiling. Full article

In this article, we describe multiple analytical strategies that were first developed for forensic purposes, on a set of three bone samples collected in 2011. We analyzed a single bone sample (patella) collected from the artificially mummified body of the Baron Pasquale Revoltella (1795–1869), as well two femurs which allegedly belonged to the Baron’s mother (Domenica Privato Revoltella, 1775–1830). Likely due to the artificial mummification procedures, the inner part of the Baron’s patella allowed the extraction of high-quality DNA yields, which were successfully used for PCR-CE and PCR-MPS typing of autosomal, Y-specific, and mitochondrial markers. The samples extracted from the trabecular inner part of the two femurs yielded no typing results by using the SNP identity panel, whereas the samples extracted from the compact cortical part of the same bone samples allowed genetic typing, even by the employment of PCR-CE technology. Altogether, 10/15 STR markers, 80/90 identity SNP markers, and HVR1, HVR2, and HVR3 regions of the mtDNA were successfully typed from the Baron’s mother’s remains by the combined use of PCR-CE and PCR-MPS technologies. The kinship analysis showed a likelihood ratio of at least 9.1 × 10⁶ (corresponding to a probability of maternity of 99.9999999%), and thus confirmed the identity of the skeletal remains as those of the Baron’s mother. This casework represented a challenging trial for testing forensic protocols on aged bones samples. It highlighted the importance of accurately sampling from the long bones, and that DNA degradation is not blocked by freezing at −80 °C. Full article

A civil paternity investigation involving the parents of the deceased alleged father in order to establish a family relationship is presented. On the basis of the 23 autosomal short tandem repeat (aSTR) genotyping results, conclusive proof of paternity was not achieved, as the probability of paternity (W) was calculated to 0.99988. Additional genetic data of 17 classical and non-classical human leukocyte alleles (HLA) typing by next-generation sequencing (NGS) at a high-resolution level supported the hypothesis of grandpaternity over the hypothesis of coincidental paternal obligate allele (POA) sharing (total W_aSTR&HLA = 0.9999998). The present study demonstrates the utility of 17 HLA genetic markers-typing in the solution of deficiency cases of disputed parentage. Full article

Microsatellites (or short-tandem repeats (STRs)) are widely used in anthropology and evolutionary studies. Their extensive polymorphism and rapid evolution make them the ideal genetic marker for dating events, such as the age of a gene or a population. This usage requires the estimation of mutation rates, which are usually estimated by counting the observed Mendelian incompatibilities in one-generation familial configurations (typically parent(s)–child duos or trios). Underestimations are inevitable when using this approach, due to the occurrence of mutational events that do not lead to incompatibilities with the parental genotypes (‘hidden’ or ‘covert’ mutations). It is known that the likelihood that one mutation event leads to a Mendelian incompatibility depends on the mode of genetic transmission considered, the type of familial configuration (duos or trios) considered, and the genotype(s) of the progenitor(s). In this work, we show how the magnitude of the underestimation of autosomal microsatellite mutation rates varies with the populations’ allele frequency distribution spectrum. The Mendelian incompatibilities approach (MIA) was applied to simulated parent(s)/offspring duos and trios in different populational scenarios. The results showed that the magnitude and type of biases depend on the population allele frequency distribution, whatever the type of familial data considered, and are greater when duos, instead of trios, are used to obtain the estimates. The implications for molecular anthropology are discussed and a simple framework is presented to correct the naïf estimates, along with an informatics tool for the correction of incompatibility rates obtained through the MIA. Full article

PDZD7, a PDZ domain-containing scaffold protein, is critical for the organization of Usher syndrome type 2 (USH2) interactome. Recently, biallelic PDZD7 variants have been associated with autosomal-recessive, non-syndromic hearing loss (ARNSHL). Indeed, we identified novel, likely pathogenic PDZD7 variants based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines from Korean families manifesting putative moderate-to-severe prelingual ARNSHL; these were c.490C>T (p.Arg164Trp), c.1669delC (p.Arg557Glyfs*13), and c.1526G>A (p.Gly509Glu), with p.Arg164Trp being a predominantly recurring variant. Given the recurring missense variant (p.Arg164Trp) from our cohort, we compared the genotyping data using six short tandem-repeat (STR) markers within or flanking PDZD7 between four probands carrying p.Arg164Trp and 81 normal-hearing controls. We observed an identical haplotype across three out of six STR genotyping markers exclusively shared by two unrelated hearing impaired probands but not by any of the 81 normal-hearing controls, suggesting a potential founder effect. However, STR genotyping, based on six STR markers, revealed various p.Arg164Trp-linked haplotypes shared by all of the affected subjects. In conclusion, PDZD7 can be an important causative gene for moderate to severe ARNSHL in Koreans. Moreover, at least some, if not all, p.Arg164Trp alleles in Koreans could exert a potential founder effect and arise from diverse haplotypes as a mutational hot spot. Full article