Evaluation of the usefulness of insertion-null markers in critical skeletal remains.

Forensic DNA analysis in compromised skeletal remains may pose challenges due to DNA degradation, often resulting in partial or negative autosomal STRs profiles. To address this issue, alternative approaches such as mitochondrial DNA or SNPs typing may be employed; however, they are labour-intensive and costly. Insertion-null alleles (INNULs), short interspersed nuclear elements, have been suggested as a valuable tool for human identification in challenging samples due to their small amplicon size. A commercial kit including 20 INNULs markers along with amelogenin (InnoTyper® 21) has been developed. This study assesses its utility using degraded skeletal remains, comparing the results obtained (the number of detected alleles, RFU values, PHR, and the number of reportable markers) to those obtained using GlobalFiler™. Subsequently, the random match probability of the two profiles for each sample was determined using Familias version 3 to evaluate the power of discrimination of the results obtained from each kit. In every sample, InnoTyper® 21 yielded more alleles, higher RFU values, and a greater number of reportable loci. However, in most cases, both profiles were similarly informative. In conclusion, InnoTyper® 21 serves as a valuable complement to the analysis of challenging samples in cases where a poor or negative profile was obtained.

B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen.

Neutrophils use immunoglobulins to clear antigen, but their role in immunoglobulin production is unknown. Here we identified neutrophils around the marginal zone (MZ) of the spleen, a B cell area specialized in T cell-independent immunoglobulin responses to circulating antigen. Neutrophils colonized peri-MZ areas after postnatal mucosal colonization by microbes and enhanced their B cell-helper function after receiving reprogramming signals, including interleukin 10 (IL-10), from splenic sinusoidal endothelial cells. Splenic neutrophils induced immunoglobulin class switching, somatic hypermutation and antibody production by activating MZ B cells through a mechanism that involved the cytokines BAFF, APRIL and IL-21. Neutropenic patients had fewer and hypomutated MZ B cells and a lower abundance of preimmune immunoglobulins to T cell-independent antigens, which indicates that neutrophils generate an innate layer of antimicrobial immunoglobulin defense by interacting with MZ B cells.

A comparison between petrous bone and tooth, femur and tibia DNA analysis from degraded skeletal remains.

Skeletal remains are the only biological material that remains after long periods; however, environmental conditions such as temperature, humidity, and pH affect DNA preservation, turning skeletal remains into a challenging sample for DNA laboratories. Sample selection is a key factor, and femur and tooth have been traditionally recommended as the best substrate of genetic material. Recently, petrous bone (cochlear area) has been suggested as a better option due to its DNA yield. This research aims to evaluate the efficiency of petrous bone compared to other cranium samples (tooth) and postcranial long bones (femur and tibia). A total amount of 88 samples were selected from 38 different individuals. The samples were extracted by using an organic extraction protocol, DNA quantification by Quantifiler Trio kit and amplified with GlobalFiler kit. Results show that petrous bone outperforms other bone remains in quantification data, yielding 15-30 times more DNA than the others. DNA profile data presented likeness between petrous bone and tooth regarding detected alleles; however, the amount of DNA extracted in petrous bones allowed us to obtain more informative DNA profiles with superior quality. In conclusion, petrous bone or teeth sampling is recommended if DNA typing is going to be performed with environmentally degraded skeletal remains.

Identification of hereditary breast and ovarian cancer germline variants in Granada (Spain): NGS perspective.

The aim of this study was to assess the prevalence of germline variants in cancer-predisposing genes by either targeted (BRCA1/2) or multigene NGS panel in a high-risk Hereditary Breast and Ovarian Cancer (HBOC) cohort. Samples from 824 Caucasian probands were retrospectively collected and the impact of genetic diagnosis and genetic variants epidemiology in this cohort was evaluated. Performance of risk-reducing prophylactic measures, such as prophylactic mastectomy and/or prophylactic oophorectomy, was assessed through clinical follow-up of patients with a positive genetic result. Pathogenic variants predisposing to HBOC were identified in 11.9% (98/824) individuals at BRCA2 (47/98), BRCA1 (24/98), PALB2 (8/51), ATM (7/51), CHEK2 (6/51) MSH6, (2/51), RAD51C (2/51) and TP53 (2/386). Of them, 11 novel pathogenic variants and 12 VUS were identified, characterized, and submitted to ClinVar. Regarding clinical impact, the risk of developing basal or Her2 breast cancer was increased 15.7 times or 37.5 times for BRCA1 and MSH6 pathogenic variants respectively. On the contrary, the risk of developing basal or luminal A breast cancer was reduced to 81% or 77% for BRCA2 and BRCA1 pathogenic variants, respectively. Finally, 53.2% of individuals testing positive for class IV/V variants underwent prophylactic surgery (mastectomy, oophorectomy or both) being significantly younger at the cancer diagnosis than those undertaking prophylactic measures (p = 0.008). Of them, 8 carried a pathogenic/likely pathogenic variant in other genes different from BRCA1 and BRCA2, and the remaining (46.7%) decided to continue with clinical follow-up. No differences in pathogenicity or risk of developing cancer were found for BRCA1/2 between targeted and multigene sequencing strategies; however, NGS was able to resolve a greater proportion of high-risk patients.

GITAD 2020: quality assurance test through 20 years of experience.

GITAD (Grupo Iberoamericano de Trabajo en Análisis de DNA) was founded in 1998 as the first operational group of AICEF (Academia Iberoamericana de Criminalística y Estudios Forenses), formally created in 1999. The mission and the vision of GITAD are to promote the development of forensic genetics in Ibero-American countries and to achieve the maximum level of innovation and quality in each country, and with that aim, a proficiency test was developed. Since 1999, the member laboratories receive four reference samples with the objective of obtaining the genetic profile with their routine protocols, a theoretical exercise since 2003, and since 2007, it was incorporated a forensic sample, which changes every year. The consensus results and the different discrepancies are discussed in an annual meeting. This article illustrates the evolution of the proficiency test through 20 years from different points of view: the increase of participant laboratories, the evolution of the different DNA typing techniques reported by the Ibero-American participant laboratories, the challenges that the proficiency test have met, and future perspectives for a continuous improvement of the proficiency test, especially regarding its accreditation under ISO 17043.

Follow-Up Biomarkers in the Evolution of Prostate Cancer, Levels of S100A4 as a Detector in Plasma.

The management and screening of prostate cancer (PC) is still the main problem in clinical practice. In this study, we investigated the role of aggressiveness genetic markers for PC stratification. We analyzed 201 plasma samples from PC patients and controls by digital PCR. For selection and validation, 26 formalin-fixed paraffin-embedded tissues, 12 fresh tissues, and 24 plasma samples were characterized by RNA-Seq, immunochemistry, immunofluorescence, Western blot, and extracellular-vesicles analyses. We identified three novel non-invasive biomarkers; all with an increased expression pattern in patients (PCA3: p = 0.002, S100A4: p ≤ 0.0001 and MRC2: p = 0.005). S100A4 presents the most informative AUC (area under the curve) (0.735). Combination of S100A4, MRC2, and PCA3 increases the discriminatory power between patients and controls and between different more and less aggressive stages (AUC = 0.761, p ≤ 0.0001). However, although a sensitivity of 97.47% in PCA3 and a specificity of 90.32% in S100A4 was reached, the detection signal level could be variable in some analyses owing to tumor heterogeneity. This is the first time that the role of S100A4 and MRC2 has been described in PC aggressiveness. Moreover, the combination of S100A4, MRC2, and PCA3 has never been described as a non-invasive biomarker for PC screening and aggressiveness.

Combined Genome, Transcriptome and Metabolome Analysis in the Diagnosis of Childhood Cerebellar Ataxia.

Ataxia in children is a common clinical sign of numerous neurological disorders consisting of impaired coordination of voluntary muscle movement. Its most common form, cerebellar ataxia, describes a heterogeneous array of neurologic conditions with uncountable causes broadly divided as acquired or genetic. Numerous genetic disorders are associated with chronic progressive ataxia, which complicates clinical management, particularly on the diagnostic stage. Advances in omics technologies enable improvements in clinical practice and research, so we proposed a multi-omics approach to aid in the genetic diagnosis and molecular elucidation of an undiagnosed infantile condition of chronic progressive cerebellar ataxia. Using whole-exome sequencing, RNA-seq, and untargeted metabolomics, we identified three clinically relevant mutations (rs141471029, rs191582628 and rs398124292) and an altered metabolic profile in our patient. Two POLR1C diagnostic variants already classified as pathogenic were found, and a diagnosis of hypomyelinating leukodystrophy was achieved. A mutation on the MMACHC gene, known to be associated with methylmalonic aciduria and homocystinuria cblC type, was also found. Additionally, preliminary metabolome analysis revealed alterations in our patient's amino acid, fatty acid and carbohydrate metabolism. Our findings provided a definitive genetic diagnosis reinforcing the association between POLR1C mutations and hypomyelinating leukodystrophy and highlighted the relevance of multi-omics approaches to the disease.

Exosomal miRNA profile as complementary tool in the diagnostic and prediction of treatment response in localized breast cancer under neoadjuvant chemotherapy.

BACKGROUND: Breast cancer patients under neoadjuvant chemotherapy includes a heterogeneous group of patients who eventually develop distal disease, not detectable by current methods. We propose the use of exosomal miRNAs and circulating tumor cells as diagnostic and predictive biomarkers in these patients. METHODS: Fifty-three breast cancer women initially diagnosed with localized breast cancer under neoadjuvant chemotherapy were prospectively enrolled in this study. However, six of them were later re-evaluated and diagnosed as metastatic breast cancer patients by PET-CT scan. Additionally, eight healthy donors were included. Circulating tumor cells and serum exosomal miRNAs were isolated from blood samples before and at the middle of neoadjuvant therapy and exosomal miRNA levels analyzed by qPCR. RESULTS: Before neoadjuvant therapy, exosomal miRNA-21 and 105 expression levels were higher in metastatic versus non-metastatic patients and healthy donors. Likewise, higher levels of miRNA-222 were observed in basal-like (p = 0.037) and in luminal B versus luminal A (p = 0.0145) tumor subtypes. Exosomal miRNA-222 levels correlated with clinical and pathological variables such as progesterone receptor status (p = 0.017) and Ki67 (p = 0.05). During neoadjuvant treatment, exosomal miRNA-21 expression levels directly correlated with tumor size (p = 0.039) and inversely with Ki67 expression (p = 0.031). Finally, higher levels of exosomal miRNA-21, miRNA-222, and miRNA-155 were significantly associated with the presence of circulating tumor cells. CONCLUSION: Liquid biopsies based on exosomal miRNAs and circulating tumor cells can be a complementary clinical tool for improving breast cancer diagnosis and prognosis.

Population genetic data of 20 insertion-null allele markers in the Andalusian population.

Genetic data from 21 autosomal insertion-null (INNULs) markers found in the InnoTyper® 21 Human DNA Analysis (InnoGenomics®) was evaluated in 190 unrelated individuals from Andalusia. Allele frequencies and forensic parameters were estimated for the 20 INNULs. All loci were in Hardy-Weinberg equilibrium in the study population after Bonferroni correction and showed no signs of linkage between them. The combined power of discrimination and the power of exclusion for the 20 INNULs were 1-7.42352 × 10-9 and 93.60946%, respectively. These data might be useful for the research of population genetics and for individual identification and paternity testing in forensic science.

The influence of nutritional factors on prostate cancer incidence and aggressiveness.

There is an increasing evidence for a link between nutrition, lifestyle and prostate cancer (PCa) development and/or progression of disease. The objective of this study was to examine the association between dietary factors and PCa incidence and aggressiveness in a case-control study. After the analysis of the anatomic pathology, subjects were classified in patients with PCa (n = 157) and controls (n = 158). Clinical data including Gleason score, PSA values and biopsy results, were compiled. Frequencies of food consumption and sociodemographic data were also obtained. The results showed that physical activity was significantly higher in control (p < .022). It was also found that some nutritional habits offer a protective effect among studied subjects, like high nuts (p = .041) and fish (p = .041) intakes. Moreover, there was a significant reduction in risk (p = .029) in cases with a higher fruits and vegetables intakes. A decreased risk of aggressive PCa was associated with fruits, vegetables, legumes and fish intakes. However, these relationships were not statistically significant when data were adjusted for covariates. In conclusion, this study found an inverse association between PCa risk and the intake of fruits and vegetables, fish and nuts. The results suggested that a diet with higher intakes of these foods as Mediterranean diet may lower the risk of PCa in the studied population. As dietary factors are modifiable, identifying food groups or dietary patterns that modulate the risk of PCa and its aggressiveness can offer effective and practical strategies for its primary prevention.

Bioactive compounds of the Mediterranean diet and prostate cancer.

OBJECTIVE: The purpose of this review is to examine the evidence on the effects of bioactive constituents of the Mediterranean diet (MeDi) on prostate cancer (PCa) risk. METHODS: The search for articles came from extensive research in the following databases: PubMed, Scopus, and Web of Science. We used the search terms "Mediterranean diet," "lycopene," "vitamin E," "vitamin C," "Selenium," "resveratrol," "prostate cancer," and combinations, such as "lycopene and prostate cancer" or "resveratrol and prostate cancer." RESULTS: Numerous studies investigating the effect of various dietary nutrients on PCa have suggested that selenium is probably the most promising. Several studies reported reduced PCa risk associated with vitamin C and E intake, while other studies reported no association. Lycopene inhibits cell proliferation and inducts apoptosis, thus protecting against cancer. Also, it has been found in various in vivo and in vitro studies that resveratrol, inhibits PCa development. CONCLUSIONS: The high content of bioactive phytochemicals in the MeDi is of particular interest in the prevention of PCa. Further large-scale studies are required to clarify the effect of MeDi bioactive compounds on prostate health, in order to establish the role of this diet in the prevention of PCa.

Significance of EGFR Expression in Circulating Tumor Cells.

This chapter focuses on a deep description of the epidermal growth factor receptor (EGFR) expression in circulating tumor cells (CTCs) and its main role in cancer progression, genetic changes related to metastasis , and resistance to treatment. The aberrant behavior of cancer cells is caused by genetic mutations and altered patterns of gene expression. These changes can be responsible for an increase in cell motility but also an ability of CTCs to survival in different microenvironments, as well as developing therapy-resistant clones. Finally, CTCs can acquire the ability to invade distant organs, where metastatic foci can develop.

Next generation sequencing: an application in forensic sciences?

CONTEXT: Over the last few decades, advances in sequencing have improved greatly. One of the most important achievements of Next Generation Sequencing (NGS) is to produce millions of sequence reads in a short period of time, and to produce large sequences of DNA in fragments of any size. Libraries can be generated from whole genomes or any DNA or RNA region of interest without the need to know its sequence beforehand. This allows for looking for variations and facilitating genetic identification. OBJECTIVES: A deep analysis of current NGS technologies and their application, especially in forensics, including a discussion about the pros and cons of these technologies in genetic identification. METHODS: A systematic literature search in PubMed, Science Direct and Scopus electronic databases was performed for the period of December 2012 to June 2015. RESULTS: In the forensic field, one of the main problems is the limited amount of sample available, as well as its degraded state. If the amount of DNA input required for preparing NGS libraries continues to decrease, nearly any sample could be sequenced; therefore, the maximum information from any biological remains could be obtained. Additionally, microbiome typification could be an interesting application to study for crime scene characterisation. CONCLUSIONS: NGS technologies are going to be crucial for DNA human typing in cases like mass disasters or other events where forensic specimens and samples are compromised and degraded. With the use of NGS it will be possible to achieve the simultaneous analysis of the standard autosomal DNA (STRs and SNPs), mitochondrial DNA, and X and Y chromosomal markers.

Genetic variation of 17 STR loci in a Mexican Mestizo population from Mexico City.

Genetic data from 17 autosomal short tandem repeat (STR) loci found in the Powerplex® ESX 17 System (Promega, Madison, WI, USA) was evaluated in 162 unrelated Mexican Mestizo individuals from Mexico City. Allele frequencies and forensic parameters were estimated for the 17 STRs. All loci analyzed were in Hardy-Weinberg equilibrium in the studied population and showed not any signs of linkage between loci. The combined power of discrimination and the power of exclusion for the 16 aSTRs studied were 1-2.56409·10-19 and 99.999938 %, respectively. Genetic distances reveal a close relationship within different populations of Mexican Mestizos. The obtained data might be useful for population genetics research and for individual identification and paternity testing in forensic science.

Twelve Core Template Prostate Biopsy is an Unreliable Tool to Select Patients Eligible for Focal Therapy.

INTRODUCTION: To determine whether unilateral prostate cancer diagnosed at 12-core prostate biopsy harbours relevant prostate cancer foci in contralateral lobe in cases eligible for hemiablative focal therapy. MATERIAL AND METHODS: We analysed 112 radical prostatectomies of unilateral Gleason 6/7 prostate cancer based on prostate biopsy information. The presence of significant prostate cancer foci and/or the index lesion in the contralateral lobe is described. A subanalysis is performed in cases of Gleason score 6 and in cases of very-low-risk prostate cancer. RESULTS: Contralateral prostate cancer was present in 69.6% of cases, fulfilling significant prostate cancer criteria in 33% and being the index lesion in 32%. No significant differences were found when analysing the Gleason 6 group (73% contralateral prostate cancer, 34% significant prostate cancer and 35% index lesion) or the very-low-risk prostate cancer group (80% contralateral prostate cancer, 29% significant prostate cancer and 45% index lesion). CONCLUSIONS: The assumption of unilateral prostate cancer based on 12-core template prostate biopsy information is unreliable. In about one third of the cases, there will be focus of significant prostate cancer or the index lesion in the contralateral lobe. This information should be taken into account when hemiablative focal therapies are considered.

Lifestyle and dietary factors in relation to prostate cancer risk.

The aim of the present study was to determine the association between the socio-demographic, lifestyle factors, and dietary habits with the risk of prostate cancer (PC) in a case-control study of Spanish men. None of the socio-demographic, lifestyle or dietetic variables was found predictors of PC risk. Body mass index was associated with an increased risk for aggressive PC and fruit consumption with lower Gleason scores, thus less aggressive cancers. Nonetheless, after applying Bonferroni correction, these variables were not still associated with PC aggressiveness. More adequately, powered epidemiological studies that measure the effect of lifestyle and dietary intake in PC risk and aggressiveness are warranted to further elucidate the role of these modifiable factors on PC etiology.

Exploring the interplay between climate, population immunity and SARS-CoV-2 transmission dynamics in Mediterranean countries.

The relationship between SARS-CoV-2 transmission and environmental factors has been analyzed in numerous studies since the outbreak of the pandemic, resulting in heterogeneous results and conclusions. This may be due to differences in methodology, considered variables, confounding factors, studied periods and/or lack of adequate data. Furthermore, previous works have reported that the lack of population immunity is the fundamental driver in transmission dynamics and can mask the potential impact of environmental variables. In this study, we aimed to investigate the association between climate variables and COVID-19 transmission considering the influence of population immunity. We analyzed two different periods characterized by the absence of vaccination (low population immunity) and a high degree of vaccination (high level of population immunity), respectively. Although this study has some limitations, such us the restriction to a specific climatic zone and the omission of other environmental factors, our results indicate that transmission of SARS-CoV-2 may increase independently of temperature and specific humidity in periods with low levels of population immunity while a negative association is found under conditions with higher levels of population immunity in the analyzed regions.

Performance comparison of four qPCR and three autosomal STR commercial kits from degraded skeletal remains.

This research evaluates the current DNA quantification (Quantifiler™ Trio, PowerQuant®, Investigator® Quantiplex® Pro and InnoQuant® HY Fast) and autosomal STRs amplification kits (GlobalFiler™, PowerPlex® Fusion 6 C, Investigator® 24Plex QS) using 62 degraded skeletal remains from armed conflicts (petrous bone, femur, tibia, and tooth) with several parameters (autosomal small, large, and male target, degradation index, probability of degradation, number of alleles above analytical threshold, number of alleles above stochastic threshold, RFU, peak height ratio, number of reportable loci). The best qPCR/autosomal STRs amplification tandem was determined by comparing quantification results by a DNA quantity estimation based on sample average RFU. InnoQuant® HY Fast was the most sensitive kit, and no significative differences were observed among amplification kits; however, Investigator® 24 Plex QS was found to be the most sensitive in our samples. That is why InnoQuant™ and Investigator® 24Plex QS were determined to be the best tandem.

A comparison of five DNA extraction methods from degraded human skeletal remains.

Extracting DNA from degraded human remains poses a challenge for any forensic genetics laboratory, as it requires efficient high-throughput methods. While little research has compared different techniques, silica in suspension has been identified in the literature as the best method for recovering small fragments, which are often present in these types of samples. In this study, we tested five DNA extraction protocols on 25 different degraded skeletal remains. Including the humerus, ulna, tibia, femur, and petrous bone. The five protocols were organic extraction by phenol/chloroform/isoamyl alcohol, silica in suspension, High Pure Nucleic Acid Large Volume silica columns (Roche), InnoXtract™ Bone (InnoGenomics), and PrepFiler™ BTA with AutoMate™ Express robot (ThermoFisher). We analysed five DNA quantification parameters (small human target quantity, large human target quantity, human male target quantity, degradation index, and internal PCR control threshold), and five DNA profile parameters (number of alleles with peak height higher than analytic and stochastic threshold, average relative fluorescence units (RFU), heterozygous balance, and number of reportable loci) were analysed. Our results suggest that organic extraction by phenol/chloroform/isoamyl alcohol was the best performing method in terms of both quantification and DNA profile results. However, Roche silica columns were found to be the most efficient method.

Baseline extracellular vesicle miRNA-30c and autophagic CTCs predict chemoradiotherapy resistance and outcomes in patients with lung cancer.

Concurrent chemoradiotherapy (cCRT) is the mainstay of treatment for patients diagnosed with locally advanced non-small cell lung cancer (NSCLC). One significant challenge in the effectiveness of this therapy is the potential development of resistance mechanisms, where autophagy up-regulation has been proposed as a key contributing factor. However, there is a lack of reliable biomarkers to predict outcomes on these patients. Interestingly, for addressing this gap, extracellular vesicles (EVs) and circulating tumor cells (CTCs) have emerged as potential sources of such biomarkers. In this study, we investigated EV-associated miRNAs and presence of autophagic CTCs in prospectively collected serial samples from 38 patients with stage III NSCLC undergoing cCRT. Our findings revealed that non-responders exhibited low levels of baseline EV miR-375, miR-200c, and miR-30c. In particular, EV miR-30c showed high predictive value with an area under the curve of 87.2%. Low EV miR-30c and the presence of autophagic-activated CTCs emerged as independent predictive biomarkers for shorter relapse-free survival and overall survival. Furthermore, in experimental models simulating the effects of chemo- and radiotherapy, the administration of miR-30c, either through direct transfection or encapsulation into human EVs, led to the inhibition of autophagy in these cells. This is the first report demonstrating that EV miR-30c inhibits tumor autophagy and its quantification, together with autophagic-activated CTCs, could be used as biomarkers for the stratification and monitoring of patients with NSCLC undergoing cCRT, and they may hold promising potential for guiding subsequent consolidation treatment with immunotherapy or other novel therapies based on autophagy inhibitors.