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The identification of human fire victims is a challenging task in forensic medicine. The heat-induced alterations of biological tissues can make the conventional anthropological analyses difficult. Even if the DNA profile of the victim is achieved, it is possible that no match can be found in a forensic DNA database, thus hindering positive identification. In such cases, any information useful to nail down a possible identity should be collected, such as DNA methylation analysis which could provide useful investigative leads. In the present study, five age-related epigenetic markers (ELOVL2, FHL2, KLF14, C1orf132, and TRIM59) were initially analysed in blood samples of 72 living Italian individuals of known age, using a Single Base Extension (SBE) assay. An age prediction model was built by multiple linear regression including all the markers (Mean Absolute Error, MAE: 3.15 years). This model was tested on 29 blood samples collected during autopsies from burnt human remains, already identified through DNA analysis, providing a MAE of 6.92 years. The model allowed a correct prediction in 79.3% of the cases (95% prediction interval), while six cases were associated with inaccurate predictions (min-max prediction error: 9.8-37.3 years). Among the different sample variables considered to explain these results, only the DNA degradation index was a relevant factor affecting the reliability of the predictions. In conclusion, the SBE typing of blood from burnt remains proved to be a reliable tool to estimate chronological age of most of the samples, also in consideration of its cost-effectiveness and the availability of CE sequencers in every forensic genetics laboratory., (© 2024. The Author(s).)

Introduction: "Tethered cord syndrome" (TCS) refers to a congenital abnormality associated with neurological signs and symptoms. The aim of surgery is to prevent or arrest their progression. This study reports a retrospective case series of tethered cord syndrome surgeries, supported by intraoperative neurophysiological monitoring., Methods: The case series comprises 50 surgeries for tethered cord syndrome in which multimodal intraoperative neurophysiological monitoring was performed using motor evoked potentials (transcranial motor evoked potentials [TcMEPs]), tibial nerve somatosensory evoked potentials (TNSEPs), and pudendal-anal reflex (PAR). The intraoperative neurophysiological monitoring results are reported and correlated with clinical outcomes., Results: Sensitivity, specificity, and negative predictive value were high for TcMEPs and TNSEPs, while PAR exhibited low sensitivity and positive predictive value but high specificity and negative predictive value. Fisher's exact test revealed a significant correlation between changes in TcMEPs, TNSEPs, and clinical outcome ( P < 0.000 and P = 0.049 respectively), but no correlation was detected between PAR and urinary/anal function ( P = 0.497)., Conclusions: While TcMEPs and TNSEPs were found to be reliable intraoperative neurophysiological monitoring parameters during tethered cord syndrome surgery, PAR had low sensitivity and positive predictive value probably because the reflex is not directly related to bladder function and because its multisynaptic pathway may be sensitive to anesthetics. New onset muscle weakness and sensory deficits were related to postoperative changes in TcMEPs and TNSEPs, whereas changes in PAR did not predict bladder/urinary impairment. Urinary deficits may be predicted and prevented with other neurophysiological techniques, such as the bladder-anal reflex., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 by the American Clinical Neurophysiology Society.)

The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated analyses, over-amplified samples, or unnecessary examinations. In our laboratory, we use the Quantifiler ® Trio system to quantify DNA extracts from a wide range of samples extracted from traces (bloodstains, saliva, semen, tissues, etc.), including swabs from touched objects, which are very numerous in the forensic context. This method has been extensively used continuously for nine years, following an initial validation process, and is part of the ISO/IEC 17025 accredited method. In routine practice, based on the quantitative values determined from the extracts of each trace, we use a standard method or a low-copy-number method that involves repeating the amplification with the generation of a consensus genetic profile. Nowadays, when the quantification results are less than 0.003 ng/μL in the minimum extraction volume (40 μL), we do not proceed with the DNA extract analysis. By verifying the limits of the method, we make a conscious cost-benefit choice, in particular by using the least amount of DNA needed to obtain sufficiently robust genetic profiles appropriate for submission to the Italian DNA Forensic Database. In this work, we present a critical re-evaluation of this phase of the method, which is based on the use of standard curves obtained from the average values of the control DNA analysed in duplicate. Considering the various contributions to uncertainty that are difficult to measure, such as manual pipetting or analytical phases carried out by different operators, we have decided to thoroughly investigate the contribution of variability in the preparation of calibration curves to the final results. Thus, 757 samples from 20 independent experiments were re-evaluated using two different standards for the construction of curves, determining the quantitative differences between the two methods. The experiments also determined the parameters of the slope, Y-intercept, R 2 , and the values of the synthetic control probe to verify how these parameters can provide information on the final outcome of each analysis. The outcome of this revalidation demonstrated that it is preferable to use quantification ranges rather than exact quantitative limits before deciding how to analyse the extracts via PCR or forgoing the determination of profiles. Additionally, we present some preliminary data related to the analysis of samples that would not have been analysed based on the initial validation, from which genetic profiles were obtained after applying a concentration method to the extracts. Our goal is to improve the accredited analytical method, with a careful risk assessment as indicated by accreditation standards, ensuring that no source of evidence is lost in the reconstruction of a criminal event.

•New insight into prognostic factors for recovery of clinical function following posterior decompression for degenerative cervical myelopathy.•An increase of IOM amplitude of at least 50% coupled with preoperative T2-only and diffuse T2 signal changes on MRI is a positive prognostic factors for clinical improvement 6 months after surgery.•Clinical improvement at 6 months follow-up can be expected in patients with T1 hypo intensity if a diffuse border of the lesion on T2 images is present., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Marco Teli reports a relationship with Medtronic Inc that includes: consulting or advisory and travel reimbursement., (© 2022 The Authors.)

Next generation sequencing (NGS) is the emerging technology in forensic genomics laboratories. It offers higher resolution to address most problems of human identification, greater efficiency and potential ability to interrogate very challenging forensic casework samples. In this study, a trial set of DNA samples was artificially degraded by progressive aqueous hydrolysis, and analyzed together with the corresponding unmodified DNA sample and control sample 2800 M, to test the performance and reliability of the ForenSeq TM DNA Signature Prep kit using the MiSeq Sequencer (Illumina). The results of replicate tests performed on the unmodified sample (1.0 ng) and on scalar dilutions (1.0, 0.5 and 0.1 ng) of the reference sample 2800 M showed the robustness and the reliability of the NGS approach even from sub-optimal amounts of high quality DNA. The degraded samples showed a very limited number of reads/sample, from 2.9-10.2 folds lower than the ones reported for the less concentrated 2800 M DNA dilution (0.1 ng). In addition, it was impossible to assign up to 78.2% of the genotypes in the degraded samples as the software identified the corresponding loci as "low coverage" (< 50x). Amplification artifacts such as allelic imbalances, allele drop outs and a single allele drop in were also scored in the degraded samples. However, the ForenSeq TM DNA Sequencing kit, on the Illumina MiSeq, was able to generate data which led to the correct typing of 5.1-44.8% and 10.9-58.7% of 58 of the STRs and 92 SNPs, respectively. In all trial samples, the SNP markers showed higher chances to be typed correctly compared to the STRs. This NGS approach showed very promising results in terms of ability to recover genetic information from heavily degraded DNA samples for which the conventional PCR/CE approach gave no results. The frequency of genetic mistyping was very low, reaching the value of 1.4% for only one of the degraded samples. However, these results suggest that further validation studies and a definition of interpretation criteria for NGS data are needed before implementation of this technique in forensic genetics., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The article presents a case of multiple casualties following a textile factory fire. The incident required a full DVI team similar to large mass-disaster because of the specific operational aspects and identification difficulties. The autopsy results were consistent with death by fire and the toxicological investigations revealed carbon monoxide poisoning in four cases (HbCO% ranging between 88,05 and 95,77), two deaths by cyanide intoxication (with concentrations between 5,17 and 8,85 mcg/ml), and in one case there was a synergistic effect of the two substances (carbon monoxide and cyanide). The identification, carried out in accordance with INTERPOL protocols, encountered serious difficulties in the AM phase primarily due to a language barrier and the lack of any dental or medical information relating to the victims. Secondary identifiers proved to be very useful in corroborating possible identities. As a result of the combined efforts of a team of experts the identity of each victim was determined and in all cases at least one primary identifier was used in the identification process. The deployment of DVI teams composed of forensic experts from different fields of expertise and well acquainted with DVI procedures, is essential in events involving multiple casualties that may also include foreign victims. The DVI team should intervene not only in PM examinations but also in the collection of AM data for those individuals not accounted for and by helping police in contacting families of missing people.

Identification of human remains can be hindered by several factors (e.g., traumatic mutilation, carbonization or decomposition). Moreover, in some criminal cases, offenders may purposely adopt various expedients to thwart the victim's identification, including the dissolution of body tissues by the use of corrosive reagents, as repeatedly reported in the past for Mafia-related murders. By means of an animal model, namely porcine samples, we evaluated standard DNA typing as a method for identifying soft (muscle) and hard (bone and teeth) tissues immersed in strong acids (hydrochloric, nitric and sulfuric acid) or in mixtures of acids (aqua regia). Samples were tested at different time intervals, ranging between 2 and 6h (soft tissues) and 2-28 days (hard tissues). It was shown that, in every type of acid, complete degradation of the DNA extracted from soft tissues preceded tissue dissolution and could be observed within 4h of immersion. Conversely, high molecular weight DNA amenable to STR analysis could be isolated from hard tissues as long as cortical bone fragments were still present (28 days for sulfuric acid, 7 days for nitric acid, 2 days for hydrochloric acid and aqua regia), or the integrity of the dental pulp chamber was preserved (7 days, in sulfuric acid only). The results indicate that DNA profiling of acid-treated body parts (in particular, cortical bone) is still feasible at advanced stages of corrosion, even when the morphological methods used in forensic anthropology and odontology can no longer be applied for identification purposes., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

In a standard paternity testing, mother, child, and alleged father are analyzed with STR markers using commercially available kits. Since Italian civil legislation does not have thresholds to confirm a paternity, paternity is practically proven when likelihood ratio increases prior probability of paternity to posterior, accepted by court as sufficient. However, in some cases the number of markers included in a commercial kit may be insufficient to conclusively prove or disprove a relationship between individuals, especially when complex family scenarios are suspected or indirect analyses are required. Additional genetic information can increase the values of the likelihood ratio regarding the detection of true parental relationships in a pedigree, while reducing the chances of false attributions (e.g. false paternities). In these cases the introduction of a 26Plex amplification system allows to examine 23-26 additional markers depending on the commercial kit used, thus increasing the statistical power of the kinship analysis. The PCR conditions were optimized for a multiplex amplification system and a new generation CE instrument. In order to demonstrate the utility of additional STRs markers, four complex kinship cases are presented., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In a case of robbery in which the criminals passed through the garden adorned with calamondin trees (Citrus madurensis), the investigators found in the grass six calamondin fruits, some undamaged, while others apparently bitten. The fruits were collected and sent to the laboratory for DNA analysis to verify the presence of saliva and robbers' DNA profile. A specific immunochromatographic strip test for saliva confirmed the presence of human salivary α-amylase, but similar positive results were also observed for intact calamondin and other citrus fruits. Further analysis with a specific automated amylase test confirmed the absence of amylase activity. DNA quantification and typing using a specific forensic kit revealed no human DNA presence in any fruits. This case report demonstrates for the first time the occurrence of false positives when human saliva is sought on citrus fruits., (© 2014 American Academy of Forensic Sciences.)

The unequivocal tissue identification in forensic casework samples is a key step for crime scene reconstruction. Just knowing the origin of a fluid can sometimes be enough to either prove or disprove a fact in court. Despite the importance of this test, very few data are available in literature concerning human saliva identification in old forensic caseworks. In this work the stability of human α-amylase activity in aged samples is described by using three different methods integrated with DNA profiling techniques. This analytical protocol was successfully applied on 26-years old samples coming from anonymous threat letters sent to prosecutors who were working on "the Monster of Florence", a case of serial murders happened around Florence (Italy) between 1968 and 1985., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Allele frequencies for 26 short tandem repeats (STRs) were obtained from a sample of 203 unrelated individuals living in the area of Florence, Prato and Pistoia (Tuscany, Central Italy). These 26 loci are in addition to the existing 13 U.S. core loci and can help provide assistance in complex kinship testing when, for example, the alleged father is not available for testing. The results were compared with U.S. Caucasian, African American and Hispanic populations.

Mitochondrial DNA (mtDNA) U/K and J/T are sister haplogroups within the superhaplogroup R. They are both common in Europe, with a combined overall frequency similar to the one reported for H, the most common European haplogroup (40-50%). In this study, we selected 159 Italian subjects, already ascribed to U/K and J/T by RFLP typing, and assigned each mtDNA to specific clades/subclades by investigating at least one diagnostic coding region SNP. For each sister haplogroup, one multiplex PCR and one SNaPshot minisequencing reaction were set up targeting 16 U/K and 7 J/T coding region SNPs. Each mtDNA sample was clearly assigned to a specific subclade, which could be further subdivided into several minor sub-branches according to peculiar HVS I/II motifs. Such a molecular dissection of haplogroups U/K and J/T could be extremely useful to reduce the overall analysis time and labor intensive sequencing procedures in high volume forensic casework, for example when it is important to rapidly exclude samples in order to restrict the number of suspects.

A CE/UV method was developed to separate by a micellar system the four DNA bases and other five purinic-pyrimidinic compounds (5-methyl-cytosine, uracil, xanthyne, hypoxanthyne and 5-bromo-uracil). Selectivity, precision, accuracy and sensitivity were assessed and proved to be suitable for the analysis of the primary structure of DNA. This method was adopted to study 16 aged samples including two Egyptian mummies, formaldehyde-fixed paraffin-embedded tissues and other forensic specimens. Lower relative values of the four canonical unmodified DNA bases (uDNAb) and more complex pherograms were found in the aged samples when compared with the modern controls. The results of the CE analysis, together with those obtained by classical molecular methods (agarose gel electrophoresis, DNase I and RNase A assays, and UV spectrophotometry), were finally evaluated for assessing the reliability of STR typing. Since samples with low uDNAb showed no amplification or unreliable STR profiles, the uDNAb value is discussed as a further quality criterion in the evaluation of the genetic data obtained from aged samples.

Microsatellite instability (MSI) is present in hereditary conditions due to mismatch repair (MMR) gene mutations. Following MSI analysis, tumor samples are classified into MSS (stable), MSI-L (low instability), and MSI-H (high instability) based on the fraction of unstable loci. Another MSI-based classification takes into account the size difference between mutant alleles in tumor DNA compared to wild-type alleles; two types of MSI, A and B, are recognized using this approach, type A being characterized by smaller, more subtle allelic shifts compared to type B. Biallelic mutations of MMR genes are associated with pediatric cancers, including glial tumors, in Turcot syndrome type 1 (TS1). However, most TS1-associated gliomas so far analyzed did not display MSI. We investigated the frequency of MSI in a series of 34 pediatric gliomas of different grade using a panel of five mononucleotide quasimonomorphic markers. Subtle qualitative changes were observed for the majority of markers in two glioblastomas (5.9% of the total series and 33.3% of glioblastomas). In both cases, family histories were compatible with TS1, and mutations of the PMS2 and MLH1 genes were identified. In one family, the MSI patterns were compared between the glioblastoma and a colon cancer from an affected relative, showing a clear qualitative difference, with the former displaying type A and the latter type B instability, respectively. These results were confirmed using additional microsatellite markers, indicating that knowledge of the association between TS1-related glial tumors and subtle type A MSI is important for full ascertainment of TS1 patients and appropriate counselling.

Allele frequencies for the 13 CODIS markers plus D2S1358 and D19S433 loci were estimated in a sample of 148 unrelated individuals from Bolivia and parameters of forensic interest were calculated. Further, the STR data were analyzed using a distance-based method to assess the genetic relationships of this population with other ones living in Argentina, Brazil, Costa Rica, Mexico, Peru, Venezuela and three autochthonous populations living in the Beni Department of Bolivia (Quechua, Aymara and Beni population).

Objective: Vascular damage in the cavernous sinus can cause ischemic injury to the cranial nerves. An appropriate anatomical knowledge of the blood supply to the cranial nerves can help to reduce the morbidity associated with cavernous sinus surgery., Material and Methods: Three formalin-fixed and six adult cadaveric fresh heads, with common carotid arteries injected, were used for anatomical dissection in this study. A fronto-temporal craniotomy was performed and the cavernous sinus was explored according to the Dolenc technique. With microsurgical dissection and photographic documentation, we demonstrate the anatomy of the superior orbital fissure artery in the antero-medial triangle., Results: The 12 explored cavernous sinuses demonstrated the presence of two principal branches directly from the intracavernous internal carotid artery that supply the cranial nerves: the infero-lateral trunk and the meningohypophyseal trunk. The artery of the Superior Orbital Fissure (SOF), originated more often from the infero-lateral trunk, and vascularized the III, IV, VI, and VI, and ophtalmic division of the trigeminal nerve (TGN VI) at their entry in the fissure., Conclusion: In this study we demonstrate that the superior orbital fissure artery is a branch from the infero-lateral trunk which runs immediately under the reticularis layer at the level of the anteromedial triangle in the lateral wall of the cavernous sinus. The blood supply to all cranial nerves in the SOF is at risk to injury when the lateral wall of the cavernous sinus is transgressed at the anteromedial triangle since the SOF-artery runs superficially at this level.

Allele frequencies for 17 STRs, together with some parameters of forensic interest, were estimated in a sample of 835 unrelated individuals born in Tuscany, an Italian region. These data were compared with Italian, Chinese, Kosovo Albanian, Romanian and Tunisian populations, strongly represented in this area. No significant differences in single loci were detected, except for Chinese in comparison with all the other populations.

We report an unusual paternity test case showing multiple peculiarities. Using AmpFlSTR Profiler Plus and AmpFlSTR Identifiler PCR Amplification kits, the alleged father and the two children were apparently homozygous at the FGA locus, but using the PowerPlex 16 kit the three individuals were found to be heterozygous. Drop-out was caused by a single mutation event in the presumptive binding site of the reverse primer. In addition, three inconsistencies were detected between the daughter and the alleged father among 18 STR markers. The occurrence of the rare null allele at the FGA locus and case history suggested that the true father was the brother of the alleged father. Furthermore, a single-step repeat maternal mutation was also detected at D16S539. This puzzling case was solved by using multiple analytical approaches, including the use of different primer pairs, the use of a high number of STR markers, and the characterization of the mutation causing the "null allele."

Sequence variation of the hypervariable segments (HVS) I/II of mitochondrial DNA (mtDNA) and the haplogroup affiliation were determined in a sample of 271 Italian subjects. This analysis showed that 42% of the individuals could be ascribed to H, the most frequent haplogroup in European Caucasian populations. This fraction was then screened for specific single nucleotide polymorphisms located in the coding region to identify H subclades H1-H15. We set up two multiplex polymerase chain reactions and specific SNaPshot assays to investigate the frequency distribution of these subgroups in our population sample and to examine their usefulness in discriminating among commonly shared HVS I/II sequences. This allowed the assignment of a large portion of the mtDNAs ( approximately 70%) to specific subhaplogroups, with H1 and H5 being the most represented. About two-thirds of the individuals sharing common HVS I/II sequences were subdivided and ascribed to specific H subhaplogroups with a significant reduction of the frequencies of the most common mtDNA haplotypes. Haplogroup H subtyping could thus be extremely useful in forensic identification when many samples have to be analysed and compared, avoiding excessive time-consuming and labor-intensive sequencing analysis.

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.

Objects: The authors report their experience of gamma knife radiosurgery (GKR) in a large series of pediatric cerebral arteriovenous malformations (cAVMs). The advantages, risks and failures of this approach are presented and discussed., Methods: Gamma knife radiosurgery was performed on 63 children aged < or =16 years. Haemorrhage was the clinical onset in 50 out of 63 cases. The mean pre-GK cAVM volume was 3.8 cm(3). Fifty-eight out of 63 cAVMs were Spetzler-Martin grades I-III. Most lesions (47 out of 63) were in eloquent or deep-seated brain regions., Conclusion: Gamma knife radiosurgery-related complications occurred in 2 out of 47 cases with an available follow-up (1 had transient and 1 permanent morbidity). No bleeding occurred during the latency period. In 39 children with >36-month follow-up, complete cAVM occlusion was angiographically documented in 31, with a 3- and 4-year actuarial obliteration rate of 72 and 77% respectively. High rates of complete obliteration and very low frequency of permanent morbidity with no bleeding during the latency period encourage widespread application of GKR in the treatment of pediatric cAVMs.

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.

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.

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.

We present a paternity testing case in which a double incompatibility was found for two short tandem repeat (STR) markers, human fibrinogen alpha (FGA) and Penta E. Analysis of the trio (mother, father, and daughter) included the amplification with a battery of 15 autosomal short tandem repeats (STR) by using a commercially available PowerPlex 16 System kit, and the detection with an ultraviolet-automatic sequencer. The biological paternity was confirmed with 12 additional markers. Reanalysis of the trio for the same markers with different primers was carried out by using an infrared automated sequencer and infrared-fluorescent primers. High paternity index confirmed that the observed inconsistencies were due to a double mutation, which was confirmed by sequence analysis at FGA and Penta E loci. Amplification and detection results obtained by the infrared-protocol showed consistent results with those obtained by ultraviolet-protocol and a commercially available kit. This has been our first case of double mutation at FGA and Penta E in a paternity testing. The use of our approach, based on two amplification and detection formats and on the sequence analysis, confirmed the observed meiotic paternal mutations.

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.

Eight Y-chromosomal short tandem repeats (STRs), DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393 and DYS385, were typed in a population sample (n=101) of first-generation Albanian immigrants living in Italy.

Allele frequencies for the 13 STRs of the Combined DNA Index System (CODIS) core were obtained from a sample of 188 unrelated individuals living in the area of Florence, Prato and Pistoia (Tuscany, Central Italy).

A sample of 1176 males from 10 Italian regions have been typed for DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, and DYS385. Individual haplotype data are available on line. A low degree of variation is present among regions. Use of this database is specifically recommended for forensic applications in Italy.

Here we show the Y-haplotype database consisting in the loci DYS19, DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, YCAII and DXYS156Y of 107 males living in Toscany (central Italy).

We used an infrared (IR) automated fluorescence monolaser sequencer for the analysis of 13 autosomal short tandem repeat (STR) systems (TPOX, D3S1358, FGA, CSF1PO, D5S818, D7S820, D8S1179, TH01, vWA, D13S317, D16S359, D18S51, D21S11) and the X-Y homologous gene amelogenin system. These two systems represent the core of the combined DNA index systems (CODIS). Four independent multiplex reactions, based on the polymerase chain reaction (PCR) technique and on the direct labeling of the forward primer of every primer pair, with a new molecule (IRDye800), were set up, permitting the exact characterization of the alleles by comparison with ladders of specific sequenced alleles. This is the first report of the whole analysis of the STRs of the CODIS core using an IR automated DNA sequencer. The protocol was used to solve paternity/maternity tests and for population studies. The electrophoretic system also proved useful for the correct typing of those loci differing in size by only 2 bp. A sensibility study demonstrated that the test can detect an average of 10 pg of undegraded human DNA. We also performed a preliminary study analyzing some forensic samples and mixed stains, which suggested the usefulness of using this analytical system for human identification as well as for forensic purposes.

Early menopause in the fragile X carriers has been well documented in several reports. All surveys demonstrated that 13-25% of fragile X carriers experienced premature ovarian failure (POF), defined as menopause before the age of 40 years. In 1995 we started screening two groups of subjects as a part of a Fragile X Research Program: 1) women previously diagnosed as fragile X carriers from the register of our center and 2) women with POF and without a family history of fragile X or other forms of mental retardation. In this study we report the preliminary data collected from 75 fragile X families; in 30 of them, POF was present in one or several subjects, all of whom had a fragile X premutation. None of the women with a full mutation experienced POF in our series of patients. We also identified 89 families without a family history of fragile X or mental retardation, and there were 108 subjects who experienced POF, of which 6.5% had a fragile X premutation. This is 70-fold higher than the background prevalence of fragile X premutation in the Italian population and suggests an association with POF. These data confirm the results of other surveys.

In this paper we describe a new primer pair for the short tandem repeat (STR) D12S391 which makes it possible to obtain considerably shorter amplification fragments (125-173 bp), compared to the previously published primers (205-253 bp). The primers were tested on 70 samples with known genotypes, and no differing results were found. In sensitivity studies, forensic casework samples and DNA quality studies, we proved that the new primers can improve the efficiency of the amplification. Moreover, the resolution of this locus on denaturing PAGE followed by silver staining was dramatically improved. This improvement was found to be most valuable for typing the rare.3 variants known for this locus. We also present and propose a new method for silver staining denaturing acrylamide gels.

The short tandem repeat systems (STRs) D12S391 and CSF1P0 were amplified by the polymerase chain reaction (PCR) on blood samples from 100 to 158 unrelated Austrians, Italians, Yemenians and Egyptians. The samples were analyzed by both native and denaturing electrophoresis and two primer pairs were tested for the CSF1PO locus. Except for the CSF1PO data on the Egyptians, no deviations from the Hardy-Weinberg equilibrium were detected. For D12S391, no significant differences were found between the two Arab populations and between the two European populations, but the differences between both Arab populations and the Italians were significant. For CSF1PO, differences were only observed between the Yemenians and all three other populations. No evidence of linkage disequilibrium between the two STRs was found. The observation of a D12S391 allele consisting of only 14 repeats was confirmed by sequencing.

A sample of 265 subjects from central Italy was analyzed at the HumCD4 locus by polymerase-chain-reaction (PCR). Phenotypes were identified by comparison with a sequenced ladder, after high-resolution horizontal polyacrylamide gel electrophoresis (PAGE) followed by silver staining. A set of representative alleles was sequenced by Taq-cycle-sequencing with dye terminator labeling and capillary gel electrophoresis strategies. Eight common alleles--5, 6, 7, 8, 9, 10, 11, 12--and a rare larger 14, never before described in Caucasians, were found. Allele and genotype frequencies were similar to those described in former studies on Caucasians, with a prevalency of alleles number 5, 6, 10. Sequence analysis showed that the polymorphism is due to a pentameric TTTTC basic motif, tandemly repeated, and that from allele number 10 onwards the fourth repeat presents a T to C translation (CTTTC). Instead, allele number 9 may exist in two forms, because 75% of alleles examined in this study presented the CTTTC motif at the fourth position, while the remaining 25% had the basic repeat structure.

The short tandem repeat TPOX was studied using two different pairs of primers and three different electrophoretic methods with the aim of optimizing and standardizing the typing conditions for this locus. A genetic population study was subsequently conducted on two population samples from Central Italy (151 individuals) and from Austria (153 individuals) and compared using an R x C contingency table. With the aim of using this system for forensic samples, differences in sensitivity between the methods utilized were studied and several parameters of forensic interest for the two populations (PD, MEC, MEP, pM, PIC) were calculated. A new multiplex system for the loci CSF1PO, TPOX and CD4 is also presented.