AIM： To evaluate the role of genetic factors in the pathogenesis of Crohn＇s disease （CD） and ulcerative colitis （UC）, we investigated the single nucleotide polymorphisms （SNPs） of NOD2/CARD15 （R702W, Gg08R an...AIM： To evaluate the role of genetic factors in the pathogenesis of Crohn＇s disease （CD） and ulcerative colitis （UC）, we investigated the single nucleotide polymorphisms （SNPs） of NOD2/CARD15 （R702W, Gg08R and L1007finsC）, and Toll-like receptor 4 （TLR4） genes （D299G and T399I） in a selected inflammatory bowel disease （IBD） population coming from Southern Italy. METHODS： Allele and genotype frequencies of NOD2/ CARD15 （R702W, Gg08R and L1007finsC） and TLR4 （D299G and T399I） SNPs were examined in 133 CD patients, in 45 UC patients, and in 103 healthy controls. A genotype-phenotype correlation was performed. RESULTS： NOD2/CARD15 R702W mutation was significantly more frequent in CD （9.8%） than in controls （2.4%, P = 0.001） and in UC （2.3%, P = 0.03）. No significant difference was found between UC patients and control group （P 〉 0.05）. In CD and UC patients, no significant association with G908R variant was found. L1007finsC SNP showed an association with CD （9.8%） compared with controls （2.9%, P = 0.002） and UC patients （2.3%, P = 0.01）. Moreover, in CD patients, G908R and L1007finsC mutations were significantly associated with different phenotypes compared to CD wild-type patients. No association of IBD with the TLR4 SNPs was found in either cohort （allele frequencies： D299G-controls 3.9%, CD 3.7%, UC 3.4%, P 〉 0.05; T399I-controls 2.9%, CD 3.0%, UC 3.4%, P 〉 0.05）. CONCLUSION： These findings confirm that, in our IBD patients selected from Southern Italy, the NOD2/ CARD15, but not TLR4 SNPs, are associated with increased risk of CD.展开更多
AIM: Crohn's disease(CD)and ulcerative colitis(UC)are multifactorial diseases with a significant genetic background.Apart from CARD15/NOD2 gene, evidence is accumulating that molecules related to the innate immune...AIM: Crohn's disease(CD)and ulcerative colitis(UC)are multifactorial diseases with a significant genetic background.Apart from CARD15/NOD2 gene, evidence is accumulating that molecules related to the innate immune response such as CD14 or Toll-like receptor 4 (TLR4), are involved in their pathogenesis. In further exploring the genetic background of these diseases, we investigated the variations in the CARD15/NOD2 gene (Arg702Trp,Gly908Arg and Leu1007fsinsC), and polymorphisms in the TLR4 gene (Asp299Gly and Thr399Ile) as well as in the promoter of the CD14 gene (T/C at position -159) in Greek patients with CD and UC.METHODS: DNA was obtained from 120 patients with CD,85 with UC and 100 healthy individuals. Genotyping was performed by allele specific PCR or by PCR-RFLP analysis.RESULTS: The 299Gly allele frequency of the TLR4 gene and the T allele and TT genotype frequendes of the CD14 promoter were significantly higher in CD patients only compared to healthy individuals (P = 0.026＜0.05; P = 0.0048＜0.01 and P= 0.047＜0.05 respectively). Concerning the NOD2/CARD15mutations the overall presence in CD patients was significantly higher than that in UC patients or in controls.Additionally, 51.67% of the CD patients were carriers of a TLR4 and/or CD14 polymorphic allele and at least one variant of the NOD2/CARD15, compared to 27% of the UC patients. It should be pointed out that both frequencies significantly increased as compared with the 10% frequency of multiple carriers found in healthy controls. A possible interaction of the NOD2/CARD15 with TLR4 and especially CD14, increased the risk of developing inflammatory bowel disease (IBD).CONCLUSION: Our results indicate that co-existence of a mutation in either the TLR4 or CD14 gene, and in NOD2/CARD15is associated with an increased susceptibility to developing CD compared to UC, and to developing either CD or UC compared to healthy individuals.展开更多
AIM: To evaluate the effect of promoter region polymorphisms of toll-like receptor(TLR)2-196 to-174 del and TLR4-1607T/C(rs10759932) on m RNA and protein expression in tumor tissue and of TLR4+896A/G(rs4986790) on col...AIM: To evaluate the effect of promoter region polymorphisms of toll-like receptor(TLR)2-196 to-174 del and TLR4-1607T/C(rs10759932) on m RNA and protein expression in tumor tissue and of TLR4+896A/G(rs4986790) on colorectal cancer(CRC) risk.METHODS: The TLR2-196 to-174 del polymorphism was investigated using allele-specific polymerase chain reaction(PCR) and the TLR4-1607T/C and TLR4+896A/G by PCR-restriction fragment length p o l y m o r p h i s m( R F L P). W e g e n o t y p e d 4 3 4 D N A samples from 194 CRC patients and 240 healthy individuals. The m RNA relative quantification(RQ) was performed in 40 tumor tissue samples by quantitative PCR Taq Man assay, using specific probes for TLR2 and TLR4 genes, and ACTB and GAPDH reference geneswere used as endogenous controls. Protein expression was analyzed by immunohistochemistry with specific primary antibodies.RESULTS: No association was found for TLR4-1607T/C and TLR4+896A/G by three statistical models(logadditive, dominant and recessive). However, based on dominant and log-additive models, the polymorphic variant TLR2-196 to-174 del 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 m RNA expression was increased in tumor tissue(RQ = 2.36) when compared to adjacent normal tissue(RQ = 1; P < 0.0001), whereas the TLR4 m RNA 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 m RNA expression. In addition, the TLR2-196 to-174 del variant carriers showed m RNA relative expression 2.19 times higher than wild-genotype carriers. The TLR2 protein expression was also higher for the TLR2-196 to-174 del 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 m RNA(P = 0.56) and protein expression(P = 0.26).CONCLUSION: Our findings suggest that TLR2-196 to-174 del polymorphism increases TLR2 m RNA expression and is associated with higher CRC risk, indicating an important role in CRC genetic susceptibility.展开更多
文摘AIM： To evaluate the role of genetic factors in the pathogenesis of Crohn＇s disease （CD） and ulcerative colitis （UC）, we investigated the single nucleotide polymorphisms （SNPs） of NOD2/CARD15 （R702W, Gg08R and L1007finsC）, and Toll-like receptor 4 （TLR4） genes （D299G and T399I） in a selected inflammatory bowel disease （IBD） population coming from Southern Italy. METHODS： Allele and genotype frequencies of NOD2/ CARD15 （R702W, Gg08R and L1007finsC） and TLR4 （D299G and T399I） SNPs were examined in 133 CD patients, in 45 UC patients, and in 103 healthy controls. A genotype-phenotype correlation was performed. RESULTS： NOD2/CARD15 R702W mutation was significantly more frequent in CD （9.8%） than in controls （2.4%, P = 0.001） and in UC （2.3%, P = 0.03）. No significant difference was found between UC patients and control group （P 〉 0.05）. In CD and UC patients, no significant association with G908R variant was found. L1007finsC SNP showed an association with CD （9.8%） compared with controls （2.9%, P = 0.002） and UC patients （2.3%, P = 0.01）. Moreover, in CD patients, G908R and L1007finsC mutations were significantly associated with different phenotypes compared to CD wild-type patients. No association of IBD with the TLR4 SNPs was found in either cohort （allele frequencies： D299G-controls 3.9%, CD 3.7%, UC 3.4%, P 〉 0.05; T399I-controls 2.9%, CD 3.0%, UC 3.4%, P 〉 0.05）. CONCLUSION： These findings confirm that, in our IBD patients selected from Southern Italy, the NOD2/ CARD15, but not TLR4 SNPs, are associated with increased risk of CD.
基金Supported by the EU Project "Sacrohn" N. QLK2-CT-2000-00928.
文摘AIM: Crohn's disease(CD)and ulcerative colitis(UC)are multifactorial diseases with a significant genetic background.Apart from CARD15/NOD2 gene, evidence is accumulating that molecules related to the innate immune response such as CD14 or Toll-like receptor 4 (TLR4), are involved in their pathogenesis. In further exploring the genetic background of these diseases, we investigated the variations in the CARD15/NOD2 gene (Arg702Trp,Gly908Arg and Leu1007fsinsC), and polymorphisms in the TLR4 gene (Asp299Gly and Thr399Ile) as well as in the promoter of the CD14 gene (T/C at position -159) in Greek patients with CD and UC.METHODS: DNA was obtained from 120 patients with CD,85 with UC and 100 healthy individuals. Genotyping was performed by allele specific PCR or by PCR-RFLP analysis.RESULTS: The 299Gly allele frequency of the TLR4 gene and the T allele and TT genotype frequendes of the CD14 promoter were significantly higher in CD patients only compared to healthy individuals (P = 0.026＜0.05; P = 0.0048＜0.01 and P= 0.047＜0.05 respectively). Concerning the NOD2/CARD15mutations the overall presence in CD patients was significantly higher than that in UC patients or in controls.Additionally, 51.67% of the CD patients were carriers of a TLR4 and/or CD14 polymorphic allele and at least one variant of the NOD2/CARD15, compared to 27% of the UC patients. It should be pointed out that both frequencies significantly increased as compared with the 10% frequency of multiple carriers found in healthy controls. A possible interaction of the NOD2/CARD15 with TLR4 and especially CD14, increased the risk of developing inflammatory bowel disease (IBD).CONCLUSION: Our results indicate that co-existence of a mutation in either the TLR4 or CD14 gene, and in NOD2/CARD15is associated with an increased susceptibility to developing CD compared to UC, and to developing either CD or UC compared to healthy individuals.
文摘目的明确TOLL样受体2(TLR2)与micro RNA144(mi R-144)作用之间的关系。方法利用不同浓度的mi R-144的模拟物(mimics)和抑制剂(inhibitor)瞬时转染大鼠巨噬细胞系NR8383细胞,RT-q PCR检测mi R-144和TLR2及其下游分子TNF-α的表达。利用大鼠肝脏c DNA为模板,PCR获取目的片段(即含mi R-144野生及突变结合位点的TLR2 m RNA的3'UTR区);用SacⅠ、XbaⅠ双酶切pmir GLO报告基因载体和含mi R-144野生及突变结合位点的TLR2 m RNA的3'UTR区,构建携带上述片段的双荧光素酶报告基因,并通过PCR、双酶切、DNA测序鉴定构建的重组质粒,即pmir-TLR2-3'UTR及pmir-mutant-TLR2-3'UTR;并用mi R-144的mimics与双荧光素酶报告基因共转染明确mi R-144与TLR2 m RNA的3'UTR区的靶向关系。结果瞬时转染100 nmol/L mi R-144 mimics后,NR8383细胞中mi R-144表达显著升高,而TLR2及其下游分子TNF-α表达显著下降;而100 nmol/L mi R-144 inhibitor作用则相反。PCR和双酶切DNA测序结果证实pmir-TLR2-3'UTR及pmir-mutant-TLR2-3'UTR重组载体构建成功;用100 nmol/L mi R-144 mimics与空载体及上述两个构建体分别共转染HEK 293T细胞后,pmir-TLR2-3'UTR转染组相对荧光素酶活性显著降低。结论 mi R-144通过靶向结合TLR2 m RNA的3'UTR区负性调节TLR2及其下游促炎因子的表达。
基金Supported by Grants from Brazilian agencies FAPESP,No.2012/15036-8and CNPq,No.304870/2012-9
文摘AIM: To evaluate the effect of promoter region polymorphisms of toll-like receptor(TLR)2-196 to-174 del and TLR4-1607T/C(rs10759932) on m RNA and protein expression in tumor tissue and of TLR4+896A/G(rs4986790) on colorectal cancer(CRC) risk.METHODS: The TLR2-196 to-174 del polymorphism was investigated using allele-specific polymerase chain reaction(PCR) and the TLR4-1607T/C and TLR4+896A/G by PCR-restriction fragment length p o l y m o r p h i s m( R F L P). W e g e n o t y p e d 4 3 4 D N A samples from 194 CRC patients and 240 healthy individuals. The m RNA relative quantification(RQ) was performed in 40 tumor tissue samples by quantitative PCR Taq Man assay, using specific probes for TLR2 and TLR4 genes, and ACTB and GAPDH reference geneswere used as endogenous controls. Protein expression was analyzed by immunohistochemistry with specific primary antibodies.RESULTS: No association was found for TLR4-1607T/C and TLR4+896A/G by three statistical models(logadditive, dominant and recessive). However, based on dominant and log-additive models, the polymorphic variant TLR2-196 to-174 del 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 m RNA expression was increased in tumor tissue(RQ = 2.36) when compared to adjacent normal tissue(RQ = 1; P < 0.0001), whereas the TLR4 m RNA 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 m RNA expression. In addition, the TLR2-196 to-174 del variant carriers showed m RNA relative expression 2.19 times higher than wild-genotype carriers. The TLR2 protein expression was also higher for the TLR2-196 to-174 del 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 m RNA(P = 0.56) and protein expression(P = 0.26).CONCLUSION: Our findings suggest that TLR2-196 to-174 del polymorphism increases TLR2 m RNA expression and is associated with higher CRC risk, indicating an important role in CRC genetic susceptibility.
