Frameshift Mutations In Genes Research Articles

Gene Clusters Located on Two Large Plasmids Determine Spore Crystal Association (SCA) in Bacillus thuringiensis Subsp. finitimus Strain YBT-020

Crystals in Bacillus thuringiensis are usually formed in the mother cell compartment during sporulation and are separated from the spores after mother cell lysis. In a few strains, crystals are produced inside the exosporium and are associated with the spores after sporulation. This special phenotype, named ‘spore crystal association’ (SCA), typically occurs in B. thuringiensis subsp. finitimus. Our aim was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020. Plasmid conjugation experiments indicated that the SCA phenotype in this strain was tightly linked with two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed. Six fragments from BAC clones were screened from this library and discovered to cover the full length of pBMB26; four others were found to cover pBMB28. Using fragment complementation testing, two fragments, each of approximately 35 kb and located on pBMB26 and pBMB28, were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable to the SCA phenotype. Gene disruption and frame-shift mutation analyses revealed that two of the five genes from pBMB28, which showed low similarity to crystal proteins, determined the location of crystals inside the exosporium. Gene disruption revealed that the three remaining genes, similar to spore germination genes, contributed to the stability of the SCA phenotype in strain YBT-020. Our results thus identified the genes determining the SCA phenotype in B. thuringiensis subsp. finitimus.

A cotton gene encoding novel MADS-box protein is preferentially expressed in fibers and functions in cell elongation

Cotton fibers, as natural fibers, are widely used in the textile industry in the world. In order to find genes involved in fiber development, a cDNA (designated as GhMADS11) encoding a novel MADS protein with 151 amino acid residues was isolated from cotton fiber cDNA library. The deduced protein shares high similarity with Arabidopsis AP1 and AGL8 in MADS domain. However, the GhMADS11 protein (being absent of the partial K-domain and normal C-terminus) is shorter than AP1 and AGL8 by the reason of gene frameshift mutation during evolution. The experimental results revealed that GhMADS11 was not a transcriptional activator, and it did not form homodimer. GhMADS11 transcripts were specifically accumulated in elongating fibers, but no or very low signals of its expression were detected in other tissues of cotton. Overexpression of GhMADS11 in fission yeast promotes atypical cell elongation by 1.4-2.0-fold. Furthermore, morphological analysis indicated that the transformed cells expressing GhMADS11m, a MIKC-type derivative of GhMADS11 by the site-directed mutation, displayed the same phenotype as that of the transformed cells with GhMADS11. The concurrence of these data sets suggested that GhMADS11 protein may function in fiber cell elongation, and its MADS domain and partial K-domain are sufficient for this function.

Frameshift mutations of vacuolar protein sorting genes in gastric and colorectal cancers with microsatellite instability

Vacuolar protein sorting plays crucial roles in the traffic of molecules between cellular organelles. Although involvement of vacuolar protein sorting proteins in cancer is known, genetic alterations of VPS genes have not been reported in cancers. We found that VPS4B, VPS13A, VPS13B, VPS13C, VPS33A, VPS35, VPS37B, VPS37D, VPS41, and VPS54 have mononucleotide repeats in their coding sequences. To see whether these genes are mutated in cancers with microsatellite instability, we analyzed the mononucleotide repeats in 30 gastric cancers with high microsatellite instability, 13 gastric cancers with low microsatellite instability, and 45 gastric cancers with stable microsatellites and 40 colorectal cancers with high microsatellite instability, 14 colorectal cancers with low microsatellite instability, and 45 colorectal cancers with stable microsatellites by single-strand conformation polymorphism. We found mutations of VPS13A, VPS13B, VPS13C, VPS33A, VPS35, VPS37B, VPS41, and VPS54 in 9, 3, 12, 3, 5, 9, 2, and 2 cancers, respectively, all in cancers with high microsatellite instability. The gastric cancers and colorectal cancers with high microsatellite instability harbored one or more mutations of the VPS genes in 53.3% and 50.0%, respectively. Loss of Vps13A expression was observed in 30% of the gastric cancers and 35% of the colorectal cancers, whereas loss of Vps35 was observed in 55% of the gastric cancers and 55% of the colorectal cancers. Our data indicate that frameshift mutations of VPS genes and losses of expression of Vps13A and Vps35 proteins are common in gastric cancers and colorectal cancers with high microsatellite instability and suggest that these alterations might contribute to development of cancers with high microsatellite instability by deregulating vacuolar protein sorting proteins.

Juvenile parkinsonism associated with heterozygous frameshift ATP13A2 gene mutation

We report a case of levodopa-responsive juvenile parkinsonism (JP) associated with a heterozygous ATP13A2 gene frameshift mutation. The clinical phenotype of our case is more severe when compared with other published reports of symptomatic heterozygous ATP13A2 mutation carriers. To our knowledge, this is the youngest reported patient with JP associated with a heterozygous ATP13A2 mutation. Our findings expand the clinical phenotypic spectrum of JP associated with heterozygous ATP13A2 mutation.

Microsatellite Instability (MSI) In High Risk Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukaemia (AML) Cells Promotes Frameshift Mutations In DNA Repair Genes: Indications for PARP Inhibitor Therapy.

AbstractAbstract 1194Despite major advances in the biology and pathogenesis of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) identification of the most effective and safest form of treatment continue to present a formidable challenge particularly in older patients. Older patients (>70 years) that constitute the majority of MDS/AML patients are often resistant to chemotherapy, achieve short lived remission and are not candidates for stem cell transplantation. Therefore the emphasis is to prolong survival or improve the quality of life. Currently, a number of therapeutic strategies are being evaluated and these include treatment with DNA methyltransferase or histone deacetylase inhibitors. Seminal work in breast cancer have shown that inhibitors of poly ADP ribose polymerase (PARP) activity can selectively target tumour cells through exploitation of inherent DNA repair defects. MDS/AML are characterized by genomic instability (GI) and single nucleotide polymorphism arrays (SNPA) karyotyping show that loss of heterozygosity (LOH) and uniparental disomy (UPD) are common in MDS/AML and it has been suggested that the underlying cause of this GI is a defect in double strand DNA repair. We have demonstrated that non homologous end joining, a major pathway for the repair of double strand DNA breaks is overactive and associated with extensive joining errors in primary AML cells. Hence, potentially MDS/AML patients are candidates for PI therapy. We have also shown more recently, that 15% of MDS/AML primary patient cells and cell lines are sensitive to PARP inhibitors (PI) through exploitation of homologous recombination DNA repair defects. To further elucidate the mechanisms that underlie PI sensitivity in MDS/AML we tested for microsatellite instability (MSI) in MDS/AML cell lines and high risk MDS patients and the presence of frameshift mutations in specific DNA repair genes that confer PI sensitivity. MSI is a change in length of a microsatellite allele caused by insertion or deletion of nucleotides that are misincorporated during DNA replication and not removed by the mismatch repair pathway. Using fluorescent PCR analysis, PI sensitive cell lines, P39, KG-1 and Molm-13 showed MSI-high (instability at ≥ 2 loci) at 5 mononucleotide microsatellites, in contrast to 12 PI insensitive cell lines that showed no MSI at these loci. We also show using fluorescent PCR and DNA sequencing that these MSI positive cell lines demonstrate MSI (monoallelic 1–2 base pair [bp] deletion) in the coding region microsatellites of DNA repair genes, Ataxia telancgiectasia mutated gene (ATM), CTiP, and MRE11. Monoallelic 1–2 bp base pair deletions at these loci produced frameshift mutations that induced aberrant gene splicing transcripts in ATM and MRE11 and a markedly truncated CTiP gene transcript. No MSI was detected in DNA repair genes CHK1, RAD50, PTEN, BLM and ATR in these cell lines and no mutations were observed at any DNA repair gene microsatellite in the 13 PI insensitive cell lines. We then determined MSI in high risk MDS patients with or without monosomy 7 (-7/del7q). 13 of 63 (21%) high risk MDS patients showed MSI (9 MSI-low and 4 MSI-high). Of the 13 MSI positive patients, 7 (4 MSI-high, 3 MSI-low) had monosomy 7 and other complex chromosomal abnormalities (Group 1, 54%), 2 (MSI-low) patients had isolated monosomy 7 (Group 2, 15%) whilst 4 patients (MSI-low) had normal cytogenetics (Group 3, 30%). Constitutional DNA from these patients did not show MSI at these loci. Significantly, however, Group 3 with MSI and normal cytogenetics all had widespread UPD and cryptic chromosome changes determined by SNPA. Strikingly, thus all 13 patients with MSI possessed chromosomal abnormalities, both gross and cryptic. Furthermore, 12 patients (19%) found to be cytogenetically normal and lacking UPD and genomic aberrations by SNPA did not show MSI. We have also identified that 3 patients with MSI-high (Group 1) and 1 patient with MSI-low (Group 3) had a monoallelic 1 bp deletion in the CTiP exon coding microsatellite. 1 bp deletion within the coding exon of CTiP resulted in an abbreviated CTiP gene transcript. In conclusion, we have made the important correlation between MSI and subsequent frameshift mutations in specific DNA repair genes with the gross and cryptic chromosomal changes observed in MDS/AML. Identification of a cohort of MDS/AML patients with MSI would herald a significant advancement for the selection of candidates for PI therapy. Disclosures:No relevant conflicts of interest to declare.

Novel somatic frameshift mutations of genes related to cell cycle and DNA damage response in gastric and colorectal cancers with microsatellite instability.

Microsatellite instability (MSI) in sporadic gastric cancer (GC) and colorectal cancer (CRC) causes frameshift mutations in gene sequences that contribute to cancer pathogenesis. Many mutations have already been identified in these two cancer types, but some are still undiscovered. We analyzed seven genes (cell cycle control and DNA damage signaling/repair-related genes) with seven or more mononucleotide repeats in 30 GC samples with high MSI (MSI-H), 15 GC samples with low MSI (MSI-L), 45 GC samples that were microsatellite stable (MSS), 33 CRC samples with MSI-H, 15 CRC samples with MSI-L, and 45 CRC samples that were MSS. Single-strand conformation polymorphism (SSCP) and DNA sequencing were used for the analysis. We found somatic frameshit mutations of the KNTC1 (6.7% GC, 12.1% CRC), ZC3H13 (3.3% GC, 15.2% CRC), CENPH (6.7% GC), TOPBP1 (3.0% CRC), NDCO80 (3.0% CRC), RIF1 (6.7% GC), and NBS1 (3.3% GC, 3.0% CRC) genes in the cancers with MSI-H. Mutations were detected in MSI-H, but not in MSI-L or MSS samples. Novel frameshift mutations occurred in seven genes in GC and CRC with MSI-H. The results of our study suggest that the mutations might contribute to the development of GC and CRC with MSI by deregulation of the cell cycle and DNA damage signaling/repair.

Evaluation of a rapid molecular screening approach for the detection of toxigenic Clostridium difficile in general and subsequent identification of the tcdC Δ117 mutation in human stools

We have developed and validated a rapid molecular screening protocol for toxigenic Clostridium difficile, that also enables the identification of the hypervirulent epidemic 027/NAP1 strain. We describe a multiplex real-time PCR assay, which detects the presence of the tcdA and tcdB genes directly in stool samples. In case of positive PCR results, a separate multiplex real-time PCR typing assay was performed targeting the tcdC gene frame shift mutation at position 117. We prospectively compared the results of the screening PCR with those of a cytotoxicity assay (CTA), and a rapid immuno-enzyme assay for 161 stool samples with a specific request for diagnosis of C. difficile infection (CDI). A total of 16 stool samples were positive by CTA. The screening PCR assay confirmed all 16 samples, and gave a PCR positive signal in eight additional samples. The typing PCR assay detected the tcdC Δ117 mutation in 2/24 samples suggesting the presence of the epidemic strain in these samples. This was confirmed by PCR ribotyping and sequencing of the tcdC gene. Using CTA as the “gold standard”, the sensitivity, specificity, positive predictive value, and negative predictive value, for the screening PCR were 100%, 94.4%, 66.7%, and 100%, respectively. In conclusion, PCR may serve as a rapid negative screening assay for patients suspected of having CDI, although the low PPV hamper the use of PCR as a standalone test. However, PCR results may provide valuable information for patient management and minimising the spread of the epidemic 027/NAP1 strain.

Mutational Analysis of ASPP1 and ASPP2 Genes, a p53-related Gene, in Gastric and Cololorectal Cancers with Microsatellite Instability

To the editor, In most human cancers, functions of p53 tumor suppressor are inactivated by many mechanisms, most commonly by somatic mutation.1 Alternatively, p53 is inactivated by genes whose protein products interact with p53.1 Apoptosis-stimulating protein of p53-1 (ASPP1) and -2 (ASPP2) promote p53-mediated apoptosis by enhancing its transactivation function.2 ASPP2+/- mice suffer from an increased incidence of tumorsm,3 suggesting it as a haploinsufficient tumor suppressor gene. However, neither of ASPP1 or ASPP2 mutation has been reported in cancers. Microsatellite instability (MSI) is characterized by length alterations in repeated DNA sequences, and 10-30% of colorectal cancer (CRC) and gastric cancer (GC) are categorized as MSI-positive cancers.4 In a public database, we found an A7 repeat in exon 8 of ASPP1 and an A7 repeat in exon 6 of ASPP2. To see whether these A7 are mutated in GC and CRC with MSI, we analyzed them by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) as described previously.5 We analyzed 32 high-MSI (MSI-H) and 13 low-MSI (MSI-L) GC, and 38 MSI-H and 15 MSI-L CRC. We used two primer pairs that amplified each A7 of ASPP1 and the ASPP2. Tumor and normal DNA from microdisscted cells were amplified by the PCR. Radioisotope ([32P]dCTP) was incorporated into the PCR for detection by SSCP. Direct DNA sequencing was performed in the cancers with mobility shifts in the SSCP. The PCR-SSCP analysis of ASPP2 identified aberrant bands in one GC with MSI-H (3.1%) and two CRC with MSI-H (5.3%), but none in those with MSI-L (Fig. 1). Normal DNA of the patients showed no shifts, indicating the mutations had risen somatically. Direct DNA sequencing identified an identical ASPP2 deletion mutation in the A7 repeat (c.576delA), which would result in a premature stop (p.Val193fsX1) and resembled a typical loss-of-function mutation. For ASPP1, there was no aberrantly migrating SSCP band in the cancer tissues. Fig. 1 Mutation of ASPP2 in a gastric carcinoma with high-microsatellite instability. (A) PCR product of ASPP2 from a colon carcinoma shows aberrant bands (arrows in lane T) as compared to single-strand conformation polymorphism from normal tissue (lane N) of ... As a possible mechanism for p53 inactivation, we analyzed ASPP1 and ASPP2 mutations in their repeat sequences and found some of the cancers with MSI-H harbored ASPP2 frameshift mutation. In colon, tumors with MSI have frameshift mutations in specific target genes, and fewer mutations are found in K-RAS and p53.4 Although the incidence of the ASPP2 mutation is not high, our data indicate that the ASPP2 mutation could partially explain a p53-inactivating mechanism in GC and CRC with MSI. Four ankylin repeats (amino acids 930-1047) and an SH3 domain (amino acids 1064-1114) of ASPP2 are essential for the interaction of ASPP2 with p53.2,3,6 The frameshift mutation identified in this study (p.Val193fsX1) would remove ankylin repeats and SH3 domain, and pro-apoptotic function of ASPP2 may be decreased in the affected cancers. Our data suggest that decreased p53 functions by the ASPP2 mutation might possibly contribute to pathogenesis in MSI-H cancers. Whether inactivation of ASPP genes further contributes to GC and CRC development in addition to the frameshift mutation of ASPP2, mutational analysis of entire coding region of ASPP1 and ASPP2 genes is required in future studies.

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 mononucleotide repeats in dual specificity tyrosine phosphatase genes in gastric and colon carcinomas with microsatellite instability

Coordinated protein phosphorylation and dephosphorylation are crucial in the regulation of cell signaling, and disruption of the coordination is known to play important roles in cancer development. Recent reports revealed that classical protein tyrosine phosphatase (PTP)-encoded genes are somatically mutated in human colorectal cancer. However, data on dual specificity phosphatase (DPTP) gene mutations in human cancers are lacking. By analyzing a public genomic database, we found that five DPTP genes, CDC14A, MTM1, MTMR3, SSH1, and SSH2, have mononucleotide repeats in their coding DNA sequences. To see whether these genes are mutated in cancers with microsatellite instability (MSI), we analyzed the mononucleotide repeats in 26 gastric cancers (GC) with MSI (MSI-H), 12 GC with low MSI (MSI-L), 45 GC with stable MSI (MSS), 33 colorectal cancers (CRC) with MSI-H, 14 CRC with MSI-L, and 45 CRC with MSS by single-strand conformation polymorphism (SSCP). We found CDC14A and MTMR3 mutations in five and one cancer (s), respectively. These mutations were detected in MSI-H cancers, but not in MSI-L or MSS cancers. The GC and CRC with MSI-H harbored the mutations in 15% and 6%, respectively. The CDC14A and MTMR3 mutations detected in the GC and CRC were deletion or duplication mutations of one base in the nucleotide repeats that would result in premature stops of the amino acid syntheses. Our data show that frameshift mutations of DPTP genes in MSI-H cancers occur at moderate frequencies. The data suggested that alterations in the CDC14A and MTMR3 genes may play a role in the development of GC and CRC with MSI-H by deregulating phosphatase functions possibly together with mutations of classical PTP genes.

Abstract 5078: Sustained Acting Atrial Natriuretic Peptide: A Novel Natriuretic Peptide With Potent Blood Pressure Lowering and Renal Enhancing Properties in a Canine Model of Acute Angiotensin Induced Hypertension

Background Sustained Acting Atrial Natriuretic Peptide (SA-ANP) is a 40 AA peptide consisting of native ANP (1–28) with a 12 AA C-terminus extension which we synthesized based upon a familial ANP gene frameshift mutation. SA-ANP possesses markedly greater and more sustained mean arterial pressure (MAP) lowering, diuretic/natriuretic, angiotensin-aldosterone suppressing, and cGMP activating properties compared to ANP in normal dogs consistent with a super agonist of the ANP receptor (NPR-A) ( JACC in press ). Here we evaluated SA-ANP in a model of acute hypertension with the hypothesis that SA-ANP would have potent MAP lowering and renal enhancing properties. Methods Integrated physiological studies were performed in a canine model of ANG induced hypertension (20 ng/kg/min continuous infusion) before, during, and up to 3 hours following a 45 minute infusion of SA-ANP (30 pmol/kg/min) (n=6) or vehicle (n=6). Results MAP is shown in Figure 1 . SVR significantly decreased and despite a significant reduction in MAP both renal blood flow (RBF) and GFR were significantly increased following SA-ANP infusion. There were significant reductions in proximal and distal tubule sodium reabsorption resulting in a significant increase (>50 fold) in diuresis and natriuresis with SA-ANP. These renal actions were associated with significant increases in plasma and urine cGMP and a significant inhibition of aldosterone activation. Discussion We report for the first time in an acute model of hypertension that SA-ANP reduced MAP while enhancing RBF, GFR, and sodium/water excretion and thus may serve as a potential therapeutic agent in the treatment of hypertension in the setting of volume overload.

Rapid detection of the Clostridium difficile ribotype 027 tcdC gene frame shift mutation at position 117 by real-time PCR and melt curve analysis

The emergence of the hypervirulent strain Clostridium difficile PCR ribotype 027 has increased the necessity for rapid C. difficile typing tests for clinical and epidemiological purposes. We developed a rapid real-time polymerase chain reaction (PCR) test for the detection of C. difficile. As the target, we chose the tcdC gene, which encodes for a negative regulator in toxin production. A deletion at position 117 of the tcdC gene, which is associated with severe tcdC truncation, is well conserved in all PCR ribotype 027 isolates. Probe sequences of the real-time PCR test were designed to result in distinct melt profiles for sequence variations at positions 117 to 120 of the tcdC gene. The tcdC gene deletion at position 117 was easily detected with real-time PCR and melt curve analysis in all C. difficile ribotype 027 isolates. In five non-027 strains and 46 hospitalised patient samples, melt curve analysis detected no deletion. PCR results were confirmed by DNA sequencing. The combination of real-time PCR and melt curve analysis is a rapid and accurate method for the detection of C. difficile DNA and simultaneous screening for the tcdC gene deletion at position 117, which is closely related to the C. difficile PCR ribotype 027 strain.

Compensation of loss of protein function in microsatellite-unstable colon cancer cells (HCT116): A gene-dependent effect on the cell surface glycan profile

Tumors that display a high level of microsatellite instability (MSI-H) accumulate somatic frameshift mutations in several genes. The compensation of this loss of function by transfection represents a suitable approach to tie respective gene deficiency to alterations in cellular characteristics. In view of the emerging significance of cell surface glycans as biochemical signals for presentation/activity of various receptors/integrins and for susceptibility to adhesion/growth-regulatory tissue lectins, we examined the glycophenotype in the MSI-H colon cancer cell line HCT116 for activin type 2 receptor (ACVR2), absent in melanoma 2 (AIM2), and transforming growth factor beta-type 2 receptor (TGFBR2) known to be associated with MSI colorectal carcinogenesis. A panel of probes specific for functional carbohydrate epitopes including human lectins was used to trace changes in cell surface levels, thereby initiating glycan analysis related to MSI. In particular, the presence of core substitutions and branching in N-glycans, the sialylation status of N- and O-glycans, and the presence of Le(a/x)-epitopes were profiled. Transient transfection affected the glycophenotype, depending on the nature of the gene and the probe. The TGFBR2 presence reduced binding of probes specific for a core substitution and increased branch length in N-glycosylation, even reaching a P-value of 0.0016. ACVR2/AIM2 influenced core 1 mucin-type O-glycosylation differentially, upregulation by ACVR2, and downregulation by AIM2. These alterations of cell surface glycosylation by gene products that are not directly associated with the machinery for glycan generation direct attention to pursue analysis of glycosylation in MSI tumor cells on the level of target glycoproteins and open the way for functional studies.

Frameshift mutations of autophagy‐related genes ATG2B, ATG5, ATG9B and ATG12 in gastric and colorectal cancers with microsatellite instability

Mounting evidence indicates that alterations of autophagy processes are directly involved in the development of many human diseases, including cancers. Autophagy-related gene (ATG) products are main players in the autophagy process. In humans there are 16 known ATG genes, of which four (ATG2B, ATG5, ATG9B and ATG12) have mononucleotide repeats with seven or more nucleotides. Frameshift mutations of genes with mononucleotide repeats are features of cancers with microsatellite instability (MSI). It is not known whether ATG genes with mononucleotide repeats are altered by frameshift mutations in gastric and colorectal carcinomas with MSI. For this, we analysed the mononecleotide repeats in ATG2B, ATG5, ATG9B and ATG12 in 32 gastric carcinomas with high MSI (MSI-H), 13 gastric carcinomas with low MSI (MSI-L), 43 colorectal carcinomas with MSI-H and 15 colorectal carcinomas with MSI-L by a single-strand conformation polymorphism (SSCP) analysis. We found ATG2B, ATG5, ATG9B and ATG12 mutations in 10, 2, 13 and 0 cancers, respectively. The mutations were detected in MSI-H cancers but not in MSI-L cancers. Gastric and colorectal cancers with MSI-H harboured one or more ATG mutations in 28.1% and 27.9%, respectively. Our data indicate that frameshift mutations in ATG genes with mononucleotide repeats are common in gastric and colorectal carcinomas with MSI-H, and suggest that these mutations may contribute to cancer development by deregulating the autophagy process.

Frameshift mutation at a mononucleotide repeat in CASP1 is rare in gastric and colorectal carcinomas with microsatellite instability

Caspase-1 was initially identified as an activating enzyme for the proteolytic maturation of pro-interleukin-1β [ [1] Soung Y.H. Jeong E.G. Ahn C.H. Kim S.S. Song S.Y. Yoo N.J. et al. Mutational analysis of caspase 1, 4, and 5 genes in common human cancers. Hum Pathol. 2008; 39: 895-900 Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar ]. In addition, it is well known that caspase-1 overexpression induces apoptotic cell death. Evasion of apoptosis is one of the acquired capabilities of cancers and is considered a hallmark of cancer. Inactivation of apoptosis by somatic mutations of genes encoding proapoptotic proteins, including CASP8 and BAX, has been reported in human cancers [ 2 Kim H.S. Lee J.W. Soung Y.H. Park W.S. Kim S.Y. Nam S.W. et al. Inactivating mutations of caspase-8 gene in colorectal carcinomas. Gastroenterology. 2003; 125: 708-715 Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar , 3 Soung Y.H. Lee J.W. Kim S.Y. Jang J. Park Y.G. Park W.S. et al. CASPASE-8 gene is inactivated by somatic mutations in gastric carcinomas. Cancer Res. 2005; 65: 815-821 PubMed Google Scholar , 4 Mori Y. Sato F. Selaru F.M. Olaru A. Perry K. Kimos M.C. et al. Instabilotyping reveals unique mutational spectra in microsatellite-unstable gastric cancers. Cancer Res. 2002; 62: 3641-3645 PubMed Google Scholar , 5 Mori Y. Yin J. Rashid A. Leggett B.A. Young J. Simms L. et al. Instabilotyping: comprehensive identification of frameshift mutations caused by coding region microsatellite instability. Cancer Res. 2001; 61: 6046-6049 PubMed Google Scholar ]. Frameshift mutations of genes containing mononucleotide repeats are features of colorectal and gastric cancers with microsatellite instability (MSI). There is a polyguanine (G8) in BAX gene that is frequently mutated in cancers with MSI [ 4 Mori Y. Sato F. Selaru F.M. Olaru A. Perry K. Kimos M.C. et al. Instabilotyping reveals unique mutational spectra in microsatellite-unstable gastric cancers. Cancer Res. 2002; 62: 3641-3645 PubMed Google Scholar , 5 Mori Y. Yin J. Rashid A. Leggett B.A. Young J. Simms L. et al. Instabilotyping: comprehensive identification of frameshift mutations caused by coding region microsatellite instability. Cancer Res. 2001; 61: 6046-6049 PubMed Google Scholar ]. The BAX mutant has a decreased apoptotic activity and is considered to contribute to cancer development.

Frameshift mutations of Wnt pathway genes AXIN2 and TCF7L2 in gastric carcinomas with high microsatellite instability

Frameshift mutations of genes with mononucleotide repeats are features of colorectal and gastric cancers with microsatellite instability (MSI). Deregulation of Wnt pathway is involved in the mechanisms of cancer development, and mutations of the Wnt-pathway genes have frequently been detected in cancers, indicating somatic mutations are important deregulation mechanisms of the Wnt signaling in cancer development. Both AXIN2 and TCF7L2 genes in the Wnt pathway possess mononucleotide repeats in their coding sequences and are considered as candidate tumor suppressor genes. The aim of this study was to see whether AXIN2 and TCF7L2 are altered by frameshift mutations in gastric carcinomas with MSI. For this, we analyzed human AXIN2 exon 8 and TCF7L2 exon 14 in 32 gastric carcinomas with high MSI, 13 gastric carcinomas with low MSI, and 47 gastric carcinomas without MSI by a single-strand conformation polymorphism analysis. Overall, we detected 9 AXIN2 and 6 TCF7L2 frameshift mutations in the mononucleotide repeats in the cancers with MSH-H, and all of them were found in MSH-H cancers (AXIN2, 28.1%; TCF7L2, 18.8%). Of the 32 high MSI cancers, 13 cancers (40.6%) harbored at least one of AXIN2 and TCF7L2 mutation, whereas 19 cancers (59.4%) harbored neither. The present data indicate that frameshift mutations in both AXIN2 and TCF7L2 genes are common in gastric carcinomas with high MSI and suggest that these mutations may contribute to development of gastric cancers with high MSI by deregulating the Wnt signaling in the affected cancer cells.

New insights into the evolution of chorionic gonadotrophin

The glycoprotein hormones luteinizing hormone (LH) and chorionic gonadotrophin (CG) are crucial for reproduction, as LH induces sex hormone production and ovulation, and CG is essential for the establishment of pregnancy and fetal male sexual differentiation. Both consist of two heterodimeric peptides of which the alpha-subunit is common to both hormones whereas the beta-subunit is hormone-specific. The CGB gene was derived from LHB by gene duplication and frame shift mutation that led to a read-through into the formerly 3'-untranslated region, giving rise to the carboxyl-terminal peptide. Owing to nucleotide changes within the 5'-region of CGB, a new transcriptional start site and regulatory region was gained. These changes led to the specific expression of CGB in the placenta and its decrease in the pituitary. Recent findings on gonadotrophins led to an extended model for the sequence of events in the evolution of the CGB gene in primates and its tissue-specific expression.

Specific Clinical and Biological Features Characterize Inflammatory Bowel Disease–Associated Colorectal Cancers Showing Microsatellite Instability

Microsatellite instability (MSI) due to mismatch repair (MMR) deficiency has been reported to occur at variable frequencies in inflammatory bowel disease-associated intestinal neoplasias (IBD-Ns). We investigated a large series of IBD-N for associations between MSI and several biologic and clinical parameters related to tumors, patients, and their treatment. A total of 277 IBD-Ns in 205 patients were screened for MSI. Biologic and clinical variables of patients with high levels of DNA microsatellite instability high (MSI-H) were collected and compared with those associated with 33 MSI-H non-IBD colorectal cancers (CRCs). A total of 27 IBD-Ns from 17 patients were found to be MSI-H. Compared with sporadic MSI-H CRCs, patients presented with a younger age at diagnosis, and there was no female predominance and no right-sided predominance. Unlike sporadic MSI-H CRCs, MSI-H IBD-Ns presented with heterogeneous mismatch repair defects involving MLH1, MSH2, MSH6, or PMS2, and a low frequency of MLH1 promoter methylation. They exhibited frequent BRAF mutations and frameshift mutations in genes containing coding repeat sequences. The mechanisms underlying MMR deficiency in MSI-H IBD-Ns are different from those in sporadic MSI-H tumors and seem to be more related to those observed in hereditary MSI-H tumors. However, BRAF mutations were observed in MSI-H IBD-Ns, similar to sporadic MSI-H tumors, but unlike hereditary MSI-H tumors. Finally, the mutational events in target genes for instability are the same in MSI-H IBD-N tumors as in non-IBD sporadic and hereditary colorectal MSI-H cancers, indicating a colon-related repertoire of target gene alterations.

Microsatellite instability and mutation analysis of candidate genes in urothelial cell carcinomas of upper urinary tract

A subset of upper urinary tract urothelial cell carcinomas (UUC), arising sporadically or as a manifestation of hereditary non-polyposis colorectal cancer, displays microsatellite instability (MSI). MSI tumours are characterized by defective mismatch repair and accumulation of frameshift mutations in numerous genes harbouring repeats in their coding sequences. We have evaluated the incidence of MSI in UUC and the intratumoral distribution of mutations in 13 candidate target genes. A total of 58 unselected UUC were screened for MSI using the panel of five mononucleotide markers recently recommended by the National Cancer Institute for a precise MSI assessment. Four tumours displayed MSI (7%), among which at least three had alterations in the genes MSH3, BAX, MRE11, RAD50. Mutations in genes involved in key cellular pathways (ATR, DNA-PKcs, MBD4, TCF-4, MSH6, and BLM) were further detected. BAX and MRE11 mutations tend to present homogeneously within the three MSI UUC. Immunohistochemistry (MLH1, MSH2, MSH6) showed that loss of mismatch repair protein expression occurred in all MSI UUC defining the gene defect and that MRE11 and RAD50 mutations were associated with their concomitant loss expression. In conclusion, MSI UUC represent a small proportion of UUC in which BAX and MRE11 mutations are frequent and may play a role early in UUC tumorigenesis.

Interplay between GCN2 and GCN4 expression, translation elongation factor 1 mutations and translational fidelity in yeast

Genetic screens in Saccharomyces cerevisiae have identified the roles of ribosome components, tRNAs and translation factors in translational fidelity. These screens rely on the suppression of altered start codons, nonsense codons or frameshift mutations in genes involved in amino acid or nucleotide metabolism. Many of these genes are regulated by the General Amino Acid Control (GAAC) pathway. Upon amino acid starvation, the kinase GCN2 induces the GAAC cascade via increased translation of the transcriptional activator GCN4 controlled by upstream open reading frames (uORFs). Overexpression of the GCN2 or GCN4 genes enhances the sensitivity of translation fidelity assays that utilize genes regulated by GCN4, such as the suppression of a +1 insertion by S.cerevisiae translation elongation factor 1A (eEF1A) mutants. Paromomycin and the prion [PSI+], which reduce translational fidelity, do not increase GCN4 expression to induce the suppression phenotype and in fact reduce derepression. eEF1A mutations that reduce translation, however, reduce expression of GCN4 under non-starvation conditions. These eEF1A mutants also reduce HIS4 mRNA expression. Taken together, this system improves in vivo strategies for the analysis of translational fidelity and further provides new information on the interplay among translation fidelity, altered elongation and translational control via uORFs.