Huijuan Wang

Association analysis of CYP2A6 genotypes and haplotypes with 5-fluorouracil formation from tegafur in human liver microsomes

AIM Tegafur is primarily converted to 5-fluorouracil (5-FU) by CYP2A6 in the human liver to exert its antitumor effect. Our objective was to comprehensively investigate the effects of CYP2A6 genetic polymorphisms on tegafur bioactivation activity. MATERIALS & METHODS Using a set of over 45 Chinese livers, the association between CYP2A6 genetic variations and 5-FU formation rates from tegafur, as well as CYP2A6 mRNA and protein levels, was determined. RESULTS A total of 20 polymorphic variants and 20 haplotypes of CYP2A6 were identified. From genotype/haplotype-phenotype association tests, we demonstrated that CYP2A6*4 was the main allele responsible for the decreased 5-FU formation from tegafur and CYP2A6 expression in this population. By contrast, haplotype 14 (a novel CYP2A6*1B allele) was associated with increased microsomal 5-FU formation activity and CYP2A6 expression, and this may be attributed to the combined effects of three single variants (g.22C>T, g.1620T>C and a gene conversion in the 3´-UTR) included in this haplotype. CONCLUSION We concluded that CYP2A6*4 and the novel CYP2A6*1B variant were the major genetic determinants of interindividual variability in 5-FU formation from tegafur in Chinese livers. Original submitted 2 November 2010; Revision submitted 3 December 2010.

Tissue-specific selection of optimal reference genes for expression analysis of anti-cancer drug-related genes in tumor samples using quantitative real-time RT-PCR

Gene transcription analysis in clinical tumor samples can help with diagnosis, prognosis, and treatment of cancers. We aimed to identify the optimal reference genes for reliable expression analysis in various tumor samples by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Using a one-step TaqMan-based qRT-PCR, 5 commonly used reference genes (ACTB, GAPDH, RPLPO, GUSB, and TFRC) and 10 anticancer drug-related genes (TYMS, RRM1, TUBB3, STMN1, TOP2A, EGFR, VEGFR2, HER2, ERCC1, and BRCA1) were analyzed in 327 tissue samples from lung, rectal, colon, gastric, esophageal, and breast tumors. According to the expression stability assessments obtained by using three programs (geNorm, NormFinder, and BestKeeper) and a comprehensive ranking method, the optimal reference genes for lung, gastric, esophageal, and breast tumors were RPLPO, GAPDH, ACTB, and ACTB, respectively. For rectal tumors, a combination of the 3 most stable genes (GUSB, ACTB, and RPLPO) was suitable for qRT-PCR, whereas for colon tumors, a combination of the 4 most stable genes (GAPDH, ACTB, GUSB, and RPLPO) was optimal for qRT-PCR. Based on the expression data of target genes normalized against selected reference genes, the principal component analysis revealed 4 expression patterns in 6 different tissues. One pattern was observed in gastric, rectal, and colon tumor tissues, which are gastrointestinal tumors. Expressions in the breast, lung, and esophageal tissues were separately represented as one pattern. Our results could facilitate the practice of personalized cancer medicine based on the gene expression profile of the patients.

Expression Profile Analysis of Zinc Transporters (ZIP4, ZIP9, ZIP11, ZnT9) in Gliomas and their Correlation with IDH1 Mutation Status

BACKGROUND Zinc transporters have been considered as essential regulators in many cancers; however, their mechanisms remain unknown, especially in gliomas. Isocitrate dehydrogenase 1(IDH1) mutation is crucial to glioma. This study aimed to investigate whether zinc transporters are correlated with glioma grade and IDH1 mutation status. MATERIALS AND METHODS IDH1 mutation status and mRNA expression of four zinc transporters (ZIP4, ZIP9, ZIP11, and ZnT9) were determined by subjecting a panel of 74 glioma tissue samples to quantitative real-time PCR and pyrosequencing. The correlations between the expression levels of these zinc transporter genes and the grade of glioma, as well as IDH1 mutation status, were investigated. RESULTS Among the four zinc transporter genes, high ZIP4 expression and low ZIP11 expression were significantly associated with higher grade (grades III and IV) tumors compared with lower grade (grades I and II) counterparts (p<0.0001). However, only ZIP11 exhibited weak correlation with IDH1 mutation status (p=0.045). Samples with mutations in IDH1 displayed higher ZIP11 expression than those without IDH1 mutations. CONCLUSIONS This finding indicated that zinc transporters may interact with IDH1 mutation by direct modulation or action in some shared pathways or genes to promote the development of glioma. Zinc transporters may play an important role in glioma. ZIP4 and ZIP11 are promising molecular diagnostic markers and novel therapeutic targets. Nevertheless, the detailed biological function of zinc transporters and the mechanism of the potential interaction between ZIP11 and IDH1 mutation in gliomagenesis should be further investigated.

Rapid and reliable genotyping of HLA-B*58:01 in four Chinese populations using a single-tube duplex real-time PCR assay

AIM HLA-B*58:01 is strongly associated with allopurinol-induced severe cutaneous adverse reactions. This study aimed to develop a new and convenient method for HLA-B*58:01 genotyping and to investigate HLA-B*58:01 distribution in different Chinese populations. MATERIALS & METHODS Combining of sequence-specific primers and TaqMan probe, a single-tube duplex real-time PCR assay for HLA-B*58:01 typing was established. RESULTS The HLA-B*58:01 genotyping result in Buyei (n = 100) by real-time PCR showed 100% concordance with those by sequence-based typing. The prevalence of HLA-B*58:01 carrier in Buyei (17%, n = 100) was significantly higher than those in Northern Han (4%, n = 100), Tibetan (5.1%, n = 99) and Uighur (2%, n = 50) populations (p < 0.05). CONCLUSION The newly developed reliable assay was appropriate for HLA-B*58:01 detection prior to allopurinol administration in clinical settings.

Mitochondrial DNA Levels in Blood and Tissue Samples from Breast Cancer Patients of Different Stages

AIMS Alterations in mitochondrial DNA (mtDNA) have been implicated in carcinogenesis and tumor progression. We here evaluated the diagnostic and prognostic potential of mtDNA as a biomarker for breast cancer. METHODS Using multiplex real-time polymerase chain reaction, nuclear DNA (nDNA) and mtDNA levels in serum, buffy coat, tumor, and tumor-adjacent tissue samples from 50 breast cancer patients were determined and assessed for associations with clinicopathological features. To evaluate mtDNA as a biomarker for distinguishing between the four sample types, we created receiver operating characteristic (ROC) curves. RESULTS The mtDNA levels in buffy coat were significantly lower than in other sample types. Relative to tumor-adjacent tissue, reduced levels of mtDNA were identified in buffy coat and tumor tissue but not in serum. According to ROC curve analysis, mtDNA levels could be used to distinguish between buffy coat and tumor-adjacent tissue samples with good sensitivity (77%) and specificity (83%). Moreover, mtDNA levels in serum and tumor tissue were positively associated with cancer TMN stage. CONCLUSIONS The mtDNA levels in blood samples may represent a promising, non-invasive biomarker in breast cancer patients. Additional, large-scale validation studies are required to establish the potential use of mtDNA levels in the early diagnosis and monitoring of breast cancer.

CLPTM1L genetic polymorphisms and interaction with smoking and alcohol drinking in lung cancer risk: a case-control study in the Han population from northwest China.

AbstractGenetic variants of cleft lip and palate trans-membrane 1-like (CLPTM1L) genes in the p15.33 region of chromosome 5 were previously identified to influence susceptibility to lung cancer. We examined the association of single nucleotide polymorphisms (SNPs) in CLPTM1L genes with lung cancer and explored their potential effects on the relationship between environmental risk factors (smoking, drinking) and lung cancer in a Chinese Han population.We genotyped 9 single nucleotide polymorphisms (SNPs) of CLPTM1L in a case–control study with 228 lung cancer cases and 301 controls from northwest China. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by unconditional logistic regression.We identified that the minor alleles of rs451360, rs402710, and rs31484 in CLPTM1L were associated with a 0.52-fold, 0.76-fold, and 0.70-fold decreased risk of lung cancer in allelic model analysis, respectively. In the genetic model analysis, we found rs402710 and rs401681 were associated with decreased lung cancer risk. Further stratification analysis showed that rs380286 displayed a significantly decreased lung cancer risk (OR = 0.65, P = 0.041) in the non-drinkers. In addition, Haplotype “GTTATCTGT” was found to be associated with decreased lung cancer risk (OR = 0.50, P = 0.033).Our results verified that genetic variants of CLPTM1L contribute to lung cancer susceptibility in the northwest Chinese Han population. Additionally, we found that consumption of alcohol may interact with CLPTM1L polymorphisms to contribute to overall lung cancer susceptibility.

A multiplex allele-specific real-time polymerase chain reaction assay for HLA-B*13:01 genotyping in four Chinese populations.

Human leukocyte antigen HLA‐B*13:01 is identified currently as a marker of individual susceptibility to drug‐induced hypersensitivity reaction, such as dapsone‐induced hypersensitivity reactions (DIHRs) and trichloroethylene‐induced dermatitis. Therefore, screening for the HLA‐B*13:01 allele can assist clinics in identifying patients at risk of developing DIHRs. By combining the allele‐specific primers with TaqMan probes, we established a single tube, triplex real‐time PCR to detect HLA‐B*13:01. The reliability of this assay was validated by the comparison of genotyping results with those by sequence‐based typing (SBT). With this assay, the distribution of HLA‐B*13:01 in a total of 350 blood samples from four ethnic groups: Han, Tibetan, Uighur, and Buyei were determined. A 100% concordance was observed between the results with the established real‐time PCR and SBT in 100 samples. The detection limit of this assay was 0.016 ng genomic DNA. The prevalence of HLA‐B*13:01 carriers were 11%, 8%, 1%, and 2% in the Buyei (n = 100), Northern Han (n = 100), Tibetan (n = 100), and Uighur (n = 50) populations, respectively. The multiplex real‐time PCR assay provided a fast and reliable method for accurate detection of HLA‐B*13:01 allele prior to dapsone administration in clinical practice and onset of the reaction after exposure to trichloroethylene.

Genetic polymorphism of pharmacogenomic VIP variants in the Deng people from the Himalayas in Southeast Tibet

Abstract Little is known about polymorphic distribution of pharmacogenes among ethnicities, including the Deng people. In this study, we recruited 100 unrelated, healthy Deng people and genotyped them with respect to 76 different single-nucleotide polymorphisms by the PharmGKB database. Our results first indicated that the polymorphic distribution of pharmacogenes of the Deng people is most similar to CHD, suggesting that Deng people have a closest genetic relationship with CHD. Our data will enrich the database of pharmacogenomics and provide a theoretical basis for safer drug administration and individualized treatment plans, promoting the development of personalized medicine.

Association analysis of UGT1A genotype and haplotype with SN-38 glucuronidation in human livers

AIM 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, is mainly eliminated hepatically through glucuronidation by UGT1A1 and UGT1A9 enzymes. This study comprehensively investigates the effects of UGT1A1 and UGT1A9 genetic polymorphism on SN-38 glucuronidation activity. MATERIALS & METHODS Genetic polymorphisms and combinational haplotypes of UGT1A1 and UGT1A9, SN-38 glucuronidation activities, and protein levels of UGT1A1 and UGT1A9 were determined using a set of over 45 Chinese livers. RESULTS UGT1A1 reduced function variants UGT1A1*6, *28, *60 and *1B exhibited additive effect. The number of UGT1A1 reduced function alleles was associated with decreased SN-38G formation rates and UGT1A protein levels. UGT1A9 I399C>T and UGT1A9*1b, which were highly linked, were associated with increased SN-38 glucuronidation activity and UGT1A protein levels. However, further analysis based on UGT1A9-1A1 haplotypes confirmed that their increased effect was partly due to their close linkage with UGT1A1 reduced function alleles. CONCLUSION UGT1A1 genetic polymorphisms have a more important function in human liver SN-38 glucuronidation activity than UGT1A9. Original submitted 7 November 2013; Revision submitted 30 January 2014.

Rapid and Reliable Genotyping of HLA-B*57:01 in Four Chinese Populations using a Single-tube Duplex Real-time PCR Assay

Abstract HLA-B*57:01 is strongly associated with severe adverse drug reaction induced by the anti-HIV drug abacavir (ABC) and antibiotic flucloxacillin. This study was dedicated to establishing a new method for HLA-B*57:01 genotyping and investigating the HLA-B*57:01 distribution pattern in four Chinese populations. A single-tube duplex real-time polymerase chain reaction (PCR) system was established by combining the amplification refractory mutation system and TaqMan probe. The reliability of this assay was validated by comparing the genotyping results with those by sequence-based typing. With this assay, the distribution of HLA-B*57:01 in 354 blood samples from four ethnic groups, namely, Han, Tibetan, Uighur, and Buyei, was determined. A 100% concordance was observed between the results of real-time PCR and sequence-based typing in 50 Uighur samples. As low as 0.016 ng DNA that carried HLA-B*57:01 could be detected with this assay. HLA-B*57:01 carriers identified in 100 Northern Han Chinese, 104 Buyeis...