Clarice Sampaio Alho

Effect of CD14 −260C>T polymorphism on the mortality of critically ill patients

The CD14 receptor seems to be an important part of the innate immune system. A mutant CD14 can produce a reduced signal in response to infection, as a result of which an adequate inflammatory innate response is not induced, leading to a systemic infection. Defects in the innate immunity increase patient susceptibility to systemic infections and can produce a deregulated inflammatory response causing sepsis, organ failure or death in critically ill patients. We evaluated the CD14 −260C>T polymorphism genotyping as a genetic tool for risk evaluation of critically ill patients admitted to an intensive care unit (ICU) in Southern Brazil. We monitored the patients daily during their entire ICU and post‐ICU (hospital) stay (measured from the ICU admission day to a maximum of 224 days). A total of 85 patients, aged 19–95 years (mean = 56 years, median = 58 years), were included in this study. Patient mortality was 58.8%. The genotypic (TT = 0.27, TC = 0.41, CC = 0.32) and allelic (T = 0.48, C = 0.52) frequencies did not differ from the values expected by the Hardy–Weinberg model and genotype distribution was random for all clinical characteristics at ICU admission. We found a statistically significant difference favouring the survival of patients with TT genotype (P = 0.042), suggesting that this CD14 gene polymorphism could be a candidate for further study in the search for a complementary prognostic tool for patient risk evaluation. Our study describes, for the first time, the effect of the CD14 gene polymorphism in critically ill Brazilian patients. Our data suggest that patients carrying the TT genotype have a better survival outcome.

CD14 expression in the first 24h of sepsis: effect of -260C>T CD14 SNP.

Sepsis is defined as systemic inflammation caused by infection. The membrane bound CD14 (mCD14) or the soluble form (sCD14) play a crucial role facing Gram-negative and Gram-positive sepsis since they are pattern recognition receptors of the innate immune response enabling cells to produce inflammatory cytokines against bacterial infections. A −260C>T single nucleotide polymorphism (SNP) was detected in the promoter modulating the CD14 gene expression. We hypothesized that the CD14 expression depends of the genetic inheritance of −260C>T CD14 SNP and it is modulated by sepsis condition. We investigated human CD14 expression on early sepsis diagnosis (in vivo) and after LPS stimulation (in vitro), and determined the −260C>T CD14 SNP. We found that TT homozygotes showed higher mCD14 density (p = 0.0207), but not different sCD14 levels when compared to the CT+CC genotypes. Monocyte mCD14 density and sCD14 serum levels in our sample of early 14 septic patients were significantly higher than normal 30 controls (p<0.0001). Our results suggest that the −260TT CD14 genotype is associated with higher monocyte mCD14, but not sCD14 expression, and that in the first 24 h after sepsis diagnosis, both monocyte mCD14 density and sCD14 levels are elevated, similarly to what is observed in vitro upon challenge with LPS.

Evaluation of DNA methylation markers and their potential to predict human aging

We present epigenetic methylation data for two genetic loci, GRIA2, and NPTX2, which were tested for prediction of age from different donors of biofluids. We analyzed 44 saliva samples and 23 blood samples from volunteers with ages ranging from 5 to 72 years. DNA was extracted and bisulfite modified using commercial kits. Specific primers were used for amplification and methylation profiles were determined by pyrosequencing. Methylation data from both markers and their relationship with age were determined using linear regression analysis, which indicates a positive correlation between methylation and age. Older individuals tend to have increased methylation in both markers compared to younger individuals and this trend was more pronounced in the GRIA2 locus when compared to NPTX2. The epigenetic predicted age, calculated using a GRIA2 regression analysis model, was strongly correlated to chronological age (R2 = 0.801), with an average difference of 6.9 years between estimated and observed ages. When using a NPTX2 regression model, we observed a lower correlation between predicted and chronological age (R2 = 0.654), with an average difference of 9.2 years. These data indicate these loci can be used as a novel tool for age prediction with potential applications in many areas, including clinical and forensic investigations.

Developmental validation studies of epigenetic DNA methylation markers for the detection of blood, semen and saliva samples.

Determining the type and origin of body fluids in a forensic investigation can provide important assistance in reconstructing crime scenes. A set of epigenetic markers, ZC3H12D, BCAS4 and cg06379435, have been developed to produce unique and specific patterns of DNA methylation that can be used to identify semen, saliva, and blood, respectively. To ensure the efficacy of these markers, developmental validation studies were performed to determine the conditions and limitations of this new tool for forensic analysis. DNA was extracted from human samples and bisulfite modified using commercial bisulfite modification kits. Specific primers were used to amplify the region of interest and the methylation profile of the CpG sites were determined by pyrosequencing. The percent methylation values at each CpG site were determined in multiple samples and averaged for each tissue type. The versatility of these new markers is presented by showing the results of validation studies on sensitivity, human specificity, stability and mixture resolution. When testing the markers using different organisms, we did obtain positive results for certain non-human primate samples, however, all other tested species were negative. The lowest concentration consistently detected varied from 0.1 to 10ng, depending on the locus, indicating the importance of primer design and sequence in the assay. The method also proved to be effective when inhibitors were present in the samples or when samples were degraded by heat. Simulated case- samples were also tested. In the case of mixtures of different cell types, the overall methylation values varied in a consistent and predictable manner when multiple cell types were present in the same sample. Overall, the validation studies demonstrate the robustness and effectiveness of this new tool for body fluid identification.

TNF -308G > A promoter polymorphism (rs1800629) and outcome from critical illness

BACKGROUND The susceptibility to adverse outcome from critical illness (occurrence of sepsis, septic shock, organ dysfunction/failure, and mortality) varies dramatically due to different degrees of inflammatory response. An over expression of tumor necrosis factor alpha (TNF-α) can lead to the progression of the inflammatory condition. OBJECTIVE We assessed the relationship of the genotype distribution of -308G >A TNF-α polymorphism with regard to the development of sepsis, septic shock, higher organ dysfunction or mortality in critically ill patients. METHODS Observational, hospital-based cohort study of 520 critically ill Caucasian patients from southern Brazil admitted to the general ICU of São Lucas Hospital, Porto Alegre, Brazil. Patients were monitored daily from the ICU admission day to hospital discharge or death, measuring SOFA score, sepsis, and septic shock occurrences. The -308G >A TNF-α SNP effect was analyzed in the entire patient group, in patients with sepsis (349/520), and in those who developed septic shock (248/520). RESULTS The genotypic and allelic frequencies were -308GG = 0.72; -308GA = 0.27; -308AA = 0.01; -308G = 0.85; -308A = 0.15. No associations were found with sepsis, septic shock, organ dysfunction, and/or mortality rates among the TNF-α genotypes. Our results reveal that the -308G >A TNF-α SNP alone was not predictive of severe outcomes in critically ill patients. CONCLUSION The principal novel input of this study was the larger sample size in an investigation with -308G > A TNF-α SNP. The presence of -308A allele is not associated with sepsis, septic shock, higher organ dysfunction or mortality in critically ill patients.

The Influences of CD14 −260C>T Polymorphism on Survival in ICU Critically Ill Patients

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) patients 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.

Implication of the G145C polymorphism (rs713598) of the TAS2r38 gene on food consumption by Brazilian older women

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.

High-resolution melt analysis of DNA methylation to discriminate semen in biological stains.

The goal of this study was to develop a method for the detection of semen in biological stains using high-resolution melt (HRM) analysis and DNA methylation. To perform this task, we used an epigenetic locus that targets a tissue-specific differentially methylated region for semen. This specific locus, ZC3H12D, contains methylated CpG sites that are hypomethylated in semen and hypermethylated in blood and saliva. Using this procedure, DNA from forensic stains can be isolated, processed using bisulfite-modified polymerase chain reaction (PCR), and detected by real-time PCR with HRM capability. The method described in this article is robust; we were able to obtain results from samples with as little as 1 ng of genomic DNA. Samples inhibited by humic acid still produced reliable results. Furthermore, the procedure is specific and will not amplify non-bisulfite-modified DNA. Because this process can be performed using real-time PCR and is quantitative, it fits nicely within the workflow of current forensic DNA laboratories. As a result, it should prove to be a useful technique for processing trace evidence samples for serological analysis.

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.

TGFA/TAQ I polymorphism in nonsyndromic cleft lip and palate patients from Rio Grande Do Sul, Brazil.

Objective: To test the TGFA/Taq I polymorphism in the development of nonsyndromic cleft lip and palate. Design And Setting: The research was based on a case-control study, including nonsyndromic cleft lip and palate patients (140 individuals) and a control sample of unaffected individuals (142) to ascertain the absence or presence of genic mutation at the TGFA locus. Interventions: The DNA of carriers of nonsyndromic cleft lip with or without cleft palate was obtained by buccal swab, and the DNA of the control group was extracted from peripheral blood leucocytes. TGFA/Taq I polymorphism was determined genetically by polymerase chain reaction using specific primers and fragment digestion with Taq I restriction enzyme. Results: No significant association was detected when patients and controls were compared with the genotype for TGFA/Taq I polymorphism. Conclusion: Mutations in TGFA gene have no association with nonsyndromic cleft lip and palate in the sample from Rio Grande do Sul. Therefore, based on this study, it is not possible to determine the role played by TGFA in the expression of cleft lip and palate.