Single-strand Conformation Polymorphism Assay Research Articles

Genetic diversity analysis of grapevine rupestris stem pitting-associated virus from grapevine by colony PCR-SSCP and -RFLP.

We have developed methods for detecting the genetic diversity of grapevine rupestris stem pitting-associated virus (GRSPaV) based on restriction fragment length polymorphism (RFLP) and single stranded conformational polymorphism (SSCP) in the 905 nt 3' sequence. The amplicons were cloned from six grapevine cultivars, and colony polymerase chain reaction (colony PCR) using recombination bacteria was subsequently analyzed by RFLP and SSCP. Four haplotypes of SSCP and six haplotypes of Sac IRFLPs were defined. The two methods had a40% discrepancy rate in showing the degree of diversity. All clones were sequenced and were used to construct aphylogenetic tree with seven previously reported GRSPaV sequences. In the tree, all the newly acquired sequences were divided into three clusters, I, II, and III, which corresponded to haplotypes I, II, and III of SSCP, respectively. Haplotype IV of SSCP was grouped into cluster II. Arecombination analysis showed that haplotype IV has undergone arecombination event. Together, these results indicate that the SSCP assay is useful for the rapid identification of genetic diversity of GRSPaV. This is the first report of an analysis of the large fragment of GRSPaV by colony PCR-SSCP. Keywords: grapevine; grapevine rupestris stem pitting-associated virus (GRSPaV); RFLP; SSCP; genetic diversity analysis.

Cancer of unknown primary with EGFR mutation successfully treated with targeted therapy directed by clinical next-generation sequencing: a case report

BackgroundCancer of unknown primary (CUP) is usually treated with nonselective and empirical chemotherapy; however, its prognosis is generally poor, with a median survival of less than a year. Thus, clinicians eagerly await the development of more effective treatment strategies. In recent years, advances in next-generation sequencing (NGS) have made it possible to analyze comprehensively the genome of individual cancers. NGS has identified many genomic alterations, some of which are potential molecular targets of specific agents. We report a case of CUP that was successfully treated with targeted therapy directed by the genomic data obtained from an NGS-based multiplex assay.Case presentationA 52-year-old Asian woman with right hip joint pain underwent fluorodeoxyglucose-positron emission tomography/computed tomography, which showed multiple metastatic tumors in her right hip joint, thyroid gland, lung, and vertebrae. Brain magnetic resonance imaging showed multiple cerebral metastases. Additional tests, including pathology examination and conventional epidermal growth factor receptor (EGFR) gene mutation analysis (single-strand conformation polymorphism assay), could not identify the primary origin of the tumors, so the patient was diagnosed with CUP. After empirical chemotherapy for CUP, an NGS-based multiplex assay performed using a resected specimen of thyroid tumor detected the EGFR mutation c.2573 T > G p.Leu858Arg (L858R). Her treatment was changed to erlotinib, an EGFR tyrosine-kinase inhibiter, which dramatically shrank the tumors and decreased her serum carcinoembryonic antigen level. She achieved long-term disease control and survived for 2 years and 9 months from the first diagnosis.ConclusionThis case might support the strategy that NGS-based multiplex assays could identify actionable molecular targets for individual patients with CUP.

Application of biological and single-strand conformation polymorphism assays for characterizing potential mild isolates of Citrus tristeza virus for cross protection

Citrus tristeza virus (CTV) by killing millions of citrus cultivars grown on sour orange rootstock worldwide has become one of the most dangerous viral pathogen. Characterization of 12 CTV isolates was analyzed by biological indexing. Infected samples of citrus were collected from citrus growing areas of Pakistan and CTV was detected by symptoms on indicator plants and confirmed by direct tissue blot immunoassay (DTBIA). CTV positive samples were graft inoculated on six biological indicator hosts in the study. A standardized protocol was deployed to study biological characteristics of these isolates. All biological indicators induced mild and from mild to moderate reactions against all of the CTV isolates tested. About two isolates produced stem-pitting symptoms from moderate to severe on Mexican lime. CTV strains were further characterized and confirmed by the analysis of p25 gene of CTV isolates using single-strand conformation polymorphism (SSCP) assay. SSCP analysis revealed that most isolates confined only one predominant sequence variant. SSCP profiles of PCR amplified products from CTV isolates showed bands patterns corresponding to mild and sever strain. Three isolates (4MF, 8KBS and 10GS) from different regions and cultivars were identified as potential source of mild strains for cross protection. These results are the best base for mild strain cross protection (MSCP) in the country.

Molecular, serological and biological characterizations of Potato Leaf Roll Virus in infected potato plants in Egypt, and its effects on plant cell organelles

Potato Leaf Roll Virus (PLRV) is one of the most serious viruses infecting potato plant (Solanum tuberosum L.) in Egypt. Indirect Enzyme Linked Immunosorbent Assay (Indirect- ELISA) results revealed that 70 % of the collected samples were infected with PLRV. A multiplex Polymerase Chain Reaction (PCR) was carried out using three different sets of primers, specific for both PLRV and Potato virus Y (PVY) isolates. For confirmation; the movement coat protein (MP) gene was isolated from the infected plant tissues, and a band with molecular size 336 bp was obtained using Reverse transcription-Polymerase chain reaction (RT-PCR). The DNA sequence of the Egyptian PLRV-Banha -MP gene was deposited in GenBank under an accession number of KR002119. Moreover, sequence analysis revealed that the Egyptian PLRV isolate was closely related to a New Zealand isolate of PLRV (GU002341), with identity of 100%. Transmission electron microscope (TEM) examination of PLRV showed isometric particles, with approximate size of 24-30 nm. The cytopathological examination of the potato plant infected with PLRV revealed many cellular effects such as; partially degraded and deformed chloroplast, starch with an increased size and color change, in addition to nucleus and cytoplasmic bridge. It could be concluded that PLRV is present in Egypt, infecting most of the potato cultivars. Moreover, four different strains of PLRV were detected based on the Single strand conformation polymorphism (SSCP) of the MP gene. The aims of the current study were to identify the PLRV infection of potato plant in Egypt using; molecular, serological and biological methods, in addition to studying the effect of this virus on potato cell organelles. This is the first record of the presence of four different strains of PLRV infecting potato in Egypt, using SSCP assay.

Genetic polymorphisms and protein expression of P53 and BRCA1 in preneoplastic and neoplastic rat mammary glands.

Breast cancer is the most common type of cancer and the leading cause of cancer-related deaths among women in the United Arab Emirates and worldwide. Although many factors contribute to the high incidence of breast cancer, a considerable number of cases are related to environmental factors. In the present study, breast cancer was induced in female rats using a single dose, 80mg/kg bodywt, of the environmental carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The aim of the present study, was to characterize some of the molecular changes that occur during breast cancer development in the DMBA-treated rat model. Mammary gland tissues of control and DMBA-treated rats were processed for: i)immunohistochemical probing using anti-BRCA1 antibody to characterize and correlate the localization of this cellcycle protein during progression to cancer, ii)western blotting to analyze the alteration of p53 protein expression in preneoplastic and neoplastic lesions of the mammary glands, and iii)polymerase chain reactions using primers specific for BRCA1 and P53 genes followed by single stranded conformational polymorphism (SSCP) or restriction fragment length polymorphism (RFLP) assays to detect possible mutations in these genes during development of breast cancer. Microscopic examination revealed a wide range of preneoplastic and neoplastic lesions providing a sequence representing the multistep process of breast cancer formation in DMBA-treated rats. Probing for BRCA1 protein revealed a gradual defect in its translocation from the cytoplasm to the nucleus during breast cancer progression. In control rats, BRCA1 was present in the nuclei of terminal duct epithelial cells. However, in the preneoplastic lesions, BRCA1 was localized in both the cytoplasm and nuclei of the epithelial duct cells. In all malignant lesions, BRCA1 was mostly found in the cytoplasm. Western blotting revealed initial downregulation in the expression of p53 protein during breast cancer development. However, with progression towards malignancy, upregulation of p53 was observed. These changes were associated with polymorphism in p53 gene, which was detected in exon5 using SSCP assay. However, using RFLP and BamHI to digest the PCR products of exon11 of BRCA1 gene revealed no detectable polymorphisms. In conclusion, molecular characterization of the early changes that occur during development of breast cancer provides some clues for better understanding of its pathogenesis.

Analysis of molecular alterations in laterally spreading tumors of the colorectum.

Colorectal laterally spreading tumors (LSTs) are classified into LST-Gs and LST-NGs, according to macroscopic findings. In the present study, we determined the genetic and epigenetic alterations within colorectal LSTs and protruding adenomas. A crypt isolation method was used to isolate DNA from tumors and normal glands of 73 macroscopically verified colorectal LSTs (histologically defined adenomas; 38 LST-Gs and 35 LST-NGs) and 36 protruding adenomas. The DNA was processed using polymerase chain reaction (PCR) microsatellite assays, single-strand conformation polymorphism (SSCP) assays, and pyrosequencing to detect chromosomal allelic imbalance (AI),mutations in APC, KRAS, and TP53, and the methylation of MLH1, MGMT, CDKN2A, HPP1, RASSF2A, SFRP1, DKK1, ZFP64, and SALL4 genes. In addition, methylation status was examined using the following set of markers: MIN1, MINT2, MINT31, MLH1, and CDKN2A (with classification of negative/low and high). Microsatellite instability (MSI) was also examined. 5q AI and methylation of the SFRP1 and SALL4 genes were common molecular events in both LST-Gs and LST-NGs. Neither MSI nor mutations in BRAF ware observed in the LSTs. TP53 mutations were rarely found in LSTs. The frequencies of KRAS and APC mutations and the methylation levels of ZFP64, RASSF2A, and HPP1 genes were significantly higher in LST-Gs than in LST-NGs. Protruding adenomas showed alterations common to LST-Gs. Negative/low methylation status was common among the three types of tumors. Combined genetic and epigenetic data suggested that the molecular mechanisms of tumorigenesis were different between LST-Gs and LST-NGs.

A SNP in the 3′-untranslated region of AMPKγ1 may associate with serum ketone body and milk production of Holstein dairy cows

AMPK is the key switch for providing the energy balance between cellular anabolic and catabolic processes. In this study, we aimed to screen the PRKAG1 (AMPKγ1) gene in high, moderate, and low producing Holstein dairy cows. A sample of 100 pregnant dairy cows, comprising 41 high, 33 moderate, and 26 low milk yields were selected from three large dairy herds in Isfahan province of Iran. Body condition score (BCS) was estimated before parturition while beta hydroxyl butyric acid (BHBA) as a measure of ketone bodies was measured at the fifth day postpartum. In addition, using three primer pairs covering exons 2–11 and 3′-UTR of the PRKAG1 gene, a random sample of 10 high milk yield dairy cows were amplified and sequenced. The sequencing results showed the presence of a T12571C mutation in intron 6 and a T14280C mutation in the 3′-untranslated region (UTR) of the PRKAG1 gene. Following a PCR reaction for amplification of the 3′-UTR amplicons, single strand conformation polymorphism (SSCP) assay was implemented for discrimination of the mutation in the studied population. Then, we evaluated if the mutation associates with the BCS, serum BHBA level, and production traits. The experimental analysis showed that the mutated allele significantly increased the BHBA level, BCS, as well as milk and protein yield. Bioinformatic study revealed that this 3′-UTR mutation distorts the target site of mir-423-5p microRNA which is one of the most highly expressed microRNAs in the bovine mammary gland, liver, and kidney. Given the role of AMPK in energy metabolism, the newly identified 3′-UTR mutation highlights the importance of AMPK and suggests a role of miRNAs for regulation of cellular metabolism, metabolism disorders, and production traits in Holstein dairy cows.

Mutational and expressional analysis ofERBB3gene in common solid cancers

ERBB3 is a member of EGFR family receptor tyrosine kinases, genetic alterations of which are common and therapeutically targeted in human cancers. Recently, somatic mutations of ERBB3 gene, including recurrent mutation in exon 3 altering Val104, were reported in gastric cancers (GC) and colorectal cancers (CRC), strongly suggesting its role in the development of GC and CRC. To examine whether the recurrent ERBB3 mutations of exon 3 occur in GC and CRC, and other malignancies as well, we analyzed the ERBB3 in 1677 cancer tissues by a single-strand conformation polymorphism (SSCP) assay. We identified ERBB3 mutations altering the Val104 mutations in GC (0.5%) and CRC (2.2%). However, we did not find the ERBB3 mutations in the other cancers besides GC and CRC. We observed that an increased intensity of phosphorylated ERBB3 (pERBB3) in GC and CRC. Of note, all of the cancers with ERBB3 mutations displayed an increased intensity of pERBB3 immunostaining. Our data indicate that the recurrent ERBB3 mutations altering Val104 occur predominantly in GC and CRC. Also, the data suggest that ERBB3 is altered in GC and CRC by various ways, including somatic mutations and increased expression that might play roles in tumorigenesis.

Minor role of BRCA2 mutation (Exon2 and Exon11) in patients with early-onset breast cancer amongst Iranian Azeri-Turkish women.

Breast cancer is the most common cancer in women. Every year, one million new cases are reported worldwide, representing 18% of the total number of cancer in women. Hereditary BRCA1 and BRCA2 mutations account for about 60% of inherited breast cancer and are the only known causes of hereditary breast cancer syndrome. The aim of this study was to determine the frequency of BRCA2 (exon2 and exon11) gene mutation in patients with early-onset breast cancer among Iranian Azeri-Turkish women. We obtained clinical information, family history and peripheral blood from 110 women under the age of 45 with early-onset breast cancer for scanning germline mutations in the exon2 and 11 of BRCA2 genes which comprises over 50% of the gene. Single-strand conformation polymorphism assay was used in order for screening potential mutations on amplified regions followed by direct sequencing analysis to determine the genotypes. Overall, 11 sequence variants were identified in this study group, including four homozygotes and seven heterozygotes silent substitution of c.3807T to C, p.Val1269Val (rs543304). Mutations in BRCA2 were surprisingly infrequent in the early onset breast cancer patients among Iranian Azeri-Turkish women.

Absence of p53 gene mutations in mice colon pre-cancerous stage induced by o-nitrotoluene.

p53 gene is one of the most frequently mutated genes found in the human colonic tumors. Mice have been used as an experimental model to study the pathogenesis of colon cancer in humans. The alterations in cancer genes and proteins found in the mouse large intestinal tumors included mutations which are hallmarks of human colon cancer, probably contributed to the pathogenesis of the large intestinal carcinomas in mice following o-nitrotoluene (o-nt) exposure. Detection of p53 gene mutations in colon precancerous stage. In this study, mice colon precancerous stage induced by o-nt were examined for the presence of point mutations in highly conserved coding region (exons 5-8) and outside it (exons 10, 11) using a single-strand conformation polymorphism assay (SSCP). SSCP analysis showed no differences in banding patterns between the normal negative control group and o-nt-induced precancerous stage in mice colon. The results from the present study indicate that point mutations in the p53 gene, in the coding region (exons 5-8) and outside it (exons 10, 11), are not involved in the development of the colon precancerous stage induced by o-nt in mice.

Identification of complete linkage disequilibrium in the DSG4 gene and its association with wool length and crimp in Chinese indigenous sheep.

The desmoglein 4 (DSG4) gene is a potential candidate in the search for genes that may affect wool traits, because of its function. This study aimed to screen for polymorphisms in partial exon 16 and 3ꞌUTR of the sheep desmoglein 4 DSG4 gene, and to test its possible association with wool length and crimp associated with fur. Overall, 326 sheep were scanned via single-strand conformational polymorphism assay, through three pairs of primers. The breeds included Tan, Han, and TanxHan from China, Polled Dorset from Australia, and Suffolk from Britain genotypes AA, BB, and AB for primer2 and genotypes DD, EE, and DE for primer3 were detected in native breeds. Six SNPs and 3-bp insertion/deletions were found in exon 16, of which 4 lead to amino acid substitutions. In addition, 1 SNP was found in 3ꞌUTR. The DSG4 genotype was found to be strongly associated with all wool traits that were considered in this study (P < 0.01). Sheep with the genotype MM had a higher least square mean compared to sheep with the genotype WW or WM with respect to birth scapular wool length (P < 0.01), crimp number of birth scapular wool crimp (P < 0.01), crimp number of weaning scapular wool crimp (P < 0.01), and crimp number of weaning rump wool crimp (P < 0.01, P < 0.05). In conclusion, our study is the first to demonstrate that the DSG4 gene may be a candidate, or major gene, which influences important wool traits.

NIPBL, a Cohesion Loading Factor, Is Somatically Mutated in Gastric and Colorectal Cancers with High Microsatellite Instability

Timely segregation of sister chromatids during the cell cycle is essential for maintaining euploidy [1]. Aberrant segregation into daughter cells promotes aneuploidy and chromosome instability, which are features of many tumors [1]. Chromatid cohesion, a physical linkage of replicated sister chromatids, is mediated by a multiprotein complex called cohesin [1]. NIPBL is a cohesion loading factor that is required for cohesin to interact with chromosomes [2, 3]. NIPBL mutations (missense, nonsense, frameshift and deletion mutations) account for approximately 60 % of Cornelia de Lange syndrome, a congenital disorder with multisystem developmental abnormalities [3]. In Cornelia de Lange syndrome, mutated NIPBL interferes with its interaction with its partners in the cohesion complex [3]. Components in the cohesin complex and cohesin regulatory proteins are frequently altered at expression and mutation levels in human cancers [1]. For example, cohesin protector SGOL1 down-regulation leads to chromosomal instability in colorectal cancers (CRC) [4]. Somatic mutations of cohesion regulatory factor PDS5B have been identified in prostate cancers [5]. These data suggest that cohesin-related genes could be considered potential tumor suppressor genes (TSG) [1]. Somatic mutations of the NIPBL gene have been found in CRC with stable miscrosatellite instability (MSI) that may underlie chromosomal instability in CRC [6]. Together, these data suggest a possibility that NIPBL is a candidate TSG. In a public genome database (http://genome.cse.ucsc. edu/), we found that the human NIPBL gene had mononucleotide repeats in its coding sequences that could be targets for frameshift mutation in cancers with high MSI (MSI-H) [7]. To data, however, the data on NIPBL somatic mutation in gastric (GC) and CRC with MSI-H are lacking. To see whether the mononucleotide repeats in the NIPBL gene are mutated in GC and CRC, we analyzed two A7 and one A8 repeats in NIPBL exon 10 by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay with three primer pairs. For this, we used 32 GC with MSI-H, 59 GC with MSS, 47 CRC with MSI-H and 53 CRC with MSS in methacarnfixed tissues. All of the patients of the cancers were Koreans. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosinstained slides by microdissection [8]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP. On the SSCP, we observed aberrant bands of the NIPBL gene in 11 cancers (Fig. 1). DNA from normal tissue Min Sung Kim and Chang Hyeok An contributed equally to this study.

Detection of New Point Mutations of BRCA1 and BRCA2 in Breast Cancer Patients

This study included 20 selected female patients with breast cancer, 30 of their female relatives (sisters and daughters), and 10 healthy females as a control group. Genomic DNA was extracted from peripheral blood lymphocytes of all the subjects, and the polymerase chain reaction was carried out using specific primers for BRCA1 (exons 2 and 8) and BRCA2 (exons 9, 11, and 21). The mutations were detected using a single-strand conformation polymorphism assay and heteroduplex analysis. Finally, the sample variants and their controls were sequenced. Mutations were detected in 44% of the study population, with 18% found in the BRCA1 gene and 26% attributed to BRCA2. Five sequence variants were identified, including two frameshift mutations, one nonsense mutation, and two missense mutations. Therefore, we conclude that germline mutations in two major genes, BRCA1 and BRCA2, may have an important influence on the predisposition and development of familial breast cancer.

Evaluation of the role of the antioxidant silymarin in modulating the in vivo genotoxicity of the antiviral drug ribavirin in mice

Ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a widely used broad-spectrum antiviral drug. Recently, several reports revealed genotoxic effects of ribavirin in vivo and in vitro, which were correlated with the production of reactive oxygen species (ROS). This study aimed to evaluate the genotoxicity of ribavirin and to investigate the role of the natural antioxidant silymarin to modulate this genotoxicity. Male albino mice (age, 8–10 weeks) were injected intraperitoneally (i.p.) with ribavirin at three dose levels (20, 75 and 130mg/kgbw) either in a single injection (acute treatment) or in multiple injections on five consecutive days (sub-acute treatment). Other comparable groups were treated with silymarin (70mg/kgbw) 1h before the injection with ribavirin. Mice were sacrificed at different sampling times (24, 48 and 72h) after the last ribavirin treatment. Micronucleus (MN) and single-strand conformation polymorphism (SSCP) assays were used to assess genotoxic and cytotoxic effects of ribavirin and to evaluate the protective effect of the pre-treatment with silymarin. Our results reveal genotoxic and cytotoxic effects of ribavirin in the MN assay. Pre-treatment with silymarin reduced the toxicity of ribavirin. In the SSCP assay, ribavirin treatment did not induce any mutations in the two selected sites in the D-loop of mitochondrial DNA (mtDNA).

Rare somatic mutation of pro-apoptotic BAX and BAK genes in common human cancers

BAX and BAK are both pro-apoptotic Bcl-2 proteins and are essential for the pathway of intrinsic apoptosis. Apoptosis in cancer cells is frequently inactivated by somatic mutations. The aim of the study was to see whether somatic mutations of BAX and BAK genes are characteristics of common human cancers. We analyzed somatic mutation of BAX and BAK genes in 47 gastric, 47 colorectal, 47 breast, 47 lung and 47 prostate carcinomas, and 47 acute leukemias by a polymerase chain reaction and single-strand conformation polymorphism assay. We identified BAX gene mutations in one colon (2.1%) and three gastric (6.4%) cancers. All of the mutations were frameshift mutations in the G8 repeat sequences and were detected in cancers with high microsatellite instability (36.4%). There was no evidence of BAX mutation in the other cancers, nor was somatic mutation of the BAK gene detected in the cancers. Our data indicate that somatic mutation of BAX and BAK genes are rare in the common cancers (besides the cancers with high microsatellite instability) and suggest that neither BAX nor BAK mutation may causally be implicated in their tumorigenesis.

Evaluation of DNA extraction methods of rumen microbial populations

The dynamism of microbial populations in the rumen has been studied with molecular methods that analyze single nucleotide polymorphisms of ribosomal RNA gene fragments (rDNA). Therefore DNA of good quality is needed for this kind of analysis. In this work we report the evaluation of four DNA extraction protocols (mechanical lysis or chemical lysis with CTAB, ethylxanthogenate or DNAzol(®)) from ruminal fluid. The suitability of two of these protocols (mechanical lysis and DNAzol(®)) was tested on single-strand conformation polymorphism (SSCP) of rDNA of rumen microbial populations. DNAzol(®) was a simple method that rendered good integrity, yield and purity. With this method, subtle changes in protozoan populations were detected in young bulls fed with slightly different formulations of a supplement of multinutritional blocks of molasses and urea. Sequences related to Eudiplodinium maggi and a non-cultured Entodiniomorphid similar to Entodinium caudatum, were related to major fluctuating populations in an SSCP assay.

Expression and Mutation Analyses of Fas, FLIP and Bcl-2 in Granulosa Cell Tumor of Ovary

Aims and background Mounting evidence indicates that evasion of apoptosis contributes to tumor pathogenesis. Although both Fas and Bcl-2 are crucial in apoptosis of normal ovarian cells, their roles in ovarian tumors, especially stromal tumors, are largely unknown. The aim of this study was to explore mutation of the Fas gene and expression of the apoptosis-related proteins Fas, FLICE-like inhibitory protein (FLIP) and Bcl-2 in granulosa cell tumor (GCT) of the ovary. Methods We analyzed the expression of Fas, FLIP and Bcl-2 in 20 GCT tissues by immunohistochemistry. We also analyzed somatic mutations of the Fas gene in the same GCT tissues by polymerase chain reaction and single-strand conformation polymorphism assay. Results Expression of Fas was evident in 12 GCTs (60%), but the remaining 8 GCTs showed no or markedly decreased Fas immunostaining. Expression of FLIP was identified in 30% of the GCT samples and expression of Bcl-2 in 75%. All GCTs with positive Fas expression (n = 12) showed either FLIP or Bcl-2 expression. The GCTs were found to carry no somatic Fas mutations. Conclusions Our data show that alterations of the apoptosis-related proteins Fas, FLIP and Bcl-2 are common in GCT, and suggest that expression of FLIP and Bcl-2 and loss of Fas expression might play role in the pathogenesis of GCT, possibly by inhibiting apoptosis.

Mutational analysis of DNMT3A gene in acute leukemias and common solid cancers

DNMT3A, a DNA methyltransferase that functions for de novo methylation, is important in development and many cellular processes related to tumorigenesis. Somatic mutations of DNMT3A gene, including recurrent mutations in its Arg-882, were recently reported in acute myelogenous leukemia (AML), strongly suggesting its role in development of AML. To see whether DNMT3A mutation occurs in other malignancies as well, we analyzed DNMT3A in 916 cancer tissues from 401 hematologic malignancies (AML, acute lymphoblastic leukemias (ALL), multiple myelomas and lymphomas) and 515 carcinomas (lung, breast, prostate, colorectal and gastric carcinomas) using a single-strand conformation polymorphism (SSCP) assay. We identified DNMT3A mutations, especially the Arg-882 mutations, in adulthood AML (9.4%). In addition, we found DNMT3A mutations in pre-B-ALL and three lung cancers at lower frequencies. Allelic loss of DNMT3A was frequently observed in most cancer types analyzed, including lymphomas (48.1%), gastric cancers (23.5%) and lung cancers (18.3%) irrespective of DNMT3A mutation. Also, loss of DNMT3A expression was common in lung cancers (46.4%), and was associated with the allelic loss. Our data indicate that DNMT3A gene is mutated mainly in AML, but it occurs in other cancers, such as ALL and lung cancer, despite the lower incidences. Also, the data suggest that DNMT3A is altered in many cancer types by various ways, including somatic mutations, allelic loss and loss of expression that might play roles in tumorigenesis.

Abstract 2191: Mutational analysis of DNMT3A gene in acute leukemias and common solid cancers

Abstract DNMT3A, a DNA methyltransferase that functions for de novo methylation, is important in development and many cellular processes related to tumorigenesis. Somatic mutations of DNMT3A gene, including recurrent mutations in its Arg-882, were recently reported in acute myelogenous leukemia (AML), strongly suggesting its role in development of AML. To see whether DNMT3A mutation occurs in other malignancies as well, we analyzed DNMT3A in 916 cancer tissues from 401 hematlogoic malignancies (AML, acute lymphoblastic leukemias (ALL), multiple myelomas and lymphomas) and 515 carcinomas (lung, breast, prostate, colorectal and gastric carcinomas) by a single-strand conformation polymorphism (SSCP) assay. We identified DNMT3A mutations, especially the Arg-882 mutations, in adulthood AML (9.4%). In addition, we found DNMT3A mutations in a pre-B-ALL and three lung cancers at lower frequencies. Allelic loss of DNMT3A was frequently observed in most cancer types analyzed, including lymphomas (48.1%), gastric cancers (23.5%) and lung cancers (18.3%) irrespective of DNMT3A mutation. Also, loss of DNMT3A expression was common in lung cancers (46.4%) and was associated with the allelic loss. Our data indicate that DNMT3A gene is mutated mainly in AML but it occurs in other cancers such as ALL and lung cancer despite the lower incidences. Also, the data suggest that DNMT3A is altered in many cancers types by various ways, including somatic mutations, allelic loss and loss of expression that might play roles in tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2191. doi:1538-7445.AM2012-2191

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.