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.

Assessment protocol of mesothelioma and relevance of SEM-EDS analysis through a case studies of legal medicine of Brescia (Italy).

INTRODUCTION: This study evaluates the assessment protocol that allows the correlation between the development of mesothelioma to a specific exposure, with particular focus on investigations with Scanning Electron Microscope with Energy Dispersion Spectroscopy. METHODS: This retrospective study includes 80 subjects who died from mesothelioma in the period 2001-2019. A judicial autopsy was performed for each case to confirm cause of death and correlate the disease with specific asbestos exposure. In 28 cases investigations were carried out to determine the pulmonary load of the asbestos fibres and corpuscles in the lung tissue through microscopic investigations, in order to confirm the suspicion of occupational exposure. RESULTS: Our data agree with the scientific literature reported, but it is interesting to underline how the present study uses a different systematic approach than others, which are mainly based on epidemiological and environmental studies without considering the lung content of fibres and corpuscles. CONCLUSION: It would be desirable that the use of the microscopic analysis was introduced in the evaluation protocol: it should always be carried out if the suspicion of asbestos-related disease is raised and not only as a possible integration to the less expensive anamnestic evaluation, even more so if the work or personal history should be suggestive of exposure to asbestos fibres.

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.

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.

Social egg freezing under public health perspective: Just a medical reality or a women's right? An ethical case analysis.

In recent years, a social trend toward delaying childbearing has been observed in women of reproductive age. A novel technomedical innovation was commercialized for non-medical reasons to healthy, ostensibly fertile women, who wished to postpone motherhood for various reasons such as educational or career demands, or because they had not yet found a partner. As a consequence, these women may be affected by age-related infertility when they decide to conceive, and fertility preservation techniques can be obtained through the so-called social egg freezing. This paper examines, from an ethical point of view, the impact of social egg freezing under some aspects that can involve policy making and resources allocation in public health. Due to the increasing demand for this procedure, some debated issues regard if it is reasonable to include social egg freezing in Public Healthcare System and consequently how to manage the storage of cryopreserved oocytes also from individual donors, how to support these egg banks and how to face, in the future, with the possibility that egg freezing will play a role in enabling childbearing for gays, lesbians, and unmarried persons. Social freezing may be advertised to harmonise gender differences, but we wonder if it is the proper solution to the problem or if it could also create further challenges. An ethical argumentation on these topics should address some questions that will be discussed.

X-chromosome polymorphism on DXS8378, DXS7132, HPRTB and DXS7423 loci in 130 individuals from Brescia (northern Italy).

One hundred and thirty unrelated individuals (80 females and 50 males) from Brescia (northern Italy) were typed for DXS8378, DXS7132, HPRTB and DXS7423 loci. Allele, genotype and haplotype frequencies were calculated. A comparison between our population data and others from Caucasians and Asians populations was performed.

Validation of a large Italian Database of 15 STR loci.

Results from a collaborative exercise with proficiency testing conducted by 20 Italian laboratories on the 15 loci included in the Identifiler kit were analyzed by allele sharing methods and by standard population genetics tests. The validated database, including about 1500 subjects, was merged with that of a previous exercise conducted on nine loci, and the resulting allele frequencies, subdivided by Italian region, were published on-line.

Population data for 12 Y-chromosome STRs in a sample from Brescia (northern Italy).

Twelve Y-chromosome STRs--DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439--were typed in a population sample (n=104) of unrelated males from Brescia (northern Italy). A total of 91 haplotypes were identified by the 12 Y-STR loci. The haplotype diversity (98.68%), discrimination capacity (87.50%) and gene diversity were calculated.

Allele sharing in first-degree and unrelated pairs of individuals in the Ge F I AmpFlSTR Profiler Plus database.

Eleven Italian forensic laboratories participated in a population study based on the AB Profiler Plus loci with proficiency testing. The validated database, including 1340 individuals, is available on-line. Tests for Hardy-Weinberg equilibrium, gametic unbalance, and heterogeneity of gene frequency were generally not significant. Gene frequencies at each locus were consistent with those of two previously published Italian studies, but different from a third. Individuals of each subsample were paired, and the total number of alleles shared across the nine loci was determined in each pair. The analysis was replicated over the total sample. In addition, two samples of mother-child pairs (N=315) and full-sib pairs (N=91) were subjected to allele sharing analysis. The resulting distributions were sufficiently distinct from the sample of unrelated pairs as to be of practical usefulness.

Population data for 15 autosomal STRs loci and 12 Y chromosome STRs loci in a population sample from the Sardinia island (Italy).

One hundred twenty-five unrelated individuals (69 females and 56 males) from Sassari (Northern Sardinia) and Orgosolo (Central Sardinia) were typed for 15 STRs loci. The 56 males were typed for 12 Y chromosome STRs loci too. Frequency distribution is described.

Mixed stains from sexual assault cases: autosomal or Y-chromosome short tandem repeats?

We analyzed forensic DNA samples from four cases of sexual assault, using the Y-chromosome-specific human DNA markers and a panel of autosomal short tandem repeats (STRs). The presence of male contribution was evaluated by the analysis of the Amelogenin locus. A panel of tetrameric Y-STR (DYS19, DYS390, DYS391, DYS392, DYS385, DYS389I and II) was used in further analysis of samples, increasing the efficiency of the forensic genetic analyses. It was possible to identify a partial or full Y-profile of the rapists in different DNA mixtures when genetic profile could not be detected by autosomal STRs. However, in the case of male/male DNA mixture, only the victim's Y-profile could be obtained because the DNA of the offenders was present in low amounts. When the mixture contained different male/male proportion of DNA, only the full profile of the major component could be detected. In cases where male/female DNA mixed stains contained a sufficient amount of male DNA, the analysis of autosomal STRs was adequate enough to identify the full profile of the rapist. Our experience shows that the main advantage of the Y-STR approach is its ability to detect the male component in the mixed stains when the DNA of the male contributor is present only in a very small amount.

Haplotype studies support slippage as the mechanism of germline mutations in short tandem repeats.

Germline mutations of human short tandem repeat (STR) loci are expansions or contractions of repeat arrays which are not well understood in terms of the mechanism(s) underlying such mutations. Although polymerase slippage is generally accepted as a mechanism capable to explain most features of such mutations, it is still possible that unequal crossing over plays some role in those events, as most studies in humans could not exclude unequal crossing over (UCO). Crossing over can be studied by analyzing haplotypes using flanking markers. To check for UCO in mutations, we have analyzed 150 paternity cases for which more than the usual trio (mother, child, and father) were available for testing by analyzing 16 STR loci. In a total of 4900 parent-child allele transfers four mutations were observed at different loci (D8S1179, D18S51, D21S11, and SE33/ACTBP2). To identify the mutated allele and to check for UCO, we typed at least four informative loci flanking the mutated locus and used the pedigree data to establish haplotypes. By doing so we were able to exclude UCO in each case. Moreover, we were able to identify the mutations as one-repeat contractions/expansions. Our data thus support slippage as the mechanism of germline mutations in STRs.

Identification of forensic samples by using an infrared-based automatic DNA sequencer.

We have recently introduced a new protocol for analyzing all core loci of the Federal Bureau of Investigation's (FBI) Combined DNA Index System (CODIS) with an infrared (IR) automatic DNA sequencer (LI-COR 4200). The amplicons were labeled with forward oligonucleotide primers, covalently linked to a new infrared fluorescent molecule (IRDye 800). The alleles were displayed as familiar autoradiogram-like images with real-time detection. This protocol was employed for paternity testing, population studies, and identification of degraded forensic samples. We extensively analyzed some simulated forensic samples and mixed stains (blood, semen, saliva, bones, and fixed archival embedded tissues), comparing the results with donor samples. Sensitivity studies were also performed for the four multiplex systems. Our results show the efficiency, reliability, and accuracy of the IR system for the analysis of forensic samples. We also compared the efficiency of the multiplex protocol with ultraviolet (UV) technology. Paternity tests, undegraded DNA samples, and real forensic samples were analyzed with this approach based on IR technology and with UV-based automatic sequencers in combination with commercially-available kits. The comparability of the results with the widespread UV methods suggests that it is possible to exchange data between laboratories using the same core group of markers but different primer sets and detection methods.