4893

NRAS

ALPS4|N-ras|NRAS1|NS6;neuroblastoma RAS viral (v-ras) oncogene homolog;NRAS;neuroblastoma RAS viral (v-ras) oncogene homolog

GTPase NRas|N-ras protein part 4|transforming protein N-Ras|v-ras neuroblastoma RAS viral oncogene homolog

1p13.2

protein-coding

Ras proto-oncogene mutations have been implicated in the pathogenesis of many malignancies, including leukemia. While both human and animal studies have linked several chemical carcinogens to specific ras mutations, little data exist regarding the association of ras mutations with parental exposures and risk of childhood leukemia. Using data from a large case-control study of childhood acute lymphoblastic leukemia (ALL; age <15 years) conducted by the children's cancer Group, we used a case-case comparison approach to examine whether reported parental exposure to hydrocarbons at work or use of specific medications are related to ras gene mutations in the leukemia cells of children with ALL. DNA was extracted from archived bone marrow slides or cryopreserved marrow samples for 837 ALL cases. We examined mutations in K-ras and N-ras genes at codons 12, 13, and 61 by PCR and allele-specific oligonucleotide hybridization and confirmed them by DNA sequencing. We interviewed mothers and, if available, fathers by telephone to collect exposure information. Odds ratios (ORs) and 95% confidence intervals (CIs) were derived from logistic regression to examine the associationof parental exposures with ras mutations. A total of 127 (15.2%) cases had ras mutations (K-ras 4.7% and N-ras 10.68%). Both maternal (OR 3.2, 95% CI 1.7-6.1) and paternal (OR 2.0, 95% CI 1.1-3.7) reported use of mind-altering drugs were associated with N-ras mutations. Paternal use of amphetamines or diet pills was associated with N-ras mutations (OR 4.1, 95% CI 1.1-15.0); no association was observed with maternal use. Maternal exposure to solvents (OR 3.1, 95% CI 1.0-9.7) and plastic materials (OR 6.9, 95% CI 1.2-39.7) during pregnancy and plastic materials after pregnancy (OR 8.3, 95% CI 1.4-48.8) were related to K-ras mutation. Maternal ever exposure to oil and coal products before case diagnosis (OR 2.3, 95% CI 1.1-4.8) and during the postnatal period (OR 2.2, 95% CI 1.0-5.5) and paternal exposure to plastic materials before index pregnancy (OR 2.4, 95% CI 1.1-5.1) and other hydrocarbons during the postnatal period (OR 1.8, 95% CI 1.0-1.3) were associated with N-ras mutations. This study suggests that parentalexposure to specific chemicals may be associated with distinct ras mutations in children who develop ALL.

BACKGROUND: Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessedPIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as thesehave not been previously reported. METHODS: Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutationswithin the C2, helical and kinase domain "hot spots" where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations toassess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. RESULTS: We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studiedto date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model. CONCLUSION: These data suggest that activating mutationsin the Ras/Raf-MAPK/PI3K signaling cascades occur infrequently in neuroblastoma.Further, despite compelling evidence for MYC and RAS cooperation in vitro and invivo to promote tumourigenesis, activation of RAS signal transduction does not constitute a preferred secondary pathway in neuroblastomas with MYCN deregulation in either human tumors or murine models.

Rhabdomyosarcoma (RMS) is a common childhood solid tumor, resulting from dysregulation of the skeletal myogenesis program. Two major histological subtypes occur in childhood RMS, embryonal and alveolar. While chromosomal rearrangementsaccount for the majority of alveolar tumors, the genetic defects underlying the pathogenesis of embryonal RMS remain largely undetermined. A few studies performed on small series of embryonal tumors suggest that dysregulation of RAS function may be relevant to disease pathogenesis. To explore further the biological and clinical relevance of mutations with perturbing consequences on RAS signaling in embryonal RMS, we investigated the prevalence of PTPN11, HRAS, KRAS, NRAS, BRAF, MEK1, and MEK2 mutations in a relatively large cohort of primary tumors. While HRAS and KRAS were found to be rarely mutated, we identified somatic NRAS lesions in 20% of cases. ALL mutations were missense andaffected codon 61, with the introduction of a positive charged amino acid residue representing the most common event. PTPN11 was found mutated in one tumor specimen, confirming that somatic defects in this gene are relatively uncommon in RMS, while no mutation was observed in BRAF and MEK genes. Although no clear association of mutations with any clinical variable was observed, comparison of the outcome between mutation-positive and mutation-negative cases indicated a trend for a higher percentage of patients exhibiting a better outcome in the former. Our findings provide evidence that dysregulation of RAS signaling is a major event contributing to embryonal RMS pathogenesis.#CI- Copyright 2009 Wiley-Liss, Inc.

Precise data on the incidence of transforming ras oncogenes in pediatric tumors and the correlations with the histopathological properties of the tumors are very limited. Additionally the presence of ras activation in medulloblastomas has notbeen investigated so far. Using a combination of techniques including in vitro gene amplification by polymerase chain reaction (PCR) and detection of single base mutations by sequence-specific oligonucleotides we studied N-ras activation(mutations at codon 12, 13, and 61) in 32 medulloblastomas. DNA was isolated from 20 microns sections of formalin-fixed paraffin-embedded tissue. mutations were found in 3 out of 32 examined medulloblastomas. In ALL cases only mutations of codon 61 were found: two of three mutations were C to A mutations at position 1 of the codon 61 (leading to a substitution of a glutamine residue for a lysine) and one was A to T mutation at position 3 in the same codon (glutamine-histidine). Our results indicate 10% incidence N-ras mutation in medulloblastoma, higher than in other CNS tumors studied so far. The main advantages of the procedure described are its greatly improved sensitivity, the increased speed with which tumor samples can be analyzed, and the possibility ofusing paraffin-embedded sections to analyze various rare tumors in retrospect.

Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative disorder of early childhood characterized by mutations of the RAS-RAF-MAP kinase signaling pathway. We report the case of a child with a diagnosis of JMML carrying two mutations of NRAS gene (c.37G>C and c.38G>A) independently occurring in long-term culture initiating cells. However,only the former was consistently found in more mature hematopoietic cells, suggesting that cancer transformation may lead to the loss of a mutation. This case also indicates that molecular analysis on cell types other than peripheral blood leukocytes may be useful to obtain relevant biological information on JMMLpathogenesis.#CI- Copyright (c) 2011 Wiley Periodicals, Inc.

The recessive autosomal hereditary disease, xeroderma pigmentosum (XP), is characterized by a high incidence of tumors in sun-exposed skin. The defect in early steps of excision repair of XP cells leads to hypermutability towards UV-mimicking agents. DNA from eight XP tumors were screened for activated transforming genes using 3T3 transfection. In two skin tumors isolated from a XPchild, an activated N-ras oncogene was detected. Synthetic oligonucleotide probes were used to characterize the mutation in the ras gene. Both tumors were found to be mutated in the 61st N-ras codon from gln to his. The mutation was accompaniedby an increase in the level of N-ras specific mRNA and in one transformant, by the alteration of the p21 protein. In the same tumors, c-myc amplification and over transcription, and Ha-ras gene rearrangement and amplification were also detected. Analysis of other XP tumors with eleven different oncogene probes revealed an amplification of the Ha-ras gene in 6 out of 10 cases. The normal skin fibroblasts from XP patients show normal pattern levels of N-ras, c-myc andHa-ras sequences. The hypothesis is proposed that the presence of several oncogene alterations in the same tumor could be due to the high amount of UV-induced DNA lesions found in the exposed skin cells, in the absence of efficient repair.

Medulloblastoma is a malignant brain tumor of early childhood whose cells resemble the primitive neuroepithelial cells found normally in the developing nervous system. Medulloblastoma may be caused by mutational events affecting primitive neuroepithelial cells and preventing their differentiation into postmitotic neurons. The human ras genes, H-ras, K-ras and N-ras, are members ofa family of proto-oncogenes that are targets for mutational changes that convertthese normal genes into active, transforming oncogenes. Here we report that the N-ras oncogene is activated in the human medulloblastoma cell line TE 671 by a mutation at the third position of codon 61. A point mutation at this location corresponds to a substitution of histidine for glutamine in the N-ras gene product, p21. The oncogenic activation was shown by focus-formation in NIH 3T3 cell transfection assays. The location of the mutation was established using oligonucleotide hybridization assays enhanced through in vitro amplification of N-ras coding sequences using the Taq polymerase chain reaction. N-ras activationmay be one of the mutational events that subvert normal neuroectodermal differentiation and lead to medulloblastoma in children.

Using the polymerase chain reaction-single strand conformation polymorphism method and direct sequencing, 12 acute myeloid leukemia (AML) cell lines and 108fresh childhood myeloid tumor specimens, including 67 AML, 29 myelodysplastic syndrome (MDS), and 12 juvenile chronic myelocytic leukemia (JCML) were examinedfor mutation in H-, K-, and N-RAS genes. The mutation was found in eight of the 120 samples (6.7%), which consisted of four cell lines (33.3%) and four fresh myeloid tumors (3.7%). The frequency of the mutation in the cell lines was apparently higher than that in fresh myeloid tumors. K-RAS gene mutations were found in two of the 67 fresh AML specimens (3%). Interestingly, these two patients had 11q23 translocations. The N-RAS gene mutation was found in one of the 29 specimens (3.4%) of MDS and in one of the 12 specimens (8.3%) of JCML. ALL mutations were found in codon 12, 13 or 61 of the N-RAS and K-RAS genes. Frequency of mutation of RAS genes in fresh myeloid malignancies was very low. These findings suggest that mutation of RAS genes does not play an important role in the development of childhood myeloid malignancies.

The role of tumor suppressor genes and oncogenes in the development of Ewing's sarcoma has not yet been fully clarified. In this study, we analyzed the frequency of p53 tumor suppressor gene mutation in exons 4-8 by PCR-SSCP and direct sequencing, and the expression of p53-protein in Ewing's sarcoma (ES) by using immunohistochemistry. The overexpression of MDM2, which acts as a functional inactivator of p53, was studied by immunohistochemistry. In addition,a screening for point mutations in the hot spot regions codon 12 and 13 of exon 1 and codon 61 of exon 2 of ras-genes (H-ras, N-ras, K-ras) was performed. In one case, a p53 gene mutation could be confirmed in codon 238 of exon 7 (1/24). Overexpression of MDM2 was found in five cases; in ras-genes, no mutations were detected. Compared with other highly malignant mesenchymal pediatric tumors suchas osteosarcomas, mutations of p53 and ras in Ewing's sarcomas are an extraordinarily rare event. However, their frequency is comparable to that of PNET, suggesting that the low incidence of these mutations in ES and PNET could be group-specific for tumors of neuroectodermal genesis.