Single-strand Conformation Polymorphism Assay Research Articles

Mutational analysis of tumour suppressor gene NF2 in common solid cancers and acute leukaemias

Germline mutation of NF2 gene is a feature of neurofibromatosis type 2 familial cancer syndrome. Also, somatic point mutations of NF2 mutation have been reported in tumours originated from nerve structures. A recent study revealed that NF2 gene was mutated in renal cell carcinoma (RCC) as well, suggesting a possibility that NF2 gene might be somatically mutated in other human cancers. The aim of this study was to explore whether NF2 genes are somatically mutated, and contribute to tumorigenesis in common human cancers. For this, we analysed the entire coding region of NF2 gene in 45 colorectal carcinomas, 45 gastric, 45 breast, 45 lung, 45 hepatocellular (HCC), 45 prostate carcinomas, and 45 acute leukaemias by a single-strand conformation polymorphism assay. Overall, we found NF2 mutations in one HCC (1/45; 2.2%) (hepatitis B virus-related HCC), one lung carcinoma (1/45; 2.2%) (squamous cell carcinoma), and one acute leukaemia (1/45; 2.2%) (acute myelogenous leukaemia minimally differentiated). All of the mutations were missense mutations that would substitute amino acids in the NF2 protein (p.A238 V, p.A451T and p.R467K). Our data indicate that somatic mutation of NF2 gene is not prevalent in common human cancers, and its mutation somatically occurs in a minor fraction of HCC, lung cancer and acute leukaemia. These data suggest that somatic mutation of NF2 tumour suppressor gene may not play a central role in development of common cancers.

Somatic mutation of PHF6 gene in T-cell acute lymphoblatic leukemia, acute myelogenous leukemia and hepatocellular carcinoma

Introduction. Somatic mutations in plant homeodomain finger protein 6 (PHF6) gene have recently been reported in T-cell acute lymphoblastic leukemia (T-ALL), strongly suggesting its role in the pathogenesis of human cancers. Materials and methods. To see whether the PHF6 mutation occurs in other malignancies, we analyzed entire coding sequences of PHF6 in 231 hematologic malignancies [105 acute myelogenous leukemias (AML), 66 pre-B-ALL, 23 T-ALL, one undifferentiated acute leukemia and 36 multiple myelomas] by single-strand conformation polymorphism assay. Also, we analyzed the mutation in 236 solid cancers, including 41 lung, 39 hepatocellular (HCC), 36 breast, 40 colorectal, 40 gastric and 40 prostate carcinomas. Results. In the hematologic malignancies, there were 11 PHF6 mutations that were detected not only in T-ALL (34.7%) (five adult and three childhood T-ALL), but also in two AML (1.9%) (one acute monocytic leukemia and one AML minimally differentiated). In addition, there was a PHF6 mutation in the HCC (2.6%). The PHF6 mutations were detected in both male and female patients, and consisted of six frameshift, three nonsense and two intron mutations. Conclusion. Our data suggest that PHF6 mutation might play a role in tumorigenesis not only of T-ALL, but also of AML and HCC.

Absence of Paxillin Gene Mutation in Lung Cancer and other Common Solid Cancers

Mounting evidence indicates that deregulated cell adhesion is involved in the mechanisms of cancer pathogenesis. A recent study showed that the paxillin gene (PXN) encoding a focal adhesion protein was somatically mutated in lung cancers. The aim of this study was to confirm the presence of PXN mutations in lung cancers as well as in other common solid cancers. We analyzed somatic PXN mutations in 45 lung, 45 gastric, 45 colorectal, 45 breast, 45 liver and 45 prostate cancers by polymerase chain reaction and single-strand conformation polymorphism assay. Neither lung nor other cancers were found to be associated with somatic mutations of PXN. In contrast to the previous report, our study revealed that PXN mutation was absent in lung cancers and other common solid cancers, suggesting that PXN mutation may not play a principal role in solid cancer development.

Differential contributions to the transcriptome of duplicated genes in response to abiotic stresses in natural and synthetic polyploids

Polyploidy has occurred throughout plant evolution and can result in considerable changes to gene expression when it takes place and over evolutionary time. Little is known about the effects of abiotic stress conditions on duplicate gene expression patterns in polyploid plants. We examined the expression patterns of 60 duplicated genes in leaves, roots and cotyledons of allotetraploid Gossypium hirsutum in response to five abiotic stress treatments (heat, cold, drought, high salt and water submersion) using single-strand conformation polymorphism assays, and 20 genes in a synthetic allotetraploid. Over 70% of the genes showed stress-induced changes in the relative expression levels of the duplicates under one or more stress treatments with frequent variability among treatments. Twelve pairs showed opposite changes in expression levels in response to different abiotic stress treatments. Stress-induced expression changes occurred in the synthetic allopolyploid, but there was little correspondence in patterns between the natural and synthetic polyploids. Our results indicate that abiotic stress conditions can have considerable effects on duplicate gene expression in a polyploid, with the effects varying by gene, stress and organ type. Differential expression in response to environmental stresses may be a factor in the preservation of some duplicated genes in polyploids.

Development of a new DHPLC assay for genotyping UGT1A1(TA)n polymorphism associated with Gilbert’s syndrome

Gilbert's syndrome is the most common hereditary disorder of bilirubin metabolism. The causative mutation in Caucasians is almost exclusively a (TA) dinucleotide insertion in the UGT1A1 promoter. Affected individuals are homozygous for the variant promoter and have 7 TA repeats instead of 6. Promoters with 5 and 8 TA repeats also exist but are extremely rare in Caucasians. The aim of our study was to develop denaturing high-performance liquid chromatography (DHPLC) assay for genotyping UGT1A1(TA)n polymorphism and to compare it with a previously described single-strand conformation polymorphism (SSCP) assay. Fifty DNA samples with common genotypes ((TA)6/6, (TA)6/7, (TA)7/7) as well as 7 samples with one of the following rare genotypes- (TA)5/6, (TA)5/7, (TA)6/8 or (TA)7/8 were amplified by polymerase chain reaction (PCR) and genotyped by DHPLC using sizing mode. All samples were previously genotyped by SSCP assay which was validated by sequencing analysis. All samples with either common or rare genotypes showed completely concordant results between DHPLC and SSCP assays. Our results show that sizing DHPLC assay is more efficient compared to classical SSCP assay due to shorter time of genotyping analysis, ability of genotyping increased number of samples per day, higher robustness, reproducibility and cost-effectiveness with no loss of accuracy in detection of all UGT1A1(TA)n genotypes. We developed a new DHPLC assay which is suitable for accurate, automated, highthroughput, robust genotyping of all UGT1A1(TA)n polymorphism variants, compared to a labour intensive and time-consuming SSCP assay.

A fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp.

The present study aimed to establish a fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp. Based on the sequences of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA, we designed a set of genus-specific primers for the amplification of Fasciola ITS-2, with an estimated size of 140 bp. These primers were labelled by fluorescence dyes, and the PCR products were analyzed by capillary electrophoresis under non-denaturing conditions (F-PCR-SSCP). Capillary electrophoresis analysis of the fluorescence-labelled DNA fragments displayed three different peak profiles that allowed the accurate identification of Fasciola species: one single peak specific for either Fasciola hepatica or Fasciola gigantica and a doublet peak corresponding to the "intermediate" Fasciola. Validation of our novel method was performed using Fasciola specimens from different host animals from China, Spain, Nigeria, and Egypt. This F-PCR-SSCP assay provides a rapid, simple, and robust tool for the identification and differentiation between Fasciola spp.

Presymptomatic breast cancer in Egypt: role of BRCA1 and BRCA2 tumor suppressor genes mutations detection

BackgroundBreast cancer is one of the most common diseases affecting women. Inherited susceptibility genes, BRCA1 and BRCA2, are considered in breast, ovarian and other common cancers etiology. BRCA1 and BRCA2 genes have been identified that confer a high degree of breast cancer risk.ObjectiveOur study was performed to identify germline mutations in some exons of BRCA1 and BRCA2 genes for the early detection of presymptomatic breast cancer in females.MethodsThis study was applied on Egyptian healthy females who first degree relatives to those, with or without a family history, infected with breast cancer. Sixty breast cancer patients, derived from 60 families, were selected for molecular genetic testing of BRCA1 and BRCA2 genes. The study also included 120 healthy first degree female relatives of the patients, either sisters and/or daughters, for early detection of presymptomatic breast cancer mutation carriers. Genomic DNA was extracted from peripheral blood lymphocytes of all the studied subjects. Universal primers were used to amplify four regions of the BRCA1 gene (exons 2,8,13 and 22) and one region (exon 9) of BRCA2 gene using specific PCR. The polymerase chain reaction was carried out. Single strand conformation polymorphism assay and heteroduplex analysis were used to screen for mutations in the studied exons. In addition, DNA sequencing of the normal and mutated exons were performed.ResultsMutations in both BRCA1 and BRCA2 genes were detected in 86.7% of the families. Current study indicates that 60% of these families were attributable to BRCA1 mutations, while 26.7% of them were attributable to BRCA2 mutations. Results showed that four mutations were detected in the BRCA1 gene, while one mutation was detected in the BRCA2 gene. Asymptomatic relatives, 80(67%) out of total 120, were mutation carriers.ConclusionsBRCA1 and BRCA2 genes mutations are responsible for a significant proportion of breast cancer. BRCA mutations were found in individuals with and without family history.

Mutational Analysis of MITOSTATIN, a Candidate Tumor-Suppressor Gene, at a Mononucleotide Repeat in Gastric and Colorectal Carcinoma

To the editor, Human MITOSTATIN gene encodes a mitochondrial protein, which promotes apoptosis and inhibits cell growth.1 The MITOSTATIN gene is somatically mutated in cancer cell lines (gastric, prostate and pancreas cell lines) and is down-regulated in bladder and breast cancer tissues.1 These data suggest that MITOSTATIN gene may be a tumor suppressor gene, inactivation of which plays an important role in cancer development. Although somatic mutations of MITOSTATIN gene have been identified in cancer cell lines, its mutation status has not yet been studied in cancer tissues. Frameshift mutations of genes containing mononucleotide repeats are features of gastric (GC) and colorectal carcinomas (CRC) with microsatellite instability (MSI).2 Many cancer-associated genes, such as TGFBR2, BAX, IGFR2 and TCF4, have been found to harbor mutations at mono- or dinucleotide repeats in the coding sequences in these cancers with MSI.2 There is a polyadenine tract (A8) in the exon 5 of the MITOSTATINgene, but the frameshift mutation at this site is not known. To see whether the mononucleotide repeat A8 in the exon 5 of MITOSTATIN gene is mutated in GC and CRC, we analyzed the exon 5 by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay. For the mutation analysis, methacarn-fixed tissues of 42 GC and 51 CRC with MSI were used in this study. These cancers consisted of 31 GC with high MSI (MSI-H), 11 GC with low MSI (MSI-L), 37 CRC with MSI-H and 14 CRC with MSI-L. Malignant cells and normal cells from the same patients were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle affixed to a micromanipulator.3 Genomic DNA each from tumor cells and corresponding normal cells were amplified with a primer pair by PCR. Radioisotope ([32P]dCTP) was incorporated into the PCR products for detection by SSCP autoradiogram. After SSCP, migration of the PCR products on the SSCP was analyzed by visual inspection. Direct DNA sequencing reactions were performed in the cancers with the mobility shifts in the SSCP. Other procedures of the PCR and SSCP were described in our previous studies.3,4 On the SSCP, all of the PCR products from the cancers were clearly seen. The PCR-SSCP analysis identified aberrant bands in one (3.2%) of the 31 GC with MSI-H, but not in the other cancers. DNA from normal tissues from the same patients showed no evidence of the mutation in SSCP, indicating the mutations had risen somatically. A direct DNA sequence analysis of the GC with the aberrant SSCP bands led to identification of a MITOSTATIN deletion mutation within the A8 repeat sequences (Fig. 1). The mutation was c.522delA, which would result in premature stops of the amino acid synthesis (p.Lys174AsnfsX26). We repeated the experiments twice, including PCR, SSCP and DNA sequencing analysis to ensure the specificity of the results, and found that the data were consistent. Fig. 1 Frameshift mutation of MITOSTATIN in a gastric carcinoma with microsatellite instability. Direct DNA sequence analysis of MITOSTATIN exon 5 from a gastric adenocarcinoma shows a heterozygous A deletion in tumor tissue relative to normal tissue. Failure of apoptosis allows survival of cells and plays an important role in development of cancers.3 As a mechanism of apoptosis resistance in cancer cells, somatic mutations of pro-apoptosis genes have been reported in many human cancers, and many of the mutations were proven to inactivate cell death.3,5 Because the main tumor suppressor function of MITOSTATIN appears to depend on apoptosis activity, we analyzed frameshift mutation of MITOSTATIN gene at a mononucleotide repeat that may inactivate the apoptosis. However, we detected only one MITOSTATIN mutation in the cancers with MSI, indicating that the mutation at the repeat is rare in GC and CRC with MSI. In the earlier study1 that first disclosed tumor suppressor functions of MITOSTATIN, the authors found that loss of expression and mutation of the gene in solid tumors and cell lines, respectively. Our and the earlier data1 suggest that MITOSTATIN may be inactivated by various mechanisms. To see whether loss of function of MITOSTATIN is a feature of human cancers, the status of MITOSTATIN gene should be further analyzed at various levels in many cancer types.

Mutational analysis of caspase genes in prostate carcinomas

Evasion of apoptosis is one of the hallmarks of cancer. Of the components of apoptosis machinery, caspases are the main executioners of apoptosis that initiate and propagate the apoptosis, and finally degrade target molecules. Caspase-encoding genes have been reported to harbor inactivating mutations in many human cancers. However, mutational status of caspase genes in prostate carcinomas has not been identified. The aim of this study was to explore whether caspase genes are somatically mutated in prostate carcinomas. For this, we analyzed entire coding regions of 11 human caspase-encoding genes (CASP1-10 and 14) in 45 prostate carcinoma tissues by a single-strand conformation polymorphism (SSCP) assay. In this study, however, we detected no somatic mutation of CASP genes in the prostate carcinomas by the SSCP. This is the first report on systematic evaluation of caspase-encoding gene mutations in human prostate carcinomas, and our data indicate that CASP genes may not be mutated in prostate carcinomas. The data suggest that apoptosis evasion in prostate carcinoma may be dependent on other mechanisms besides genetic alteration of caspase-encoding genes.

Oncogenic NRF2 mutations in squamous cell carcinomas of oesophagus and skin

Nuclear factor erythroid-related factor 2 (NRF2) encodes a transcription factor that induces expression of cytoprotective proteins upon oxidative stress and oncogenic NRF2 mutations have been found in lung and head/neck cancers that inactivate KEAP1-mediated degradation of NRF2. The aim of this study was to catalogue NRF2 mutations in other human cancers. For this, we analysed 1145 cancer tissues from carcinomas from oesophagus, skin, uterine cervix, lung, larynx, breast, colon, stomach, liver, prostate, urinary bladder, ovary, uterine cervix, and kidney, and meningiomas, multiple myelomas, and acute leukaemias by single-strand conformation polymorphism (SSCP) assay. We detected NRF2 mutations in oesophagus (8/70; 11.4%), skin (1/17; 6.3%), lung (10/125; 8.0%), and larynx (3/23; 13.0%) cancers. Of note, all of the 22 mutations except one were found in squamous cell carcinomas (SCCs) (95.5%). The mutations were observed within or near DLG and ETGE motifs that are important in NRF2 and KEAP1 interaction. All of the oesophageal SCCs and skin SCCs with the NRF2 mutations showed increased NRF2 expression in the nuclei. However, none of the SCCs from oesophagus and skin harboured KEAP1 mutation. Our study demonstrated here that NRF2 mutation occurs not only in lung and head/neck cancers, but also in oesophageal and skin cancers. Our data suggest that the NRF2 mutation plays a role in the development of SCC and is a feature of SCC.

Mutational analysis of hypoxia‐related genes HIF1α and CUL2 in common human cancers

Hypoxia is a general feature of solid cancer tissues. Hypoxia upregulates hypoxia-inducible factor 1alpha (HIF1alpha) that transactivates downstream genes and contributes to cancer pathogenesis. HIF1alpha is upregulated not only by hypoxia but also by genetic alterations in HIF1alpha-related genes, including VHL. Cullin 2 (CUL2) interacts with the trimeric VHL-elongin B-elongin C complex and plays an essential role in the ubiquitinated degradation of HIF1alpha. The aim of this study was to explore whether HIF1alpha and CUL2 genes are somatically mutated, and contribute to HIF1alpha activation in common human cancers. For this, we have analyzed the coding region of oxygen-dependent degradation domain of HIF1alpha in 47 colon, 47 gastric, 47 breast, 47 lung, and 47 hepatocellular carcinomas, and 47 acute leukemias by a single-strand conformation polymorphism assay. In addition, we analyzed mononucleotide repeat sequences (A8) in CUL2 in 55 colorectal and 45 gastric carcinomas with microsatellite instability (MSI). We found one HIF1alpha mutation (p.Ala593Pro) in the hepatocellular carcinomas (1/47; 2.1%), but none in other cancers. We found two CUL2 frameshift mutations in colon cancers (p.Asn292MetfsX20), which were exclusively detected in high MSI cancers (4.9%; 2/41). Our data indicate that somatic mutation of HIF1alpha is rare in common cancers, and somatic mutation of CUL2 occurs in a fraction of colorectal cancers (colorectal cancers with high MSI). The data suggest that neither HIF1alpha nor CUL2 mutation may play a central role in HIF1alpha activation in gastric, colorectal, breast, lung and hepatocellular carcinomas, and acute leukemias.

Hepatitis C virus quasispecies in plasma and peripheral blood mononuclear cells of treatment naïve chronically infected patients

Peripheral blood mononuclear cells (PBMCs) from 45 treatment naïve, HIV-negative, chronically hepatitis C virus (HCV)-infected patients were analyzed for the presence of HCV RNA. Viral RNA was detected in 73% of the studied patients. Single-strand conformation polymorphism assays and sequence analysis of the HCV 5'untranslated regions amplified from RNA recovered from both Plasma and PBMCs suggested virus compartmentalization in 57.6% of patients studied. In summary, our study presents evidence that HCV RNA can be found in PBMCs of treatment naïve chronically infected patients that are not immunocompromised or co-infected with the human immunodeficiency virus.

Mutational analysis of CASP1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 14 genes in gastrointestinal stromal tumors

Deregulation of apoptosis is one of the hallmarks of cancer, and inactivation of cancer cell apoptosis has been reported in many cancers. Caspases, the main executioners during apoptosis and inflammation, have been reported to harbor inactivating mutations in several cancers. The aim of this study was to explore whether CASP1 to 10 and 14 genes that encode caspase 1 to 10 and 14 are somatically mutated in gastrointestinal stromal tumor. We analyzed the entire coding region and all splice sites of all 11 human CASP genes for the detection of somatic mutations in 22 gastrointestinal stromal tumors by a single strand conformation polymorphism assay. We found a recurrent CASP4 mutation (c.1093C>G [p.L365V]) in 4 gastrointestinal stromal tumors, but there were no mutations in the other 10 CASPs. The CASP4 mutation was a missense mutation and was predicted to substitute amino acids in the small protease subunit of caspase 4. Overall, the gastrointestinal stromal tumor tissues harbored a CASP mutation in 18.2% (4/22). Our data indicate that somatic mutation of the CASP4 gene is common in gastrointestinal stromal tumor and suggest a possibility that CASP4 mutation might lead to alteration of apoptotic or inflammatory function and contribute to the pathogenesis of some gastrointestinal stromal tumors.

Absence of FLT3 and JAK2 (V617F) Mutations in Langerhans Cell Histiocytosis

Langerhans cell histocytosis (LCH) is a neoplasm of histiocytic cells believed to be derived from cells of the dendritic system, but the disease pathogenesis is unknown. FMS-like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase that is expressed on the surface of hematopoietic stem cells and plays an important role in normal hematopoiesis. Recently, FLT3 has been shown to play a role in regulating dendritic cell development and elevated levels FLT3-ligand was found in the serum of LCH patients. Therefore we evaluated 14 cases of LCH for FLT3 mutations by PCR analysis of genomic DNA, obtained from formalin-fixed paraffin-embedded tissue samples, followed by PAGE for deletions or internal tandem duplications using a commercially available kit (InVivoScrib Technologies, San Diego, CA). There were 7 males and 7 females with a mean age of 25.1. The tissues involved included bones, lymph nodes, bone marrow, breast, lungs and various soft tissues. No mutations in FLT3 were identified in any of the 14 patient samples. In the same patient population we also looked at the JAK2 (V617F) mutation, which has been found in patients with myeloproliferative disorders other than chronic myeloid leukemia and which has not yet been evaluated in LCH by a single strand conformation polymorphism assay using a commercially available kit (Ipsogen, New Haven, CT). There was no JAK2 V617F mutation in any of the 14 cases. These results suggest that FLT3 and JAK2 V617F mutations do not play a role in the pathogenesis of LCH.

Variation for homoeologous gene silencing in hexaploid wheat

The absence of expression of individual members of a homoeologous set of genes in a polyploid is a well-established phenomenon. However, the extent to which such 'homoeologous silencing' can vary between individual genotypes within a species is unexplored. We have used the single-strand conformation polymorphism assay to identify homoeologue non-expression at 15 single-copy genes across a panel of 16 wheat varieties, representative of the genetic diversity present in modern northern European winter wheat (Triticum aestivum). There was no evidence for any homoeologous silencing at seven of the fifteen genes, but in the remaining eight, at least one of the three homoeologues varied qualitatively for expression in either the root or the seedling leaf. The identity of the non-expressed homoeologue was generally consistent, but when the expression profiles of eight informative genes were compared, only two varieties shared the same pattern of silencing. A small-scale study suggested that silencing patterns were largely inherited across self-pollinated generations, and some evidence is presented for the epigenetic segregation of these patterns in a population bred from parents having contrasting silencing profiles. Epigenetic variation exerts a significant effect on phenotype, so given the ubiquity and variability in homoeologous silencing observed in wheat, we suggest that it is likely to play a considerable role in generating phenotypic variation. Thus epigenetic profiling may need to be incorporated as part of the analytical tool kit for predictive wheat breeding.

Mutational Analysis of WTX Gene in Wnt/ β-Catenin Pathway in Gastric, Colorectal, and Hepatocellular Carcinomas

A recent study of Wilms' tumors discovered a new X chromosome gene, Wilms' tumor gene on the X chromosome (WTX), which was found to harbor small deletions and point mutations. WTX protein negatively regulates Wnt/ beta-catenin signaling, and is considered a tumor-suppressor gene. One of the questions about the WTX gene is whether the genetic alterations of the WTX gene are specific to only Wilms' tumors. To see whether somatic point mutations of WTX occur in other malignancies, we analyzed the WTX gene for the detection of mutations in 141 cancer tissues by a single-strand conformation polymorphism assay. The cancer tissues consisted of 47 gastric adenocarcinomas, 47 colorectal adenocarcinomas, and 47 hepatocellular carcinomas. Overall, we detected one WTX mutation in the colorectal carcinomas (1/47; 2.1%), but there was no WTX mutation in other cancers analyzed. The detected mutation was a missense mutation (c. 1117G > A (p.Ala373Thr)). Although the WTX mutation is common in Wilms' tumors, our data indicate that it is rare in colorectal, gastric, and hepatocellular carcinomas. The data also suggest that deregulation of Wnt/ beta-catenin signaling by WTX gene mutation may be a rare event in the pathogenesis of colorectal, gastric, and hepatocellular carcinomas.

Comparison of genetic diversity and pathogenicity of fusarium head blight pathogens from China and Europe by SSCP and seedling assays on wheat

The genetic diversity and pathogenicity of isolates of Fusarium graminearum and F. asiaticum isolated from wheat heads in China were examined and compared with those of isolates of F. graminearum, F. asiaticum and F. meridionale from Europe, USA and Nepal. Genetic diversity was assessed by SSCP (single strand conformation polymorphism) and AFLP (amplified fragment length polymorphism) analysis and by molecular chemotyping. SSCP analysis of the Fg16F/Fg16R PCR amplicon differentiated F. graminearum, F. asiaticum and F. meridionale and revealed three haplotypes among sequence‐characterized amplified region (SCAR) type 1 F. graminearum isolates. AFLP analysis showed a high level of genetic diversity and clustered the majority of Chinese isolates in one group along with other isolates of Asian origin. The second cluster contained F. graminearum isolates from China, Europe and the USA. Of the Chinese isolates, 79% were F. asiaticum and 81% of these were of the 3‐AcDON chemotype, with only 9·5% of either chemotype 15‐AcDON or NIV. All the Chinese and USA isolates of F. graminearum were 15‐AcDON, whereas among the isolates from Europe, 21% were NIV and 8% were 3‐AcDON chemotype. No evidence was found for possible differences in aggressiveness between F. graminearum and F. asiaticum. Highly aggressive isolates were present in each region and no evidence was found for any association between aggressiveness and geographical origin or chemotype among the isolates examined. No difference was observed in pathogenicity towards wheat seedlings between Chinese isolates and those from Europe, the USA or Nepal.

Mutational analysis of caspase 1, 4, and 5 genes in common human cancers

Mounting evidence indicates that deregulation of apoptosis is involved in the mechanisms of cancer development. Mutations of genes encoding caspases, the executioners of apoptosis, have been detected in human cancers, indicating inactivation of apoptosis by the mutations of caspase is an important mechanism in cancer development. The aim of this study was to see whether genes encoding human caspases 1, 4, and 5 are mutated in human cancers. We analyzed the entire coding region and all splice sites of human caspase 1, 4, and 5 genes for the detection of somatic mutations in 337 human cancers, including 103 colorectal, 54 gastric, 60 breast, 60 hepatocellular, and 60 lung carcinomas by a single-strand conformation polymorphism assay. We detected 2 (0.6%) caspase-1, 2 (0.6%) caspase-4, and 15 (4.4%) caspase-5 mutations in the 343 cancers. The mutations were detected in 11 gastric carcinomas (2 caspase-1 and 9 caspase-5 mutations), 6 colorectal carcinomas (2 caspase-4 and 4 caspase-5 mutations), 1 breast carcinoma (1 caspase-5 mutation), and 1 lung carcinoma (1 caspase-5 mutation). The mutations consisted of 11 mutations in exons and 8 mutations in noncoding sequences. The 11 mutations in the exons consisted of 3 missense, 1 silent, and 7 frameshift mutation(s). Of note, most (6/9) of the caspase-5 mutations in the coding sequences were detected in microsatellite instability (MSI)-positive cancers. These data indicate that somatic mutations of caspase-1 and caspase-4 genes are rare in common solid cancers. In addition, the data indicate that caspase-5 gene is commonly mutated in the MSI-positive cancers, and suggest that inactivation of caspase-5 may play a role in the tumorigenesis of MSI-positive cancers.

Mutational analysis of oncogenic AKT E17K mutation in common solid cancers and acute leukaemias

Mounting evidence indicates that alterations of AKT signalling play important roles in cancer development. An earlier study discovered an oncogenic AKT1 gene mutation (AKT1 E17K) in breast, colorectal and ovarian cancers. The aim of this study was to see whether the AKT1 E17K mutation is common in breast, colorectal, lung, gastric and hepatocellular carcinomas and acute leukaemias. We analysed the presence of the AKT1 E17K mutation in 731 cancer tissues by a single-strand conformation polymorphism assay. In addition, we analysed the corresponding sequences of AKT1 E17K in AKT2 and AKT3 genes. Overall, we detected the four AKT1 E17K mutations in the breast cancers (4/93; 4.3%), but none in other cancers. There was no AKT2 or AKT3 mutation in the cancers. This study demonstrated that the AKT1 E17K mutation occurs in breast cancers at a low frequency, and that it is rare in other common cancers, including colorectal, lung, gastric and hepatocellular carcinomas and acute leukaemias. Despite the confirmed oncogenic function of the AKT1 E17K, the rare incidences of the mutation suggest that it may not play a crucial role in the development of the most common types of human cancers.

Mutational Analysis of the Tumor Suppressor WTX Gene in Non-small Cell Lung Cancer

Purpose: In a recent study of Wilms' tumors, a new X chromosome gene, Wilms' tumor gene on the X chromosome (WTX), was discovered that was found to harbor small deletions and point mutations. The WTX protein negatively regulates Wnt/ β-catenin signaling, and is considered to be a tumor suppressor gene. One of the questions about the WTX gene is whether the genetic alterations of the WTX gene are specific only to Wilms' tumors. The aim of this study was to explore whether the WTX gene mutation is a characteristic of human non-small cell lung cancer (NSCLC). Materials and Methods: In the current study, we analyzed the part of the WTX gene encoding the N-terminal of WTX, where most of the WTX point mutations have been detected in Wilms' tumors. Forty-eight NSCLC tissues were analyzed by a single-strand conformation polymorphism assay and DNA sequencing. Results: SSCP analysis revealed no evidence of somatic mutations in the DNA sequences encoding the N-terminal of the WTX gene in the 48 NSCLC tissues. Conclusion: The data presented here indicate that the WTX gene may not be somatically-mutated in human NSCLCs, and suggest that NSCLCs may not utilize mutational events of the WTX gene in the process of pathogenesis. (J Lung Cancer 2008;7(1):22�� 24)