Nasopharyngeal colonization by pathogenic bacteria: effect of polymorphisms in innate immune genes of young children

Nasopharyngeal bacteria can asymptomatically colonize the nasopharynx of infants and young children but are also associated with the development of respiratory infections and diseases. Such nasopharyngeal bacteria include Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae and Staphylococcus aureus. The host defense against invading pathogens is largely relies germline-encoded pattern recognition receptors (PRR), which are expressed on the cells of innate immunity, and different cytokines. These include toll-like receptors (TLR), mannose-binding lectin (MBL) and different cytokines such as IL-17A. Single nucleotide polymorphisms (SNP) in these receptors and cytokines have been reported. The aim of this study was to investigate genetic polymorphisms in the genes for TLR2, 3 and 4, MBL as well as for IL-17A and their associations with nasopharyngeal pathogenic bacterial colonization during a two-year follow-up. The study revealed that polymorphisms in TLRs, MBL2 and IL17A are associated with the nasopharyngeal bacterial colonization in young children. Healthy young (2.6 months of age) children with variant types of MBL2, TLR2 R753Q or TLR4 D299G had an increased risk to be colonized by S. pneumonia, S. aureus or M. catarrhalis, respectively. Moreover, variant types of MBL2 in healthy children with might facilitate human rhinovirus (HRV)-induced S. pneumoniae colonization at 2.6 months of age. The polymorphism of TLR4 D299G was shown to be associated with M. catarrhalis colonization throughout the whole two-year follow-up (2.6, 13 and 24 months of age) and also with the bacterial load of this pathogen. Also, the polymorphism of IL17A G152A was shown to be associated with increased risk to be colonized by S. pneumoniae at 13 and 24 months of age. Furthermore, the results suggest that IL17A G152A has an effect on production of serum IL-17A already at young age. In conclusion, the results of this study indicate that polymorphisms in the key PRRs and IL17A seem to play an important role to colonization of S. pneumoniae, M. catarrhalis, and S. aureus in healthy young Finnish children. The nasopharyngeal colonization by these pathogenic bacteria may further promote the development of respiratory infections and may be related to development of asthma and allergy in the later life of children. These findings offer a possible explanation why some children have more respiratory infections than other children and provide a rational basis for future studies in this field.

Analysis of renin-angiotensin-aldosterone system gene polymorphisms in resistant hypertension

Essential hypertension is a disease multifactorially triggered by genetic and environmental factors. The contribution of genetic polymorphisms of the renin-angiotensin-aldosterone system and clinical risk factors to the development of resistant hypertension was evaluated in 90 hypertensive patients and in 115 normotensive controls living in Southwestern Brazil. Genotyping for insertion/deletion of angiotensin-converting enzyme, angiotensinogen M235T, angiotensin II type 1 receptor A1166C, aldosterone synthase C344T, and mineralocorticoid receptor A4582C polymorphisms was performed by PCR, with further restriction analysis when required. The influence of genetic polymorphisms on blood pressure variation was assessed by analysis of the odds ratio, while clinical risk factors were evaluated by logistic regression. Our analysis indicated that individuals who carry alleles 235-T, 1166-A, 344-T, or 4582-C had a significant risk of developing resistant hypertension (P < 0.05). Surprisingly, when we tested individuals who carried the presumed risk genotypes A1166C, C344T, and A4582C we found that these genotypes were not associated with resistant hypertension. However, a gradual increase in the risk to develop resistant hypertension was detected when the 235-MT and TT genotypes were combined with one, two or three of the supposedly more vulnerable genotypes - A1166C (AC/AA), C344T (TC/TT) and A4582C (AC/CC). Analysis of clinical parameters indicated that age, body mass index and gender contribute to blood pressure increase (P < 0.05). These results suggest that unfavorable genetic renin-angiotensin-aldosterone system patterns and clinical risk variables may contribute to increasing the risk for the development of resistant hypertension in a sample of the Brazilian population.

Influence of the polymorphisms of the α-major regulatory element HS-40 on in vitro gene expression

The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

Impact of β-2 Thr164Ile and combined β-adrenergic receptor polymorphisms on prognosis in a cohort of heart failure outpatients

Genetic polymorphisms of adrenergic receptors (ARs) have been associated with the development, progression, and prognosis of patients with heart failure (HF), with few data for the Brazilian population. We evaluated the role of the β2-AR Thr164Ile polymorphism at codon 164 on prognosis in a prospective study on 315 adult Brazilian HF patients, predominantly middle-aged Caucasian men in functional class I-II, with severe left ventricular systolic dysfunction. Genomic DNA was extracted from peripheral blood and β2-AR164 genotypes were detected by PCR followed by restriction fragment length analysis. During a median follow-up of 3 years, 95 deaths occurred and 57 (60%) were HF-related. Unexpectedly, Ile164 carriers (N = 12) had no HF-related events (log-rank P value = 0.13). Analysis using genotype combination with β1-AR polymorphisms at codons 49 and 389 identified patients with favorable genotypes (Thr164Ile of β2-AR, Gly49Gly of β1-AR and/or Gly389Gly of β1-AR), who had lower HF-related mortality (P = 0.01). In a Cox proportional hazard model adjusted for other clinical characteristics, having any of the favorable genotypes remained as independent predictor of all-cause (hazard ratio (HR): 0.41, 95%CI: 0.17-0.95) and HF-related mortality (HR: 0.12, 95%CI: 0.02-0.90). These data show that the β2-AR Thr164Ile polymorphism had an impact on prognosis in a Brazilian cohort of HF patients. When combined with common β1-AR polymorphisms, a group of patients with a combination of favorable genotypes could be identified.

Education, tobacco smoking, alcohol consumption, and IL-2 and IL-6 gene polymorphisms in the survival of head and neck cancer

The association of education, tobacco smoking, alcohol consumption, and interleukin-2 (IL-2 +114 and -384) and -6 (IL-6 -174) DNA polymorphisms with head and neck squamous cell carcinoma (HNSCC) was investigated in a cohort study of 445 subjects. IL-2 and IL-6 genotypes were determined by real-time PCR. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (95%CI) of disease-specific survival according to anatomical sites of the head and neck. Mean age was 56 years and most patients were males (87.6%). Subjects with 5 or more years of schooling had better survival in larynx cancer. Smoking had no effect on HNSCC survival, but alcohol consumption had a statistically significant effect on larynx cancer. IL-2 gene +114 G/T (HR = 0.52; 95%CI = 0.15-1.81) and T/T (HR = 0.22; 95%CI = 0.02-3.19) genotypes were associated with better survival in hypopharynx cancer. IL-2 +114 G/T was a predictor of poor survival in oral cavity/oropharynx cancer and larynx cancer (HR = 1.32; 95%CI = 0.61-2.85). IL-2 -384 G/T was associated with better survival in oral cavity/oropharynx cancer (HR = 0.80; 95%CI = 0.45-1.42) and hypopharynx cancer (HR = 0.68; 95%CI = 0.21-2.20), but an inverse relationship was observed for larynx cancer. IL-6 -174 G/C was associated with better survival in hypopharynx cancer (HR = 0.68; 95%CI = 0.26-1.78) and larynx cancer (HR = 0.93; 95%CI = 0.42-2.07), and C/C reduced mortality in larynx cancer. In general, our results are similar to previous reports on the value of education, smoking, alcohol consumption, and IL-2 and IL-6 genetic polymorphisms for the prognosis of HNSCC, but the risks due to these variables are small and estimates imprecise.

Association of MICA gene polymorphisms with liver fibrosis in schistosomiasis patients in the Dongting Lake region

Major histocompatibility complex class I chain-related A (MICA) is a highly polymorphic gene located within the MHC class I region of the human genome. Expressed as a cell surface glycoprotein, MICA modulates immune surveillance by binding to its cognate receptor on natural killer cells, NKG2D, and its genetic polymorphisms have been recently associated with susceptibility to some infectious diseases. We determined whether MICA polymorphisms were associated with the high rate of Schistosoma parasitic worm infection or severity of disease outcome in the Dongting Lake region of Hunan Province, China. Polymerase chain reaction-sequence specific priming (PCR-SSP) and sequencing-based typing (SBT) were applied for high-resolution allele typing of schistosomiasis cases (N = 103, age range = 36.2-80.5 years, 64 males and 39 females) and healthy controls (N = 141, age range = 28.6-73.3 years, 73 males and 68 females). Fourteen MICA alleles and five short-tandem repeat (STR) alleles were identified among the two populations. Three (MICA*012:01/02, MICA*017 and MICA*027) showed a higher frequency in healthy controls than in schistosomiasis patients, but the difference was not significantly correlated with susceptibility to S. japonicum infection (Pc > 0.05). In contrast, higher MICA*A5 allele frequency was significantly correlated with advanced liver fibrosis (Pc < 0.05). Furthermore, the distribution profile of MICA alleles in this Hunan Han population was significantly different from those published for Korean, Thai, American-Caucasian, and Afro-American populations (P < 0.01), but similar to other Han populations within China (P > 0.05). This study provides the initial evidence that MICA genetic polymorphisms may underlie the severity of liver fibrosis occurring in schistosomiasis patients from the Dongting Lake region.

Prevalence of ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms in Brazilian breast cancer-unaffected women

Polymorphisms of hormone receptor genes have been linked to modifications in reproductive factors and to an increased risk of breast cancer (BC). In the present study, we have determined the allelic and genotypic frequencies of the ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms and investigated their relationship with mammographic density, body mass index (BMI) and other risk factors for BC. A consecutive and unselected sample of 750 Brazilian BC-unaffected women enrolled in a mammography screening program was recruited. The distribution of PGR PROGINS genotypic frequencies was 72.5, 25.5 and 2.0% for A1A1, A1A2 and A2A2, respectively, which was equivalent to that encountered in other studies with healthy women. The distribution of ERα genotypes was: ERα-397 PvuII C/T: 32.3% TT, 47.5% TC, and 20.2% CC; ERα-351 XbaI A/G: 46.3% AA, 41.7% AG and 12.0% GG. ERα haplotypes were 53.5% PX, 14.3% Px, 0.3% pX, and 32.0% px. These were significantly different from most previously published reports worldwide (P < 0.05). Overall, the PGR PROGINS genotypes A2A2 and A1A2 were associated with fatty and moderately fatty breast tissue. The same genotypes were also associated with a high BMI in postmenopausal women. In addition, the ERα-351 XbaI GG genotype was associated with menarche ≥12 years (P = 0.02). ERα and PGR polymorphisms have a phenotypic effect and may play an important role in BC risk determination. Finally, if confirmed in BC patients, these associations could have important implications for mammographic screening and strategies and may be helpful to identify women at higher risk for the disease.

Association among genetic predisposition, gut microbiota, and host immune response in the etiopathogenesis of inflammatory bowel disease

Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is a chronic disorder that affects thousands of people around the world. These diseases are characterized by exacerbated uncontrolled intestinal inflammation that leads to poor quality of life in affected patients. Although the exact cause of IBD still remains unknown, compelling evidence suggests that the interplay among immune deregulation, environmental factors, and genetic polymorphisms contributes to the multifactorial nature of the disease. Therefore, in this review we present classical and novel findings regarding IBD etiopathogenesis. Considering the genetic causes of the diseases, alterations in about 100 genes or allelic variants, most of them in components of the immune system, have been related to IBD susceptibility. Dysbiosis of the intestinal microbiota also plays a role in the initiation or perpetuation of gut inflammation, which develops under altered or impaired immune responses. In this context, unbalanced innate and especially adaptive immunity has been considered one of the major contributing factors to IBD development, with the involvement of the Th1, Th2, and Th17 effector population in addition to impaired regulatory responses in CD or UC. Finally, an understanding of the interplay among pathogenic triggers of IBD will improve knowledge about the immunological mechanisms of gut inflammation, thus providing novel tools for IBD control.

A hierarchical Bayesian model for predicting the functional consequences of amino-acid polymorphisms

Genetic polymorphisms in deoxyribonucleic acid coding regions may have a phenotypic effect on the carrier, e.g. by influencing susceptibility to disease. Detection of deleterious mutations via association studies is hampered by the large number of candidate sites; therefore methods are needed to narrow down the search to the most promising sites. For this, a possible approach is to use structural and sequence-based information of the encoded protein to predict whether a mutation at a particular site is likely to disrupt the functionality of the protein itself. We propose a hierarchical Bayesian multivariate adaptive regression spline (BMARS) model for supervised learning in this context and assess its predictive performance by using data from mutagenesis experiments on lac repressor and lysozyme proteins. In these experiments, about 12 amino-acid substitutions were performed at each native amino-acid position and the effect on protein functionality was assessed. The training data thus consist of repeated observations at each position, which the hierarchical framework is needed to account for. The model is trained on the lac repressor data and tested on the lysozyme mutations and vice versa. In particular, we show that the hierarchical BMARS model, by allowing for the clustered nature of the data, yields lower out-of-sample misclassification rates compared with both a BMARS and a frequen-tist MARS model, a support vector machine classifier and an optimally pruned classification tree.

The role played by the interaction between genetic factors and attachment in the stress response in infancy

Background: The importance of understanding which environmental and biological factors are involved in determining individual differences in physiological response to stress is widely recognized, given the impact that stress has on physical and mental health. Methods: The child-mother attachment relationship and some genetic polymorphisms (5-HTTLPR, COMT and GABRA6) were tested as predictors of salivary cortisol and alpha amylase concentrations, two biomarkers of hypothalamic-pituitary-adrenocortical (HPA) axis and sympathetic adrenomedullary (SAM) system activity, during the Strange Situation (SS) procedure in a sample of more than 100 healthy infants, aged 12 to 18 months. Results: Individual differences in alpha amylase response to separation were predicted by security of attachment in interaction with 5-HTTLPR and GABRA6 genetic polymorphisms, whereas alpha amylase basal levels were predicted by COMT x attachment interaction. No significant effect of attachment, genetics and their interaction on cortisol activity emerged. Conclusions: These results help to disentangle the role played by both genetic and environmental factors in determining individual differences in stress response in infancy. The results also shed light on the suggestion that HPA and SAM systems are likely to have different characteristic responses to stress.

CYP1A2*1C, CYP2E1*5B, and GSTM1 polymorphisms are predictors of risk and poor outcome in head and neck squamous cell carcinoma patients

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic markers between Biomphalaria glabrata snails susceptible and resistant to Schistosoma mansoni infection

The analysis of the genetic variability related to susceptibility to Schistosoma mansoni infection in the vector of the genus Biomphalaria is important in terms of a better understanding of the epidemiology of schistosomiasis itself, the possible pathological implications of this interaction in vertebrate hosts, and the formulation of new strategies and approaches for disease control. In the present study, the genetic variability of B. glabrata strains found to be resistant or susceptible to S. mansoni infection was investigated using DNA amplification by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). The amplification products were analyzed on 8% polyacrylamide gel and stained with silver. We selected 10 primers, since they have previously been useful to detect polymorphism among B. glabrata and/or B. tenagophila. The results showed polymorphisms with 5 primers. Polymorphic bands observed only in the susceptible strain. The RAPD-PCR methodology represents an adequate approach for the analysis of genetic polymorphisms. The understanding of the genetic polymorphisms associated to resistance may contribute to the future identification of genomic sequences related to the resistance/susceptibility of Biomphalaria to the larval forms of S. mansoni and to the development of new strategies for the control of schistosomiasis.

Polymorphisms of the adiponectin gene in gestational hypertension and pre-eclampsia

Adiponectin is a hormone involved in energy homeostasis by regulating glucose and lipid metabolism. In addition, the adiponectin gene (ADIPOQ) has polymorphisms that can modulate the circulating concentration of adiponectin. Abnormal adiponectin levels have been associated with pre-eclampsia (PE); however, the influence of genetic polymorphisms on the development of hypertensive disorders of pregnancy is unclear. The aim of this study was to examine whether ADIPOQ polymorphisms are associated with gestational hypertension (GH) and/or PE. We studied 401 pregnant women: 161 healthy pregnant (HP), 113 pregnant with GH and 127 pregnant with PE. ADIPOQ polymorphisms -11391G>A (rs17300539), -11377C>G (rs266729), 45T>G (rs2241766) and 276G>T (rs1501299) were genotyped by allelic discrimination assays using real-time PCR. Haplotypes were inferred using the PHASE 2.1 program. We observed that the genotypic frequencies of the -11377C>G polymorphism were different in PE compared with HP (P<0.0125), with the CT genotype being more commonly found in PE patients than in HP women (P<0.0125). However, allelic frequencies of this single-nucleotide polymorphism were similar between PE and HP (P>0.0125). No difference was observed when GH and HP groups were compared (both P>0.0125). In addition, we found no difference in genotype or allele distributions for the -11391G>A, 45T>G and 276G>T polymorphisms when we compared GH or PE with HP (all P>0.0125). In conclusion, we found a modest association between the CG genotype of the -11377C>G polymorphism and PE.Journal of Human Hypertension advance online publication, 27 June 2013; doi:10.1038/jhh.2013.53.

Frequencies of-308G/A (TNFA) and-509C/T (TGFB1) polymorphisms in sickle cell anemia patients from Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Polymorphisms in endothelial nitric oxide synthase gene in early and late severe preeclampsia

Preeclampsia (PE) is characterized by hypertension and proteinuria, occurring after the 20th week of pregnancy in women who have had no previous symptoms. The disease progresses with generalized vasoconstriction and endothelial dysfunction. Clinically, it is important to diagnose the severe form of the disease (sPE), in which blood pressure and proteinuria are much higher. Recently, the gestational age (GA) of the onset of PE has led to the classification of this disease as early (GA <34 weeks) and late (GA >= 34 weeks). Several genetic polymorphisms affecting endothelial nitric oxide synthase (eNOS) levels or function were described, including G894T (Glu298Asp), VNTR b/a (variable-number 27-bp tandem repeat) and T-786C (promoter) polymorphisms. Thus, the aim of this study was to compare the distribution of G894T, VNTR b/a and T-786C polymorphisms and their haplotypes in Brazilian early and late sPE, as well as in normotensive pregnant. A total of 201 women were evaluated, 53 with early sPE, 45 with late sPE and 103 as normotensive pregnant women. The frequency of 894T allele was higher in late sPE vs normotensive pregnant, and 894TT genotype was higher in late sPE vs early sPE and normotensive pregnant. For VNTR b/a polymorphism, higher frequencies of aa genotype and a allele were observed in early sPE vs late sPE and normotensive pregnant. Besides, the frequency of haplotype T-b-C was higher in late sPE vs early sPE and normotensive pregnant. Considering the results found for eNOS polymorphisms, it is possible to suggest that the functional alterations induced by these two polymorphisms may influence the time of severe PE onset, although both alterations are putatively associated with low NO bioavailability. However, other studies are necessary to validate these findings and clarify this issue. (C) 2014 Elsevier Inc. All rights reserved.