A single nucleotide polymorphism, rs129679860, in the IL28B locus is associated with the viral kinetics and a sustained virological response in a chronic, monoinfected hepatitis C virus genotype-1 Brazilian population treated with pegylated interferon-ribavirin

Single nucleotide polymorphisms (SNPs) in the interleukin (IL)28B locus have been associated with a sustained virological response (SVR) in interferon-ribavirin (IFN-RBV)-treated chronic hepatitis C virus (HCV)-infected patients in European and African populations. In this study, the genotype frequency of two IL28B SNPs (rs129679860 and rs8099917) in a cohort of chronic HCV-monoinfected patients in Brazil was evaluated and the SNP sufficient to predict the treatment response outcome was determined. A total of 66 naïve genotype-1 chronic HCV-infected patients were genotyped and the associated viral kinetics and SVR were assessed. The overall SVR was 38%. Both the viral kinetics and SVR were associated with rs129679860 genotypes (CC = 62% vs. CT = 33% vs. TT = 18%, p = 0.016). However, rs8099917 genotypes were only associated with SVR (TT = 53% vs. TG = 33% vs. GG = 18%; p = 0.032). In this population, the analysis of a single SNP, rs12979860, successfully predicts SVR in the IFN-RBV treatment of HCV.

Response to treatment in Brazilian patients with chronic hepatitis C is associated with a single-nucleotide polymorphism near the interleukin-28B gene

A single-nucleotide polymorphism (SNP) upstream of interleukin (IL)28B was recently identified as an important predictor of the outcome of chronic hepatitis C patients treated with pegylated interferon plus ribavirin (PEG-IFN/RBV). The aim of this study was to investigate the association between the IL28B gene polymorphism (rs12979860) and virological response in chronic hepatitis C patients. Brazilian patients (n = 263) who were infected with hepatitis C virus (HCV) genotype 1 and were receiving PEG-IFN/RBV were genotyped. Early virological response (EVR) (12 weeks), end-of-treatment response (EOTR) (48 weeks), sustained virological response (SVR) (72 weeks) and relapse were evaluated using conventional and quantitative polymerase chain reaction (PCR) assays. The frequency of the C allele in the population was 39%. Overall, 43% of patients experienced SVR. The IL28B CC genotype was significantly associated with higher treatment response rates and a lower relapse rate compared to the other genotypes [84% vs. 58% EVR, 92% vs. 63% EOTR, 76% vs. 38% SVR and 17% vs. 40% relapse rate in CC vs. other genotypes (CT and TT), respectively]. Thus, the IL28B genotype appears to be a strong predictor of SVR following PEG-IFN/RBV therapy in treatment-naïve Brazilian patients infected with HCV genotype 1. This study, together with similar research examining other SNPs, should help to define adequate protocols for the treatment of patients infected with HCV genotype 1, especially those with a poor prognosis.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Alternative genotyping method for the single nucleotide polymorphism A2959G (AF159246) of the bovine CAST gene

The objective of this work was to genotype the single nucleotide polymorphism (SNP) A2959G (AF159246) of bovine CAST gene by PCR-RFLP technique, and to report its use for the first time. For this, 147 Bos indicus and Bos taurus x Bos indicus animals were genotyped. The accuracy of the method was confirmed through the direct sequencing of PCR products of nine individuals. The lowest frequency of the meat tenderness favorable allele (A) in Bos indicus was confirmed. The use of PCR-RFLP for the genotyping of the bovine CAST gene SNP was shown to be robust and inexpensive, which will greatly facilitate its analysis by laboratories with basic structure.

A cost-effective melting temperature assay for the detection of single-nucleotide polymorphism in the MBL2 gene of HIV-1-infected children

We report a fast (less than 3 h) and cost-effective melting temperature assay method for the detection of single-nucleotide polymorphisms in the MBL2 gene. The protocol, which is based on the Corbett Rotor Gene real time PCR platform and SYBR Green I chemistry, yielded, in the cohorts studied, sensitive (100%) and specific (100%) PCR amplification without the use of costly fluorophore-labeled probes or post-PCR manipulation. At the end of the PCR, the dissociation protocol included a slow heating from 60º to 95ºC in 0.2ºC steps, with an 8-s interval between steps. Melting curve profiles were obtained using the dissociation software of the Rotor Gene-3000 apparatus. Samples were analyzed in duplicate and in different PCR runs to test the reproducibility of this technique. No supplementary data handling is required to determine the MBL2 genotype. MBL2 genotyping performed on a cohort of 164 HIV-1-positive Brazilian children and 150 healthy controls, matched for age and sex and ethnic origin, yielded reproducible results confirmed by direct sequencing of the amplicon performed in blind. The three MBL2 variants (Arg52Cys, Gly54Asp, Gly57Glu) were grouped together and called allele 0, while the combination of three wild-type alleles was called allele A. The frequency of the A/A homozygotes was significantly higher among healthy controls (0.68) than in HIV-infected children (0.55; P = 0.0234) and the frequency of MBL2 0/0 homozygotes was higher among HIV-1-infected children than healthy controls (P = 0.0296). The 0 allele was significantly more frequent among the 164 HIV-1-infected children (0.29) than among the 150 healthy controls (0.18; P = 0.0032). Our data confirm the association between the presence of the mutated MBL2 allele (allele 0) and HIV-1 infection in perinatally exposed children. Our results are in agreement with the literature data which indicate that the presence of the allele 0 confers a relative risk of 1.37 for HIV-1 infection through vertical transmission.

Single-nucleotide polymorphism analysis of GH, GHR, and IGF-1 genes in minipigs

Tibetan (TB) and Bama (BM) miniature pigs are two popular pig breeds that are used as experimental animals in China due to their small body size. Here, we analyzed single-nucleotide polymorphisms (SNPs) in gene fragments that are closely related to growth traits [growth hormone (GH), growth hormone receptor (GHR), and insulin-like growth factor (IGF)-1)] in these pig breeds and a large white (LW) control pig breed. On the basis of the analysis of 100 BMs, 108 TBs, and 50 LWs, the polymorphic distribution levels of GH, GHR, and IGF-1 were significantly different among these three pig breeds. According to correlation analyses between SNPs and five growth traits - body weight (BW), body length (BL), withers height (WH), chest circumference (CC), and abdomen circumference (AC) - three SNP loci in BMs and four SNP loci in TBs significantly affected growth traits. Three SNP sites in BMs and four SNP sites in TBs significantly affected growth traits. SNPs located in the GH gene fragment significantly affected BL and CC at locus 12 and BL at locus 45 in BMs, and also BW, WH, CC, and AC at locus 45 and WH and CC at locus 93 in TBs. One SNP at locus 85 in the BM GHR gene fragment significantly affected all growth traits. All indices were significantly reduced with a mixture of alleles at locus 85. These results provide more information regarding the genetic background of these minipig species and indicate useful selection markers for pig breeding programs.

Identification of snails within the Bulinus africanus group from East Africa by multiplex SNaPshotTManalysis of single nucleotide polymorphisms within the cytochrome oxidase subunit I

Identification of populations of Bulinus nasutus and B. globosus from East Africa is unreliable using characters of the shell. In this paper, a molecular method of identification is presented for each species based on DNA sequence variation within the mitochondrial cytochrome oxidase subunit I (COI) as detected by a novel multiplexed SNaPshotTM assay. In total, snails from 7 localities from coastal Kenya were typed using this assay and variation within shell morphology was compared to reference material from Zanzibar. Four locations were found to contain B. nasutus and 2 locations were found to contain B. globosus. A mixed population containing both B. nasutus and B. globosus was found at Kinango. Morphometric variation between samples was considerable and UPGMA cluster analysis failed to differentiate species. The multiplex SNaPshotTM assay is an important development for more precise methods of identification of B. africanus group snails. The assay could be further broadened for identification of other snail intermediate host species.

Prevalence of the STK15 F31I polymorphism and its relationship with mammographic density

Several studies have identified the single nucleotide polymorphism STK15 F31I as a low-penetrance risk allele for breast cancer, but its prevalence and risk association in the Brazilian population have not been determined. The goal of this study was to identify the frequency of this polymorphism in the Brazilian setting. Considering the high degree of admixture of our population, it is of fundamental importance to validate the results already reported in the literature and also to verify the relationship between this variant and breast cancer risk. A total of 750 women without breast cancer were genotyped using the TaqMan PCR assay for STK15 F31I polymorphism. Clinical information was obtained from review of the medical records and mammographic density from the images obtained using the BI-RADS System. The estimated risk of developing cancer was calculated according to the Gail model. The genotypic frequencies observed in this study were 4.5, 38.7, and 56.6%, respectively, for the STK15 F31I AA, AT and TT genotypes. The AT and AA genotypes were encountered significantly more often in premenopausal women with moderately dense, dense and heterogeneously dense breast tissue (P = 0.023). In addition, the presence of the TT genotype was significantly associated with age at menarche ≥12 years (P = 0.023). High mammographic density, associated with increased breast cancer risk, was encountered more frequently in premenopausal women with the risk genotypes STK15 F31I AA and AT. The genotypic frequencies observed in our Brazilian sample were similar to those described in other predominantly European populations.

Effect of estrogen receptor-alpha (ESR1) gene polymorphism on high density lipoprotein levels in response to hormone replacement therapy

Studies have shown that estrogen replacement therapy and estrogen plus progestin replacement therapy alter serum levels of total, LDL and HDL cholesterol levels. However, HDL cholesterol levels in women vary considerably in response to hormone replacement therapy (HRT). A significant portion of the variability of these levels has been attributed to genetic factors. Therefore, we investigated the influence of estrogen receptor-alpha (ESR1) gene polymorphisms on HDL levels in response to postmenopausal HRT. We performed a prospective cohort study on 54 postmenopausal women who had not used HRT before the study and had no significant general medical illness. HRT consisted of conjugated equine estrogen and medroxyprogesterone acetate continuously for 1 year. The lipoprotein levels were measured from blood samples taken before the start of therapy and after 1 year of HRT. ESR1 polymorphism (MspI C>T, HaeIII C>T, PvuII C>T, and XbaI A>G) frequencies were assayed by restriction fragment length polymorphism. A general linear model was used to describe the relationships between HDL levels and genotypes after adjusting for age. A significant increase in HDL levels was observed after HRT (P = 0.029). Women with the ESR1 PvuII TT genotype showed a statistically significant increase in HDL levels after HRT (P = 0.032). No association was found between other ESR1 polymorphisms and HDL levels. According to our results, the ESR1 PvuII TT genotype was associated with increased levels of HDL after 1 year of HRT.

Single nucleotide polymorphisms at 15 codons of the prion protein gene from a scrapie-affected herd of Suffolk sheep in Brazil

Scrapie is a transmissible spongiform encephalopathy of sheeps and goats, associated with the deposition of a isoform of the prion protein (PrPsc). This isoform presents an altered conformation that leads to aggregation in the host's central nervous and lymphoreticular systems. Predisposition to the prion agent infection can be influenced by specific genotypes related to mutations in amino acids of the PrPsc gene. The most characterized mutations occur at codons 136, 154 and 171, with genotypes VRQ being the most susceptible and ARR the most resistant. In this study we have analyzed polymorphisms in 15 different codons of the PrPsc gene in sheeps from a Suffolk herd from Brazil affected by an outbreak of classical scrapie. Amplicons from the PrPsc gene, encompassing the most relevant altered codons in the protein, were sequenced in order to determine each animal's genotype. We have found polymorphisms at 3 of the 15 analyzed codons (136, 143 and 171). The most variable codon was 171, where all described alleles were identified. A rare polymorphism was found at the 143 codon in 4% of the samples analyzed, which has been described as increasing scrapie resistance in otherwise susceptible animals. No other polymorphisms were detected in the remaining 12 analyzed codons, all of them corresponding to the wild-type prion protein. Regarding the risk degree of developing scrapie, most of the animals (96%) had genotypes corresponding to risk groups 1 to 3 (very low to moderate), with only 4% in the higher risks group. Our data is discussed in relation to preventive measures involving genotyping and positive selection to control the disease.

High baseline serum total and LDL cholesterol levels are associated with MDR1 haplotypes in Brazilian hypercholesterolemic individuals of European descent

The MDR1 gene encodes the P-glycoprotein, an efflux transporter with broad substrate specificity. P-glycoprotein has raised great interest in pharmacogenetics because it transports a variety of structurally divergent drugs, including lipid-lowering drugs. The synonymous single-nucleotide polymorphism C3435T and the nonsynonymous single-nucleotide polymorphism G2677T/A in MDR1 have been indicated as potential determinants of variability in drug disposition and efficacy. In order to evaluate the effect of G2677T/A and C3435T MDR1 polymorphisms on serum levels of lipids before and after atorvastatin administration, 69 unrelated hypercholesterolemic individuals from São Paulo city, Brazil, were selected and treated with 10 mg atorvastatin orally once daily for four weeks. MDR1 polymorphisms were analyzed by PCR-RFLP. C3435T and G2677T polymorphisms were found to be linked. The allelic frequencies for C3435T polymorphism were 0.536 and 0.464 for the 3435C and 3435T alleles, respectively, while for G2677T/A polymorphism allele frequencies were 0.580 for the 2677G allele, 0.384 for the 2677T allele and 0.036 for the 2677A allele. There was no significant relation between atorvastatin response and MDR1 polymorphisms (repeated measures ANOVA; P > 0.05). However, haplotype analysis revealed an association between T/T carriers and higher basal serum total (TC) and LDL cholesterol levels (TC: 303 ± 56, LDL-C: 216 ± 57 mg/dl, respectively) compared with non-T/T carriers (TC: 278 ± 28, LDL-C: 189 ± 24 mg/dl; repeated measures ANOVA/Tukey test; P < 0.05). These data indicate that MDR1 polymorphism may have an important contribution to the control of basal serum cholesterol levels in Brazilian hypercholesterolemic individuals of European descent.

Effects of 174 G/C polymorphism in the promoter region of the interleukin-6 gene on plasma IL-6 levels and muscle strength in elderly women

We investigated the effect of -174 G/C single-nucleotide polymorphism in the promoter region of the IL6 gene on plasma IL-6 levels and muscle strength, and the relationship between IL-6 levels and muscle strength in elderly women. The sample consisted of 199 elderly residents (73.0 ± 7.8 years old) from rest homes and the community in Belo Horizonte, MG, Brazil. -174 G/C polymorphism was determined by direct sequencing of the product by PCR, and plasma IL-6 concentrations were measured by ELISA. Muscle strength in the knee joint was evaluated using a Biodex System 3 Pro® isokinetic dynamometer. ANCOVA was used to determine the effect of polymorphism on IL-6 levels and muscle strength, and the Pearson correlation coefficient to assess the relationship between IL-6 levels and muscle strength. -174 G/C polymorphism was associated with the plasma IL-6 levels of elderly women (P < 0.01) since homozygotes for the G allele showed high IL-6 levels (GG 3.85 pg/mL, GC + CC 2.13 pg/mL). There was no association of polymorphism on muscle strength (P > 0.05). No association was found between IL-6 levels and knee extensor muscle (r = 0.087, P = 0.306) or flexor (r = -0.011, P = 0.894) strength. An interaction between -174 G/C polymorphism and housing conditions of the sample of elderly women was identified, with the effect of genotype on IL-6 levels being higher in the institutionalized elderly. These results support the evidence that -174 G/C polymorphism of the IL6 gene associates with individual variability of plasma IL-6 levels in elderly women.

Single nucleotide polymorphisms predisposing to asthma in children of Mauritian Indian and Chinese Han ethnicity

Our objective was to investigate the distributions of six single nucleotide polymorphisms (SNPs) MS4A2 E237G, MS4A2 C-109T, ADRB2 R16G, IL4RA I75V,IL4 C-590T, and IL13 C1923T in Mauritian Indian and Chinese Han children with asthma. This case-control association study enrolled 382 unrelated Mauritian Indian children, 193 with asthma and 189 healthy controls, and 384 unrelated Chinese Han children, 192 with asthma and 192 healthy controls. The SNP loci were genotyped using polymerase chain reaction (PCR)-restriction fragment length polymorphism for the Chinese Han samples and TaqMan real-time quantitative PCR for the Mauritian Indian samples. In the Mauritian Indian children, there was a significant difference in the distribution of IL13 C1923T between the asthma and control groups (P=0.033). The frequency of IL13 C1923T T/T in the Mauritian Indian asthma group was significantly higher than in the control group [odds ratio (OR)=2.119, 95% confidence interval=1.048-4.285]. The Chinese Han children with asthma had significantly higher frequencies ofMS4A2 C-109T T/T (OR=1.961, P=0.001) and ADRB2 R16G A/A (OR=2.575, P=0.000) than the control group. The IL13 C1923T locus predisposed to asthma in Mauritian Indian children, which represents an ethnic difference from the Chinese Han population. TheMS4A2 C-109T T/T and ADRB2 R16G A/Agenotypes were associated with asthma in the Chinese Han children.

Alterations in lipid transfer to High-Density Lipoprotein (HDL) and activity of paraoxonase-1 in HIV+ patients

HIV+ patients often develop alterations of the plasma lipids that may implicate in development of premature coronary artery disease. High-density lipoprotein (HDL) has an important role in preventing atherogenesis and the aim of this study was to investigate aspects of HDL function in HIV+ patients. HIV+ patients (n = 48) and healthy control subjects (n = 45) of both sexes with similar age were studied. Twenty-five were not being treated with antiretroviral agents, 13 were under reverse transcriptase inhibitor nucleosidic and non-nucleosidic (NRTI+NNRTI) and 10 were under NRTI + protease inhibitors (NRTI+PI) treatment. Paraoxonase 1 (PON1) activity and the transfer of free and esterified cholesterol, tryglicerides and phospholipids from a lipidic nanoemulsion to HDL were analyzed. In comparison with healthy controls, HIV+ patients presented low PON-1 activity and diminished transfer of free cholesterol and tryglicerides. In contrast, phospholipid transfer was increased in those patients, whereas the transfer of cholesteryl esters was unchanged. NRTI+NNRTI increases the transfer of cholesteryl esters and triglycerides but in NRTI+PI there was no difference in respect to non-treated HIV+ patients. HDL from HIV+ patients has smaller antioxidant properties, as shown by lower PON-1 activity, and the transfer of lipids to this lipoprotein fraction is also altered, suggesting that HDL function is defective in those patients.

Genetic polymorphism for IFNγ +874T/A in patients with acute toxoplasmosis

INTRODUCTION: A single nucleotide polymorphism (SNP) in the gene encoding gamma interferon influences its production and is associated with severity of infectious diseases. This study aimed to evaluate the association of IFNγ+874T/A SNP with duration of disease, morbidity, and development of retinochoroiditis in acute toxoplasmosis. METHODS: A case-control study was conducted among 30 patients and 90 controls. RESULTS: Although statistical associations were not confirmed, A-allele was more common among retinochoroiditis cases and prolonged illness, while T-allele was more frequent in severe disease. CONCLUSIONS: Despite few cases, the results could indicate a relation between IFNγ+874T/A single nucleotide polymorphism and clinical manifestations of toxoplasmosis.