TaqMan Polymerase Chain Reaction Research Articles

Polymorphisms of the FOXF1 and MHC locus genes in individuals undergoing esophageal acid reflux assessments.

Gastroesophageal reflux disease (GERD) may lead to Barrett's esophagus (BE). Previously, a large genome-wide association study found two germline markers to be associated with BE, FOXF1 rs9936833 (C allele) and MHC rs9257809 (A allele). This study evaluated whether these two polymorphisms are associated with gastroesphageal acid reflux as measured by 24-hour pH testing. Patients with acid reflux symptoms referred for esophageal manometry and 24-hour pH monitoring at University Health Network (Toronto, ON) were enrolled. DNA extracted from blood was genotyped using a Taqman Polymerase Chain Reaction (PCR) assay. DeMeester scores of ≥14.7 or prior evidence of reflux esophagitis on endoscopy defined individuals with esophageal acid reflux. Logistic regression analysis, adjusted for clinical risk factors, was used to calculate odds ratios with 95% confidence intervals for each polymorphism in relation to the presence of acid reflux. Of 182 patients, the median age was 50years and 62% were female; 95 (52%) met the definition of GERD. In the multivariable analysis, both FOXF1 rs9936833 (OR = 1.82; 95%CI: 1.12-2.96; P = 0.02) and MHC rs9257809 (OR = 9.36; 95%CI: 2.92-29.99; P < 0.001) remained significantly associated with presence of acid reflux. When both polymorphisms were placed in the same model, the adjusted ORs were 2.10 (95%CI: 1.24-3.53; P = 0.005) and 10.95 (95%CI: 3.32-36.09; P < 0.001), respectively. The association for risk allele C in FOXF1 rs9936833 and risk allele A in MHC rs9257809 with the presence of acid reflux suggests a potential pathophysiologic mechanism for the role of genetic influences in BE development.

EPIDEMIOLOGY STUDY OF VIRAL NERVOUS NECROSIS (VNN) IN MALAYSIAN GROUPERS: SEQUENCE ANALYSIS STRATEGIES AND QUASISPECIES DETERMINANT’S

EPIDEMIOLOGY STUDY OF VIRAL NERVOUS NECROSIS (VNN) IN MALAYSIAN GROUPERS: SEQUENCE ANALYSIS STRATEGIES AND QUASISPECIES DETERMINANT’S

Clinical Significance of MYD88 Mutation in Patients with Diffuse Large Cell Lymphoma

Mutation of the MYD88 has recently been identified in activated B cell like diffuse large B cell lymphoma (DLBCL) and enhanced cell proliferation systems such as JAK-STAT and NF-kB signaling pathways. However, much remains unclear about its clinical significance. In this study, we developed a highly sensitive and an automatic method utilizing guanine-quenching probes (QP) to detect mutation and investigated the relationship between MYD88 L265P mutation and clinical significance. We amplify a DNA fragment including the mutation to intend for by PCR and associate it with Q-probe with complementary sequence, using the temperature that Q-probe dissociates varying according to a conformity degree of the complementarity sequence. We judge it by detecting the fluorescence to be provided by dissociation. Results were obtained from 1ul of DNA solution(10ng) within 90 min by the method. Detected mutations were identical between QP method and allele-specific PCR (AS-PCR).Eighty-nine patients with a diagnosis of de novo DLBCL made between 1999 and 2014, and treated with CHOP or R-CHOP therapy. We retrospectively analyzed the outcome of 89 patients (age range; 21-88 and 59% were female). The median follow-up time was 4.4 y. Survival analyses were performed using the Kaplan-Meier method. None of the patients had a known history of human immunodeficiency virus infection. MYD88 L265P mutation was both assessed by Q-probe system that can detect low levels of mutant DNA and allele-specific TaqMan polymerase chain reaction assay. We performed the direct sequence method using 3130 Applied Biosystem Genetic Analyzer as antithesis. The cell-of-origin was determined based on immunohistochemical (IHC) stains for CD10, BCL-6 and MUM-1 by Hans' algorithm.MYD88 L265Pmutation was detected in 25.8% (23/89) in various tissues of DLBCL. MYD88 mutations occurred more frequently in males (P<0.05), cases without B symptoms (P<0.05). MYD88 mutation was infrequent in DLBCL arising in lymph nodes (10.6%), but more frequently found in extranodal sites such as testes (83%, 5/6), nasal (75%,9/12), central nervous system (50%,2/4), and leg (100%,1/1). In agreement with recent studies, we found no mutated cases among gastric cases. As somatic mutations in MYD88 was reported to be the most frequent alterations found in non-GCB type, we further analyzed GCB or non-GCB type by IHC. MYD88 mutations were predominantly observed in the non-GCB type (74%, 17/23), compared with 26%, 6/23 in GCB type. Overall survival (OS) for 3 years were 84.2% and 70.2% in patients with wild-type MYD88 and in MYD88 mutation group (P=0.366), respectively. Progression-free survival (PFS) for 3 years, 76.9% and 64.3% in patients with wild type and in mutated group (P=0.156), respectively. However, all four cases with CNS relapse had this mutation, 2 originated from testis, and remained 2 from lymph nodes. Our results confirm the remarkable site-specific occurrence of MYD88 mutation. In addition, Q-probe system for detection of MYD88 mutation was very useful because of its sensitivity and in the case who obtained only a small amount of biopsy specimen. MYD88 L265Ppromotes survival of malignant lymphoid cells through several mechanisms. Further large scale study should be necessary for more understanding of biological and clinical significance of DLBCL patients with MYD88 mutation. DisclosuresNo relevant conflicts of interest to declare.

High Rate of β-Globin DNA Detection Validates Self-Sampling in Herpes Simplex Virus Shedding Studies.

Self-sampling is a convenient, feasible, and acceptable way of collecting genital specimens, but the veracity of reported self-collection is difficult to verify. We investigated whether a host gene, β-globin, can be used to confirm adequacy of self-collected mucosal and skin genital specimens in studies of genital herpes simplex virus type 2 (HSV-2) infection. Herpes simplex virus type 2-seropositive adults self-collected daily anogenital and oral swabs. Mucosal samples were tested for HSV DNA using a real-time quantitative polymerase chain reaction assay. A real-time Taqman polymerase chain reaction detecting the β-globin gene was used to quantify host cells. One hundred twelve participants collected 5559 genital and 2002 oral swabs. Sixty (54%) were women, 65% were HSV-2 seropositive, and 35% were HSV-1 and HSV-2 seropositive by Western blot. β-globin DNA was detected in 99% and 93% of swabs obtained from women and men, respectively. The quantity of β-globin DNA detected was significantly higher when HSV was present in genital swabs in women (0.1 log10 copies/mL; P = 0.001) and in men (0.6 log10 copies/mL; P < 0.001), but not in oral swabs in women (0.2 log10 copies/mL; P = 0.08) or men (0.0 log10 copies/mL; P = 0.70). Human β-globin DNA detection rate was high, and the quantity obtained significantly increased with genital, but not oral HSV shedding. The high rate of β-globin DNA detection is consistent with high adherence to study procedures in longitudinal studies of genital herpes shedding.

Association of NR3C1/Glucocorticoid Receptor gene SNP with azoospermia in Japanese men.

The molecular pathogenesis of non-obstructive azoospermia (NOA) is unclear. Our aim was to identify the genetic susceptibility for NOA in Japanese men by using a combination of transcriptome network analysis and SNP genotyping. We searched for candidate genes using RNA transcriptome network analysis of 2611 NOA-related genes that we had previously reported. We analyzed candidate genes for disease linkage with single nucleotide polymorphisms (SNP) in the genomes of 335 Japanese men with NOA and 410 healthy controls using SNP-specific real-time polymerase chain reaction TaqMan assays. Three candidate genes (NR3C1, YBX2, and BCL2) were identified by the transcriptome network analysis, each with three SNP. Allele frequency analysis of the nine SNP indicated a significantly higher frequency of the NR3C1 rs852977 G allele in NOA cases compared with controls (corrected P = 5.7e-15; odds ratio = 3.20; 95% confidence interval, 2.40-4.26). The other eight candidate polymorphisms showed no significant association. The NR3C1 rs852977 polymorphism is a potential marker for genetic susceptibility to NOA in Japanese men. Further studies are necessary to clarify the association between the NR3C1 polymorphism and alterations of glucocorticoid signaling pathway leading to male infertility.

Three patients treated with daclatasvir and asunaprevir for recurrent hepatitis C after liver transplantation: Case report

We previously reported our data on telaprevir or simeprevir used in combination with pegylated interferon (PEG IFN) and ribavirin (RBV) for the treatment of recurrent hepatitis C virus (HCV) genotype 1 infection after liver transplantation (LT). Here, we report three patients who achieved viral responses with no effect on the blood concentrations of immunosuppressive agents following daclatasvir and asunaprevir treatment. The first patient was a 57-year-old man with HCV-related liver cirrhosis who failed to respond to PEG IFN/RBV after living donor LT. He had been treated with 1mg/day of tacrolimus. The second was a 63-year-old man with HCV-related liver cirrhosis and hepatocellular carcinoma who failed to respond to PEG IFN/RBV after living donor LT. He had been treated with 1mg/day of tacrolimus. The third was a 61-year-old man with HCV-related liver cirrhosis. He had been treated with mycophenolate mofetil (MMF). Serum HCV RNA became undetectable by TaqMan polymerase chain reaction test after 4weeks of daclatasvir and asunaprevir treatment in all patients, and no remarkable fluctuations in blood concentration were observed either in tacrolimus or in MMF during 24weeks of therapy. No adverse events were observed, and all patients received the full dose of daclatasvir and asunaprevir over 24weeks. Serum HCV RNA remained negative at 12weeks after the end of treatment in all patients. The daclatasvir and asunaprevir treatment showed a remarkable viral response with little effect on blood levels of immunosuppressive agents for recurrent HCV genotype 1 infection after LT.

Liver Transplant Patient Carriers of Polymorphism Cyp3a5*1 Donors May Need More Doses of Tacrolimus From the First Month After Transplantation

BackgroundThe aim of this work was to evaluate the CYP3A5:CYP3A5*1/CYP3A5*3 (6986A>G) polymorphism related to the pharmacokinetic characteristics of tacrolimus during the first 3 months after transplantation, analyzing both donor and recipient genotype, in liver transplant patients. MethodsThis retrospective, single-center, cohort study included patients who had been treated with tacrolimus monotherapy with or without corticoids (n = 67). Donors and recipients were genotyped for the CYP3A5*3 allele polymorphism (6986A>G) by use of a TaqMan polymerase chain reaction technique. The presence or absence of the *1 allele (“minor-allele”) was analyzed for correlation with the tacrolimus dose-normalized ratio during the 3 months after transplantation. ResultsThe following observations were obtained in the population studied: (1) Frequency of the minor allele*1 was much lower both in recipients (11.9% versus 88.1%) and donors (19.4% versus 80.6%), with no statistically significant differences between both distributions. (2) Recipient genotype for CYP3A5*1/*3-polymorphism had no influence in tacrolimus pharmacokinetics, with no differences between carriers and non-carriers of the minor-allele*1. (3) However, from the first month after transplantation, patients with grafts from donor carriers of minor allele*1 had lower concentration-dose ratios compared with patients with grafts from donor non-carriers of that allele (71.1 versus 119.3 and 90.5 versus 126.3, for 30 and 90 days after transplantation, respectively; P < .05). ConclusionsThe presence of the CYP3A5-6986A>G-polymorphism in the donor affects tacrolimus pharmacokinetics in the recipient, although the difference was statistically significant only for the first month after transplantation. This means that in liver transplant patients receiving grafts from donors carrying the CYP3A5*1-polymorphism, a larger dose of tacrolimus from the first month after transplantation would be needed. The evidence provided in this study showed no effect of the recipient genotype.

Effects of microRNA-155 on mice with lipopolysaccharide-induced acute lung injury

To study the effects of microRNA (miR)-155 on mice with acute lung injury induced by lipopolysaccharide (LPS). According to the random number table, 30 C57 mice were divided into group LPS, group miR-155 inhibitor, group miR control, group miR-155 mimics, normal control group, with 6 mice in each group. Mice in group LPS and the other three treatment groups were given 5 µl LPS (20 µg/µl, in the dose of 4 mg/kg) to reproduce acute lung injury model. Half an hour after injury, group LPS, group miR-155 inhibitor, group miR control, group miR-155 mimics were injected with saline, miR-155 inhibitor, miR control and miR-155 mimics through trachea. Mice in normal control group were received no treatment. All mice were anesthetized after 24 h, while lung tissue samples were harvested for histomorphological observation, detection of mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) with SYBR Green real-time polymerase chain reaction (PCR), detection of miR-155 in group miR-155 inhibitor, group miR control, group miR-155 mimics by Taqman PCR. The protein expression of cyclooxygenase-2 (COX-2) was determined by western blot. Data were statistically analyzed. The relative mRNA expression values of TNF-α were 24.49 ± 3.64, 38.92 ± 3.40, 36.00 ± 3.86, 12.56 ± 2.06, 1.04 ± 0.41 in group LPS, group miR-155 inhibitor, group miR control, group miR-155 mimics, normal control group. The relative mRNA expression values of IL-1β were 55.96 ± 4.87, 62.90 ± 6.41, 59.99 ± 6.48, 25.33 ± 3.50, 1.11 ± 0.44 in group LPS, group miR-155 inhibitor, group miR control, group miR-155 mimics, normal control group. The differences of TNF-α and IL-1β in group miR-155 mimics were statistically significant by compared with group LPS, group miR-155 inhibitor, group miR control (P < 0.05). The differences were not statistically significant among group LPS, group miR-155 inhibitor, group miR control. Western blot analysis showed the relative expression values of COX-2 were 0.94 ± 0.06, 0.96 ± 0.09, 0.91 ± 0.03, 0.79 ± 0.04, 0.63 ± 0.07 in group LPS, group miR-155 inhibitor, group miR control, miR-155 mimics and normal control group. The value in normal control group was significantly lower than other groups (P < 0.05). Using the miR-155 can effectively alleviate the lung inflammation in mice with acute lung injury induced by LPS.

Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay

Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 102 and 3.3 × 105 cells/l in river water and 72.1–5.7 × 106 cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors.

A single FTO gene variant rs9939609 is associated with body weight evolution in a multiethnic extremely obese population that underwent bariatric surgery

ObjectivesThe rs9939609 single nucleotide polymorphism (SNP) in the fat mass and obesity-associated (FTO) gene is involved in obesity. Few studies have been conducted on patients who underwent bariatric surgery. The aim of this study was to evaluate the influence of FTO SNPs on body weight, body composition, and weight regain during a 60-mo follow-up period after bariatric surgery. MethodsThe rs9939609 was genotyped in 146 individuals using a real-time polymerase chain reaction TaqMan assay. Data for lifestyle, comorbidities, body weight, body mass index (BMI), excess weight loss (EWL), and body composition were obtained before and 6, 12, 18, 24, 36, 48, and 60 mo after surgery. Data were analyzed by comparing two groups of patients according to rs9939609 FTO gene polymorphism. Mixed-regression models were constructed to evaluate the dynamics of body weight, BMI, and EWL over time in female patients. ResultsNo differences were observed between the groups during the first 24 mo after surgery. After 36, 48, and 60 mo, body weight, fat mass, and BMI were higher, whereas fat-free mass and EWL were lower in the FTO-SNP patient group. Weight regain was more frequent and occurred sooner in the FTO-SNP group. ConclusionsThere is a different evolution of weight loss in obese carriers of the FTO gene variant rs9939609 after bariatric surgery. However, this pattern was evident at only 2 y postbariatric surgery, inducing a lower proportion of surgery success and a greater and earlier weight regain.

IL23R gene polymorphism with juvenile idiopathic arthritis and its association with serum IL-17A.

Interleukin 23 (IL-23) and its receptor (IL-23R) seem to play a major role in differentiation of CD4+ T cells into Th17 cells, induction of IL-17 production, and activation of inflammatory pathways. Recent studies have suggested the association of IL-23R polymorphisms with bone and articular inflammation in diseases such as ankylosing spondylitis and rheumatoid arthritis. The aim of this study was to determine the association between IL-23R polymorphisms and juvenile idiopathic arthritis (JIA). A case-control study on 55 patients with JIA and 78 healthy controls was performed. All samples were genotyped for eight single nucleotide polymorphisms (SNPs) of IL23R (rs1004819, rs2201841, rs10889677, rs1495965, rs7517847, rs10489629, rs11209026 and rs1343151), using real-time polymerase chain reaction Taqman genotyping technique. Forty-two patients and 42 healthy controls were chosen randomly to measure the level of serum IL-17A using enzyme-linked immunosorbent assay. Although the heterozygous genotype of rs1004819 (GA) showed a weak, but statistically significant protective effect on polyarticular subtype (P=0.03), none of the selected SNPs were associated with JIA overall. Indeed the analysis of haplotypes did not show any significant association with JIA. Serum IL-17A level was not significantly different among patients and healthy controls and between JIA subtypes, as well. Moreover, there was no significant correlation between SNPs and serum IL-17A concentration. This is the first study of the IL-23R gene in Iranian patients with JIA. Our results did not show any strong association between IL-23R polymorphisms and JIA disease or serum IL-17A levels. The only association was seen between rs1004819 and polyarticular JIA. Further larger studies may help clarify the role, if any, of the IL-23/IL-17 pathway in the pathogenesis of JIA.

East asian variant of aldehyde dehydrogenase 2 is associated with coronary spastic angina: possible roles of reactive aldehydes and implications of alcohol flushing syndrome.

Coronary spastic angina (CSA) is a common disease among East Asians, including Japanese. The prevalence of alcohol flushing syndrome associated with deficient activity of the variant aldehyde dehydrogenase 2 (ALDH2*2) genotype is prevalent among East Asians. We examined whether CSA is associated with the ALDH2*2 genotype in Japanese. The study subjects consisted of 202 patients in whom intracoronary injection of acetylcholine was performed by angiography on suspicion of CSA (119 men and 83 women; mean age, 66.2±11.4 years). They were divided into CSA (112 patients) and control groups (90 patients). ALDH2 genotyping was performed by the direct application of the TaqMan polymerase chain reaction system on dried whole blood. Clinical and laboratory data were examined using conventional methods. The frequencies of male sex, ALDH2*2 genotype carriers, alcohol flushing syndrome, tobacco smoking, and the plasma level of uric acid were higher (P<0.001, P<0.001, P<0.001, P<0.001, and P=0.007, respectively) and the plasma high-density lipoprotein cholesterol levels were lower (P<0.001) in the CSA group than in the control group. The multivariable logistic regression analysis revealed that ALDH2*2 genotype and smoking were significantly associated with CSA (P<0.001 and P=0.024, respectively). East Asian variant ALDH2*2 genotypes and, hence, deficient ALDH2 activity were associated with CSA in Japanese. These data support further investigation of treatment targeting aldehydes for CSA.

Susceptibility to male infertility: replication study in Japanese men looking for an association with four GWAS-derived loci identified in European men.

A previous genome-wide association study in European men identified four single nucleotide polymorphism (SNP) loci associated with male infertility. Our aim was to replicate, if possible, the association of these SNPs with Japanese male infertility. We genotyped four SNPs (rs5911500, rs10246939, rs2059807, and rs11204546) in 517 Japanese patients with male infertility and 369 fertile controls using SNP-specific real-time polymerase chain reaction TaqMan assays. Subsequently, we divided patients with male infertility into azoospermia (n = 417) and oligospermia subgroups (n = 70). The four SNPs previously identified in European men showed no significant association with collective male infertility in our Japanese cohort. However, allele frequency analysis did indicate a significantly higher frequency of the rs11204546 C allele of the OR2W3 gene in the oligospermia subset of infertility patients compared with controls (p = 0.0037; odds ratio = 1.74; 95% confidence interval, 1.21-2.53). Although this study was somewhat limited by overall sample size, the OR2W3 gene polymorphism rs11204546 was significantly associated with oligospermia in Japanese men, suggesting that OR2W3 might be involved in genetic susceptibility to Japanese male infertility as well as in European males.

Two Patients Treated With Simeprevir Plus Pegylated-Interferon and Ribavirin Triple Therapy for Recurrent Hepatitis C After Living Donor Liver Transplantation: Case Report

We previously reported our data on telaprevir (TVR) used in combination with pegylated-interferon and ribavirin (PEG-IFN/RBV) for the treatment of recurrent hepatitis C virus (HCV) genotype 1 infection after liver transplantation (LT). TVR substantially increases the blood levels of immunosuppressive agents such as cyclosporine and tacrolimus for drug-drug interactions. On the other hand, the effect of simeprevir (SMV) on the blood levels of these immunosuppressive agents is unclear. We report 2 patients who achieved viral responses with little effect on the blood levels of cyclosporine and tacrolimus using SMV plus PEG-IFN/RBV treatment. The first was a 71-year-old woman with HCV-related liver cirrhosis and hepatocellular carcinoma who failed to respond to PEG-IFN/RBV after living donor LT. She was treated with 40 mg/d of cyclosporine, and received SMV plus PEG-IFN/RBV treatment. The second was a 65-year-old man with HCV-related liver cirrhosis who failed to respond to PEG-IFN/RBV after living donor LT. He was treated with 3 mg/d of tacrolimus, and received SMV plus PEG-IFN/RBV treatment. Serum HCV RNA became undetectable using TaqMan polymerase chain reaction (PCR) test after 4 weeks of treatment in both patients, and no remarkable fluctuation in blood concentration was observed either in cyclosporine or tacrolimus during the 12 weeks of SMV treatment. Completion of 12-week SMV triple therapy was followed by PEG-IFNα2b plus RBV, and both patients achieved sustained virological response 12 weeks after the end of treatment. SMV plus PEG-IFNRBV treatment showed a remarkable viral response with little effect on blood levels of immunosuppressive agents for recurrent HCV genotype 1 infection after LT.

An association study between DLGAP1 rs11081062 and EFNA5 rs26728 polymorphisms with obsessive-compulsive disorder in a Chinese Han population.

BackgroundA recent genome-wide association study indicated that the single nucleotide polymorphisms (SNPs) rs11081062 in DLGAP1 and rs26728 in EFNA5 were associated with obsessive–compulsive disorder (OCD) in Caucasians. The present case–control association study assessed the global relevance of these two SNPs with respect to OCD subtypes in a Chinese Han population.MethodsWe recruited 320 OCD patients and 431 age- and sex-matched controls from a Chinese Han population. rs11081062 and rs26728 SNPs were genotyped by real-time TaqMan polymerase chain reaction, and the chi-squared test was used to compare allele and genotype frequencies of variants between the two groups.ResultsNo significant differences were found in allele or genotype frequencies of DLGAP1 rs11081062 and EFNA5 rs26728 between the OCD and control groups. Moreover, consistently negative results were observed when classifying by sex, onset age, and comorbidity. However, on analyzing OCD subphenotypes, significant associations were observed between rs11081062 and the presence of contamination obsessions and cleaning compulsions (χ2=7.724, P=0.021 by genotype; χ2=3.745, P=0.053 by allele; and χ2=0.821, P=0.365 by genotype, χ2=27.809, P=0.000 by allele, respectively), and rs26728 with the presence of repeating compulsions (χ2=8.285, P=0.004 by genotype; χ2=7.512, P=0.006 by allele).ConclusionAlthough we found no association between DLGAP1 rs11081062 and EFNA5 rs26728 SNPs with OCD in a Chinese Han population, obvious associations were observed with OCD subphenotypes. Therefore, it appears to be useful to divide OCD into more homogeneous subphenotypes to help understand the complex genetic basis of this disorder. Further investigations are needed to replicate these findings using larger sample sizes, different populations, and other polymorphisms.

Multiplex qRT-PCR for the Detection of Western Equine Encephalomyelitis, St. Louis Encephalitis, and West Nile Viral RNA in Mosquito Pools (Diptera: Culicidae).

Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk.

A single-nucleotide polymorphism in serine-threonine kinase 11, the gene encoding liver kinase B1, is a risk factor for multiple sclerosis.

We identified a family in which five siblings were diagnosed with multiple sclerosis (MS) or clinically isolated syndrome. Several women in the maternal lineage have comorbidities typically associated with Peutz Jeghers Syndrome, a rare autosomal-dominant disease caused by mutations in the serine-threonine-kinase 11 (STK11) gene, which encodes liver kinase B1. Sequence analysis of DNA from one sibling identified a single-nucleotide polymorphism (SNP) within STK11 intron 5. This SNP (dbSNP ID: rs9282860) was identified by TaqMan polymerase chain reaction (PCR) assays in DNA samples available from two other siblings. Further screening was carried out in samples from 654 relapsing-remitting MS patients, 100 primary progressive MS patients, and 661 controls. The STK11-SNP has increased frequency in all female patients versus controls (odds ratio = 1.66, 95% CI = 1.05, 2.64, p = .032). The STK11-SNP was not associated with disease duration or onset; however, it was significantly associated with reduced severity (assessed by MS severity scores), with the lowest scores in patients who also harbored the HLA-DRB1*1501 allele. In vitro studies showed that peripheral blood mononuclear cells from members of the family were more sensitive to the mitochondrial inhibitor metformin than cells from MS patients with the major STK11 allele. The increased association of SNP rs9282860 in women with MS defines this variant as a genetic risk factor. The lower disease severity observed in the context of HLA-DRB1*1501 combined with limited in vitro studies raises the provocative possibility that cells harboring the STK11-SNP could be targeted by drugs which increase metabolic stress.

The novel association between red complex of oral microbe and body mass index in healthy Japanese: a population based cross-sectional study.

Microbiota has been thought to be one of important environmental factors for obesity or Type 2 diabetes mellitus. Among oral microbe, Porphyromonas gingivalis, Treponema denticola and Tannellera forsythia are known as risk factors, so called red complex, for periodontitis. Red complex could also be a risk factor for obesity. However, recent study indicated that obesity was not improved by periodontal therapy. Thus, we performed a cross sectional study to reveal the association of oral microbe with body mass index in a healthy population. Healthy individuals were randomly recruited. The infections of oral microbe were identified by Taqman polymerase chain reaction. The relationships between number of red complex and body mass index or waist circumference were analyzed. Two hundred and twenty-two apparently healthy Japanese were enrolled. BMI and waist circumference as well as age, periodontitis, number of brushing teeth were significantly associated with the number of red complex after adjusting covariance. The effect size of body mass index or waist circumference was 0.023 (p = 0.028) or 0.024 (p = 0.024), respectively. Body mass index and waist circumference were independently associated with the number of red complex among apparently healthy Japanese. The current observation implies the possibility that oral microbe was associated with obesity in healthy population.

Relationship between zinc finger protein 36 (ZFP36) gene polymorphisms and obstructive sleep apnea.

Recent data have indicated that inflammation may have an important correlation with obstructive sleep apnea (OSA). Studies have indicated a relationship between OSA and TNF-α gene polymorphisms. Zinc finger protein 36 (ZFP36) regulates TNF-α mRNAs. However, ZFP36 gene polymorphisms have not been investigated in OSA. Therefore, we conducted the present case-control study to assess whether variances in ZFP36 gene polymorphisms account for differences in TNF-α levels in patients with moderate-to-severe OSA. This case-control study aims to investigate the relationship between genetic variations in the ZFP36 gene and moderate-to-severe OSA. Three common single nucleotide polymorphisms of the ZFP36 gene (rs251864, rs3746083, and rs17879933) were evaluated in a group of patients with moderate-to-severe OSA (N = 408) and in a control group (N = 394) by using TaqMan polymerase chain reaction analysis. The moderate-to-severe OSA group and the control group exhibited significant differences in the distributions of rs251864 and rs17879933 genotypes and alleles (P < 0.05). TNF-α levels were significantly different not only among the three rs251864 genotypes but also between the II genotype and the DD + ID genotypes of rs17879933. However, no significant differences in sleep apnea parameters in the three ZFP36 gene polymorphisms were observed. Logistic regression analyses demonstrated that TNF-α and the three ZFP36 gene polymorphisms were not independently associated with OSA. ZFP36 might be involved in TNF-α regulation. However, ZFP36 gene variants were not independent risk factors for moderate-to-severe OSA.

TLR2 and TLR4 polymorphisms influence mRNA and protein expression in colorectal cancer.

To evaluate the effect of promoter region polymorphisms of toll-like receptor (TLR)2-196 to -174del and TLR4-1607T/C (rs10759932) on mRNA and protein expression in tumor tissue and of TLR4+896A/G (rs4986790) on colorectal cancer (CRC) risk. The TLR2-196 to -174del polymorphism was investigated using allele-specific polymerase chain reaction (PCR) and the TLR4-1607T/C and TLR4+896A/G by PCR-restriction fragment length polymorphism (RFLP). We genotyped 434 DNA samples from 194 CRC patients and 240 healthy individuals. The mRNA relative quantification (RQ) was performed in 40 tumor tissue samples by quantitative PCR TaqMan assay, using specific probes for TLR2 and TLR4 genes, and ACTB and GAPDH reference genes were used as endogenous controls. Protein expression was analyzed by immunohistochemistry with specific primary antibodies. No association was found for TLR4-1607T/C and TLR4+896A/G by three statistical models (log-additive, dominant and recessive). However, based on dominant and log-additive models, the polymorphic variant TLR2-196 to -174del was associated with increased CRC risk [dominant: odds ratio (OR) = 1.72, 95%CI: 1.03-2.89; P = 0.038 and log-additive: OR =1.59, 95%CI: 1.02-2.48; P = 0.039]. TLR2 mRNA expression was increased in tumor tissue (RQ = 2.36) when compared to adjacent normal tissue (RQ = 1; P < 0.0001), whereas the TLR4 mRNA showed a basal expression (RQ = 0.74 vs RQ = 1, P = 0.452). Immunohistochemistry analysis of TLR2 and TLR4 protein expression was concordant with the findings of mRNA expression. In addition, the TLR2-196 to -174del variant carriers showed mRNA relative expression 2.19 times higher than wild-genotype carriers. The TLR2 protein expression was also higher for the TLR2-196 to -174del variant carriers [117 ± 10 arbitrary unit (a.u.) vs 95 ± 4 a.u., P = 0.03]. However, for the TLR4 -1607T/C polymorphism no significant difference was found for both mRNA (P = 0.56) and protein expression (P = 0.26). Our findings suggest that TLR2-196 to -174del polymorphism increases TLR2 mRNA expression and is associated with higher CRC risk, indicating an important role in CRC genetic susceptibility.