Reaction-single Strand Conformation Polymorphism Analysis Research Articles

Genetic analysis of microsomal epoxide hydrolase gene and its association with lung cancer risk

The human microsomal epoxide hydrolase (EH) gene contains polymorphic alleles, which may be linked to increased risk for tobacco-related lung cancer. The purpose of this study is to screen new polymorphisms and determine whether these polymorphisms can be used to predict individual susceptibility to lung cancer. The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis was used to screen for polymorphisms in the coding region of the EH gene. Eleven polymorphisms, including previously reported polymorphisms, were identified and the prevalence of these variants was assessed in at least 50 healthy Caucasians and African-Americans. Among the 11 polymorphisms, the prevalence of the amino acid-changing EH polymorphisms in codons 43, 113 and 139 was examined in 182 Caucasian incident cases with primary lung cancer, as well as in 365 frequency-matched controls to examine the role of EH polymorphisms in lung cancer risk. A significant increase in lung cancer risk was observed for predicted high EH activity genotypes (odds ratio (OR) 2.3, 95% confidence interval (CI) 1.2-4.3) as compared with low EH activity genotypes. This association was more pronounced among patients with lung adenocarcinoma (OR 4.7, 95% CI 1.7-13.1). These results suggest that the EH polymorphism plays an important role in lung cancer risk and is linked to tobacco smoke exposure.

Somatic VHL alteration and its impact on prognosis in patients with clear cell renal cell carcinoma

4602 Backgrounds: Somatic inactivation of the von Hippel-Lindau (VHL) gene is the most frequent genetic event observed in clear cell renal cell carcinomas (CC-RCCs). However, the prognostic relevance of somatic VHL alteration and its target, hypoxia inducible factor (HIF)-1α, has not been defined. We investigated the genetic changes in the VHL gene and HIF-1α and studied their clinical implications in patients with sporadic CC-RCC. Methods: Patients who underwent nephrectomy were eligible if they had pathologically confirmed CC-RCC that was not associated with VHL disease or familial RCC. Tumor tissues were selected from paraffin blocks on the basis of hematoxylin and eosin (H&E)-stained sections. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis was performed to detect VHL mutations and genetic changes in HIF-1α, which were followed by automated direct sequencing. VHL hypermethylation was examined by methylation-specific PCR. Results: A total of 56 patients were enrolled and somatic VHL alterations were detected in 16 (29%); intragenic mutation in eight, hypermethylation in five, and both alteration in three. The mutation types were missense in five patients, silent in three, nonsense in two, and frameshift in one. Somatic VHL alterations were not significantly associated with progression-free survival (PFS) or overall survival (OS). However, patients with a ‘loss-of-function’ VHL mutation showed significantly decreased PFS (P=0.016) and OS (P=0.046). Although the association between VHL alteration and response to immunotherapy was not significant (P=0.486), The CC-RCCs with missense mutation seem to have had better responses to immunotherapy. The Pro582Ser change in HIF-1α was detected in six patients (11%) and was positively correlated with the development of metastases (P=0.023). Concluions: This study did not show an association between somatic VHL alteration and prognosis in patients with sporadic CC-RCC. However, it suggests that the therapeutic and prognostic implication of somatic VHL alteration may be different according to the mutational subtype and that the Pro582Ser change in HIF-1α may contribute to the development of metastases. No significant financial relationships to disclose.

Somatic VHL alteration and its impact on prognosis in patients with clear cell renal cell carcinoma

Somatic inactivation of the von Hippel-Lindau (VHL) gene is the most frequent genetic event observed in clear cell renal cell carcinoma (CC-RCC). However, the prognostic relevance of somatic VHL alteration and its target, hypoxia inducible factor (HIF)-1alpha has not been defined. We investigated the genetic changes in the VHL gene and HIF-1alpha and studied their clinical implications in patients with sporadic CC-RCC. Patients who underwent nephrectomy were eligible if they had pathologically confirmed CC-RCC not associated with VHL disease or familial RCC. Tumor tissues were selected from paraffin blocks on the basis of hematoxylin and eosin (H&E)-stained sections. Polymerase chain reaction-single strand conformation polymorphism analysis was performed to detect VHL mutations and genetic changes in HIF-1alpha, which were followed by automated direct sequencing. VHL hypermethylation was examined by methylation-specific PCR. A total of 56 patients were enrolled and somatic VHL alterations were detected in 16 patients (29%); intragenic mutation in eight, hypermethylation in five, both alterations in three. The mutation types were missense in five patients, silent in three, nonsense in two, and frameshift in one. Somatic VHL alterations were not significantly associated with progression-free survival (PFS) or overall survival (OS). However, patients with 'loss-of-function' VHL mutation showed significantly decreased PFS (P=0.016) and OS (P=0.046). Although the association between VHL alteration and response to immunotherapy was not significant (P=0.486), patients with missense mutation seem to have better response to immunotherapy. The Pro582Ser change in HIF-1alpha was detected in six patients (11%) and was positively correlated with the development of metastases (P=0.023). This study did not show an association between somatic VHL alteration and prognosis in patients with sporadic CC-RCC. However, it suggests that the therapeutic and prognostic implication of somatic VHL alteration may be different according to the mutational subtype and that the Pro582Ser change in HIF-1alpha may contribute to the development of metastases.

TP53 Mutations and Outcome in Osteosarcoma: A Prospective, Multicenter Study

Mutations of the TP53 gene have been associated with resistance to chemotherapy as well as poor prognosis in many different malignancies. This is the first prospective study of the prognostic value of somatic TP53 mutations in patients with newly diagnosed extremity osteosarcoma. One hundred ninety-six patients with high-grade, nonmetastatic osteosarcoma of the extremities were enrolled from seven tertiary care institutions and observed prospectively for tumor recurrence (median follow-up duration, 44 months). All patients received neoadjuvant or adjuvant chemotherapy and surgery. Tumors were analyzed for the presence of TP53 mutations by polymerase chain reaction single-strand conformation polymorphism analysis and direct DNA sequencing. The association of the status of the TP53 gene with the risk of systemic recurrence was examined using survival analyses with traditional and histologic markers as prognostic factors. Patient age was the only factor that varied with TP53 gene status (P = .05). No relationship was identified between TP53 status and systemic relapse (relative risk, 1.24; P = .41). Analyses based on missense or nonsense mutations gave similar results (P > .10). In multivariate analysis, large (> 9 cm) tumor size (relative risk, 1.9; P = .006) and poor histologic response (< or = 90% necrosis) to chemotherapy (relative risk, 2.14; P = .02) were the only significant independent predictors of systemic outcome. We found no evidence that TP53 mutations predict for development of metastases in patients with high-grade osteosarcoma. Identification of other genes that influence chemotherapy response and clinical outcome in osteosarcoma is needed to facilitate further improvements in patient outcomes.

BRAF mutation associated with dysregulation of apoptosis in human colorectal neoplasms

To understand the role of BRAF dysfunction in the carcinogenesis and progression/development of colorectal tumors, the authors investigated genetic alterations in the BRAF gene in human colorectal neoplasms as well as the effects of an RAS inhibitor in BRAF-mutant cells. Seven colon cancer cell lines and 116 colorectal tumors (34 adenomas and 82 adenocarcinomas) were analyzed. Genetic alterations in the BRAF and K-ras genes were examined using polymerase chain reaction-single strand conformation polymorphism and direct sequencing analyses. The growth-inhibitory and apoptosis-inducing effects of the FTI-277 RAS inhibitor in colon cancer cell lines were analyzed as well. An immunohistochemical study was also performed to investigate the correlations between the clinicopathologic parameters involved in the Ki-67 labeling index and the number of apoptotic bodies in tumor cells. FTI-277 did not suppress the proliferation of BRAF-mutant cells (WiDr and TCO), but remarkably inhibited the growth of K-ras mutant cells (LoVo). Interestingly, LoVo cells underwent apoptosis by FTI-277 in a dose-dependent manner, whereas WiDr cells were resistant to this agent. In tumor samples, BRAF mutations were found in 1 (3.0%) of 33 adenomas and 6 (7.2%) of 83 adenocarcinomas. No tumor exhibited mutations in both the BRAF and K-ras genes. Neither BRAF nor K-ras mutations correlated with the Ki-67 labeling index immunohistochemically. However, the number of apoptotic bodies was significantly decreased in the BRAF-mutant tumors. Mutation in the BRAF gene may contribute to colorectal carcinogenesis by upregulating the antiapoptotic role of the RAS/RAF/MEK/ERK pathway.

Polymerase chain reaction-single strand conformational polymorphism analysis of rearranged during transfection proto-oncogene in Chinese familial hirschsprung’s disease

To investigate the relationship between mutations of rearranged during transfection (RET) proto-oncogene and Chinese patients with Hirschsprung's disease (HD), and to elucidate the genetic mechanism of familial HD patient at the molecular level. Genomic DNA was extracted from venous blood of probands and their relatives in two genealogies. Polymerase chain reaction (PCR) products, which were amplified using specific primers (RET, exons 11, 13, 15 and 17), were electrophoresed to analyze the single-strand conformational polymorphism (SSCP) patterns. The positive amplified products were sequenced. Forty-eight sporadic HD patients and 30 normal children were screened for mutations of RET proto-oncogene simultaneously. Three cases with HD in one family were found to have a G heterozygous insertion at nucleotide 18,974 in exon 13 of RET cDNA (18,974insG), which resulted in a frameshift mutation. In another family, a heterozygosity for T to G transition at nucleotide 18,888 in the same exon which resulted in a synonymous mutation of Leu at codon 745 was detected in the proband and his father. Eight RET mutations were confirmed in 48 sporadic HD patients. Mutations of RET proto-oncogene may play an important role in the pathogenesis of Chinese patients with HD. Detection of mutated RET proto-oncogene carriers may be used for genetic counseling of potential risk for HD in the affected families.

Common polymorphisms of the PPAR-?2 (Pro12Ala) and PGC-1? (Gly482Ser) genes are associated with the conversion from impaired glucose tolerance to type 2 diabetes in the STOP-NIDDM trial

We investigated the effects of the common polymorphisms in the peroxisome proliferator-activated receptor gamma2 (PPAR-gamma2; Pro12Ala) and in PPAR-gamma coactivator 1alpha (PGC-1alpha; Gly482Ser) genes on the conversion from impaired glucose tolerance to type 2 diabetes in participants in the STOP-NIDDM trial. This trial aimed to study the effect of acarbose in the prevention of type 2 diabetes. Genotyping was performed in 770 study subjects whose DNA was available. The Gly482Ser variant in the PGC-1alpha gene was determined with the polymerase chain reaction amplification, Hpa II enzyme digestion, and gel electrophoresis. The Pro12Ala polymorphism of the PPAR-gamma2 gene was determined by the polymerase chain reaction-single-strand conformation polymorphism analysis. The Pro12Pro genotype of the PPAR-gamma2 gene predicted the conversion to diabetes in women in the acarbose group (odds ratio 2.89, 95% CI 1.20 to 6.96; p=0.018). The 482Ser allele of the PGC-1alpha gene had a significant interaction with the mode of treatment (p=0.012), and in the placebo group the 482Ser allele was associated with a 1.6-fold higher risk for type 2 diabetes compared to the Gly482Gly genotype (95% CI 1.06 to 2.33; p=0.023). Acarbose prevented the development of diabetes independently of the genotype of the PPAR-gamma2 gene, but only the carriers of the 482Ser allele of the PGC-1alpha gene were responsive to acarbose treatment. We conclude that the Pro12Pro genotype of the PPAR-gamma2 gene and the 482Ser allele of the PGC-1alpha gene are associated with the conversion from impaired glucose tolerance to type 2 diabetes in the STOP-NIDDM trial.

Mutations of the PTPN11 Gene in Childhood Hematological Malignancies.

The PTPN11 gene encodes SHP2, which is a protein tyrosine phosphatase functioning as signal transducer downstream to growth factors and cytokine receptors and its function is mediated, at least in part, through the Ras/Raf/ERK cascade in hematopoietic and non-hematopoietic cells. Recently, it has been reported that germline mutations in PTPN11 cause Noonan syndrome and a gain-of-function somatic mutations in PTPN11 has also been reported in juvenile myelomonocytic leukemia (JMML). To investigate the prevalence of mutations in PTPN11 in childhood acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), we screened the hot spot of PTPN11, exon 3, in 80 cell lines, including 14 AML, 5 chronic myeloid leukemia, 14 T-cell ALL and 47 B-precursor ALL cell lines, and 207 fresh samples, including 20 myelodysplastic syndrome (MDS), 3 JMML, 85 AML, 57 B-precursor ALL patients as well as 42 infant leukemia patients (6 AMLs, 35 ALLs, 1 mixed lineage leukemia), using polymerase chain reaction-single strand conformation polymorphism and direct sequencing analyses. In exon 3, mutations were observed in 2 of 14 (14.3%) AML cell lines (Asp61Val and Glu76Gly). A missense mutation (Ala72Val) was detected in 1 of 85 (1.2%) AML patients (a 9-year-old girl with FAB-M0). In 20 MDSs, a missense mutation (Asp61Val) (5%) was identified in a RAEB-T patient. In 3 JMMLs, 2 missense mutations (66.7%) were detected; one was Gly60Ala, and the other was Glu76Val. In 42 infant leukemia patients, we detected only one missense mutation (Ala72Asp) (2.4%) in an AML-M5b patient. However, we did not detect any mutations of exon 3 in 61 ALL cell lines and 92 ALL patients including 35 infant ALL patients. The screening for other coding regions is still undergoing although more than 90% of the reported mutations were clustered in exon 3. In conclusion, our data suggest that the PTPN11 mutations are involved in myeloid hematological malignancies rather than ALL.

Alterations of APC, c- met, and p53 genes in tumor tissue and serum of patients with gastric cancers

Background Gastric cancer is one of the most significant causes of cancer-related death worldwide. A genetic model consisting of sequential accumulations of alterations in specific genes for gastric cancer has been proposed. Materials and methods The significance of adenomatous polyposis coli (APC) and p53 gene mutations in cancer tissues and their paired serum of 34 gastric cancer patients was investigated using polymerase chain reaction single-strand conformation polymorphism analysis (PCR-SSCP), followed by direct sequencing. c- met mRNA expression was evaluated by reverse-transcription PCR (RT-PCR). Additionally, analyses were carried out to detect the serum carcinoembryonic antigen (CEA) levels, and their correlation to these three molecular markers. Finally, serum molecular markers and their correlation to the presence of postoperative recurrence/metastasis were analyzed. Results Of all, 32.4% of patients presented mutations in APC and p53, respectively, and 58.8% presented the overexpression in c- met, overall, at least one of these genetic alterations in 79.4% of tumor tissues. Comparison of three molecular markers showed that the individual detection rate in the serum of patients with tumors harboring the same abnormalities was 18.2, 70.0, and 36.4% for APC, c- met, and p53 genes, respectively. In general, 59.3% of serum from cancerous tissues with gene alterations was demonstrated as positive, whereas all healthy volunteers’ sera remained negative. Regarding gene alterations in tumor tissues, c- met overexpression was significantly related to the tumor size ( P = 0.017), depth of tumor invasion ( P = 0.007), lymph-node metastasis ( P < 0.001), and TNM stage ( P = 0.001). In the serum, c- met overexpression was closely associated with lymph-node metastasis ( P = 0.008) and TNM stage ( P = 0.016). The overall positive tumor gene detection rate in the serum was prominently correlated to the serum CEA levels ( P = 0.038). In addition, a significantly higher postoperative metastasis/recurrence rate in patients harboring gene mutations with serum molecular markers than those without serum molecular markers was also demonstrated ( P = 0.014). Conclusions Our findings suggest that serum molecular markers can be detected in a substantial proportion of gastric cancer patients, and these may offer an auxiliary approach in the noninvasive detection and prognosis of gastric cancer.

Molecular detection of APC, K- ras, and p53 mutations in the serum of colorectal cancer patients as circulating biomarkers.

Early detection of tumor DNA in serum/plasma prior to the development of recurrence or metastases could help improve the outcome of patients with colorectal cancer (CRC) after tumor resection. Recent advances in the detection of tumor DNA in the serum/plasma has opened up numerous new areas for investigation and new possibilities for molecular diagnosis. APC and K- ras mutations are considered to be early-stage developments of CRCs, whereas p53 mutations are thought to be relatively late events in the tumorigenesis of CRCs. The aim of this study was to search for the presence of genetic mutations in the DNA extracted from the serum of CRC patients and healthy subjects. We simultaneously evaluate the significance of APC, K- ras, and p53 gene mutations in cancer tissues and their paired serum samples of 104 CRC patients by polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP) followed by direct sequencing. Additionally, analysis was carried out to detect the serum carcinoembryonic antigen (CEA) levels in CRC patients. Overall, we found at least one of the gene mutations in tumor tissues from 75% (78/104) of the CRC patients. Comparison of the three molecular markers showed that the detection rates in the serum were 30.4%, 34.0%, and 34.2% for APC, K- ras, and p53 genes, respectively. Of these patients, 46.2% (36/78) were identified as having positive serum results, whereas all healthy controls remained negative. The overall positive tumor DNA detection rates in the serum were 0% (0/7) for Dukes' A classification, 22.4% (11/49) for Dukes' B, 48.7% (19/39) for Dukes' C, and 66.7% (6/9) for Dukes' D. The detection rate increased as the tumor stage progressed ( p = 0.012). Concurrently, a significant difference was observed between lymph node metastases and positive serum tumor DNA detection ( p < 0.001). A significantly higher postoperative metastasis/recurrence rate in patients harboring gene mutations with serum tumor DNA than those without serum tumor DNA was also demonstrated ( p < 0.001). However, no significant correlation between the postoperative metastasis/recurrence and serum CEA levels was observed ( p = 0.247). These data suggest that the identification of circulating tumor DNA using the molecular detection of APC, K- ras, and p53 gene mutations is a potential tool for early detection of postoperative recurrence/metastases. Moreover, these genes may be potential molecular markers of poor clinical outcome in CRC patients.

Simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by polymerase chain reaction-single strand conformation polymorphism and sequence analysis of the RNA polymerase gene ( rpoB)

Interspecies variations and mutations associated with rifampin resistance in rpoB of Mycobacterium allow for the simultaneous identification of rifampin-resistant Mycobacterium tuberculosis and nontuberculous mycobacteria by PCR-SSCP analysis and PCR- sequencing. One hundred and ten strains of rifampin-susceptible M. tuberculosis, 14 strains of rifampin-resistant M. tuberculosis, and four strains of the M. avium complex were easily identified by PCR-SSCP. Of another seven strains, which showed unique SSCP patterns, three were identified as rifampin-resistant M. tuberculosis and four as M. terrae complex by subsequent sequence analysis of their rpoB DNAs (306 bp). These results were concordant with those obtained by susceptibility testing, biochemical identification, and 16S rDNA sequencing.

High frequency of G/C transversion on p53 gene alterations in breast cancers from Taiwan

p53 gene mutation is a very frequent event in many human cancers and is associated with a poor clinical outcome in breast cancer patients. Analysis of p53 gene mutations can also provide clues to the etiology of tumor formation. The present study was conducted to investigate the p53 mutations in patients with breast cancer from Taiwan. Tumor samples from 119 patients undergoing mastectomy for breast cancer were evaluated. The mutational status of the p53 gene (exons 5–8) was screened by polymerase chain reaction-single strand conformation polymorphism analysis followed by direct sequencing. Of all 119 cases of breast carcinoma, 26 mutations of the p53 gene were found in 22 cases (18.5%). Among these mutations, 78% (20/26) were point mutations with the majority of those being missense mutations (75%, 15 of 20 mutations) and the other 22% (6/26) were frameshift mutations. No significant correlation between p53 mutations and clinicopathological features was found, including HER2 status. Moreover, our results disclosed distinct mutation spectra in excess transversions to transitions (15/21, 71.4% vs. 6/21, 28.6%) with GC to CG dominant (6/15, 40%). Mutation hot spots we identified at codons 167, 185, 186, 210, 265 and 295 have rarely been documented in the literature. These findings showed that p53 gene mutation might contribute to the pathogenesis of breast carcinoma. Furthermore, the different mutation spectrum with high transversions in G:C to C:G may imply that the exogenous mutagens outweigh the endogenous processes in breast cancer in patients in Taiwan.

Delineation of Clavispora lusitaniae clinical isolates by polymerase chain reaction-single strand conformation polymorphism analysis of the ITS1 region: a retrospective study comparing five typing methods

Strain delineation of the emerging opportunistic pathogen Clavispora lusitaniae was studied using 12 strains, including two strains of known opposite mating type, CBS 6936 (h+) and CBS 5094 (h-), and 10 strains isolated between 1998 and 2001 from immunocompromized patients. This retrospective study assessed the occurrence of C. lusitaniae subtypes within and among hospitals, and in outpatients who were regularly screened for fungal infections in the course of radio-chemotherapy. Strain typing was accomplished for the first time using single strand conformation polymorphism (SSCP) analysis of amplicons of the ribosomal DNA internal transcribed spacer (ITS) 1 and 2 regions. The results were compared with those produced by three pulsed-field gel electrophoresis (PFGE) methods and PCR fingerprinting with the minisatellite-specific primer M13. Karyotyping separated 7-9 chromosomes, not 6-8 as previously reported. Pulsotyping of SfiI and NotI digested chromosomes grouped isolates in five and four distinct clusters, respectively. All methods revealed strain heterogeneity, though not as extensive as previously recorded. SSCP analysis of the ITS1 region generated five subtypes, based on a sequencing-confirmed nucleotide polymorphism. The discriminatory power of this method was high. All strains displayed a homogeneous SSCP pattern for the ITS2 region. ITS1 PCR-SSCP appears to allow rapid and reliable delineation of C. lusitaniae strains. Pending examination of a larger sample size and interlaboratory study, this protocol can be recommended for rapid prospective identification of hospital outbreaks.

Mutation of the von Hippel-Lindau (VHL) gene in human colorectal carcinoma: association with cytoplasmic accumulation of hypoxia-inducible factor (HIF)-1alpha.

We screened for mutations in the von Hippel-Lindau (VHL) tumor suppressor gene and examined the relationship of these mutations with expression of hypoxia-inducible factor (HIF)-1alpha protein in human colorectal carcinomas. DNAs were extracted from 130 paraffin-embedded tumor specimens and subjected to polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis followed by direct sequencing. We identified 13 mutations in the coding sequence of VHL, 12 of which were unique events. Three mutations were located in exon 2 and the others in exon 3. These mutations were detected in 10 of 88 (11.4%) tumors tested. Furthermore, seven of the 13 (53.8%) VHL mutants immunohistochemically showed high HIF-1alpha expression. The mean percentage of cells with strong cytoplasmic HIF-1alpha expression was 67.5% in tumors with VHL mutations, and this level was significantly higher than that in tumors without mutations (50.8%, P < 0.05). These findings suggest that mutations in VHL may play a role in colorectal carcinoma via activation of the HIF-related transcriptional cascade.

Loss of heterozygosity at chromosome 9p21 is a frequent finding in enteropathy-type T-cell lymphoma.

Enteropathy-type T-cell lymphoma (ETL) and ulcerative jejunitis (UJ) are rare disorders often occurring in patients with coeliac disease. The genetic events associated with the accumulation of intraepithelial lymphocytes in coeliac disease and tumour development are largely unknown. Deletions at chromosome 9p21, which harbours the tumour suppressor genes p14/ARF, p15/INK4b, and p16/INK4a, and 17p13, where p53 is located, are associated with the development and progression of lymphomas. To examine whether deletions at 9p21 and 17p13 play a role in ETL, 22 cases of ETL and seven cases of UJ were screened for loss of heterozygosity (LOH) by tissue microdissection and polymerase chain reaction (PCR) analysis for microsatellite markers. Furthermore, p53 and p16 protein expression was examined by immunohistochemistry. In addition, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis for detection of mutations in exons 5-8 of the p53 gene was performed in five cases of ETL and three cases of UJ. LOH was found in at least one microsatellite marker at the 9p21 locus in 8 of 22 (36%) ETLs, but not in UJ. Five of nine (56%) tumours composed of large cells showed LOH at 9p21, as opposed to two of eight (25%) tumours with small- or medium-sized cell morphology. The region spanning the p14/p15/p16 gene locus was most frequently affected (five cases); LOH at these markers coincided with loss of p16 protein expression in all of these cases. p53 overexpression was demonstrated in all ETLs examined and in four of seven cases of UJ. However, no alterations of the p53 gene were detected by LOH or PCR-SSCP analysis. The results of this study show that LOH at chromosome 9p21 is frequent in ETL, especially in tumours with large cell morphology; this finding suggests that gene loss at this locus may play a role in the development of ETL.

Variable Number of Tandem Repeat of the 5'-Flanking Region of Type-C Human Natriuretic Peptide Receptor Gene Influences Blood Pressure Levels in Obesity-Associated Hypertension

The gene for natriuretic peptides receptor C (NPRC), which is a candidate susceptibility gene for essential hypertension (EH) or obese hypertension, plays a key role in the regulation of plasma levels and biological effects of natriuretic peptides. The aims of the present study were to find new genetic markers in the 5'-flanking region of the NPRC gene and to assess relationships between variants and phenotypes of EH, including EH in obese patients. Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, we discovered a novel six-nucleotide repeat polymorphism located 4 base pairs (bp) upstream of the major transcriptional initiation site. We performed association analysis of this variable number of tandem repeat (VNTR) in 242 EH patients and 212 normotensive controls (NT). Although no significant difference in overall frequency of VNTR was found between NT and EH groups, the blood pressure level of EH patients with the 5/6 genotype was significantly higher in obese subjects. This suggests that the VNTR of the 5'-flanking region of the NPRC gene influences blood pressure levels in obesity-associated hypertension.

Absence of &amp;beta;&lt;i&gt;-catenin&lt;/i&gt; Mutations in Mouse Hepatoblastomas Induced by &lt;i&gt;N&lt;/i&gt;, &lt;i&gt;N&lt;/i&gt;-dimethylformamide

Abnormal β-catenin protein accumulation in cytoplasm or nucleus with somatic mutation of β-catenin has been reported to be very frequent in hepatoblastomas (HBs) both in humans and in experimental animals. Recently we noted HBs developing in an evaluation of the carcinogenicity of N, N-dimethylformamide (DMF) in a 104 week mouse inhalation study1 performed by the Japan Bioassay Research Center. In the present study we evaluated β-catenin mutations in mouse HBs induced by DMF, which is considered to be a non-genotoxic carcinogen. Immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analyses were performed using formalin-fixed and paraffin-embedded sections. No abnormal β-catenin accumulation or no apparent mutation was detected, and these findings point to functional normality of the Wnt signal pathway in these HBs induced by DMF. Our results raise questions as to the general nature of any relationship between HB and abnormal β-catenin accumulation.

Mutational analysis of the macrophage scavenger receptor 1 (MSR1) gene in primary lung cancer.

Allelic deletion at chromosome 8p21-25 is an early and frequent event in the carcinogenesis and development of various cancers. To facilitate investigation of alterations of the macrophage scavenger receptor 1 (MSR1), which is located on 8p22, and to determine the role of this gene in human carcinogenesis and tumor progression, we determined intronic primers designed to amplify the coding region. Since frequent deletion of 8p21-23 has been previously reported in lung cancer, we searched for mutations throughout the coding sequence of the MSR1 gene within a panel of genomic DNA samples obtained from 30 primary lung cancers. Our approach, which involved polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct DNA sequencing, revealed nucleotide variants of the MSR1 gene in only one of the 30 cases examined, with this sample displaying both a 6 bp deletion and a thymine-to-cytosine substitution, the latter occurring within intron 7. The 6 bp deletion was located at a DNA microsatellite region and the thymine-to-cytosine substitution seemed to be a polymorphism. These results suggest that the MSR1 gene is not commonly mutated in lung cancer and not important in susceptibility to lung cancer. Further studies may focus on alternative mechanisms through which the MSR1 gene might be inactivated, such as aberrant DNA methylation, and/or pursue analyses of other genes on 8p21-23 for mutational events. Nevertheless, the panel of intronic PCR primer pair sequences presented here will facilitate future studies to determine the full spectrum and frequency of genetic events that may affect expression/activity of the MSR1 gene in human tumors.

Somatic mutation of the Caspase-5 gene in human lung cancer.

Using cDNA array-based gene expression profiling, we previously found reduced expression of the Caspase-5 gene in highly metastatic subpopulations of a lung cancer cell line. The Caspase-5 gene contained poly(A) repeats in its coding region, an area that has been reported to be mutated in both endometrial and gastrointestinal tumors displaying evidence of microsatellite instability. In order to determine the contribution of Caspase-5 gene inactivation to lung cancer development and progression, the mutational status of the Caspase-5 poly(A) tract in 30 primary lung cancers with distant metastasis and 30 lung cancer cell lines was determined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Three somatic mutations of the Caspase-5 gene were found in two out of 30 lung cancer tissues, although no mutations were found in other genes that also contain small nucleotide repeats, such as hMSH3, hMSH6 and BAX. The results of the present study, combined with our prior cDNA array-based gene expression profiling data, suggest that Caspase-5 might be a suppressor gene of highly metastatic potential in lung cancer.

High frequency of activated K-ras codon 15 mutant in colorectal carcinomas from Taiwanese patients.

Colorectal carcinogenesis is regarded as a multistep process resulting from accumulation of genetic alterations, including activation of protooncogenes and inactivation of tumor suppressor genes via signal transduction trigger the stage-wise progression to malignancy. The reported incidence of K-ras mutation detected in general tissue samples ranges from 21-60% in primary colorectal cancers (CRC). To assess the prevalence and spectrum of K-ras mutations in Taiwanese patients with CRC, we analyzed 65 CRC patients by polymerase chain reaction-single strand conformation polymorphism analysis, followed by direct sequencing. K-ras mutations were detected in 43.1% (28 of 65) of the tumors. The mutational hot spots were located at codons 12, 13, 15 and 20, especially with the highest frequency at codon 15. To understand whether the codon 15 mutations in CRC were associated with activation of K-ras oncogene and the alterations of its biocharacteristics, mutant K-ras genes were cloned from tumor tissues and then inserted into expression vector pBKCMV to construct the prokaryotic expression plasmid pK15MCMV. Mutant K-ras genes were expressed at high levels in E. coli and the mutant K-ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. The purified K-ras protein from E. coli was then measured for its intrinsic GTPase activity and the extrinsic GTPase activity in the presence of GTPase-activating protein for ras. We found that the extrinsic GTPase activity of the codon 15 mutant K-ras proteins (p21(K-ras15M)) in the presence of GAP is much lower than that of the wild-type K-ras protein (p21 BN), whereas the intrinsic GTPase activity is nearly the same as that of the wild-type K-ras protein. The results indicated that mutation at the codon 15 of K-ras gene indeed decreased GTPase activity in CRC, however, its association with tumorigenesis of CRC needs be clarified by further studies.