Forensic characterization of Brazilian regional populations through massive parallel sequencing of 124 SNPs included in HID ion Ampliseq Identity Panel.

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.

Reduced frequency of two activating KIR genes in patients with sepsis.

Natural killer (NK) cell activity is regulated by activating and inhibitory signals transduced by killer cell immunoglobulin-like receptors (KIR). Diversity in KIR gene repertoire among individuals may affect disease outcome. Sepsis development and severity may be influenced by genetic factors affecting the immune response. Here, we examined sixteen KIR genes and their human leucocyte antigen (HLA) class I ligands in critical patients, aiming to identify patterns that could be associated with sepsis. Male and female patients (ages ranging between 14 and 94years-old) were included. DNA samples from 211 patients with sepsis and 60 controls (critical care patients with no sepsis) collected between 2004 and 2010 were included and genotyped for KIR genes using the polymerase chain reaction method with sequence-specific oligonucleotide (PCR-SSO), and for HLA genes using the polymerase chain reaction method with sequence-specific primers (PCR-SSP). The frequencies of activating KIR2DS1 and KIR3DS1 in sepsis patients when compared to controls were 41.23% versus 55.00% and 36.49% versus 51.67% (p=0.077 and 0.037 respectively before Bonferroni correction). These results indicate that activating KIR genes 2DS1 and 3DS1 may more prevalent in critical patients without sepsis than in patients with sepsis, suggesting a potential protective role of activating KIR genes in sepsis.

Effect of 593C>T GPx1 SNP alone and in synergy with 47C>T SOD2 SNP on the outcome of critically ill patients.

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.

Higher frequency of septic shock in septic patients with the 47C allele (rs4880) of the SOD2 gene.

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.

Polymorphic variants in exon 8 at the 3' UTR of the HLA-G gene are associated with septic shock in critically ill patients.

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_+3142 G_+3187A haplotype and septic shock (P = 0.047) was observed. Septic patients who carried the +2960IN_+3142G_+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.

Ewing's sarcoma: analysis of single nucleotide polymorphism in the EWS gene.

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.

Very low frequencies of Toll-like receptor 2 supposed-2029T and 2258A (RS5743708) mutant alleles in southern Brazilian critically ill patients: would it be a lack of worldwide-accepted clinical applications of Toll-like receptor 2 variants?

Toll-like receptor 2 (TLR2) is a recognition receptor for the widest repertoire of pathogen-associated molecular patterns. Two polymorphisms of TLR2 could be linked to reduced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation and to increased risk of infection (supposed-2029C>T and 2258G>A). We investigated the supposed-2029C>T and 2258G>A TLR2 polymorphisms in 422 critically ill patients of European origin from southern Brazil (295 with sepsis and 127 without sepsis) and reviewed 33 studies on these polymorphisms, conducting a quality assessment with a score system. Among our patients we found only one heterozygote (1/422) for the supposed-2029C>T and none for the 2258G>A (0/422) single nucleotide polymorphism (SNP). We were unable to find a clinical application of supposed-2029T and 2258A allele analyses in our southern Brazilian population. Our review detected that current TLR2 SNP assays had very controversial and contradictory results derived from reports with a variety of investigation quality criteria. We suggest that, if analyzed alone, the supposed-2029C>T and 2258G>A TLR2 SNP are not good candidates for genetic markers in studies that search for direct or indirect clinical applications between genotype and phenotype. Future efforts to improve the knowledge and to provide other simultaneous genetic markers might reveal a more effective TLR2 effect on the susceptibility to infectious diseases.