Simple and rapid detection of uncoupling protein-2 - 866G/A polymorphism by mutagenically separated polymerase chain reaction

BACKGROUND: Uncoupling protein-2 (UCP2), a recently identified member of the mitochondrial transporter superfamily, is a candidate gene for obesity. A common G/A polymorphism in the UCP2 promoter region is associated with enhanced adipose tissue mRNA expression in vivo. METHODS: We developed a rapid and simple method, mutagenically separated polymerase chain reaction (MS-PCR) for genotyping UCP2 - 866G/A polymorphism. Two reverse mutagenic allele-specific primers of different lengths for the UCP2 - 866G/A polymorphic site were paired with the same forward primer in the same PCR reaction. RESULTS: Agarose gel electrophoresis (3.5%) showed at least one of the two allelic products and provided a within-assay quality control to exclude false-negative results. The 203-bp fragment of the PCR products was A allele-specific and the 183-bp fragment was G allele-specific. The frequencies of the UCP2 - 866G/A genotypes in 72 Japanese subjects were AA: 21 (29.2%), AG: 32 (44.4%), and GG: 19 (26.4%). The results were confirmed by the PCR-RFLP genotyping method, in which a 360-bp fragment of PCR products was cut into 290- and 70-bp fragments by the restriction enzyme MluI when the G allele was present. This Japanese group showed a higher frequency of the AA genotype, which is associated with a low prevalence of obesity, than Caucasian populations. CONCLUSIONS: The MS-PCR technique is a simple, rapid, and reliable method for genotyping UCP2 - 866G/A polymorphism.     

The mutagenically separated polymerase chain reaction is a rapid and reliable method for genotyping of the tumour necrosis factor-alpha promoter polymorphism (-308 G/A)

BACKGROUND: The tumour necrosis factor-alpha (TNF alpha) promoter polymorphism (-308 G/A) has been shown to be associated with the susceptibility to and/or the severity of diverse diseases such as infections, autoimmunity, and malignancies. We developed a genotyping technique based on the mutagenically separated polymerase chain reaction (MS-PCR) which may be useful in the clinical risk assessment. METHODS: Different length allele-specific primers and an unspecific complementary strand primer were used in a one-tube assay. At least one PCR product was generated in a single reaction obviating the need for an internal control amplification. Introduction of additional base substitutions into the allele-specific primers led to a clear-cut separation between the alleles through the reduction of cross-reactions during amplification. The only post-PCR step required was the separation of allelic PCR products by size upon agarose gel electrophoresis. RESULTS: The allele frequencies in 300 German healthy Caucasians were 0.84 for TNF1 (-308 G) and 0.16 for TNF2 (-308 A) in accordance with published data obtained with the conventional RFLP method. No significant deviation from Hardy-Weinberg equilibrium was observed. The specificity of MS-PCR was confirmed by sequence-based typing. CONCLUSIONS: MS-PCR is a rapid, reliable, and cost-effective technique for genotyping of the TNF alpha promoter polymorphism (-308 G/A).     

小儿脓毒症Toll样受体信号途径转导分子及调节因子的变化研究

目的 探讨Toll样受体(TLRs)信号途径转导分子及调节因子在小儿0脓毒症异常炎症免疫反应发病机制中的可能作用.方法 选择脓毒症患儿10例、严重脓毒症患儿13例及同期健康体检儿童17例.采用实时荧光定量聚合酶链反应(real-time PCR)检测前炎症细胞因子[肿瘤坏死因子-a(TNF-a)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)],以及TLRs信号途径转导分子和调节因子的mRNA表达;采用酶联免疫吸附法(ELISA)检测前炎症细胞因子蛋白水平.结果 与健康对照组比较,脓毒症组TNF-a、IL-1β和IL-6的mRNA和蛋白表达均增加,TLRs信号途径转导分子TLR2、TLR4、髓样细胞分化蛋白-88(MyD88)、TNF相关因子6(TRAF6)、IL-1受体相关激酶4(IRAK4)、转化生长因子-β活化激酶1(TAKl)、TAK1结合蛋白2(TAB2)的mRNA表达以及TLRs信号途径正性调节因子TLR4相关蛋白(PRAT4B)、信号转换接头蛋白2(STAP2)的mRNA表达均明显增高(P均<0.01),且严重脓毒症组较脓毒症组升高更显著(p均<0.01).脓毒症组TLRs信号途径负性调节因子IL-1受体相关激酶3(IRAK-M)、核转录因子-kB抑制性锌指蛋白(Triad3A)的mRNA表达均明显增高(P均<0.01),严重脓毒症组表达则明显低于脓毒症组(P均<0.01).结论 TLRs信号途径转导分子/调节因子异常表达可能是脓毒症时全身炎症反应的原因之一.     

Can the interleukin-6 response to endotoxin be predicted? Studies of the influence of a promoter polymorphism of the interleukin-6 gene, gender, the density of the endotoxin receptor CD14, and inflammatory cytokines

OBJECTIVES: To evaluate whether the -174 G/C promoter polymorphism of the interleukin-6 gene, gender, the monocyte density of the endotoxin receptor CD14, or the inflammatory cytokines tumor necrosis factor-alpha or interleukin-1beta influence the interleukin-6 response of whole blood to endotoxin. DESIGN: Analysis of interleukin-6 release from endotoxin-stimulated human whole blood. SETTING: Medical research laboratory. PATIENTS: Healthy human blood donors. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The interleukin-6 -174 G/C and the tumor necrosis factor -308 G/A promoter polymorphisms were determined by real-time polymerase chain reaction assay by using specific fluorescence labeled hybridization probes. Monocyte CD14 expression was assessed by flow cytometry. After incubation of whole blood with endotoxin, plasma concentrations of interleukin-6, tumor necrosis factor-alpha, and interleukin-1beta were measured by means of chemiluminescence. The interleukin-6 concentrations were lower (p = .005) in individuals who were CG heterozygotes compared with individuals homozygous for the C or the G. The difference between C and G homozygotes was not significant (p = .67). The interleukin-6 response was enhanced in men compared with women (p = .015). There was no correlation between interleukin-6 concentrations and monocyte CD14 density. Interleukin-6 concentrations correlated with the concentrations of tumor necrosis factor-alpha (r = .59, p = .01) and interleukin-1beta (r = .47, p = .01). There was no linkage between the tumor necrosis factor -308 and the interleukin-6 -174 polymorphisms. CONCLUSIONS: The interleukin-6 response to endotoxin was influenced by gender and correlated with the concentrations of more proximal cytokine tumor necrosis factor-alpha and interleukin-1beta. The interleukin-6 -174 G/C promoter polymorphism can only partly predict the interleukin-6 response of human whole blood to endotoxin stimulation, and the results were different from previous reporter gene assays that reported higher interleukin-6 concentrations for the G allele. Tumor necrosis factor -308 G homozygotes produce the lowest tumor necrosis factor concentrations. The number of tumor necrosis factor -308 G homozygotes was not higher among interleukin-6 -174 heterozygotes, and thus this cannot account for their significantly smaller interleukin-6 production.     

分子信标毛细管电泳技术联合检测哮喘患者白细胞介素13基因多态性

目的　利用分子信标杂交和毛细管电泳理论 ,建立一种新的基因突变检测方法 ,应用于疾病的基因诊断。探讨白细胞介素 (IL) 13外显子 4单核苷酸多态性 (SNP)A2 0 4 4G与中国北方人群哮喘易感性的相关关系。方法　扩增 32例有明显支气管哮喘家族史的患者和 2 0名健康对照者的IL 13exon 4 ,所得PCR产物与分子信标杂交 ,利用毛细管电泳检测杂交产物 ,并进行序列测定。结果分子信标和毛细管电泳 ,联合检测支气管哮喘患者IL 13exon 4第 2 0 4 4位基因型 ,为A的频率是31 2 % ,为G是 6 8 8% ,与测序结果相同。经统计学分析 ,支气管哮喘患者与健康对照者的等位基因频率差异具有显著意义 (χ2 =2 4 3,P <0 0 1)。结论　分子信标毛细管电泳联合技术高效、特异、灵敏 ,可对PCR产物进行分析 ,适用于碱基突变的检测。IL 13 2 0 4 4位A/G多态性与支气管哮喘的发病密切相关。     

Study of disease-relevant polymorphisms in the TLR4 and TLR9 genes: a novel method applied to the analysis of the Portuguese population

Toll-like receptors (TLRs) are cellular receptors that mediate recognition of microbial challenges and the subsequent inflammatory response. Genetic variations within these inflammation-associated genes may alter host-pathogen defence mechanisms affecting susceptibility towards infectious diseases. Taking into account the significance of these genes, we developed a simple and rapid method based in the bi-directional PCR amplification of specific alleles (Bi-PASA) for genotyping known sequence variants in TLR4 (Asp299Gly and Thr399Ile) and TLR9 (T-1237C) genes. This method allows genotype determination in a single reaction and is amenable to large-scale analysis. We used Bi-PASA to characterize the distribution of these polymorphisms in the Portuguese population. A total of 388 randomly selected blood donors of Portuguese origin (203 females and 185 males) were genotyped and allele frequencies were determined. Among the tested individuals, 11.1% and 10.8% were heterozygous for Asp299Gly and Thr399Ile, respectively. In what concerns the T-1237C variation in TLR9, the variant allele was present in 19.4% of the individuals tested. Besides confirming the usefulness of the Bi-PASA in polymorphism analysis, the data presented provide valuable information on TLR polymorphisms in the Portuguese population that can be used to stratify risk patients with increased susceptibility to infection.     

白细胞介素6基因启动子区两个突变位点多态性检测方法的建立

目的建立一种快速检测IL-6基因启动子区-597G／A、-572G／C多态性的双重实时荧光PCR方法。方法采用1对引物，2对荧光标记探针，结合荧光共振能量转移原理和熔点曲线分析技术，检测IL-6基因启动子区-572G／C，-597G／A2个位点多态性。结果用建立的双重实时荧光PCR方法对123名健康查体者进行检测，发现中国汉族人IL-6基因启动子区-572位点有3种基因型，分别为GG，GC，CC型；-597位点仅发现4名为GA型，其余均为GG型，尚未发现从型。结论双重实时荧光PCR法简便快速，与其他方法比较具有准确、经济的特点，适合临床基因快速分型。     

Real-time genotyping of cytochrome P4501A1 A4889G and T6235C polymorphisms

Single nucleotide polymorphisms, such as the cytochrome P450 1A1 (CYP1A1) A4889G and T6235C variants, have been reported to be associated with an increased cancer risk. In order to study their role in molecular-epidemiological studies, we developed a single-step procedure for genotyping these two CYP1A1 polymorphisms using real-time polymerase chain reaction (PCR) and subsequent melting curve analysis. Genotypes of 300 unrelated Caucasians, without prior history of cancer, were determined by real-time PCR and compared to genotypes obtained by restriction fragment length polymorphism PCR. In the population examined, the allele frequency of the CYP1A1 G allele at position 4889 was 0.042, and the frequency of the T allele at position 6235 was 0.10. Five percent of samples disagreed between methods. Sequence analysis of discordant samples revealed that the differences are likely attributed to incomplete digestion of amplicons by the RFLP method. These findings confirm the reliability and utility of real-time PCR as a method for large-scale genotyping.     

Genotyping of Helicobacter pylori strains from gastric biopsies by multiplex polymerase chain reaction. How advantageous is it?

Recent application of multiplex polymerase chain reaction (PCR) for genotyping Helicobacter pylori direct from biopsies revealed variable results (detection of amplicons from DNA extracted by boiling biopsies, variable size amplicons and deletions, uniform intensity of amplicon bands). We aimed to look at how applicable the technique is for determining cagA and vacA genotypes and to correlate the results with the severity of the disease. H. pylori strains from 52 patients (35 duodenal ulcers [DUs], 7 gastric ulcers [GUs], 10 gastritis) were included. Three antral biopsies were obtained for Campylobacter-like organism (CLO) and PCR. Primers for cagA, vacA s1s2, and m1m2 alleles were used. No PCR amplicons were detected from boiling biopsies; thus, DNA was extracted by QIAamp kit. H. pylori was positive in 84.6% of the patients (85.7% DU, 100% GU, and 70% gastritis). The cagA gene was detected in 86.6% DU, 71.4% GU, and 57.0% gastritis patients. The vacA allelic distribution among cagA-positive strains was 80.7% s1m1 in DU and 60.0% in GU patients, whereas 75.0% of gastritis had s1m2. No variability in the amplicon sizes was found, and the intensity of the amplicon bands was not uniform. A deleted band of approximately 420 bp below the m1 band was detected in strains from 2 DU and 1 GU patients. Although the multiplex PCR is a rapid and an effective tool for detecting several genes in a single-step system, one has to adjust for optimization of the technique when genotyping H. pylori direct from biopsies. A significant association was found between the cagA-positive vacA-s1m1 genotype and peptic ulcers.     

Polymorphisms of the inflammatory system and risk of ischemic cerebrovascular events.

BACKGROUND: Chronic and acute infections are associated with an increased risk of stroke. The inflammatory response can be influenced by functional polymorphisms in components of the immune system. We hypothesized that these polymorphisms may also modulate the risk of ischemic cerebrovascular events. METHODS: We determined the frequency of polymorphisms in tumor necrosis factor-alpha[(TNF-alpha) G(-376)A, G(-244)A, G(-238)A, G(-308)A], Toll-like receptor 4 [(TLR4) Gly299Asp and Thr399Ile], interleukin-1-receptor antagonist [(IL-1-RA) intron 2 variable-number tandem repeat], monocyte differentiation antigen CD14 receptor C(-260)T, and interleukin-6 [(IL-6) G(-174)C] genes in 404 patients with acute stroke or transient ischemic attack before the age of 60 years and in 415 healthy individuals. We also tested for interactions between genotypes, recent febrile episodes and stroke risk. RESULTS: None of the polymorphisms was associated with an increased risk of stroke after adjustment for age and gender. Following multivariate adjustment, carriers of the TNF-alpha (-308)A allele, the IL-1-RA 2* allele or the IL-6 (-174)C allele appeared to have an increased risk of stroke in association with a febrile episode prior to strokes. CONCLUSION: In our study none of the investigated polymorphisms of the inflammatory system was associated with the risk of acute cerebrovascular events before the age of 60 years. However, post-hoc analyses indicate that some polymorphisms seem to contribute to the risk of stroke in combination with fever.     

Factors predicting perioperative cytokine response in patients undergoing liver transplantation

OBJECTIVES: An exaggerated production of proinflammatory cytokines during liver transplantation stimulates the inflammatory process within the graft, and eventually promotes liver failure. This study was conducted to evaluate factors predicting perioperative response of proinflammatory cytokines during liver transplantation. DESIGN: Prospective, consecutive entry study of liver transplant candidates. SETTING: University hospital. PATIENTS: Thirty liver transplant recipients. INTERVENTIONS: Arterial blood samples were obtained perioperatively. MEASUREMENTS: Interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha were measured by ELISA. Endotoxin was determined by a chromogenic endotoxin-specific method. MAIN RESULTS: The peak concentrations of IL-1beta and IL-6 in the patients with complications were significantly higher than those in the patients without complications. The peak concentration of IL-1beta was significantly correlated with the level of bilirubin at admission and the intraoperative blood product requirement. The peak concentration of IL-6 was significantly correlated with the admission bilirubin and the intraoperative blood product requirement. A multivariate regression model revealed that the serum bilirubin and the intraoperative blood product requirement were the independent factors that influenced the peak concentration of IL-1beta or IL-6. The severely jaundiced patients had a significantly higher plasma concentration of endotoxin at the end of the anhepatic phase. In addition, there was a tendency for these patients to have a higher postoperative peak concentration of endotoxin. CONCLUSIONS: Serum level of bilirubin may be a potent preoperative factor influencing perioperative cytokine response in patients undergoing liver transplantation. An enhanced perioperative response of endotoxin seen in severely jaundiced patients suggests the clinical implication of endotoxin removal during the anhepatic phase in liver transplant surgery.     

Innate immunity SNPs are associated with risk for severe sepsis after burn injury

OBJECTIVE: To analyze allelic association with clinical outcome in a cohort of burn patients. PATIENTS: Two hundred twenty-eight individuals with burns > or =15% total body surface area without significant non-burn related trauma who survived >48 hours post-admission were enrolled. One hundred fifty-nine of these patients were analyzed previously. METHODS: Candidate polymorphisms within interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), cellular differentiation marker 14 (CD14) and toll-like receptor 4 (TLR4) were evaluated by logistic regression analysis for association with increased risk for severe sepsis (sepsis plus organ dysfunction or shock). RESULTS: After adjustment for age, burn size, ethnicity, gender and inhalation injury, alleles at TNF-alpha (308G, p=0.013), TLR4 (+896G, p=0.027), IL-6 (174C, p=0.040) and CD14 (159C, p=0.047) were significantly associated with an increased risk for severe sepsis. CONCLUSIONS: Carriage of variant alleles at immune response genes were associated with increased risk for severe sepsis after burn injury.     

A high-resolution melting analysis for genotyping urogenital Chlamydia trachomatis

We have developed a high-resolution melting analysis (HRMA) for the genotyping of Chlamydia trachomatis and applied it specifically to the 11 sexually transmitted infection-related genotypes: D through K and L1 through L3. The variable segment 2 (VS2) was selected as the target for HRMA genotype identification. Eleven C. trachomatis genotypes were amplified by a nested real-time polymerase chain reaction (PCR) in the presence of the LCGreen saturating dye and showed no cross-reaction with 10 pathogenic bacteria or commensals from urogenital tract. The detection limit of HRMA method was 100 elementary bodies (EB)/mL. All of the 11 genotypes can be distinguished from each other by following an HRMA workflow. Genotype F, G, H, I, J, K, L2, and L3 could be directly identified from each other, whereas D, E, and L1 could be distinguished from each other by a second analysis with fewer curves or by heteroduplex formation with a known reference strain. In the validation panel of 36 C. trachomatis-positive urogenital samples genotyped by VS1-VS2 sequencing, nested real-time VS2 PCR followed by HRMA was able to discriminate between all samples correctly. This assay requires no fluorescence-labeled probes or separate post-PCR analysis and provides a simple and rapid approach for genotyping the C. trachomatis strains that are the most commonly sexually transmitted.     

Association of IL-1Ra gene polymorphism, but no association of IL-1beta and IL-4 gene polymorphisms, with Kawasaki disease

The purpose of this study was to evaluate whether IL-1 beta (IL-1beta promoter and IL-1beta exon 5), IL-1 receptor antagonist (IL-1 Ra), and IL-4 (IL-4 promoter and IL-4 intron 3) gene polymorphisms act as markers of susceptibility to Kawasaki disease (KD), or of the severity of the disease. The study included 107 KD patients and 103 normal controls. Polymorphisms for cytokine genes were detected by polymerase chain reaction (PCR). Genotypes and allelic frequencies for cytokine gene polymorphisms in both groups were compared. No significant difference was observed in the genotypes and allelic frequencies of cytokines between patients with coronary aneurysm and without. In addition, there was no significant association in the genotype and allelic frequencies of IL-1 beta, IL-4, and IL-6 in patients with KD. The genotype I/II for IL1-Ra and the frequency of allele II for IL1-Ra are associated with a higher susceptibility to KD, and thus may be useful markers for predicting the development of KD.     

HMGB1 signals through toll-like receptor (TLR) 4 and TLR2

In response to bacterial endotoxin (e.g., LPS) or endogenous proinflammatory cytokines (e.g., TNF and IL-1beta), innate immune cells release HMGB1, a late cytokine mediator of lethal endotoxemia and sepsis. The delayed kinetics of HMGB1 release makes it an attractive therapeutic target with a wider window of opportunity for the treatment of lethal systemic inflammation. However, the receptor(s) responsible for HMGB1-mediated production of proinflammatory cytokines has not been well characterized. Here we demonstrate that in human whole blood, neutralizing antibodies against Toll-like receptor 4 (TLR4, but not TLR2 or receptor for advanced glycation end product) dose-dependently attenuate HMGB1-induced IL-8 release. Similarly, in primary human macrophages, HMGB1-induced TNF release is dose-dependently inhibited by anti-TLR4 antibodies. In primary macrophages from knockout mice, HMGB1 activates significantly less TNF release in cells obtained from MyD88 and TLR4 knockout mice as compared with cells from TLR2 knockout and wild-type controls. However, in human embryonic kidney 293 cells transfected with TLR2 or TLR4, HMGB1 effectively induces IL-8 release only from TLR2 overexpressing cells. Consistently, anti-TLR2 antibodies dose-dependently attenuate HMGB1-induced IL-8 release in human embryonic kidney/TLR2-expressing cells and markedly reduce HMGB1 cell surface binding on murine macrophage-like RAW 264.7 cells. Taken together, our data suggest that there is a differential usage of TLR2 and TLR4 in HMGB1 signaling in primary cells and in established cell lines, adding complexity to studies of HMGB1 signaling which was not previously expected.     

Multicolor real-time polymerase chain reaction genotyping of six human platelet antigens using displacing probes

BACKGROUND: Several genotyping methods for six clinically relevant human platelet antigens (HPAs) have been reported. A four-color real-time polymerase chain reaction (PCR) method using displacing probes for genotyping of the six HPAs is described. STUDY DESIGN AND METHODS: Primers and four differently fluorophor-labeled displacing probes were designed and synthesized to detect single-nucleotide polymorphisms responsible for each of the HPA-1, -2, -3, -4, -5, and -15 genotypes. Two HPA systems were analyzed in a single PCR procedure. After validation with samples of known genotypes, a total of 150 blood samples from healthy donors were genotyped. The results were compared with PCR with sequence-specific primers (SSP), PCR-restriction fragment length polymorphism (RFLP), and/or direct DNA sequencing. The frequencies of each HPA allele were calculated. RESULTS: Unequivocal real-time PCR genotyping results were obtained with minimal manual manipulation and carryover contamination. All 150 blood samples were correctly genotyped as confirmed by PCR-SSP, PCR-RFLP, and/or direct DNA sequencing. The allelic frequencies of HPA-1 through -5 and -15 among the Chinese population in Xiamen were comparable with those previously reported with Chinese living in other territories. For each specimen, genotyping of all six HPA biallelic systems was achieved in three tubes of PCR within 90 minutes and with material cost of no more than $1. CONCLUSION: Genotyping of HPA with real-time PCR using displacing probes is more rapid and reliable compared with PCR-SSP and PCR-RFLP methods and is more affordable than existing real-time PCR-based HPA genotyping assays. Thus, our approach is more suitable for routine HPA analysis and ideal for both urgent clinical testing and high-throughput screening.     

微量聚合酶链反应对人类白细胞抗原的基因分型

目的 探讨应用于移植的人类白细胞抗原（ＨＬＡ）二类基因（ＤＲＢ／ＤＱＢ）快速分型新技术。方法 采用快速ＤＮＡ抽提技术,建立了ＨＬＡ＿ＤＲＢ／ＤＱＢ微量序列特异性引物聚合酶链反应基因分型方法,对１４２份供受者ＤＮＡ进行ＨＬＡ－ＤＲＢ／ＤＱＢ基因分型,并与单克隆抗体一叔法ＨＬＡ血清学分型技术进行对比研究。结果 应用微量ＰＣＲ－ＳＳＰ方法共检出１３种ＤＲＢ１等位基因和 ＤＱＢ１等位基因,与血清学分型结果