Forensic DNA Typing From Femurs and Bones of the Foot: A Study of 3 Cases.

ABSTRACT: Evidence has been accumulating in the sense that femur may not always be the best option for DNA typing of skeletal remains. Recent studies have shown that bones of the hands and feet appear to be a superior source of preserved DNA. The current study reanalyzed DNA quantitation, degradation, and short tandem repeat typing in femurs, lateral cuneiforms, and distal foot phalanges. Data from 3 human identification cases involving corpses in an advanced decomposition state were collected. We found that in the studied cases, the femur provided equal or inferior results, recovering 84.9% of true alleles. Lateral cuneiforms (99.2%) and distal foot phalanges (96.8%) yielded higher percentages. In addition, more drop-ins and drop-outs were detected in femurs than cuneiforms and phalanges. This study adds to current findings that advocate for further investigation into bone selection for use in forensic practice. The impacts of our findings are limited by the small number of individuals studied and may not apply to old and degraded bones.

Large fragment demineralization: an alternative pretreatment for forensic DNA typing of bones.

In forensic genetics, the analysis of postmortem bones is one of the most challenging due to the low quantity of degraded endogenous DNA. The most widely used approach for sample preparation, in those cases, is pulverizing the bone. However, processing pulverized bone is extremely delicate, requiring strict laboratory conditions and operating procedures. In fact, several recent publications have focused on non-powder approaches. The objectives of this study were, thus, to validate a non-powder protocol for DNA extraction from forensic bones and an alternative pretreatment, large fragment demineralization (LFD). Thirty human femurs and tibiae received by the Legal Medicine Institute of Brescia, Italy, were included in the study. Bone powder and one transversal section of the diaphysis were sampled from each bone. DNA extraction from the powder was carried out using PrepFiler BTA (BTA), while the transversal section was submitted to the alternative demineralizing pretreatment (LFD) followed by DNA extraction using the QIAamp DNA Investigator. DNA extracts were assessed for human DNA quantity and degradation by means of a validated in-house qPCR assay and amplified with commercial kits. Inhibition assessment was carried out through Quality Sensor analysis using 24plex QS Kit. The differences in quantity, quality of human DNA, and number of alleles detected between both methods were comparable and not statistically significant. We propose the use of the LFD protocol as a complementary approach capable of confirming the genotypes or detect alleles not observed using BTA, without the need for pulverization.

DNA quantitation and degradation assessment: a quantitative PCR protocol designed for small forensic genetics laboratories.

For over 10 years, quantitative PCR (qPCR) for DNA quantitation has been reported in forensics. However, assays have not been described for small qPCR platforms. Thus, technological advancement is not always implemented in small forensic genetics laboratories. A duplex qPCR assay is reported, using a StepOne instrument and targeting a short and a long human DNA region. This study was performed according to international validation guidelines, including sensitivity, repeatability, reproducibility, precision, accuracy, contamination assessment, known and case-type samples, and degradation studies. Characterization of the genetic markers, species specificity, and population studies had already been conducted. Moreover, case-type samples were quantified, amplified using commercial kits and the number of alleles detected was recorded. Sensitivity was shown to be 10 pg/µL. Standard curve replicates demonstrated the assay is accurate, precise, as well as fairly repeatable and reproducible. The NGM Detect kit was shown to yield higher peaks than Identifiler Plus and NGM Select for degraded samples. Moreover, quality sensors were always present and proved useful. The quantification values of the large target showed a correlation with the number of alleles detected in the STR profiles for known and casework samples. The degradation index was shown to be informative, with a value of 10 or higher indicating dropout. It is suggested that after quantitation, samples with low or degraded DNA be amplified using newer amplification kits containing quality sensors to confirm that the low-quality profile was not affected by inhibition.

PowerPlex® Fusion 6C System: evaluation study for analysis of casework and database samples.

AIM: To report on the successful analysis of amplicons obtained with PowerPlex® Fusion 6C System, a highly robust 27-plex genotyping kit developed for human identification laboratories, on the Applied Biosystems® 3500 Genetic Analyzer. METHOD: We performed characterization and evaluation studies following the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines, examining several critical areas of kit performance. We report the results of sensitivity, robustness, heterozygous peak height ratio, precision, concordance, caseworks, and mixture interpretations. We tested sensitivity, using serial dilutions of control DNA. RESULTS: The minimum amount of input DNA resulting in a full profile was 125 pg. Inhibition, inducted by urea, showsed a progressively fragmentation of DNA and a full profile was obtained until 1M of inhibitor factor. To test the profile quality, casework samples were extracted with different extraction methods: Chelex®100, QIAmp DNA Micro Kit and Phenol-Chloroform extraction. The results demonstrated that extraction chemistries do not have affect on amplification performance. Concordance check was performed by typing some casework samples and comparing the typing results with those obtained with other available kits. Thus, concordance was expected and supported by the data. CONCLUSION: Reliable DNA typing results can be obtained using this new kit, demonstrating its effectiveness and utility in forensic analysis.

Y-chromosome polymorphisms and ethnic group - a combined STR and SNP approach in a population sample from northern Italy.

AIM: To find an association between Y chromosome polymorphisms and some ethnic groups. METHODS: Short tandem repeats (STR) and single-nucleotide polymorphisms (SNP) on the Y chromosome were typed in 311 unrelated men from four different ethnic groups - Italians from northern Italy, Albanians, Africans from the Maghreb region, and Indo-Pakistanis, using the AmpFlSTR® Yfiler PCR Amplification Kit and the SNaPshot Multiplex Kit. RESULTS: STRs analysis found 299 different haplotypes and SNPs analysis 11 different haplogroups. Haplotypes and haplogroups were analyzed and compared between different ethnic groups. Significant differences were found among all the population groups, except between Italians and Indo-Pakistanis and between Albanians and Indo-Pakistanis. CONCLUSIONS: Typing both STRs and SNPs on the Y chromosome could become useful in determining ethnic origin of a potential suspect.

Generation of the induced pluripotent stem cell line UNIBSi017-A from an individual with cardiospondylocarpofacial syndrome and the MAP3K7 c.737-7A > G variant.

TAK1 is a serine threonine kinase that mediates signal transduction induced by TGFß and bone morphogenetic proteins, and controls a variety of cell functions by modulating the downstream activation of NF-kkB, JNK, and p38. Heterozygous variants in the coding MAP3K7 gene cause the cardiospondylocarpofacial syndrome, characterized by various abnormalities. Skin fibroblasts derived from a patient carrying the MAP3K7 c.737-7A>G heterozygous variant were reprogrammed using Sendai viral vector system carrying the Yamanaka factors. The generated induced pluripotent stem cells (iPSC) line retained the original genotype, expressed pluripotency markers, and differentiated into cells of the three germ layers.

Genetic variation at 5 new autosomal short tandem repeat markers (D10S1248, D22S1045, D2S441, D1S1656, D12S391) in a population-based sample from Maghreb region.

AIM: To investigate allele distribution and genetic parameters of a population-based sample from Maghreb region. METHODS: Allele frequencies for 5 new autosomal short tandem repeat (STR) markers (D10S1248, D22S1045, D2S441, D1S1656, and D12S391) and several forensic parameters were determined for 95 unrelated individuals. RESULTS: The combined power of discrimination and power of exclusion for the 5 loci were high (0.9999991 and 0.9954757, respectively). Allele frequencies were compared with previously published population data. Significant differences were found between Maghreb population and all other populations at the locus D2S441. Also, significant differences were found between the Maghreb and the African American population at the D22S1045, D1S1656, and D12S391 loci, between Maghreb and Caucasian population at the D1S1656 locus, and between Maghreb and Hispanic population at the D22S1045 locus. CONCLUSIONS: Typing of the 5 new STR loci may provide a useful addition to the previously established sets of autosomal STRs.

Human identification through DNA analysis of restored postmortem teeth.

The identification of human remains using DNA analysis can be extremely challenging and its success is certainly influenced by the time elapsed since death. In that context, intact teeth have been shown to be highly successful in DNA analysis. However, restored teeth are usually available and, surprisingly, these specimens have been poorly studied. In fact, there are no reports regarding forensic DNA analysis of those types of samples in real cases. Therefore, the aim of this study was to perform DNA typing on healthy and restored teeth from exhumed human remains, which had been buried for 46 years. A powder-free DNA extraction protocol specifically designed for teeth was followed and human DNA quantitation and degradation assessment was performed using an in-house qPCR assay. Samples were amplified with commercial human identification kits for autosomal and Y chromosome markers. The obtained DNA profiles were compared to those of a previously processed femur sample as well as a buccal swab from a putative son. One healthy and one restored tooth yielded complete, concordant and compatible DNA profiles with previously typed samples from the femur and the putative son. Biostatistical calculations supported the paternity relationship with a likelihood ratio greater than 11 million. The present study highlights the use of restored teeth in a real exhumation case and the powder-free approach specifically designed for the extraction of DNA from teeth is discussed.

Discovering a double murder through skeletal remains: A case report.

Forensic examination of human remains is a complex process that relies on the contribution of multidisciplinary forensic medicine specialties. Here we present a complex forensic case regarding a double murder whose victims were found almost completely skeletonized. Post-mortem investigations allowed us to define the biological profile of the two bodies (ancestry, sex, age and stature), to discover their identity through forensic DNA analysis, and to detect peri-mortem injuries caused by firearms and stabbing weapons. Three men were recognized as involved in the crime and two of them were condemned to life imprisonment for homicide. The judges accepted the reconstruction of the crime promoted by the Prosecutor (double firearm murder).

Living with the dead: A case report and review of the literature.

The discovery of human corpses in urban domestic settings does not constitute an unusual case in criminal casework. These scenarios can be very challenging to investigate since the uninformative evidences encountered also demand a multidisciplinary effort among several specialties in the forensic sciences field. The occurrence of this incident is usually accompanied by social isolation, which is an emblematic aspect of urban modern society. The elderly population is especially susceptible to being socially isolated, which is associated with higher mortality. We present a case report of an elderly woman who had been living with her husband's dead body, contributing to the scarce literature on the "Living with the Dead" phenomenon. The use of a multidisciplinary approach and the challenges that social isolation presents to forensic sciences and the contemporary society are discussed.

Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a KCNA5 p.D322H mutation.

Atrial fibrillation (AF) is the most common sustained arrhythmia associated with several cardiac risk factors, but increasing evidences indicated a genetic component. Indeed, genetic variations of the atrial specific KCNA5 gene have been identified in patients with early-onset lone AF. To investigate the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a KCNA5 p.D322H mutation, using a commercially available non-integrating system. The generated iPSCs expressed pluripotency markers and differentiated toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the p.D322H mutation.

Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a PITX2 p.M200V mutation.

Atrial fibrillation (AF) is the most common sustained arrhythmia associated with several cardiac risk factors, but increasing evidences indicated a genetic component. Indeed, genetic variations of the specific PITX2 gene have been identified in patients with early-onset AF. To investigate the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a PITX2 p.M200V mutation, using a commercially available non-integrating expression system. The generated iPSCs expressed pluripotency markers and differentiated toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the PITX2 p.M200V mutation.

Post-mortem injuries by a dog: a case report.

Despite its unlikely occurrence, post-mortem animal depredation is not unknown to forensic pathologists. In the case at issue, the corpse of a dead woman presented extensive facial wounds, which were then traced back to the dog she owned. A small specimen of injured tissue was subjected to species diagnosis, and came back positive for human and canine antigens, which confirmed the presence of biological material of canine origin on the body. The less than usual post-mortal injury pattern described herein clearly highlights the possibility that animal depredation on a corpse may occur soon after death, and underscores the diagnostic potential posed by commonly available and low expensive testing methods such as serological species diagnosis.