AIM: To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionine-aspartate-aspartate (YMDD) motif of HBV to tyrosine-valine-aspartate-aspartate (YVDD) or tyrosine-isoleucine-aspartat...AIM: To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionine-aspartate-aspartate (YMDD) motif of HBV to tyrosine-valine-aspartate-aspartate (YVDD) or tyrosine-isoleucine-aspartate-aspartate (YIDD).METHODS: Cloned wild-type and mutant HBV sequences were used as templates to test the sensitivity and specificity of the assay. A variety of primer construction, primer concentration, dNTP concentration, and annealing temperature of primers were systematically examined. Pair primers specifi c to rtL180M and rtM204V were selected for YVDD detection. Primer specif ic to rtM204I with an additional 3’-penultimate base mismatched to both the mutant and wild-type sequence was selected for YIDD detection. We applied this assay to study YMDD mutants in 28 chronic hepatitis B patients before and after lamivudine treatment.RESULTS: We could detect as little as 0.001%-0.00001% of mutant viruses coexisting in 108-109 copies of wild-type HBV using this assay. YMDD mutants were detected in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAg-negative patients before lamivudine treatment. After treatment, two more patients in HBeAg-positive patients and seven more patients in HBeAg-negative patients developed YMDD mutations. CONCLUSION: We developed a highly sensitive and specifi c assay for detecting YMDD mutants. This assay can be applied to monitor chronic hepatitis B patients before and during lamivudine treatment.展开更多
Phosphoinositide 3-kinases(PI3Ks)are heterodimers consisting of a p110 catalytic subunit and a p85 regulatory subunit.The PlIK3CA gene,which encodes the p110a,is the most frequently mutated oncogene in cancer.Oncogeni...Phosphoinositide 3-kinases(PI3Ks)are heterodimers consisting of a p110 catalytic subunit and a p85 regulatory subunit.The PlIK3CA gene,which encodes the p110a,is the most frequently mutated oncogene in cancer.Oncogenic PIK3CA mutations activate the PI3K pathway,promote tumor initiation and development,and mediate resistance to anti-tumor treatments,making the mutant p110a an excellent target for cancer therapy.PIK3CA mutations occur in two hotspot regions:one in the helical domain and the other in the kinase domain.The PIK3CA helical and kinase domain mutations exert their oncogenic function through distinct mechanisms.For example,helical domain mutations of p110a gained direct interaction with insulin receptor substrate 1(IRs-1)to activate the downstream signaling path-ways.Moreover,p85βproteins disassociate from helical domain mutant p110a,translocate into the nucleus,and stabilize enhancer of zeste homolog 1/2(EzH1/2).Due to the funda-mental role of Pl3Ka in tumor initiation and development,Pl3Ka-specific inhibitors,repre-sented by FDA-approved alpelisib,have developed rapidly in recent decades.However,side effects,including on-target side effects such as hyperglycemia,restrict the maximum dose and thus clinical efficacy of alpelisib.Therefore,developing p110a mutant-specific inhibitors to circumvent on-target side effects becomes a new direction for targeting PIK3CA mutant can-cers.In this review,we briefly introduce the function of the Pl3K pathway and discuss how PIK3CA mutations rewire cell signaling,metabolism,and tumor microenvironment,as well as therapeutic strategies under development to treat patients with tumors harboring a PIK3CA mutation.展开更多
文摘AIM: To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionine-aspartate-aspartate (YMDD) motif of HBV to tyrosine-valine-aspartate-aspartate (YVDD) or tyrosine-isoleucine-aspartate-aspartate (YIDD).METHODS: Cloned wild-type and mutant HBV sequences were used as templates to test the sensitivity and specificity of the assay. A variety of primer construction, primer concentration, dNTP concentration, and annealing temperature of primers were systematically examined. Pair primers specifi c to rtL180M and rtM204V were selected for YVDD detection. Primer specif ic to rtM204I with an additional 3’-penultimate base mismatched to both the mutant and wild-type sequence was selected for YIDD detection. We applied this assay to study YMDD mutants in 28 chronic hepatitis B patients before and after lamivudine treatment.RESULTS: We could detect as little as 0.001%-0.00001% of mutant viruses coexisting in 108-109 copies of wild-type HBV using this assay. YMDD mutants were detected in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAg-negative patients before lamivudine treatment. After treatment, two more patients in HBeAg-positive patients and seven more patients in HBeAg-negative patients developed YMDD mutations. CONCLUSION: We developed a highly sensitive and specifi c assay for detecting YMDD mutants. This assay can be applied to monitor chronic hepatitis B patients before and during lamivudine treatment.
基金supported by the National Institutes of Health (USA)grantsSR01CA196643,R01CA264320,R01CA260629,P50CA150964,and P30CA043703 to Zhenghe Wang.
文摘Phosphoinositide 3-kinases(PI3Ks)are heterodimers consisting of a p110 catalytic subunit and a p85 regulatory subunit.The PlIK3CA gene,which encodes the p110a,is the most frequently mutated oncogene in cancer.Oncogenic PIK3CA mutations activate the PI3K pathway,promote tumor initiation and development,and mediate resistance to anti-tumor treatments,making the mutant p110a an excellent target for cancer therapy.PIK3CA mutations occur in two hotspot regions:one in the helical domain and the other in the kinase domain.The PIK3CA helical and kinase domain mutations exert their oncogenic function through distinct mechanisms.For example,helical domain mutations of p110a gained direct interaction with insulin receptor substrate 1(IRs-1)to activate the downstream signaling path-ways.Moreover,p85βproteins disassociate from helical domain mutant p110a,translocate into the nucleus,and stabilize enhancer of zeste homolog 1/2(EzH1/2).Due to the funda-mental role of Pl3Ka in tumor initiation and development,Pl3Ka-specific inhibitors,repre-sented by FDA-approved alpelisib,have developed rapidly in recent decades.However,side effects,including on-target side effects such as hyperglycemia,restrict the maximum dose and thus clinical efficacy of alpelisib.Therefore,developing p110a mutant-specific inhibitors to circumvent on-target side effects becomes a new direction for targeting PIK3CA mutant can-cers.In this review,we briefly introduce the function of the Pl3K pathway and discuss how PIK3CA mutations rewire cell signaling,metabolism,and tumor microenvironment,as well as therapeutic strategies under development to treat patients with tumors harboring a PIK3CA mutation.
