13th Codon Research Articles

Detection of RAS gene mutation and its clinical significance in children with acute lymphoblastic leukemia.

To investigate the mutation rate of the RAS gene and its clinical significance in children with acute lymphoblastic leukemia. A retrospective analysis was performed on the medical data of 120 children with newly diagnosed acute lymphoblastic leukemia, who were admitted to the Third Affiliated Hospital of Zhengzhou University from January 2015 to January 2020 and underwent next-generation sequencing. The clinical and molecular features were analyzed. The impact of RAS gene mutation on the overall survival rate was evaluated in these children. Among the 120 children, 35 (29.2%) had RAS gene mutation, 30 (25.0%) had KRAS gene mutation, and 5 (4.2%) had both NRAS and KRAS gene mutations. All NRAS mutations and 71% (25/35) of KRAS mutations were located at the 12th and 13th codons. RAS gene mutation was detected in 35 (33.3%) out of 105 children with B-lineage acute lymphoblastic leukemia, but it was not detected in those with acute T lymphocyte leukemia. Of all the children, 11 (9.2%) were lost to follow-up, and among the 109 children followed up, 16 (14.7%) died. The children with RAS gene mutation had a significantly lower 2-year overall survival rate than those without RAS gene mutation (P<0.05). The prognosis of children with RAS gene mutation combined with WT1 overexpression and WBC>50×109/L at diagnosis was worse (P<0.05). RAS gene mutation is commonly observed in children with B-lineage acute lymphoblastic leukemia and may have an adverse effect on prognosis.

The Absence of Mutations in the Exon 2 KRAS Gene in Several Ethnic Groups in North Sumatra May Not the Main Factor for Lung Cancer.

Background:Rat Sarcoma (RAS) protein encoded Guanosine Triphosphate (GTP-ase) activity, known as a switch of cell proliferation. The mutation of this protein alters the early stage of carcinogenesis and along with the interaction with other oncogene drivers and environmental factors affect the clinical characteristics and prognosis in cancer patients, particularly lung cancer.Objective:This study aims to determine the Kristen Rat Sarcoma (KRAS) mutation in lung cancer patients in North Sumatera and evaluate factors that might contribute in the development of lung cancer in the absence of KRAS mutation.Methods:This was a retrospective cohort study enrolled 44 subjects age > 18 year with the diagnosis of lung cancer. Histopathology preparation was obtained from surgery, bronchoscopy, and percutaneus needle biopsy then formed as paraffin-block. KRAS mutation was analyzed using Polymerase Chain Reaction (PCR) method with specific primer of exon 2 for evaluating the expression of RAS protein then continued with Sanger Sequencing Method at 12th and 13th codon.Results:The majority of subjects were male, age > 40 years old, bataknese, heavy smoker, with Adenocarcinoma. Almost all the subjects showed the expression of exon 2 of RAS protein in PCR examinations. However, Sequencing analysis using Bioedit Software, BLASTs and Finch T showed GGT GGC as protein base 219-224 which represented 12th and 13th Codon 12 and 13. The results interpreted there was no mutations of exon 2 of KRAS in North Sumatera Population.Conclusion:The absence of KRAS mutation in exon 2 in several ethnics in North Sumatera populations was not the main factors of lung cancer.

Frequency of somatic mutations in the KRAS gene in patients of the South Russia diagnosed with colorectal cancer.

e15081 Background: Colorectal cancer (CRC) is a common pathology in the world; the annual incidence rate reaches 1 million cases, and the annual mortality rate exceeds 500,000. The target therapy of EGFR with monoclonal drugs improves the survival of patients with this pathology. However, the effectiveness of therapy depends on the presence of mutations in genes involved in the EGFR signaling cascade, in particular KRAS. The purpose of this study was to determine the frequency of somatic mutations in the KRAS gene in tumor samples of patients from the South Russia diagnosed with colorectal cancer, as well as to analyze the effect of point mutations G12A, G12C, G12D, G12R, G12S, G12V and G13D on tumor metastasis. Methods: 744 patients diagnosed with colorectal adenocarcinoma were examined for mutations in 12th and 13th codons of KRAS. Somatic mutations in the KRAS gene were detected with reagent kit Biolink (Russia) using FFPE DNA samples of tumor tissues. Statistical analysis was performed using the method of binary logistic multiple regression and χ2-test. Results: Mutations in the KRAS gene were found in 32% of patients in the pooled sample; the most common mutation was the replacement of G12D in the KRAS gene (36%). Less common detectable mutations in the KRAS gene were G13D (18%), G12V (16%), G12A (10%), G12C (8%), G12S (10%), G12R (2%). Metastatic CRC was found in 86% of carriers of the wild-type allele and in 88% of carriers of the mutant allele. The presence of a G12V substitution almost 6 times increased the risk of metastasis in patients with CRC (OR = 5.89; 95% CI: 0.8-43.45; p = 0.049). Conclusions: Mutations in the KRAS gene were found in 32% of patients with CRC in the South of Russia. A significant association of G12V replacement with a high risk of metastasis was established. Further study of somatic mutations in the KRAS gene will allow to choose the most effective therapeutic strategies in the presence of one or another of the studied mutations.

Relationship of Ras,MAPK and CyclinD1 Pathway in Carcinogenesis of Cutaneous Pathologic Scar

Objective To detect the function of Ras/Raf/MAPK pathway and the downstream target gene CyclinD1 in the carcinogenesis of cutaneous pathologic scar.Methods(1) We adopted immunofluorescence dual staining for K-ras,H-ras and N-ras in the pathologic scar and carcinoma of scar,respectively,and observed under laser confocal microscopy;(2) Immunohistochemistry(IHC) for MAPK and CyclinD1 were used for normal skin,pathologic scar and carcinoma of scar,respectively;(3) In situ hybridization(ISH) was used for mRNA detection of MAPK and CyclinD1;(4) The 12th and 13th codon mutations of K-ras,H-ras and N-ras were detected by genetic sequencing.Results(1) The dual labeling of immunofluorescence of K-ras,H-ras and N-ras showed weak positive in pathologic scar,while the carcinoma of scar were strong positive.(2) ISH for mRNA of MAPK and CyclinD1 showed negative or weak positive in the normal epidermis and pathological scar,but strong positive in the carcinoma of scar.The expression level(positive area) and intensity(average optical density) between the carcinoma group and normal skin group or pathologic scar group were statistical different(P0.01);but without statistical difference between normal skin group and pathologic scar group(P0.05).(3) Genetic sequencing did not show mutation on the 12th or 13th codon.Conclusion(1) Ras,MAPK and CyclinD1 were not early signals for carcinogenesis of pathologic scar.(2) The 12th or 13th codon mutation of K-ras,H-ras and N-ras was not related with the carcinogenesis of pathologic scar.

Potential roles of synonymous codon usage and tRNA concentration in hosts on the two initiation regions of foot-and-mouth disease virus RNA

The open reading frame of foot-and-mouth disease virus (FMDV) contains two authentic initiation codons and the second initiation codon is often selected in high frequency. In the study, we analyzed the effects of the host-cell synonymous codon usage and the overall tRNA concentration in the hosts on the region flanked by the two initiation codons (termed as the region 1) and the same length starting from the second initiation codon (defined as the region 2). We find that low-usage codons of hosts are more selected in the region 1 than the region 2; no obvious usage bias of codon with high C/G content exists in the region 1, and the latter part (ranging from the 13th codon position to the 28th codon position) of the region 1 generally contains the codon sites with the generally lower tRNA concentration than the counterpart of the region 2. The low-usage codons of the hosts with high selection in the region 1 and the cluster codon position with low tRNA concentration in the region 1 may serve as potential factors in decreasing the translation rate of the region 1 caused by initiation from the first start codon of FMDV.

Inhibition of Translation by Consecutive Rare Leucine Codons in &lt;I&gt;E. coli&lt;/I&gt;: Absence of Effect of Varying mRNA Stability

Consecutive homologous codons that are rarely used in E. coli are known to inhibit translation to varying degrees. As few as two consecutive rare arginine codons exhibit a profound inhibition of translation when they are located in the 5' portion of a gene in E. coli. We have previously shown that nine consecutive rare CUA leucine codons cause almost complete inhibition of translation when they are placed after the 13th codon of a test message (although they do not inhibit translation when they are placed in the middle of the message). In the present work, we report that five consecutive rare CUA leucine codons exhibit approximately a threefold inhibition of translation when they are similarly placed after the 13th codon of a test message, compared to five consecutive common CUG leucine codons, in a T7 RNA polymerase-driven system. Further, by removing RNase III processing sites at the 3' ends of the mRNAs, we have manipulated the stability of the mRNAs encoding the test and control messages to see if decreasing mRNA stability might have an effect on the extent of translation inhibition by the rare leucine codons. However, the inhibition with the less stable mRNAs was similar to that with the stable mRNAs, approximately 3.4-fold, indicating that mRNA stability per se does not have a major influence on the effects of rare codons in this system.

Tissue-specific alternative splicing of mouse brain type ryanodine receptor/calcium release channel mRNA

We detected alternative splicing of the mouse brain type ryanodine receptor (RyR3) mRNA. The splicing variant was located in the transmembrane segment. The non-splicing type (RyR3-II) included a stretch of 341 bp, and that of the 13th codon was stop codon TAA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis shows that RyR3-II mRNA was expressed in various peripheral tissues and brain at all developmental stages. However, interestingly, the splicing type (RyR3-I) mRNA was detected only in the cerebrum. These findings suggest that the splicing variants RyR3-I and RyR3-II may generate functional differences of RyR3 in a tissue-specific manner.

Mouse lung tumors exhibit specific Ki-ras mutations following transplacental exposure to 3-methylcholanthrene.

A pharmacogenetic mouse model was utilized to determine the role of Cyp1a1 expression on the formation of Ki-ras mutations in lung tumors following transplacental exposure to polycyclic aromatic hydrocarbons (PAHs). A backcross between Ah responsive male B6D2F1 mice and nonresponsive female DBA mice resulted in a litter in which both responsive and nonresponsive fetuses resided in the same nonresponsive maternal environment. Pregnant mothers received a single i.p. injection of either 10 or 30 mg/kg of 3-methylcholanthrene (MC) or olive oil vehicle on day 17 of gestation. At the higher dose of MC, the responsive offspring of both sexes had significantly (P < 0.05) higher incidences of lung tumors than their nonresponsive littermates. The male responsive mice also exhibited a significantly increased liver tumor incidence over the nonresponsive mice at the P < 0.05 level. Administration of 10 mg/kg of MC caused a very low incidence of lung tumors and did not result in the appearance of macroscopically visible liver tumors. Exons 1 and 2 of the Ki-ras gene were amplified from paraffin-embedded tissue samples. The PCR products were screened by allele-specific oligonucleotide hybridization (ASO). Thirteen of 16 lung tumors (81%) screened exhibited point mutations in the 12th or 13th codon, including seven tumors that contained GGT-->GTT (GLY12-->VAL12) transversions, four which exhibited GGT-->TGT (GLY12-->CYS12) transversions, and two which contained GGC-->CGC (GLY13-->ARG13) transversions. None of the tumors had mutations at codon 61. The results obtained by ASO were confirmed by cloning and sequence analysis of the PCR products from four of these tumors. Within the subset of 16 tumors examined in this study, the same types of mutations in the Ki-ras gene were generally present in both responsive and nonresponsive mice, although G-->C transversions were found in two tumors from a single responsive female mouse. Interestingly, while both males and females exhibited the GGT-->GTT mutations at codon 12, the GGT-->TGT transversion was only found in male mice. These results are consistent with a key role for Cyp1a1 in modulating individual susceptibility to cancer formation through the formation of reactive intermediates that bind to DNA and result in activating mutations in key regulatory molecules.

A conundrum in molecular toxicology: molecular and biological changes during neoplastic transformation of human cells.

The process of multistage carcinogenesis lends itself to the concept that the effects of carcinogens are mediated through dose-related, multi-hit, linear changes. Multiple in vitro model systems have been developed that are designed to examine the cellular changes associated with the progression of cells through the different stages in the process; however, these systems may have inherent limitations due to the cell lines used for these studies, the manner of assessing the effects of the carcinogens, and the subsequent growth and differentiation of the exposed cells. Each of these variables results in increasing levels of uncertainty relative to the correlation of the events with the actual process of human tumor development. Therefore, the prediction of the ultimate effect of any carcinogen is difficult. Moreover, relationships between individual biological endpoints resulting from carcinogen treatment appear at best to be approximations. The presence of an activated carcinogen inside the cell can give rise to multiple outcomes, only some of which may be critical events. For example, site-specific modification of the 12th and 13th codons of H-ras is different than that in the adjacent 14th and 15th codons. It is interesting to speculate what effect these differences might have on a biological outcome, e.g., transformation to anchorage-independent growth. The use of different model systems to examine the effects of activated carcinogens also creates additional problems. Comparisons of in vitro transformed cells with similar cells isolated from human tumors indicate that the culture environment appears to influence the expression of a particular phenotype, in that human tumor cells in culture express many of the same parameters as those found in cells transformed with carcinogens in vitro. If the process of transformation is linear, then less aggressive phenotypes should progress to a more aggressive transformed stage. However, in carcinogen-transformed human cells, the populations exhibit phenotypic diversity in that many of the transformed cells differentiate and fail to continue to divide in culture. Historically, we have assumed only a limited role for epigenetic modulation of molecular changes that occur during progression; however, our data suggest quite strongly that nonmalignant tumor populations can be converted to a more malignant phenotype without additional mutations taking place and, conversely, malignant populations can be downregulated to a nontumorigenic phenotype. Tumor cell plasticity is not only a fundamental characteristic of diverse types of human tumors, but also appears as an integral characteristic of carcinogen-transformed cells in vitro.

7,12-Dimethylbenz [a] anthracene-Induced Mouse Keratinocyte Malignant Transformation Independent of Harvey ras Activation

Independent clones of mouse keratinocytes initiated in vitro gave rise to tumor phenotypes typical of mouse skin multistage carcinogenesis and histologically similar to human tumors of the skin, and head and neck. High-molecular-weight genomic DNAs isolated from two 7,12-dimethylbenz[a]anthracene (DMBA)-initiated murine epithelial carcinoma cell lines and one papilloma cell line were examined for transforming activity by transfection into NIH3T3 cells. DNAs from each of these cell lines resulted in the formation of foci morphologically unlike foci containing an activated c-Ha-ras oncogene. Following polymerase chain reaction amplification of the c-Ha-ras gene, Xba I restriction analysis and oligonucleotide differential hybridization did not detect 61st, 12th, or 13th codon mutations. Southern and Northern analysis confirmed that the normal c-Ha-ras gene was not activated by amplification or overexpression. These results provide evidence that 7,12-dimethylbenz[a]anthracene-induced malignant transformation of murine keratinocytes occurred independent of point mutations associated with c-Ha-ras activation. The absence of an activated c-Ha-ras oncogene in these cell lines distinguishes our model from other mouse models of carcinogenesis and may provide a model for functional genetic changes during initiation and progression of human epithelial cancers.

Prevalence of G-to-T transversions among K-ras oncogene mutations in human colorectal tumors in Yugoslavia.

Human colorectal carcinoma tissue sampled from 37 patients, routinely graded into Dukes' stages A, B and C and histologically examined for the level of differentiation, were analyzed for the presence of point mutations in the K-ras oncogene. Seventeen cases out of the 37 analyzed were found to have a mutation in either the 12th or the 13th codon of the K-ras gene, giving an overall frequency of mutation of 46%. The incidence of mutations in Dukes' stages A, B and C was 33, 46 and 58% respectively. Although the frequency of mutation appears to be similar to that reported for the USA population, the spectrum of point mutations in codons 12 and 13 of the K-ras gene in the Yugoslav population appears to differ significantly. G-to-T transversions make up 77% of all mutations present, with the distribution as follows: 18% at the first base and 59% at the second base of codons 12 and 13. G-to-A transitions at the second base is the only other mutation identified, occurring mainly in codon 13 in colorectal tumors of all 3 stages.

C-N-ras is activated infrequently in canine malignant lymphoma.

Activated c-N-ras alleles have been detected in human lymphoma specimens. The aim of the present study was to determine the frequency of c-N-ras mutational activation in canine malignant lymphoma. DNA was isolated from 28 canine malignant lymphoma specimens collected from 28 separate dogs and examined for c-N-ras mutations by polymerase chain reaction amplification and direct sequencing. The tumors were naturally occurring and derived from 20 dogs with known exposures to the phenoxy herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and from 8 dogs with no known exposure to the herbicide. An oncogenically activating mutation was found in 1 dog without known 2,4-D exposure. The mutation was a 13th codon, second position transition that would result in a glycine-to-aspartate amino acid substitution. The results of this study demonstrate that, similar to the human, c-N-ras mutations are uncommon in dogs with malignant lymphoma and that there is no association between 2,4-D exposure and activation of c-N-ras in the dog.

Characterization of benzo[a]pyrene-initiated mouse skin papillomas for Ha-ras mutations and protein kinase C levels.

The frequency and spectrum of Ha-ras mutations in benzo[a]pyrene (B[a]P)-initiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted CD-1 mouse skin papillomas were characterized by amplifying high molecular weight papilloma DNA using the polymerase chain reaction (PCR) followed by direct DNA sequencing. Analysis of 10 individual B[a]P-initiated early emergence papillomas indicated that 90% contained a Ha-ras mutation. Twenty percent of these papillomas contained a GGA-->GTA transversion in the 12th codon, 50% contained a GGC-->GTC transversion in the 13th codon and 20% contained a CAA-->CTA transversion in the 61st codon. A characteristic of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated papillomas, which contain an A-->T mutation in the 61st codon of Ha-ras, is that they exhibit a constitutive decrease in both protein kinase C (PKC) activity and PKC alpha and beta 2 isozyme levels when compared to epidermis. In the present study we found that total PKC activity, as well as PKC alpha and beta 2 isoforms, were markedly decreased in B[a]P-initiated early emergence papillomas and that this decrease was also accompanied by an altered subcellular distribution of PKC activity. The particulate/cytosolic (P/C) ratio of PKC activity in the epidermis was 0.39, whereas the P/C ratio in the papillomas was 0.77. These results demonstrate that B[a]P-initiated/TPA-promoted papillomas exhibit a high incidence of specific ras mutations and that PKC levels are constitutively decreased in these papillomas, indicating that an activated ras gene is associated with and may contribute to the observed decrease in PKC levels.

Association of Ki-ras mutation with differentiation and tumor-formation pathways in colorectal carcinoma.

The occurrence of a point mutation on the 12th and 13th codons of the Ki-ras oncogene has been investigated in 99 colorectal carcinomas in relation to 3 histological parameters: extent of differentiation, occurrence of a mucinous component within the tumor, and presence of peripheral adenomatous polyp remnants. The mutation frequency increased with each parameter: from 13% (2/15) to 44% (37/84) with differentiation, from 33% (26/79) to 65% (13/20) with mucinous character, and from 27% (15/56) to 56% (24/43) with the presence of polyp remnants. The frequency was highest in well-differentiated mucinous tumors with adenomatous remnants 83% (5/6). We suggest that Ki-ras mutation is preferentially involved in carcinomas that have developed from adenoma and that the mutation preserves differentiation and mucin secretion in these cancer cells.

RAS Mutations Are Rare Events in Philadelphia Chromosome-Negative/bcr Gene Rearrangement-Negative Chronic Myelogenous Leukemia, But Are Prevalent in Chronic Myelomonocytic Leukemia

Previous reports have indicated that mutations of the RAS oncogenes are not associated with the chronic phase of Philadelphia chromosome-positive chronic myelogenous leukemia (Ph1+ CML). However, further studies were needed to determine their association with Ph1- CML and chronic myelomonocytic leukemia (CMML). Therefore, 6 patients with Ph1- CML who were also negative for BCR rearrangements (Ph1-/BCR- CML) and 30 patients with CMML were analyzed for the presence of RAS oncogene point mutations to determine the similarities of these diseases at the molecular level. The assay used the polymerase chain reaction for amplification of the target RAS sequences and panels of specific synthetic oligonucleotide probes for hybridization to wild type and/or mutated sequences. None of the six Ph1-/BCR- CML patients had mutations in the RAS oncogenes, while 17 of 30 (57%) of the CMML patients had RAS oncogene mutations. Eighty percent of the mutations involved substitution of aspartic acid for glycine (G----A) in the 12th or 13th codons of N-ras or K-ras. Furthermore, although not statistically significant, survival studies raise the possibility of shortened survival in patients with RAS oncogene point mutations, with the average survival being 33 months for Ph1-/BCR- CML, 35 months for CMML without point mutations, and 11 months for CMML with RAS mutations. Thus, RAS mutations appear to be associated with CMML and not Ph1-/BCR- chronic phase CML, there is a high propensity for the K-ras or N-ras mutations to involve an G----A substitution in the 12th or 13th codons, and RAS mutations in CMML may relate to prognosis and require further studies.

RAS mutations are rare events in Philadelphia chromosome-negative/bcr gene rearrangement-negative chronic myelogenous leukemia, but are prevalent in chronic myelomonocytic leukemia

Previous reports have indicated that mutations of the RAS oncogenes are not associated with the chronic phase of Philadelphia chromosome- positive chronic myelogenous leukemia (Ph1+ CML). However, further studies were needed to determine their association with Ph1- CML and chronic myelomonocytic leukemia (CMML). Therefore, 6 patients with Ph1- CML who were also negative for BCR rearrangements (Ph1-/BCR- CML) and 30 patients with CMML were analyzed for the presence of RAS oncogene point mutations to determine the similarities of these diseases at the molecular level. The assay used the polymerase chain reaction for amplification of the target RAS sequences and panels of specific synthetic oligonucleotide probes for hybridization to wild type and/or mutated sequences. None of the six Ph1-/BCR- CML patients had mutations in the RAS oncogenes, while 17 of 30 (57%) of the CMML patients had RAS oncogene mutations. Eighty percent of the mutations involved substitution of aspartic acid for glycine (G----A) in the 12th or 13th codons of N-ras or K-ras. Furthermore, although not statistically significant, survival studies raise the possibility of shortened survival in patients with RAS oncogene point mutations, with the average survival being 33 months for Ph1-/BCR- CML, 35 months for CMML without point mutations, and 11 months for CMML with RAS mutations. Thus, RAS mutations appear to be associated with CMML and not Ph1-/BCR- chronic phase CML, there is a high propensity for the K-ras or N-ras mutations to involve an G----A substitution in the 12th or 13th codons, and RAS mutations in CMML may relate to prognosis and require further studies.