FASL polymorphism is associated with response to bacillus Calmette-Guérin immunotherapy in bladder cancer

Objective Deregulation of FAS/FASL system may lead to immune escape and influence bacillus Calmette-Guérin (BCG) immunotherapy outcome, which is currently the gold standard adjuvant treatment for high-risk non–muscle invasive bladder tumors. Among other events, functional promoter polymorphisms of FAS and FASL genes may alter their transcriptional activity. Therefore, we aim to evaluate the role of FAS and FASL polymorphisms in the context of BCG therapy, envisaging the validation of these biomarkers to predict response. Patients and methods DNA extracted from peripheral blood from 125 patients with bladder cancer treated with BCG therapy was analyzed by Polymerase Chain Reaction—Restriction Fragment Length Polymorphism for FAS-670 A/G and FASL-844 T/C polymorphisms. FASL mRNA expression was analyzed by real-time Polymerase Chain Reaction. Results Carriers of FASL-844 CC genotype present a decreased recurrence-free survival after BCG treatment when compared with FASL-844 T allele carriers (mean 71.5 vs. 97.8 months, P = 0.030) and have an increased risk of BCG treatment failure (Hazard Ratio = 1.922; 95% Confidence Interval: [1.064–3.471]; P = 0.030). Multivariate analysis shows that FASL-844 T/C and therapeutics scheme are independent predictive markers of recurrence after treatment. The evaluation of FASL gene mRNA levels demonstrated that patients carrying FASL-844 CC genotype had higher FASL expression in bladder tumors (P = 0.0027). Higher FASL levels were also associated with an increased risk of recurrence after BCG treatment (Hazard Ratio = 2.833; 95% Confidence Interval: [1.012–7.929]; P = 0.047). FAS-670 A/G polymorphism analysis did not reveal any association with BCG therapy outcome. Conclusions Our results suggest that analysis of FASL-844 T/C, but not FAS-670 A/G polymorphisms, may be used as a predictive marker of response to BCG immunotherapy.

Q192R polymorphism of the paraoxonase-1 gene as a risk factor for obesity in Portuguese women

Introduction - Obesity became a major public health problem as a result of its increasing prevalence worldwide. Paraoxonase-1 (PON1) is an esterase able to protect membranes and lipoproteins from oxidative modifications. At the PON1 gene, several polymorphisms in the promoter and coding regions have been identified. The aims of this study were i) to assess PON1 L55M and Q192R polymorphisms as a risk factor for obesity in women; ii) to compare PON1 activity according to the expression of each allele in L55M and Q192R polymorphisms; iii) to compare PON1 activity between obese and normal-weight women. Materials and methods - We studied 75 healthy (35.9±8.2 years) and 81 obese women (34.3±8.2 years). Inclusion criteria for obese subjects were body mass index ≥30 kg/m2 and absence of inflammatory/neoplasic conditions or kidney/hepatic dysfunction. The two PON1 polymorphisms were assessed by real-time PCR with TaqMan probes. PON1 enzymatic activity was assessed by spectrophotometric methods, using paraoxon as a substrate. Results - No significant differences were found for PON1 activity between normal and obese women. Nevertheless, PON1 activity was greater (P<0.01) for the RR genotype (in Q192R polymorphism) and for the LL genotype (in L55M polymorphism). The frequency of allele R of Q192R polymorphism was significantly higher in obese women (P<0.05) and was associated with an increased risk of obesity (odds ratio=2.0 – 95% confidence interval (1.04; 3.87)). Conclusion - 55M and Q192R polymorphisms influence PON1 activity. The allele R of the Q192R polymorphism is associated with an increased risk for development of obesity among Portuguese Caucasian premenopausal women.

Impact of the IL-18 gene polymorphism in response to antiviral therapy in chronic HCV genotype 4 patients

^a Introduction Interleukin (IL)-18 is a well-known major proinflammatory cytokine with broad biological effects. The major immunomodulatory functions of IL-18 include enhancing T cell and natural killer cell cytotoxicity. Serum levels of this cytokine were shown to increase in chronic hepatitis C patients compared to non-infected healthy people. An association between IL-18 gene promoter polymorphisms and pegylated interferon (PEG-IFN) and ribavirin treatment outcomes has been reported for individuals with chronic hepatitis C virus genotype 1 (HCV-1). In this study, HCV genotype 4 (HCV-4) patients were assessed for IL-18 gene polymorphisms and treatment outcomes or severity of liver disease because data concerning the impact of IL-18 gene polymorphisms on patients with HCV-4 infections are limited. Methods This study included 123 chronic HCV-4 Egyptian patients and 123 apparently healthy volunteer blood donors who served as a control group. HCV genotyping was performed using the line probe assay. IL-18 genotyping was performed using the TaqMan Real-Time PCR method in all 246 patient and control samples. Results In our study, all patients had HCV-4. IL-18 gene single nucleotide polymorphism (SNP) (-607C/A) genotype distributions and allele frequencies did not differ between HCV patients and normal healthy subjects or between patient groups when compared according to the therapeutic response. Moreover, the presence of an IL-18 SNP was not associated with histological disease severity. We conclude that the presence of the IL-18 SNP rs1946518 does not affect the outcome of chronic HCV-4 treatment in Egyptian patients. Conclusions The IL-18 SNP rs1946518 does not affect response to treatment in chronic HCV-4 patients.

Variant within the promoter region of the CHRNA3 gene associated with FTN dependence is not related to self-reported willingness to quit smoking.

INTRODUCTION: Common variation in the CHRNA5-CHRNA3-CHRNB4 gene region is robustly associated with smoking quantity. Conversely, the association between one of the most significant single nucleotide polymorphisms (SNPs; rs1051730 within the CHRNA3 gene) with perceived difficulty or willingness to quit smoking among current smokers is unknown. METHODS: Cross-sectional study including current smokers, 502 women, and 552 men. Heaviness of smoking index (HSI), difficulty, attempting, and intention to quit smoking were assessed by questionnaire. RESULTS: The rs1051730 SNP was associated with increased HSI (age, gender, and education-adjusted mean ± SE: 2.6 ± 0.1, 2.2 ± 0.1, and 2.0 ± 0.1 for AA, AG, and GG genotypes, respectively, p < .01). Multivariate logistic regression adjusting for gender, age, education, leisure-time physical activity, and personal history of cardiovascular or lung disease showed rs1051730 to be associated with higher smoking dependence (odds ratio [OR] and 95% CI for each additional A-allele: 1.38 [1.11-1.72] for smoking more than 20 cigarette equivalents/day; 1.31 [1.00-1.71] for an HSI ≥5 and 1.32 [1.05-1.65] for smoking 5 min after waking up) and borderline associated with difficulty to quit (OR = 1.29 [0.98-1.70]), but this relationship was no longer significant after adjusting for nicotine dependence. Also, no relationship was found with willingness (OR = 1.03 [0.85-1.26]), attempt (OR = 1.00 [0.83-1.20]), or preparation (OR = 0.95 [0.38-2.38]) to quit. Similar findings were obtained for other SNPs, but their effect on nicotine dependence was no longer significant after adjusting for rs1051730. Conclusions: These data confirm the effect of rs1051730 on nicotine dependence but failed to find any relationship with difficulty, willingness, and motivation to quit.

Characterization of polymorphisms in the mannose-binding lectin gene promoter among human immunodeficiency virus 1 infected subjects

The present study investigated the prevalence of mutations in the -550 (H/L) and -221 (X/Y) mannose-binding lectin (MBL) gene promoter regions and their impact on infection by human immunodeficiency virus 1 (HIV-1) in a population of 128 HIV-1 seropositive and 97 seronegative patients. The allele identification was performed through the sequence-specific primer polymerase chain reaction method, using primer sequences specific to each polymorphism. The evolution of the infection was evaluated through CD4+ T-lymphocyte counts and plasma viral load. The allele and haplotype frequencies among HIV-1-infected patients and seronegative healthy control patients did not show significant differences. CD4+ T-lymphocyte counts showed lower levels among seropositive patients carrying haplotypes LY, LX and HX, as compared to those carrying the HY haplotype. Mean plasma viral load was higher among seropositive patients with haplotypes LY, LX and HX than among those carrying the HY haplotype. When promoter and exon 1 mutations were matched, it was possible to identify a significantly higher viral load among HIV-1 infected individuals carrying haplotypes correlated to low serum levels of MBL. The current study shows that haplotypes related to medium and low MBL serum levels might directly influence the evolution of viral progression in patients. Therefore, it is suggested that the identification of haplotypes within the promoter region of the MBL gene among HIV-1 infected persons should be further evaluated as a prognostic tool for AIDS progression.

The INSIG2 rs7566605 polymorphism is not associated with body mass index and breast cancer risk

Background The single nucleotide polymorphism rs7566605, located in the promoter of the INSIG2 gene, has been the subject of a strong scientific effort aimed to elucidate its possible association with body mass index (BMI). The first report showing that rs7566605 could be associated with body fatness was a genome-wide association study (GWAS) which used BMI as the primary phenotype. Many follow-up studies sought to validate the association of rs7566605 with various markers of obesity, with several publications reporting inconsistent findings. BMI is considered to be one of the measures of choice to evaluate body fatness and there is evidence that body fatness is related with an increased risk of breast cancer (BC). Methods we tested in a large-scale association study (3,973 women, including 1,269 invasive BC cases and 2,194 controls), nested within the EPIC cohort, the involvement of rs7566605 as predictor of BMI and BC risk. Results and Conclusions In this study we were not able to find any statistically significant association between this SNP and BMI, nor did we find any significant association between the SNP and an increased risk of breast cancer overall and by subgroups of age, or menopausal status.

Molecular characterization of regulatory polymorphisms in the promoter region of the STAT6 gene in a Gabonese population

Parasites remain competent invaders of host immunity. Their invasion strategies have proven to impact immunorelevant genes leading to diversity among gene families. We focussed on signal transducer and activator of transcription (STAT6) factor that plays a fundamental role in signal transduction and activation of transcription. Recent studies have highlighted the role of STAT6 variants in control of infection levels. We identified and investigated regulatory single nucleotide polymorphisms (SNPs) in the promoter regions of the STAT6 gene in a group of Gabonese individuals exposed to a variety of parasitic infections. Three promoter variants were identified in 40 individual subjects. We further validated these promoter variants for their allelic gene expression using transient transfection assays. One promoter variant, rs3024944 (G/C), revealed an altered expression of the marker gene. The identification of function-altering SNPs in the promoter may facilitate studying parasite susceptibility in association studies.

Polymorphism analysis of the CTLA-4 gene in paracoccidioidomycosis patients

The CTLA-4 protein is expressed in activated T cells and plays an essential role in the immune response through its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated with autoimmune, neoplastic and infectious illnesses. This work aimed to verify possible associations between single nucleotide polymorphisms (SNPs) in CTLA-4, -318C/T in the promoter and +49A/G in exon 1 and paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. For this purpose, 66 chronic form PCM patients and 76 healthy controls had their allele, genotype and haplotype frequencies determined. The genetic admixture structure of the patients and controls was evaluated to eliminate ancestral bias. The comparison of frequencies indicated no significant differences between patients and controls that could link the SNPs to PCM. Groups were admixture matched with no difference observed in population ancestry inference, indicating that the absence of association between CTLA-4 polymorphisms and PCM could not be attributed to ancestral bias. This study showed that there was no association between the CTLA-4 SNPs -318 and +49 and the resistance or susceptibility to PCM.

Islet-brain1/C-Jun N-terminal kinase interacting protein-1 (IB1/JIP-1) promoter variant is associated with Alzheimer's disease.

Islet-brain1 (IB1) or c-Jun NH2 terminal kinase interacting protein-1 (JIP-1), the product of the MAPK8IP1 gene, functions as a neuronal scaffold protein to allow signalling specificity. IB1/JIP-1 interacts with many cellular components including the reelin receptor ApoER2, the low-density lipoprotein receptor-related protein (LRP), kinesin and the Alzheimer's amyloid precursor protein. Coexpression of IB1/JIP-1 with other components of the c-Jun NH2 terminal-kinase (JNK) pathway activates the JNK activity; conversely, selective disruption of IB1/JIP-1 in mice reduces the stress-induced apoptosis of neuronal cells. We therefore hypothesized that IB1/JIP-1 is a risk factor for Alzheimer's disease (AD). By immunocytochemistry, we first colocalized the presence of IB1/JIP-1 with JNK and phosphorylated tau in neurofibrillary tangles. We next identified a -499A>G polymorphism in the 5' regulatory region of the MAPK8IP1 gene. In two separate French populations the -499A>G polymorphism of MAPK8IP1 was not associated with an increased risk to AD. However, when stratified on the +766C>T polymorphism of exon 3 of the LRP gene, the IB1/JIP-1 polymorphism was strongly associated with AD in subjects bearing the CC genotype in the LRP gene. The functional consequences of the -499A>G polymorphism of MAPK8IP1 was investigated in vitro. In neuronal cells, the G allele increased transcriptional activity and was associated with an enhanced binding activity. Taken together, these data indicate that the increased transcriptional activity in the presence of the G allele of MAPK8IP1 is a risk factor to the onset of in patients bearing the CC genotype of the LRP gene.

A polymorphic microsatellite in the tumor necrosis factor alpha promoter identifies an allele unique to the NZW mouse strain.

We have amplified a (CA)n:(GT)n microsatellite from the TNF promoters of a panel of mouse strains using the polymerase chain reaction. The length of the microsatellites was polymorphic, with eight alleles observed among 15 inbred strains bearing seven distinct H-2 haplotypes, and four outbred strains. In B10 congenic strains, the TNF allele detected by microsatellite polymorphism segregated with the MHC, and in recombinant haplotypes (NOD, NZW), it segregated with H-2D. The TNF allele found in the NZW strain (H-2z) was distinct from those of all other haplotypes, consistent with the hypothesis that this strain may carry a genetic defect in TNF production.

Functional studies of a germ-line polymorphism at codon 47 within the p53 gene.

A rare germ-line polymorphism in codon 47 of the p53 gene replaces the wild-type proline (CCG) with a serine (TCG). Restriction analysis of 101 human samples revealed the frequency of the rare allele to be 0% (n = 69) in Caucasians and 4.7% (3/64, n = 32) among African-Americans. To investigate the consequence of this amino acid substitution, a cDNA construct (p53 mut47ser) containing the mutation was introduced into a lung adenocarcinoma cell line (Calu-6) that does not express p53. A growth suppression similar to that obtained after introduction of a wild-type p53 cDNA construct was observed, in contrast to the result obtained by introduction of p53 mut143ala. Furthermore, expression of neither p53 mut47ser nor wild-type p53 was tolerated by growing cells. In transient expression assays, both mut47ser and wild-type p53 activated the expression of a reporter gene linked to a p53 binding sequence (PG13-CAT) and inhibited the expression of the luciferase gene under the control of the Rous sarcoma virus promoter (RSVluc). In the same assay, mut143ala did not activate the expression of PG13-CAT and produced only a slight inhibitory effect on RSVluc. These findings indicate that the p53 variant with a serine at codon 47 should be considered as a rare germ-line polymorphism that does not alter the growth-suppression activity of p53.

Association studies between -1185A/G von Willebrand factor gene polymorphism and coronary artery disease

High levels of von Willebrand factor (vWF) have been associated with cardiovascular disease. The A allele of the -1185A/G polymorphism in the 5'-regulatory region of the vWF gene was associated with the highest plasma vWF levels in a normal population. To examine the association between -1185A/G polymorphism and coronary artery disease (CAD), 173 Brazilian Caucasian subjects submitted to coronary angiography were studied. Of these, 57 (33%) had normal coronary arteries (control group) and 116 (67%) had CAD (patient group). Plasma vWF levels were higher in patients (145 U/dl) than in controls (130 U/dl), but the differences were significant only for O blood group subjects. Polymerase chain reaction amplification of the 864-bp vWF promoter region followed by AccII restriction digestion was used to identify the -1185A/G genotypes. The -1185A allele frequency was 43.1% in patients and 44.7% in controls. Allele and genotype frequencies were not significantly different between patients and controls. No association was observed between the -1185A/G genotypes and plasma vWF levels in patients or controls. These results suggest that -1185A/G polymorphism is not an independent risk factor for CAD.

Novel regulatory SNPs in the promoter region of the TNFRSF18 gene in a Gabonese population

Parasites are accountable for driving diversity within immune gene families. We identified and investigated regulatory single nucleotide polymorphisms (SNPs) in the promoter regions of the tumor necrosis factor receptor superfamily member 18 (TNFRSF18) gene by direct sequencing in a group of male Gabonese individuals exposed to a wide array of parasitic diseases such as malaria, filariasis and schistosomiasis. Two new promoter variants were identified in 40 individuals. Both novel variants were heterozygous and were linked to SNP #rs3753344 (C/T), which has been described. One of the SNP variants (ss2080581728) was close to the general transcription factor site, the TATA box. We further validated these new promoter variants for their allelic gene expression using transient transfection assays. One new promoter variant with two base changes (C/T - ss2080581728/rs3753344) displayed an altered expression of the marker gene. Both novel variants remained less active at the non-induced state in comparison to the major allele. The allele frequencies observed in this study were consistent with data for other African populations. The detection and analysis of these human immune gene polymorphisms contribute to a better understanding of the interaction between host-parasite and expression of Treg activity.

MTP -493G/T gene polymorphism is associated with steatosis in hepatitis C-infected patients

The reduction of hepatic microsomal transfer protein (MTP) activity results in fatty liver, worsening hepatic steatosis and fibrosis in chronic hepatitis C (CHC). The G allele of the MTP gene promoter, -493G/T, has been associated with lower transcriptional activity than the T allele. We investigated this association with metabolic and histological variables in patients with CHC. A total of 174 untreated patients with CHC were genotyped for MTP -493G/T by direct sequencing using PCR. All patients were negative for markers of Wilson’s disease, hemochromatosis and autoimmune diseases and had current and past daily alcohol intake lower than 100 g/week. The sample distribution was in Hardy-Weinberg equilibrium. Among subjects with genotype 1, 56.8% of the patients with fibrosis grade 3+4 presented at least one G allele versus 34.3% of the patients with fibrosis grade 1+2 (OR = 1.8; 95%CI = 1.3-2.3). Logistic regression analysis with steatosis as the dependent variable identified genotypes GG+GT as independent protective factors against steatosis (OR = 0.4, 95%CI = 0.2-0.8; P = 0.01). The results suggest that the presence of the G allele of MTP -493G/T associated with lower hepatic MTP expression protects against steatosis in our CHC patients.

A novel association of a polymorphism in the first intron of adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia in Asian Indians

Adiponectin is an adipose tissue specific protein that is decreased in subjects with obesity and type 2 diabetes. The objective of the present study was to examine whether variants in the regulatory regions of the adiponectin gene contribute to type 2 diabetes in Asian Indians. The study comprised of 2,000 normal glucose tolerant (NGT) and 2,000 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin levels were measured by radioimmunoassay. We identified two proximal promoter SNPs (-11377C-->G and -11282T-->C), one intronic SNP (+10211T-->G) and one exonic SNP (+45T-->G) by SSCP and direct sequencing in a pilot study (n = 500). The +10211T-->G SNP alone was genotyped using PCR-RFLP in 4,000 study subjects. Logistic regression analysis revealed that subjects with TG genotype of +10211T-->G had significantly higher risk for diabetes compared to TT genotype [Odds ratio 1.28; 95% Confidence Interval (CI) 1.07-1.54; P = 0.008]. However, no association with diabetes was observed with GG genotype (P = 0.22). Stratification of the study subjects based on BMI showed that the odds ratio for obesity for the TG genotype was 1.53 (95%CI 1.3-1.8; P < 10(-7)) and that for GG genotype, 2.10 (95% CI 1.3-3.3; P = 0.002). Among NGT subjects, the mean serum adiponectin levels were significantly lower among the GG (P = 0.007) and TG (P = 0.001) genotypes compared to TT genotype. Among Asian Indians there is an association of +10211T-->G polymorphism in the first intron of the adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia.