AIM: To investigate the frequency of mutations in pre-core (pre-C) and basic core promoter (BCP) regions of hepatitis B virus (HBV) from Shanxi Province, and the association between mutations and disease related index...AIM: To investigate the frequency of mutations in pre-core (pre-C) and basic core promoter (BCP) regions of hepatitis B virus (HBV) from Shanxi Province, and the association between mutations and disease related indexes.METHODS: One hundred chronic hepatitis B patients treated at Shanxi Province Hospital of Traditional Chinese Medicine were included in this study. PCR-reverse dot blot hybridization and mismatch amplification mutation assay (MAMA)-PCR were used to detect the mutations in the HBV pre-C and BCP regions. HBV DNA content and liver function were compared between patients with mutant HBV pre-C and BCP loci and those with wild-type loci. The consistency between PCR-reverse dot blot hybridization and MAMA-PCR for detecting mutations in the HBV pre-C and BCP regions was assessed.RESULTS: Of the 100 serum samples detected, 9.38% had single mutations in the pre-C region, 29.17% had single mutations in the BCP region, 41.67% had mutations in both BCP and pre-C regions, and 19.79% had wild-type loci. The rates of BCP and pre-C mutations were 65.7% and 34.3%, respectively, in hepatitis B e antigen (HBeAg) positive patients, and 84.6% and 96.2%, respectively, in HBeAg negative patients. The rate of pre-C mutations was significantly higher in HBeAg negative patients than in HBeAg positive patients (χ<sup>2</sup> = 26.62, P = 0.00), but there was no significant difference in the distribution of mutations in the BCP region between HBeAg positive and negative patients (χ<sup>2</sup> = 2.43, P = 0.12). The presence of mutations in the pre-C (Wilcoxon W = 1802.5, P = 0.00) and BCP regions (Wilcoxon W = 2906.5, P = 0.00) was more common in patients with low HBV DNA content. Both AST and GGT were significantly higher in patients with mutant pre-C and BCP loci than in those with wild-type loci (P < 0.05). PCR-reverse dot blot hybridization and MAMA-PCR for detection of mutations in the BCP and pre-C regions had good consistency, and the Kappa values obtained were 0.91 and 0.58, respectively.CONCLUSION: HBeAg negative patients tend to have HBV pre-C mutations. However, these mutations do not cause increased DNA copies, but associate with damage of liver function.展开更多
Background： Epidermal growth factor receptor（EGFR） mutation is the key predictor of EGFR tyrosine kinase inhibitors（TKIs） efficacy in non-small cell lung cancer（NSCLC）. We conducted this study to verify the fea...Background： Epidermal growth factor receptor（EGFR） mutation is the key predictor of EGFR tyrosine kinase inhibitors（TKIs） efficacy in non-small cell lung cancer（NSCLC）. We conducted this study to verify the feasibility of EGFR mutation analysis in cytological specimens and investigate the responsiveness to gefitinib treatment in patients carrying EGFR mutations.Methods： A total of 210 cytological specimens were collected for EGFR mutation detection by both direct sequencing and amplification refractory mutation system（ARMS）. We analyzed EGFR mutation status by both methods and evaluated the responsiveness to gefitinib treatment in patients harboring EGFR mutations by overall response rate（ORR）, disease control rate（DCR） and progression free survival（PFS）.Results： Of all patients, EGFR mutation rate was 28.6%（60/210） by direct sequencing and 45.2%（95/210） by ARMS（P〈0.001） respectively. Among the EGFR wild type patients tested by direct sequencing, 26.7% of them were positive by ARMS. For the 72 EGFR mutation positive patients treated with gefitinib, the ORR, DCR and median PFS were 69.4%, 90.2% and 9.3 months respectively. The patients whose EGFR mutation status was negative by direct sequencing but positive by ARMS had lower ORR（48.0% vs. 80.9%, P=0.004） and shorter median PFS（7.4 vs. 10.5 months, P=0.009） as compared with that of EGFR mutation positive patients by both detection methods. Conclusions： Our study verified the feasibility of EGFR analysis in cytological specimens in advanced NSCLC. ARMS is more sensitive than direct sequencing in EGFR mutation detection. EGFR Mutation status tested on cytological samples is applicable for predicting the response to gefitinib. Abundance of EGFR mutations might have an influence on TKIs efficacy.展开更多
The purpose of the present study was to study the characteristics of epidemic growth factor receptor(EGFR)gene distribution in patients with non-small cell lung cancer(NSCLC),and to detect the mutation rate of EGFR ge...The purpose of the present study was to study the characteristics of epidemic growth factor receptor(EGFR)gene distribution in patients with non-small cell lung cancer(NSCLC),and to detect the mutation rate of EGFR gene by Sanger sequencing and amplification refractory mutation system(ARMS)-PCR.Paraffin-embedded sections of NSCLC tissues from 399 NSCLC patients diagnosed in Renmin Hospital of Wuhan University were collected,103 of them were detected for exons 18-21 mutation of EGFR by Sanger sequencing method,296 cases were detected for exons 18-21 mutation by ARMS-PCR method.DNA extraction of both groups was performed with Qiagen QLAamp DNA FFPE Tissue KIT.Comparisons of detection rates between the two methods were conducted by row X list chi-square test.The total mutation rate of EGFR gene detected by Sanger sequencing was 21.4%,exons 18-21 and combined mutation rates were 1.0%,9.7%,1.0%,7.8%and 2.0%,respectively.And the proportions were 4.7%,45.2%,4.7%,36.3%and 9.4%respectively.The total mutation rate detected by ARMS-PCR was 51.4%,exons 18-21 and combined mutation rates were 2.7%,27%,1.7%,18.2%and 1.7%,respectively.The proportions were 5.3%,52.6%,3.3%,35.5%and 3.3%respectively.Further analysis of mutation rate showed that there was significant difference between the two methods in detecting total mutation of EGFR gene(P<0.001).There were significant differences in mutation detection rates of exons 19 and 21(P<0.001,P<0.05),but there were no significant differences in other exons.And there was no significant difference in mutation detection rates between the two methods.The mutation rate of EGFR gene in NSCLC patients was 50%.And exon 19 deletion was the most common mutation type,followed by exon 21 mutation.Compared with Sanger sequencing method,ARMS method is more sensitive with significant advantages in detecting exon 19 deletions and exon 21 mutations,which can be widely used in clinical detection of EGFR gene mutations.The results of this study will further guide patients with advanced NSCLC to select TKI targeted drugs,and provide clinical diagnostic basis for targeted therapy of NSCLC patients.展开更多
Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins,The association of Single Nucleotide Polymorphism(SNP)in the calpastatin gene with meat tenderness is a...Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins,The association of Single Nucleotide Polymorphism(SNP)in the calpastatin gene with meat tenderness is an important topic in meat production.Therefore efficient procedure to investigate the SNP is necessary.The objectives of this study were to detect the SNP of calpastatin gene at domain L marker(G/C transversion)of the Kamphaengsaen beef breed(KPS cattle;n=26)by the Amplification Refractory Mutation System(ARMS)compared with the Restriction Fragment Length Polymorphism(RFLP)methods and to determine the genotypes of the KPS cattle at that marker.Genomic DNA of calpastatin gene extracted from blood of the KPS cattle was detected with ARMS and RFLP methods.The ARMS system has utilized two primer pairs to amplify the two different alleles of a polymorphism in single PCR reaction to detected single base mutation.In this method,the alleles-specific primers had a mismatch at 3'terminal base and a second deliberate mismatch at position-2 from 3'terminus.While the RFLP method detected a polymorphism using PCR-base technique follow by RsaI restriction enzyme.Amplification of the ARMS method revealed that the results were not different from the conventional method of RFLP.Analysis of genotypes revealed that the KPS cattle inherited the CC,CG and GG genotypes at domain L marker.These were reliable when verified by nucleotide sequence analysis of PCR products.The animals were genotyped and determined for tenderness phenotype with this marker that predicted variation of an intronic polymorphism at domain L of the calpastatin gene.Therefore,the ARMS method was simple,efficient technique,and suitable for detecting SNP at domain L marker of the calpastatin gene.展开更多
基金Supported by Youth Foundation of Health and Family Planning Commission of Shanxi ProvinceNo.201301024
文摘AIM: To investigate the frequency of mutations in pre-core (pre-C) and basic core promoter (BCP) regions of hepatitis B virus (HBV) from Shanxi Province, and the association between mutations and disease related indexes.METHODS: One hundred chronic hepatitis B patients treated at Shanxi Province Hospital of Traditional Chinese Medicine were included in this study. PCR-reverse dot blot hybridization and mismatch amplification mutation assay (MAMA)-PCR were used to detect the mutations in the HBV pre-C and BCP regions. HBV DNA content and liver function were compared between patients with mutant HBV pre-C and BCP loci and those with wild-type loci. The consistency between PCR-reverse dot blot hybridization and MAMA-PCR for detecting mutations in the HBV pre-C and BCP regions was assessed.RESULTS: Of the 100 serum samples detected, 9.38% had single mutations in the pre-C region, 29.17% had single mutations in the BCP region, 41.67% had mutations in both BCP and pre-C regions, and 19.79% had wild-type loci. The rates of BCP and pre-C mutations were 65.7% and 34.3%, respectively, in hepatitis B e antigen (HBeAg) positive patients, and 84.6% and 96.2%, respectively, in HBeAg negative patients. The rate of pre-C mutations was significantly higher in HBeAg negative patients than in HBeAg positive patients (χ<sup>2</sup> = 26.62, P = 0.00), but there was no significant difference in the distribution of mutations in the BCP region between HBeAg positive and negative patients (χ<sup>2</sup> = 2.43, P = 0.12). The presence of mutations in the pre-C (Wilcoxon W = 1802.5, P = 0.00) and BCP regions (Wilcoxon W = 2906.5, P = 0.00) was more common in patients with low HBV DNA content. Both AST and GGT were significantly higher in patients with mutant pre-C and BCP loci than in those with wild-type loci (P < 0.05). PCR-reverse dot blot hybridization and MAMA-PCR for detection of mutations in the BCP and pre-C regions had good consistency, and the Kappa values obtained were 0.91 and 0.58, respectively.CONCLUSION: HBeAg negative patients tend to have HBV pre-C mutations. However, these mutations do not cause increased DNA copies, but associate with damage of liver function.
文摘Background： Epidermal growth factor receptor（EGFR） mutation is the key predictor of EGFR tyrosine kinase inhibitors（TKIs） efficacy in non-small cell lung cancer（NSCLC）. We conducted this study to verify the feasibility of EGFR mutation analysis in cytological specimens and investigate the responsiveness to gefitinib treatment in patients carrying EGFR mutations.Methods： A total of 210 cytological specimens were collected for EGFR mutation detection by both direct sequencing and amplification refractory mutation system（ARMS）. We analyzed EGFR mutation status by both methods and evaluated the responsiveness to gefitinib treatment in patients harboring EGFR mutations by overall response rate（ORR）, disease control rate（DCR） and progression free survival（PFS）.Results： Of all patients, EGFR mutation rate was 28.6%（60/210） by direct sequencing and 45.2%（95/210） by ARMS（P〈0.001） respectively. Among the EGFR wild type patients tested by direct sequencing, 26.7% of them were positive by ARMS. For the 72 EGFR mutation positive patients treated with gefitinib, the ORR, DCR and median PFS were 69.4%, 90.2% and 9.3 months respectively. The patients whose EGFR mutation status was negative by direct sequencing but positive by ARMS had lower ORR（48.0% vs. 80.9%, P=0.004） and shorter median PFS（7.4 vs. 10.5 months, P=0.009） as compared with that of EGFR mutation positive patients by both detection methods. Conclusions： Our study verified the feasibility of EGFR analysis in cytological specimens in advanced NSCLC. ARMS is more sensitive than direct sequencing in EGFR mutation detection. EGFR Mutation status tested on cytological samples is applicable for predicting the response to gefitinib. Abundance of EGFR mutations might have an influence on TKIs efficacy.
基金grants from the National Natural Science Foundation of China(No.81100959)Natural Science Fund of Hubei Province(No.2015CFB185).
文摘The purpose of the present study was to study the characteristics of epidemic growth factor receptor(EGFR)gene distribution in patients with non-small cell lung cancer(NSCLC),and to detect the mutation rate of EGFR gene by Sanger sequencing and amplification refractory mutation system(ARMS)-PCR.Paraffin-embedded sections of NSCLC tissues from 399 NSCLC patients diagnosed in Renmin Hospital of Wuhan University were collected,103 of them were detected for exons 18-21 mutation of EGFR by Sanger sequencing method,296 cases were detected for exons 18-21 mutation by ARMS-PCR method.DNA extraction of both groups was performed with Qiagen QLAamp DNA FFPE Tissue KIT.Comparisons of detection rates between the two methods were conducted by row X list chi-square test.The total mutation rate of EGFR gene detected by Sanger sequencing was 21.4%,exons 18-21 and combined mutation rates were 1.0%,9.7%,1.0%,7.8%and 2.0%,respectively.And the proportions were 4.7%,45.2%,4.7%,36.3%and 9.4%respectively.The total mutation rate detected by ARMS-PCR was 51.4%,exons 18-21 and combined mutation rates were 2.7%,27%,1.7%,18.2%and 1.7%,respectively.The proportions were 5.3%,52.6%,3.3%,35.5%and 3.3%respectively.Further analysis of mutation rate showed that there was significant difference between the two methods in detecting total mutation of EGFR gene(P<0.001).There were significant differences in mutation detection rates of exons 19 and 21(P<0.001,P<0.05),but there were no significant differences in other exons.And there was no significant difference in mutation detection rates between the two methods.The mutation rate of EGFR gene in NSCLC patients was 50%.And exon 19 deletion was the most common mutation type,followed by exon 21 mutation.Compared with Sanger sequencing method,ARMS method is more sensitive with significant advantages in detecting exon 19 deletions and exon 21 mutations,which can be widely used in clinical detection of EGFR gene mutations.The results of this study will further guide patients with advanced NSCLC to select TKI targeted drugs,and provide clinical diagnostic basis for targeted therapy of NSCLC patients.
文摘Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins,The association of Single Nucleotide Polymorphism(SNP)in the calpastatin gene with meat tenderness is an important topic in meat production.Therefore efficient procedure to investigate the SNP is necessary.The objectives of this study were to detect the SNP of calpastatin gene at domain L marker(G/C transversion)of the Kamphaengsaen beef breed(KPS cattle;n=26)by the Amplification Refractory Mutation System(ARMS)compared with the Restriction Fragment Length Polymorphism(RFLP)methods and to determine the genotypes of the KPS cattle at that marker.Genomic DNA of calpastatin gene extracted from blood of the KPS cattle was detected with ARMS and RFLP methods.The ARMS system has utilized two primer pairs to amplify the two different alleles of a polymorphism in single PCR reaction to detected single base mutation.In this method,the alleles-specific primers had a mismatch at 3'terminal base and a second deliberate mismatch at position-2 from 3'terminus.While the RFLP method detected a polymorphism using PCR-base technique follow by RsaI restriction enzyme.Amplification of the ARMS method revealed that the results were not different from the conventional method of RFLP.Analysis of genotypes revealed that the KPS cattle inherited the CC,CG and GG genotypes at domain L marker.These were reliable when verified by nucleotide sequence analysis of PCR products.The animals were genotyped and determined for tenderness phenotype with this marker that predicted variation of an intronic polymorphism at domain L of the calpastatin gene.Therefore,the ARMS method was simple,efficient technique,and suitable for detecting SNP at domain L marker of the calpastatin gene.
