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Forensic DNA phenotyping (FDP) includes biogeographic ancestry (BGA) inference and externally visible characteristics (EVCs) prediction directly from an evidential DNA sample as alternatives to provide valuable intelligence when conventional DNA profiling fails to achieve identification. In this context, the application of Massively Parallel Sequencing (MPS) methodologies, which enables simultaneous typing of multiple samples and hundreds of forensic markers, has been gradually implemented in forensic genetic casework. The Precision ID Ancestry Panel (Thermo Fisher Scientific, Waltham, USA) is a forensic multiplex assay consisting of 165 autosomal SNPs designed to provide biogeographic ancestry information. In this work, a sample of 250 individuals from Rio Grande do Sul (RS) State, southern Brazil, apportioned into four main population groups (African-, European-, Amerindian-, and Admixed-derived Gauchos), was evaluated with this panel, to assess the feasibility of this approach in a highly heterogeneous population. Forensic descriptive parameters estimated for each population group revealed that this panel has enough polymorphic and informative SNPs to be used as a supplementary instrument in forensic individual identification and kinship testing regardless of ethnicity. No statistically significant deviation from Hardy-Weinberg equilibrium was observed after Bonferroni correction. However, seven loci pairs displayed linkage disequilibrium in pairwise LD testing (p < 3.70 × 10 -6 ). Interpopulation comparisons by F ST analysis, MDS plot, and STRUCTURE analysis among the four RS population groups apart and along with 89 reference worldwide populations demonstrated that Admixed- and African-derived Gauchos present the highest levels of admixture and population stratification, whereas European- and Amerindian-derived exhibit a more homogeneous genetic conformation., Competing Interests: Declaration of Competing Interest Authors declare they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

With the advent of next-generation sequencing technology, SNP markers are being explored as a useful alternative to conventional capillary electrophoresis-based STR typing. Low mutation rate and short-sized amplicons are added advantages of SNP markers over the STRs. However, to achieve a sufficient level of discrimination among individuals, a higher number of SNPs need to be characterized simultaneously. Hence, the NGS technique is highly useful to analyze a sufficiently higher number of SNPs simultaneously. Though the technique is in its nascent stage, an attempt has been made to assess its usability in the central Indian population by analyzing 124 SNPs (90 autosomal and 34 Y-chromosome) in 95 individuals. Various quality parameters such as locus balance, locus strand balance, heterozygosity balance, and noise level showed a good quality sequence obtained from the Ion GeneStudio S5 instrument. Obtained frequency of SNP alleles ranged from 0.001 to 0.377 in autosomal SNPs. rs9951171 was found to be the most informative SNP in the studied population with the highest PD and lowest MP value. The cumulative MP of 90 SNPs was found to be 4.76698 × 10 -37 . Analysis of 34 Y-chromosome SNPs reveals 11 unique haplogroups in 54 male samples with R1a1 as the most frequent haplogroup found in 22.22% of samples. Interpopulation comparison by FST analysis, PCA plot, and STRUCTURE analysis showed genetic stratification of the studied population suggesting the utility of SNP markers present in the Precision ID Identity Panel for forensic demands of the Indian population., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Real forensic casework biological evidence can be found in a myriad of different conditions and presenting very distinct features, including key elements such as degradation levels, the nature of biological evidence, mixture presence, and surface or substrate deposition, among others. Technical protocols employed by forensic DNA analysts must consider such characteristics in order to improve the chances of successfully genotyping these materials. MPS has been used as a very useful tool for forensic sample processing and genetic profile generation. However, it is not completely clear how different features encountered with real forensic samples impact sequencing quality and, consequently, profile accuracy and reliability. In this context, the present study analyzes a set of 47 real forensic casework samples, obtained from semen, saliva, blood and epithelial evidence, as well as reference oral swabs, aiming to evaluate the impact of a sample's biological nature in profiling success. All DNA extracts from samples were standardized according to sample conditions, as assessed by traditional forensic profiling methods (real-time PCR quantitation and capillary electrophoresis-coupled STR fragment analysis). Samples were separated into groups according to their biological nature, and the resultant sequencing quality was evaluated through a series of well-established statistical tests, applied specifically to six different MPS quality metrics. The results showed that certain groups of samples, especially epithelial and (to a lesser extent) saliva samples, exhibited significantly lower quality in terms of some of the evaluated metrics. A number of reasons for such unexpected behavior are discussed. In addition, a series of calculations was performed to assess the weight of genetic evidence in Brazilian samples, and reflexes in data analysis and national allele frequency database construction are discussed. Overall, the results indicate that a unified national allele frequency database can be used nationwide. Besides this, MPS genetic profiles obtained from samples with particular biological origins may benefit from meticulous manual review, and visual inspection could be important as an additional step to avoid genotyping errors or misinterpretation, leading to more trustworthy and reliable results in real criminal forensic casework analysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Use of Massive Parallel Sequencing (MPS) techniques has been investigated by forensic community aiming introduction of such methods in routine forensic casework analyses. Interesting features presented by MPS include high-throughput, ability to simultaneous genotyping of significant number of samples and forensic markers, workflow automation, among others. Emergence of single nucleotide polymorphism (SNP) as forensic relevant markers was facilitated in this process, since concurrent typing of larger marker sets is necessary for obtaining same levels of individual discrimination provided by other marker categories. In this context, HID Ion Ampliseq Identity Panel is a commercial solution with forensic purposes comprising simultaneous analysis of 90 highly informative autosomal SNPs and 34 Y -chromosome superior clade SNPs for male lineage haplotyping. SNP typing can be obtained with smaller amplicons, and this panel was designed for efficient processing of critical or challenging forensic samples. In this work, a sample of 432 individuals from all five Brazilian geopolitical regions was evaluated with this panel, in order to access feasibility of this panel use in a national basis. Results obtained for all five regions, including forensic parameters, show that this marker set can be efficiently employed for Brazilian nationals in human identification or kinship determination applications, due to high levels of genetic discriminative information content displayed by Brazilians. Interpopulation comparison studies were executed among Brazilian regional populations and 26 worldwide populations, in order to access genetic stratification occurrence. Some levels of population structure were identified, and impact on database design was discussed. Y-chromosome haplotyping of Brazilian samples revealed high levels of European ancestry in Brazilian male lineages, and utility of haplotyping in real forensic casework is addressed. Finally, genotyping and sequencing efficiency with this panel were addressed, as an effort to appraise the adequacy of this panel use in Brazilian national forensic demands., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Single nucleotide polymorphisms in the APOA5 gene have been studied for their association with metabolic syndrome. Thus, elucidating the effect of the mechanism involved in APOA5 gene polymorphisms on lipid metabolism is of great importance. In this study we aimed to determine the allelic and genotypic frequencies of -1131T>C, Ser19Trp, and intergenic APOA4/A5 and to evaluate the association between these variants with plasma lipid levels in children and adolescents from Brazil. This study included 524 healthy children and adolescents from Mother and Child Hospital in Recife, Pernambuco, Brazil. Data were obtained on medical history, drug intake, lifestyle variables, and demography. DNA from collected samples was extracted and genotyped for the three polymorphisms. In this studied population, triglycerides and very low-density protein levels were significantly high in subjects carrying the 19WW genotype (P < 0.001), demonstrating the presence of this genetic risk factor in children and adolescents.

During critical illness and sepsis there is severe antioxidant depletion, and this scenario raises the critical ill patient's mortality risk. Glutathione peroxidase (GPx) is one of the first endogenous antioxidant defense enzymes, and it works cooperatively with superoxide dismutase (SOD) and catalase (CAT) to detoxify free radicals from the cellular environment. Genetic studies are important to understand the complexity of human oxidative stress and how the organism responds to an extreme situation such as critically care conditions. Previous studies with a GPx1 single nucleotide polymorphism (593C>T SNP; rs1050450; protein variant in GPx1: Pro198Leu) showed 593T carriers and 593TT homozygotes present higher risk to develop different diseases. We assessed the relationship of the genotype distribution of GPx1 SNP in critically ill patients with their conditions (organ dysfunction, sepsis, and septic shock) and their outcome. We monitored 626 critically ill patients daily from the ICU (intensive care unit) admission to their discharge from hospital, or death. Our study revealed a significant association between 593TT GPx1 genotype and mortality; the mortality rate was higher in homozygous 593TT GPx1 (N=94) when compared with the group of subjects with genotypes 593CT or 593CC GPx1 (N=532) (52% vs. 38%, P=0.009; OR=1.79; 95% CI=1.13-2.85). Evaluating the subgroup of 293 ICU patients with sepsis, a pooled analysis including two genetic variants GPx1 and SOD2 (47C>T SNP, rs4880; protein variant in MnSOD: Ala-9Val) showed a significant difference in relation to progression to septic shock. The frequency of septic shock among septic patients with 593T GPx1 and 47C SOD2 alleles (N=122) was higher when compared with septic patients carrying other settings of genotypes (N=174) (78% vs. 66%; P=0.028; OR=1.81; 95% CI=1.03-3.18). Accepting the previously reported functional effects of these two SNPs on GPx1 and SOD2 gene expressions and, consequently, on GPx1 and MnSOD enzyme activities, we believe our results may be considered as an important contribution for the understanding of oxidative imbalance during the critical ill., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Challenging of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharides (LPS) has been shown to activate monocytes and macrophages, leading to the production of pro-inflammatory cytokines and reactive oxygen species (ROS). Manganese superoxide dismutase (MnSOD) is an important enzyme that may play a central role in the response to oxidative stress. 47C> T SNP of the SOD2 gene, the -9Val MnSOD is less efficient than the -9Ala version. We have previously characterized the cellular redox status of human PBMCs expressing either -9Ala (CC) or -9Val (TT) SOD2 and analyzed the responses of these cells to oxidative stress induced by LPS. Due to the observed alterations in the activities of these antioxidant enzymes, we decided to investigate their immunocontent and analyze the production of intracellular oxidants, as well as any resulting DNA damage. PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers per allele). We then analyzed levels of nitrite, DNA damage by comet assay, TNF-α, carboxymethyl lysine and nitrotyrosine and assessed production of intracellular reactive species by the DCFH-DA-based assay and western blots were used to analyze protein levels. Our results show that there occurs an increase in nitric oxide production in both allele groups after challenge with LPS. A significant increase in DNA damage was observed in PBMCs after an 8-h LPS challenge. Cells expressing the SOD2 47C allele quickly adapt to a more intense metabolism by upregulating cellular detoxification mechanisms. However, when these cells are stressed over a long period, they accumulate a large quantity of toxic metabolic byproducts.

The Ewing Sarcoma is an important tumor of bone and soft tissue. The SNPs Arg72Pro of TP53 and T309G of MDM2 have been associated with many cancer types and have been differently distributed among populations worldwide. Based on a case-control design, this study aimed to assess the role of these SNPs in 24 Ewing Sarcoma patients, compared to 91 control individuals. DNA samples were extracted from blood and genotyped for both SNPs by PCR-RFLP and confirmed by DNA sequencing. The results showed an association between the G allele of the T309G and Ewing Sarcoma (P=0.02). Comparing to the TT carriers, the risk of G allele carriers was 3.35 (95% CI=1.22-9.21) with P=0.02. At the genotypic level, an association of the TT genotype with the control group (P=0.03) was found. Comparing to the TT genotype, the risk of TG and GG was 2.97 (95% CI=1.03-8.58) with P=0.04 and 5.00 (95% CI=1.23-20.34) with P=0.02, respectively. No associations regarding the Arg72Pro SNP were found. Considering that the T309G has been associated with several types of cancer, including sarcomas, our results indicate that this SNP may also be important to Ewing Sarcoma predisposition.

Aim: To analyze the effect of the two different versions of the manganese superoxide dismutase gene (SOD2) on sepsis. The SOD2 gene presents the 47C>T single nucleotide polymorphism (SNP; ID: rs4880) which produces MnSOD with different activities. The -9Val MnSOD (47T allele) is less efficient than the -9Ala version (47C allele). During sepsis there are abundance of ROS, high SOD2 expression and excess of H(2)O(2) synthesis. High concentrations of H(2)O(2) could affect the sepsis scenario and/or the sepsis outcome., Methods: We determined the 47C>T single nucleotide polymorphism (SNP) frequencies in 529 critically ill patients with or without sepsis, facing outcome. To collect information on population frequencies, we obtained a pilot 47C>T genotypic and allelic frequencies in a random group of 139 healthy subjects., Results: We compared the 47C allele carriers (47CC+47CT genotypes) with 47TT homozygotes and noticed a significant association between 47C allele carriers and septic shock in septic patients (P=0.025). With an adjusted binary multivariate logistic regression, incorporating 47C>T SNP and the main clinical predictors, we showed high SOFA scores [P<0.001, OR=9.107 (95% CI=5.319-15.592)] and 47C allele [P=0.011, OR=2.125 (95% CI=1.190-3.794)] were significantly associated with septic shock outcome. With this information we presented a hypothesis suggesting that this negative outcome from sepsis is possibly explained by effects on cellular stress caused by 47C allele., Conclusion: In our population there was a significant higher frequency of septic shock in septic patients with the 47C allele of the SOD2 gene. This higher 47C allele frequency in septic patients with negative outcome could be explained by effects of higher activity MnSOD on cellular stress during the sepsis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We evaluated the response of individuals with metabolic syndrome to lifestyle modification intervention and examined the influence of the 48867A>C (Asp358Ala) IL6R (rs2228145) polymorphism on this response. Participants were randomly divided into two groups: NI, nutritional intervention; NIE, nutritional intervention and exercise practice. Intervention lasted three months and participants completed a comprehensive evaluation and had blood collected for biochemical measurements. Eighty-two sedentary individuals with at least three criteria for metabolic syndrome were included. Comparing metabolic syndrome parameters before and after intervention, a reduction of waist circumference was observed, although significant only for AA and AC genotypes. Also, a decrease in triglyceride levels was observed (significant for AA genotype individuals; for the AC genotype, only in the NIE group). Significant reduction of fasting glucose level was observed in all AA genotype individuals; for the AC genotype, only in the NI group. Systolic blood pressure showed significant reduction in AA and AC genotype individuals. After three months of lifestyle modification intervention, improvement in some of the metabolic syndrome parameters was observed, some associated with the IL6R genotype. At enrollment, participants with genotypes AA and AC showed more severe conditions regarding metabolic syndrome inclusion criteria, supporting previous reports that the A allele is a genetic risk factor. These individuals, however, had a better response to intervention compared to individuals with the CC genotype, suggesting that nutritional control and exercise practice could prevent risks associated with metabolic syndrome more efficiently in individuals bearing the A allele.

The endothelial nitric oxide synthase (eNOS) plays an important homeostatic role in the cardiovascular system (CVS) by maintaining appropriate blood pressure through production of nitric oxide. The 894TT genotype of 894G>T (Glu298Asp, rs1799983), a polymorphic variant of eNOS, has been associated with several vascular diseases. On the basis of this strong relationship, we monitored daily 585 critically ill adult patients according to their degree of CVS dysfunction and investigated their disease progression by the 894G>T genotype. To obtain information of the general population, we obtained the 894G>T genotypic and allelic frequencies in a random group of 149 healthy subjects. The patients were genotyped for the eNOS 894G>T polymorphism and daily evaluated according to their degree of CVS dysfunction through the Cardiovascular Sequential Organ Failure Assessment (SOFA) score. The mean value of the global CVS dysfunction score was significantly higher in 894TT patients (1.35 ± 0.57) than in non-894TT patients (1.23 ± 0.37; P = 0.035). This score remained significantly higher in 894TT patients, even in different patient clusters (all patients, septic, and non-septic patients) during the 1st week at the intensive care unit (1.86 ± 0.8 versus 1.63 ± 0.62, P = 0.005; 2.32 ± 0.10 versus 2.06 ± 0.08, P = 0.009; 0.84 ± 0.09 versus 0.64 ± 0.08, P = 0.027; respectively). This result shows that the mean values of the cardiovascular SOFA score were higher in 894TT patients in all subgroups. The present study provides evidence that the 894TT eNOS genotype is associated with a higher degree of CVS dysfunction in critically ill patients.

The outcome of sepsis occurs due to influence of environmental and genetic factors besides genes variants whose expression support its outcome or not. Oxidative stress is associated to the pathogenicity of sepsis, occurring when there is a reactive species overproduction associated with inflammation. The aim of this study was to characterize the cellular redox status of human peripheral blood mononuclear cells (PBMCs) with either -9Ala (AA) or -9Val (VV) SOD2 genotypes and evaluate their response to oxidative stress induced by lipopolysaccharide (LPS). The PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers for each allele) and the following assays were performed: antioxidant enzyme activities (superoxide dismutase; catalase; glutathione peroxidase), total radical-trapping antioxidant parameter, non-enzymatic antioxidant capacity (total antioxidant reactivity), and quantification of conjugated dienes (lipid peroxidation). At basal conditions (i.e., not stimulated by LPS), cells from 47C allele carriers showed higher activities of CAT and SOD, as well as higher TAR compared to 47T allele. However, when 47CC cells were challenged with LPS, we observed a higher shift toward a pro-oxidant state compared to 47TT cells. The CAT activity and lipid peroxidation were increased in cells with both alleles, but SOD activity increased significantly only in 47TT cells. These results demonstrate that SOD2 polymorphisms are associated with different cellular redox environments at both basal and LPS-stimulated states, and identification of this polymorphism may be important for a better understanding of pro-inflammatory conditions.

We aimed to investigate single nucleotide polymorphisms (SNPs) in the EWS gene breaking region in order to analyze Ewing's sarcoma susceptibility. The SNPs were investigated in a healthy subject population and in Ewing's sarcoma patients from Southern Brazil. Genotyping was performed by TaqMan® assay for allelic discrimination using Real-Time PCR. The analysis of incidence of SNPs or different SNP-arrangements revealed a higher presence of homozygote TT-rs4820804 in Ewing's sarcoma patients (p=0.02; Chi Square Test). About 300 bp from the rs4820804 SNP lies a palindromic hexamer (5'-GCTAGC-3') and three nucleotides (GTC), which were previously identified to be in close vicinity of the breakpoint junction in both EWS and FLI1 genes. This DNA segment surrounding the rs4820804 SNP is likely to indicate a breakpoint region. If the T-rs4820804 allele predisposes a DNA fragment to breakage, homozygotes (TT-rs4820804) would have double the chance of having a chromosome break, increasing the chances for a translocation to occur. In conclusion, the TT-rs4820804 EWS genotype can be associated with Ewing's sarcoma and the SNP rs4820804 can be a candidate marker to understand Ewing's sarcoma susceptibility., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Introduction: Critically ill patients are characterized as individuals hospitalized in the Intensive Care Unit (ICU) and can evolve to sepsis, septic shock or even death. Among others, genetic factors can influence the outcome of critically ill patients. HLA-G is a non-classical class Ib molecule that has limited protein variability, presenting seven isoforms generated by alternative splicing, and presents immunomodulatory properties. Polymorphisms at the 3'UTR are thought to influence HLA-G gene expression. It was previously observed that increased sHLA-G5 levels were predictive of survival among septic shock patients. We assessed the frequencies of 7 polymorphisms in exon 8 at the 3' UTR of HLA-G and associated these variants with different clinical outcomes in critically ill patients., Methods: Exon 8 at the 3' UTR of the HLA-G gene from 638 critically ill subjects was amplified by PCR and sequenced. Genotypes were identified using FinchTV software v.1.4.0 and the most probable haplotype constitution of each sample was determined by PHASE software v.2.1. Haplotype frequencies, linkage disequilibrium, heterozygosity test and Hardy-Weinberg Equilibrium were estimated using ARLEQUIN software v.3.5., Results: Among all critically ill patients, an association between carriers of the +2960IN&#95;+3142 G&#95;+3187A haplotype and septic shock (P = 0.047) was observed. Septic patients who carried the +2960IN&#95;+3142G&#95;+3187A haplotype presented an increased risk for septic shock (P = 0.031)., Conclusions: The present study showed, for the first time, an association between polymorphisms in exon 8 at the 3 'UTR of HLA-G gene and outcomes of critically ill patients. These results may be important for understanding the mechanisms involved in evolution to septic shock in critically ill patients.

To evaluate the capacity to perceive bitter taste in a sample of the elderly population of the Brazilian Federal District, and to investigate its association with the consumption profile of distinct food groups. A total of 255 female outpatients aged 60 years or older took part in this cross-sectional study. The following data were determined for all the volunteers: alimentary frequency by clinical dieticians; genotyping of the G145C polymorphism in the TAS2r38 gene; cognitive status; sensorial (visual and hearing) acuity and drugs related to ageusia or dysgeusia. Sensitivity to bitter taste was assessed using phenylthiocarbamide (PTC) in a subset. Non-parametric tests confirmed the remarkable effect of the C allele in determining sensitivity to PTC (p<0.001). C allele carriers displayed diminished consumption of type B vegetables as well as of some vegetables generally recognized as bitter: arugula (p=0.044) and chard (p=0.006). No associations were observed for the remaining food classes. The present findings suggest that the G145C genetic variation in the TAS2r38 gene modestly influenced food consumption habits of Brazilian older women. Nonetheless, the results do not rule out possible effects of past experiences on choices of elderly individuals., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

In order to analyze the effect of the two different versions of the cluster of differentiation 14 (CD14) receptor recognizing gene on survival, we determined the -260C>T single nucleotide polymorphism (SNP) frequencies in 514 critically ill patients. We compared the -260TT homozygotes with -260C allele carriers (-260CC and -260CT genotypes) and we demonstrated--260TT patients had the highest survival rate (82% vs 64%; p < 0.001; OR = 2.52, 95% CI = 1.43-4.46). We performed binary logistic regression, incorporating both -260C>T genotype groups and the main clinical predictors to exclude other risk factors that could influence the outcome from critical illness: higher age, APACHE II score, and length of stay at hospital, and the occurrence of sepsis and septic shock were risk factors to Intensive Care Unit (ICU) patient's mortality, but the -260TT genotype was protective factor toward survival (p = 0.001; OR = 3.08 95%CI = 1.54-5.98). Among septic and septic shock patients, -260TT genotype was also protective factor toward survival (p = 0.001; OR = 3.11 95%CI = 1.63-6.66 to septic patients, and p = 0.001; OR = 3.80 95%CI = 1.68-8.58 to patients with septic shock). Our results and our hypothesis suggest that the higher -260TT genotype frequency in ICU survivor patients is possibly explained by a beneficial effect on innate immunity signaling.