Chain Reaction-single Strand Conformation Polymorphism Research Articles

Reduced transcription of the Smad4 gene during pulmonary carcinogenesis in idiopathic pulmonary fibrosis

Patients with idiopathic pulmonary fibrosis (IPF) have an increased risk of developing lung cancer. To identify key molecules involved in malignant transformation in IPF, we analyzed the expression profiles of lung and lung tumor tissue from patients with lung cancer and IPF (lung cancer/IPF) using cDNA arrays and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Reduced expression of the Smad4 gene was identified in all eight tumor samples from the lung cancer/IPF patients using real-time RT-PCR. Expression levels of Smad4 were significantly lower in tumors from lung cancer/IPF patients than in those from lung cancer patients without IPF or in lung cancer cell lines (p<0.01). Mutational analysis of TGF-β type II receptor and Smad4 was performed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The methylation status of the Smad4 promoter was analyzed using methylation-specific PCR with subsequent sequence analysis. No mutations were detected in the eight tumor samples, but hypermethylated regions were detected in the Smad4 promoter in two of the eight tumors with reduced Smad4 expression. Promoter reporter assays showed that the activity of the Smad4 promoter containing the sequence of the methylated region was significantly stronger than that of the Smad4 promoter with a deleted methylated region (p<0.002). Our findings indicate that the loss of the growth inhibitory response to TGF-β signaling may be crucial in pulmonary carcinogensis or in the progression of lung cancer in IPF patients in whom TGF-β is overexpressed; hypermethylation of the Smad4 promoter region may be one mechanism by which this occurs. These findings are useful for the development of preventive measures or treatment for lung cancer patients with IPF.

Leptin gene polymorphism in Indian Sahiwal cattle by single strand conformation polymorphism (SSCP) (Short communication)

Leptin, a 16-Kilo dalton protein produced by the obesity (ob) gene, plays an important role in the regulation of feed intake, energy metabolism, growth and reproduction in cattle. The genetic variation of the leptin gene in Sahiwal cattle (Bos indicus) was investigated using an optimized non-radioactive polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of 13 amplified fragments covering almost the entire gene. Twenty-eight SSCP band patterns were detected from 10 of these fragments in a sample of 202 Sahiwal cattle. Polymorphisms were detected in the samples, indicating that Sahiwal cattle have high genetic variability in the entire leptin gene. This result opens interesting prospects for future breeding programmes and conservation strategies. These leptin gene variants can be sequenced and screened in the entire population to develop single nucleotide polymorphisms (SNPs) for association studies with different productive and reproductive performances and marker assisted selection.

Microbial diversity in Tunisian olive fermentation brine as evaluated by small subunit rRNA - Single strand conformation polymorphism analysis

The microbial diversity of a Tunisian olive fermentation brine was analysed using a culture-independent approach based on the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). SSCP patterns show a remarkably simple microbial community but higher for bacterial community than for the eukaryotic community. This study did not show the presence of archaeal populations. After PCR amplification, two small subunit (SSU) rRNA clone libraries of Bacteria and Eucarya populations were established. Three bacteria and only one eukaryotic phylotype were identified. Two dominant bacteria showed 100% phylogenetic similarity to the 16S rRNA sequences of Lactobacillus plantarum and Lactobacillus collinoides and represent 85% of bacterial community. The third bacteria phylotype was phylogenetically close related to the 16S rRNA sequence of a moderately halophilic bacterium belonging to the class gamma Proteobacteria. The dominant eukaryotic phylotype was identified as a Pichia membranaefaciens.

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility

To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 (USP26) gene and its involvement in idiopathic male infertility in China. Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. Of 41 infertile men, 9 (22.0%, P = 0.01) had changes in USP26 gene on the X chromosome. A compound mutation (364insACA; 460G right triple arrow A) was detected in 8 patients (19.5%, P = 0.01) and a 1044T right triple arrow A substitution was found in 1 patient (2.4%, P > 0.05). All three variations led to changes in the coding amino acids. Two substitutions predict some changes: 460G right triple arrow A changes a valine into an isoleucine, and 1044T right triple arrow A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.

Sensitivity and Specificity of P53 Protein Detection by Immunohistochemistry in Patients with Urothelial Bladder Carcinoma

Objective: To analyze the correlation between the genotypic and phenotypic patterns of p53 in patients with transitional cell carcinoma (TCC) of the urinary bladder. Materials and Methods: Cross-sectional study of 73 patients diagnosed with TCC. DNA was obtained from the tumor tissue to perform polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) of exons 5–9 of the p53 gene, with automatic sequencing done on any mutated samples. Immunohistochemistry (IHC) was also performed using anti-human P53 monoclonal antibody, and the diagnostic performance of this test was analyzed by a ROC curve, using the presence of p53 mutations found by PCR-SSCP as ‘gold standard’. Results: The cutoff point for defining immunopositivity was 20%. IHC had a specificity of 62.9%, and a sensitivity of 65.8%. The highest sensitivity values appeared in G3 tumors (75%) and infiltrating tumors (71.4%), and the highest specificity values were observed in G1 (77.7%) and G2 tumors (90%) and superficial tumors (66.6%). Mutations in exon 8 gave a positive result most frequently (73.7%) and were considered most relevant in terms of altering P53 function (60.9%). False negatives were documented in 28.5% of infiltrating tumors, and false positives in 33.4% of superficial tumors. Conclusions: There is a moderate correlation between p53 mutations and P53 protein overexpression, with this stronger in high-grade, infiltrating tumors, in exon 8 mutations, and when the mutation induces relevant changes in the protein structure. Although IHC is useful in routine clinical practice, the classic prognostic factors should still be considered the most important in the follow-up of these patients.

Association between peroxisome proliferator-activated receptor-γ coactivator-1α gene polymorphisms and type 2 diabetes in southern Chinese population: role of altered interaction with myocyte enhancer factor 2C

Some single nucleotide polymorphisms (SNPs) in the peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha gene have been reported to be associated with type 2 diabetes in different populations, and studies on Chinese patients yielded controversial results. The objective of this case-control study was to explore the relationship between SNPs of PGC-1alpha and type 2 diabetes in the southern Chinese population and to determine whether the common variants: Gly482Ser and Thr394Thr, in the PGC-1alpha gene have any impacts on interaction with myocyte enhancer factor (MEF) 2C. The SNPs in all exons of the PGC-1alpha gene was investigated in 50 type 2 diabetic patients using polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and direct sequencing. Thereafter, 263 type 2 diabetic patients and 282 healthy controls were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A bacterial two-hybrid system and site-directed mutagenesis were used to investigate whether Gly482Ser and Thr394Thr variants in the PGC-1alpha gene alter the interaction with MEF2C. Three frequent SNPs (Thr394Thr, Gly482Ser and Thr528Thr) were found in exons of the PGC-1alpha gene. Only the Gly482Ser variant had a different distribution between diabetic patients and healthy subjects, with the 482Ser allele more frequent in patients than in controls (40.1% vs 29.3%, P < 0.01). Even in controls, the 482Ser (A) carriers were more likely to have higher levels of total cholesterol and low-density lipoprotein cholesterol than the 482Gly (G) carriers. The 394A-482G-528A haplotype was associated with protection from diabetes, while the 394A-482A-528A was associated with the susceptibility to diabetes. The bacterial two-hybrid system and site-directed mutagenesis revealed that the 482Ser variant was less efficient than the 482Gly variant to interact with MEF2C, whereas the 394Thr (A) had a synergic effect on the interaction between 482Ser variant and MEF2C. The results suggested that the 482Ser variant of PGC-1alpha conferred the susceptibility to type 2 diabetes in the southern Chinese population. The underlying mechanism may be attributable, at least in part, to the altered interaction between the different variants (Gly482Ser, Thr394Thr) in the PGC-1alpha gene and MEF2C.

Analysis of methanogens in the bovine rumen by polymerase chain reaction single-strand conformation polymorphism

The polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method reported by Schwieger and Tebbe (1998) was used to analyze the diversity of methanogens inhabiting the rumen. Partial 16S rRNA gene fragments were amplified from DNA extracted from rumen contents by PCR with archaea-specific primers, Ar1000F and Ar1500R, or methanogen-specific primers, M301F and M915R, with one primer phosphorylated at the 5′ end. The amplified DNA fragments were analyzed by SSCP gel electrophoresis after the phosphorylated strands of the PCR products were digested with λ exonuclease. When we analyzed samples collected from the six Holstein cows used in a previous study, in which cows were given feed with or without α-cyclodextrin-horseradish oil complex (CD-HR), nine and six bands were identified in the profiles generated by PCR products amplified with archaea-specific and methanogen-specific primers, respectively. While dendrogram analysis based on SSCP gel profiles found that the methanogens from each rumen showed a particular composition of methanogens, the profiles of the methanogens isolated from two of three cows fed with CD-HR fell into the same branch in the dendrogram constructed from the profiles. Therefore, this study demonstrates the potential of the PCR-SSCP method in the methanogenic community analysis of the rumen and in investigating changes in the methanogenic community due to the addition of CD-HR to the rumen.

Analysis of the CYP1B1 gene mutation in primary congenital glaucoma patients of Hubei Han nationality

To identify novel CYP1B1 gene mutation in primary congenital glaucoma (PCG) patients of Hubei Han nationality and establish the possibility of gene diagnosis of PCG. Forty-seven patients with PCG and 100 normal subjects were studied. Genomic DNA was extracted from the peripheral leukocytes of all subjects. Mutation in exon2 and exon3 of CYP1B1 gene was detected in patients and control subjects by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), denaturing high performance liquid chromatograph (DHPLC), and direct sequencing DNA techniques. Compared to normal subjects, a novel mutation was first time identified by direct sequencing demonstrating a homozygous C-to-T transition at codon 385 (CTT to TTT) which produced L385F mutation of CYP1B1 gene in 7 of the 47 patients with PCG. The novel mutation in exon 3 of CYP1B1 found in PCG patients of Hubei Han nationality was probably pathological mutant gene by nature. It is important that further study be conducted to seek for the specific mutations of CYP1B1 gene and underlying pathological mechanism of PCG patients of Han nationality.

Fatty Acid Synthase Hyperactivation in Human Colorectal Cancer: Relationship with Tumor Side and Sex

Objective: Fatty acid synthase (FAS) is a multienzyme protein required for the conversion of acetyl coenzyme A and malonyl coenzyme A to palpitate. High levels of FAS expression have been found in many human cancers, including breast, prostate and colon. In this study, we evaluated FAS activity levels and the expression of its mRNA in normal colorectal mucosa and cancer tissue from patients operated for colorectal carcinoma. In addition, the hypothesis of a relation between FAS activity and p53 mutation status of patients was tested. Methods: Forty-two patients were enrolled in the study. FAS activity was measured by using a radiometric assay. FAS gene expression was determined using quantitative reverse-transcription polymerase chain reaction and p53 mutations by polymerase chain reaction single-strand conformation polymorphism. Results: FAS activity levels were significantly higher in cancer than in the corresponding normal mucosa. Tumors located on the left side of the colon showed higher levels of FAS activity and tumors from male patients showed higher FAS activity than tumors from females. No difference was detected in mRNA FAS levels according to tumor side and gender. Moreover, lower levels of FAS activity were detected in patients carrying the p53 mutation. Conclusions: This study suggests that biological factors including sex and gene mutation status, as well as stratification of patients with colorectal cancer into right- and left-sided subsets, may be important in patient selection for targeted therapies and for the subsequent assessment of objective therapeutic responses.

Interleukin-1β gene in esophageal, gastric and colorectal carcinomas

Interleukin (IL)-1 gene polymorphisms are associated with development of gastric atrophy and with increased risk of gastric carcinoma. A -31C to T base transition in the promoter region of this gene is involved in carcinogenic changes within the stomach, especially in Helicobacter pylori infected individuals. We examined association between IL-1 locus polymorphisms and risk of esophageal, gastric and colorectal carcinomas in Japanese patients with H. pylori infection. IL-1B and IL-1RN polymorphisms were analyzed in 136 controls, 75 patients with esophageal carcinoma, 186 patients with gastric carcinoma, 69 patients with colorectal carcinoma, and 18 patients with ulcerative colitis (UC). For IL-1B-511 and -31 polymorphisms were determined by fluorescence-based polymerase chain reaction single-strand conformation polymorphism analysis. For IL-1 receptor antagonist gene (IL-1RN), penta-allelic variable number of tandem repeats (VNTR) was determined by PCR-standard agarose gel electrophoresis. For gastric carcinoma, IL-1B-511 heterozygotes (OR, 0.48; 95% CI, 0.3-0.9; p=0.0115) and T carriers (OR, 0.52; 95% CI, 0.3-1.0; p=0.0185) had a significantly reduced risk of carcinoma. For colorectal carcinoma, IL-1B-511 heterozygotes (OR, 0.34; 95% CI, 0.2-0.7; p=0.0028) and T carriers (OR, 0.43; 95% CI, 0.2-0.9; p=0.0015) had a significantly low risk of carcinoma. No significant difference was observed in the frequencies of IL-1B-31C/T and IL-1RN genotypes between controls and the esophageal carcinoma patients. Our results shows that IL-1B-511C/T and T carrier state may indicate less risk for gastric and colorectal carcinoma in the Japanese population.

Establishment of novel human dedifferentiated chondrosarcoma cell line with osteoblastic differentiation

Dedifferentiated chondrosarcoma is a rare, highly malignant variant of chondrosarcoma in which a high-grade sarcoma coexists with a low-grade chondroid tumor. We herein review a case of dedifferentiated chondrosarcoma with an osteosarcoma omit component that occurred in the distal femur of a 38-year-old man. We established the cell line (NDCS-1) from a pleural effusion of the metastatic lung tumor. The cell line was characterized by a the G-banded karyotype, polymerase chain reaction (PCR) single-strand conformation polymorphism analysis, spectral karyotyping, and reverse transcriptase PCR (RT-PCR). The tumor exhibited complex karyotypes and a high frequency of chromosomal amplication with p53 mutation. This tumor revealed an osteoblastic and chondroblastic character in vitro and in severe combined immunodeficiency mice. The expression and phosphorylation of platelet-derived growth factor receptor-beta, which seemed to play a major role in the malignant phenotype of chondrosarcoma, was confirmed by RT-PCR and Western blotting. To our knowledge, this is the first report of the establishment of a human dedifferentiated chondrosarcoma.

Genetic polymorphism of glutamine synthetase and delta-9 desaturase in families of Pacific oyster Crassostrea gigas and susceptibility to summer mortality

Large-scale mortality events have been observed in Pacific oyster Crassostrea gigas on the west coast of France since the early 1980s, particularly during summer. In order to understand the causes of this mortality, two generations of oysters from single-pair matings were studied in three sites on the French Atlantic coast (Baie-des-Veys, Auray and Ronce-les-Bains). The present paper examines the role of two candidate genes in the susceptibility of oysters to summer mortality, and the selective pressure exerted by such mortality on their polymorphism. Glutamine synthetase (amino-acid metabolism) and delta-9 desaturase (lipid metabolism) genes were studied in the successive generations, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Observed and expected genotypic frequencies were compared. Three different alleles were detected for the glutamine synthetase gene and two for delta-9 desaturase. Allele C of glutamine synthetase seemed to be counter-selected in some second generation families. Allele B of delta-9 desaturase gene was potentially counter-selected at Auray in the families showing higher mortality, and strong selection against BB homozygotes was observed. These observations led us to conclude that any selective effect of summer mortality on allele C of glutamine synthetase gene or allele B of delta-9 desaturase gene could be mediated either directly or via linkage to other loci involved in physiological pathways affecting susceptibility.

INVOLVEMENT OF MICROBIAL POPULATIONS DURING THE COMPOSTING OF OLIVE MILL WASTEWATER SLUDGE

Olive mill waste water sludge obtained by the electro-Fenton oxidation of olive mill waste water was composted in a bench scale reactor. The evolution of microbial species within the composter was investigated using a respirometric test and by means of both cultivation-dependent and independent approaches (Polymerase Chain Reaction-Single Strand Conformation Polymorphism, PCR SSCP). During the period of high respiration rate (7-24 days), cultivation method showed that thermophilic bacteria as well as actinomycetes dominated over eumycetes. During the composting process, the PCR-SSCP method showed a higher diversity of the bacterial community than the eukaryotic one. After 60 days of composting, the compost exhibited a microbial stability and a clear absence of phytotoxicity.

Improvements of Polymerase Chain Reaction and Capillary Electrophoresis Single-Strand Conformation Polymorphism Methods in Microbial Ecology: Toward a High-throughput Method for Microbial Diversity Studies in Soil

The molecular signature of bacteria from soil ecosystems is an important tool for studying microbial ecology and biogeography. However, a high-throughput technology is needed for such studies. In this article, we tested the suitability of available methods ranging from soil DNA extraction to capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) for high-throughput studies. Our results showed that the extraction method does not dramatically influence CE-SSCP profiles, and that DNA extraction of a 0.25 g soil sample is sufficient to observe overall bacterial diversity in soil matrices. The V3 region of the 16S rRNA gene was amplified by PCR, and the extension time was found to be critical. We have also found that proofreading DNA polymerases generate a better signal in CE-SSCP profiles. Experiments performed with different soil matrices revealed the repeatability, efficiency, and consistency of CE-SSCP. Studies on PCR and CE-SSCP using single-species genomic DNA as a matrix showed that several ribotypes may migrate at the same position, and also that single species can produce double peaks. Thus, the extrapolation between number of peaks and number of species remains difficult. Additionally, peak detection is limited by the analysis software. We conclude that the presented method, including CE-SSCP and the analyzing step, is a simple and effective technique to obtain the molecular signature of a given soil sample.

Single Nucleotide Polymorphisms in the Promoter Region of the E-cadherin Gene in Gastric Cancer: Case-Control Study in a Young Mexican Population

Gastric cancer has a tendency to present at early age in the Mexican population, and it is frequently associated with a family history. A polymorphism at position -160 at the CDH1 promoter region has been reported to lead to transcriptional downregulation of the gene in vitro, with possible increase in the risk of gastric cancer. We evaluated the role of the -160A allele in the risk of gastric cancer in a young Mexican population. Peripheral blood sample of Mexican patients younger than 45 years old with diagnosis of diffuse gastric cancer were obtained. We performed DNA extraction and analyzed the frequencies of -160 promoter polymorphism of E-cadherin gene by polymerase chain reaction-single strand conformational polymorphism. These frequencies were compared with those of healthy controls. The chi2 test for association was used to test differences of the genotype frequencies between normal controls and patients with gastric cancer. Findings were considered significant at P < .05. The frequency of the -160 A allele was significantly higher (P = .002) in 39 patients with diffuse gastric cancer compared with 78 matched controls. The odds ratio associated with the A-allele was 1.98 for C/A heterozygotes (95% CI 1.01-3.98) and 6.5 for A/A homozygotes (95% CI 2.1-19.6). We found an increased risk of diffuse gastric cancer according to family history, independent of the expression of the polymorphism. The -160 C/A polymorphism of the E-cadherin has a direct effect on the risk of diffuse gastric cancer at young age in Mexican population.

Epigenetic Inactivation of the hMLH1 Gene in Progression of Gliomas

Gliomas (GLs) are characterized by highly variable biologic behavior. After surgical resection and postoperative therapy, they frequently recur with the same or higher-grade histology. Although a number of genetic aberrations have been described in different histologic types of GLs, the molecular mechanisms of histologic and clinical progression are poorly understood. In this study, we have performed longitudinal mismatch repair gene polymerase chain reaction-single strand conformation polymorphism and methylation analysis in paired samples of primary and recurrent GLs to reveal whether the inactivation of the normal DNA repair mechanism is associated with tumor progression. Polymerase chain reaction-single strand conformation polymorphism analysis of the hMLH1 gene was performed in 24 cases, in each case the samples of the first and second biopsies being evaluated simultaneously, but no alterations in the hMLH1 gene were found. Methylation analysis of the CpG sites in the hMLH1 promoter revealed a total of 4 (16.6%) hypermethylations in recurrent GLs. These results suggest that hMLH1 promoter hypermethylation may occur in low-grade GLs, associated with the development and progression of moderate malignant GL, but not with structural alterations in the hMLH1 gene. It seems that hMLH1 promoter hypermethylation is an early event in the development and progression or the clonal evolution of GLs, this gene inactivation proving stable even on tumor recurrence.

Organ‐dependent susceptibility of p53 knockout mice to 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ)

p53 knockout mice are now being frequently used to identify the carcinogenic potential of chemicals, thus it is important to precisely assess the susceptibility of the animals to various test chemicals. In the present study the susceptibility of p53 nullizygous((-/-)), heterozygous((+/-)), and wild-type((+/+)) mice to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated. Mice of all three genotypes were first fed a diet containing 100 or 300 p.p.m. IQ for 15 weeks in a short-term experiment. p53((+/-)) and ((+/+)) mice were then treated with IQ for 40 weeks and maintained without further treatment for an additional 12 weeks in the long-term experiment. In the forestomach, the incidence of squamous cell hyperplasia was significantly higher in p53((-/-)) than in ((+/-)) and ((+/+)) mice at 15 weeks and higher in ((+/-)) mice than ((+/+)) mice with long-term IQ treatment, indicating an elevated susceptibility of p53 knockout mice. In contrast, in the liver, various hepatocellular lesions developed mainly in female mice with long-term IQ exposure but no significant differences were evident between p53 knockout and wild-type mice, indicating a lack of elevated susceptibility in the knockout animals. Furthermore, polymerase chain reaction-single strand conformation polymorphism and sequencing analysis revealed relatively high (13/30) and low (1/15) incidences of p53 mutations (exons 5-8) in squamous cell hyperplasia and hepatocellular tumors, respectively. These results clearly indicate that the susceptibility of p53 knockout mice is organ-dependent, coinciding to some extent with the likelihood of p53 gene alteration in the induced tumors.

Analysis of p16 Gene Mutation, Deletion and Methylation in Patients with Arseniasis Produced by Indoor Unventilated-Stove Coal Usage in Guizhou, China

The aim of this study was to determine p16 gene mutation, deletion, and promoter 5′ CpG island hypermethylation in peripheral blood mononuclear leukocyte of patients with arseniasis as attributed to exposure to indoor unventilated coal stove. The role of the aberrant change of p16 gene in the induction and development of carcinogenesis in endemic arsenisiasis region in China was also examined. Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP), multiplex PCR (mPCR), methylation-specific PCR (MSP), and sequencing techniques were performed to detect (1) mutation of the p16 gene exon 2, (2) homozygous deletion of the p16 gene exon 1 and exon 2, and (3) hypermethylation of the promoter CpG island in peripheral blood mononuclear leukocyte of patients with arseniasis. Results showed no mutation was found in exon 2 of p16 gene. The homozygous deletion frequency of p16 gene was 5 and 15% in control and arseniasis patients, respectively. The homozygous deletion occurred mainly in exon 2, with significant deletion frequencies of 9, 13, and 20% in mild, intermediate, and severe arseniasis groups. The significant homozygous deletion frequency was 9 and 39% in noncarcinoma and carcinoma individuals. The positive rate of p16 gene promoter CpG island hyermethylation was 42 and 2% in the exposed group and the control group, respectively. The positive rate was 26, 42, and 50% in mild, intermediate, and severe arseniasis. The marked different positive rate was 22 and 56% in noncarcinoma and carcinoma individuals, respectively. In conclusion, homozygous deletion and hypermethylation of p16 gene may play an important role in the initiation and development of manifestations seen in endemic arseniasis including carcinogenesis.

Identification and Functional Characterization of JWA Polymorphisms and their Association with Risk of Gastric Cancer and Esophageal Squamous Cell Carcinoma in a Chinese Population

Recently, a novel single nucleotide polymorphism (SNP) in the promoter of the JWA gene (−76G → C) was identified that may alter the transcription activity and thus play a role in increased risk of bladder cancer. In this study, a screen for more novel variants in the JWA exons was undertaken by using polymerase chain reaction–single-strand conformation polymorphism (PCR-SSCP) followed by a PCR-restriction fragment length polymorphism (PCR-RFLP) method and evaluating the functions of newl identified JWA −76G → C using the reporter gene assay. In addition to the −76G → C polymorphism, another novel SNP (723T → G) in exon 3 of JWA was identified. In a case-control study of these two SNPs in 413 gastric cancer and 250 esophageal squamous-cell carcinoma (ESCC) patients and 814 cancer-free controls in a Chinese population, data showed that both SNPs were associated with enhanced risk of these cancers. The reporter gene assay showed that the −76C variant allele lost its response to benzo[a]pyrene (BaP) exposure, compared to the −76G allele. In addition, the JWA −76C allele was found to be associated with increased gastric and esophageal cancer risks in this study population. Further studies are needed to substantiate the biological significance and related mechanisms underlying the associations.

Functional Polymorphisms of JWA Gene are Associated with Risk of Bladder Cancer

The JWA gene is a novel cell differentiation-related gene thought to be a responsive gene in response to DNA damage and repair induced by environmental stressors. Recently, a novel single nucleotide polymorphism (SNP) was identified in the promoter of the JWA gene (−76G→C) that may alter the transcription activity and thus play a role in increased risk of bladder cancer. Further, studies were conducted to screen for more novel variants in the JWA exons by using PCR-SSCP (polymerase chain reaction–single-strand conformation polymorphism) followed by PCR-RFLP (PCR restriction fragment length polymorphism) methods. Finally, the functional relevance of the newly identified genetic variants in a hospital-based case-control study of 215 bladder cancer patients and 250 cancer-free controls was evaluated. In addition to the −76G→C polymorphism, another novel SNP (454C→A in exon2 and 723T→G in exon 3) of JWA was identified. The −76G→C allele and genotype frequencies were found to vary in different ethnic groups. The −76C allele and 454A allele were both associated with significantly increased risk of bladder cancer. In contrast, the 723GG genotype was associated with a decreased risk of bladder cancer. Furthermore, −76C and 454A together increased the risk of bladder caner using haplotype and stratification analysis. In conclusion, the three novel functional genetic polymorphisms of JWA gene, −76G→C, 454C→A, and 723T→G, appear to contribute to the etiology of bladder cancer.