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Results in Briefinstability, and served to classify MSI-tumors. Furthermore, and in agreement with the CIMPconcept, the samples with MSI and hypermethylation of several genes also carried BRAFmutations. The promoters of ADAMTS1, MAL, and MGMT were frequently methylatedboth in benign and malignant tumors, independent of microsatellite instability. In addition,MGMT was the gene with most frequent promoter hypermethylation among the normalsamples taken in distance from primary tumors, which may indicate that it is involved in thecreation of a “field effect”. From these data we conclude that methylated ADAMTS1,MGMT, and MAL are suitable as markers for early tumor detection.Paper III. “Dysregulation of RAS signaling through alterations of RAS, RAF, NF1 and/orRASSF1A in colorectal carcinomas with known microsatellite instability status”In this article, four components involved in MAP-kinase signaling were analyzed in a seriesof colorectal carcinomas. The MAP-kinase pathway is shown to be hyperactive in a largefraction of CRC due to mutations in KRAS and BRAF, but the possible role of NF1, anegative regulator of KRAS signaling, had never been examined in a series of CRC. This ismost likely due to the large size of the gene (61 exons) and the fact that no mutation hotspotsor mutation cluster region have been identified. A total of 65 colorectal carcinomaswere included in the study, in which mutations in BRAF and KRAS was assessed by directsequencing, and promoter hypermethylation of the RASSF1A was analyzed by MSP. Arepresentative subset of these tumors (n =24) was included in the NF1-analysis, which wasperformed with denaturing high-performance liquid chromatography (dHPLC), sequencing,multiple ligation-dependent probe amplification (MLPA) and real-time polymerase chainreaction (PCR). Forty percent of the carcinomas (26/65) carried a KRAS mutation, and allbut two (c.184-189delGAG and c.49insTTG) occurred in codon 12, 13 or 61 which areknown to produce a constitutively active protein. BRAF was mutated in 22% (14/64) of thecarcinomas, and all but three of the mutations were the V600E mutation which also yields aconstitutively active protein. Mutations in KRAS and BRAF were mutually exclusive, andwhile BRAF mutations were strongly associated with MSI (P = 0.006), KRAS mutationswere more common among MSS-samples (P = 0.08). We found that 31% (18/59) sampleswere hypermethylated in the promoter of RASSF1A, but there were no covariance betweenRASSF1A methylation and mutation status of either of the analyzed genes. One of the 24carcinomas analyzed for NF1 mutations contained two missense mutations and additional40
Results in Briefnine tumors displayed intronic mutations in close proximity to the intron–exon boundaries.Using MLPA, we found that another 17% (4/24) samples had a gain of parts or of the wholegene, also confirmed with real-time analysis. Furthermore, 8 of 10 samples with exonic orintronic alterations in NF1 occurred in MSI-positive tumors (P = 0.047), whereas 3 of 4duplications occurred in MSS tumors.In total we found that 74% (48/65) of the tumors most likely had an overactive RASsignaling pathway due to molecular changes of at least one of the four analyzed components.In this study, we found the NF1 mutation profile to be in contrast both to publishedgermline mutation profile of NF1 patients as well as to the somatic mutation profiles ofmalignant peripheral nerve sheath tumor taken from patients with and without the NF1disease[134-136] §§ . One may speculate whether alternative splicing of NF1 is involved incolorectal tumorigenesis.Paper IV. “Identification of RCC2 as a prognostic marker among multiple gene mutations in colorectalcancer with defect mismatch repair”Forty-one known genes with coding oligonucleotide repeats were analyzed in two series ofmicrosatellite unstable colorectal carcinomas (n = 202) in order to identify frameshiftmutations. In a previous literature survey 162 analyzed genes were recognized. Differentselection criteria narrowed down the number of genes with a potential impact on tumordevelopment to 41 which were analyzed with fragment analysis. The aim of the study was tosubclassify the MSI-tumor into those with good and those with poor prognosis based onmutation profile of one or a combination of the genes.In total, the two series of MSI-tumors carried a median number of 17 and 19 mutations. Astrong association was seen between low mutation frequency and rectal location forindividual genes (ACVR2A, ASTE1, CASP5, MARCKS, MBD4, MRE11A, MSH3, TAF1Band TFGBR2) as well as on the total level of mutations (P = 0.008). A big difference inmutation frequency between a small number of MSI-L tumors and the MSI-H tumors wasalso seen, indicating that a low degree of MSI is insufficient to induce the mutator phenotypein CRC.§§ NF1 International Mutation Database (http://www.nfmutation.org)41
Page 1 and 2: Novel genetic and epigenetic altera
Page 3 and 4: TABLE OF CONTENTSACKNOWLEDGEMENTS .
Page 5 and 6: ACKNOWLEDGEMENTSThe present work ha
Page 7 and 8: Prefacetechnology[3]. This new tech
Page 10 and 11: SummaryThe subgroup of carcinomas w
Page 12 and 13: Introduction“Epigenetic inheritan
Page 14 and 15: Introductionamino acid change it is
Page 16 and 17: Introductionmethylation during embr
Page 18 and 19: IntroductionDNA is most of the time
Page 20 and 21: IntroductionFigure 5. DNA methylati
Page 22 and 23: IntroductionFigure 6. Incidence rat
Page 24 and 25: IntroductionFigure 8. Tumor staging
Page 26 and 27: Introductioninasmuch as 80% of colo
Page 28 and 29: IntroductionInstabilities involved
Page 30 and 31: Introductionthere seems to be a fid
Page 32 and 33: Introductionsevere alterations are
Page 34 and 35: Introductionpopulation-wide screeni
Page 36 and 37: IntroductionFigure 12. Present and
Page 38 and 39: RESULTS IN BRIEFPaper Ia. “DNA hy
Page 42 and 43: Results in BriefUnivariate survival
Page 44 and 45: Discussionseveral factors, and full
Page 46 and 47: Discussionlow threshold, we increas
Page 48 and 49: DiscussionIt may seem like unnecess
Page 50 and 51: Discussionthan 96% DHPLC do not sta
Page 52 and 53: DiscussionFigure 13. Mutation detec
Page 54 and 55: DiscussionClinical impact of molecu
Page 56 and 57: Discussionmarkers with a very high
Page 58 and 59: Discussionchromosomes in metaphase[
Page 60 and 61: DiscussionThese examples underline
Page 62 and 63: Discussiongenes. One is based on mu
Page 64 and 65: CONCLUSIONSWe have identified novel
Page 66 and 67: Future PerspectivesMolecular risk a
Page 68 and 69: REFERENCES1. Breasted J (1930) The
Page 70 and 71: References29. Deng G, Chen A, Pong
Page 72 and 73: References57. Al-Sukhni W, Aronson
Page 74 and 75: References84. Kunkel TA (1993) Nucl
Page 76 and 77: ReferencesLeggett B, Levine J, Kim
Page 78 and 79: References133. Lind GE, Thorstensen
Page 80 and 81: References156. Meling GI, Lothe RA,
Page 82 and 83: ReferencesT, Song X, Day RH, Sledzi
Page 84 and 85: References196. Honda S, Haruta M, S
Page 86 and 87: ORIGINAL ARTICLESAPPENDIXAppendix I
Page 89 and 90: GASTROENTEROLOGY 2007;132:1631-1639
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Paper IbGuro E Lind, Terje Ahlquist
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Journal of Translational Medicine 2
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Paper IITerje Ahlquist, Guro E Lind
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BackgroundMost cases of colorectal
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ADAMTS1 CDKN2A CRABP1 HOXA9 MAL MGM
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pseudogene, leading to a high rate
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strands. Proc Natl Acad Sci U S A 1
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concomitant absence of transcript a
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Volume 10 Number 7 July 2008 pp. 68
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682 RAS Signaling in Colorectal Car
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Table W2. Detailed Somatic Events o
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Identification of RCC2 as a prognos
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INTRODUCTIONMicrosatellite instabil
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unselected series of primary tumors
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specificity, i.e. that they only am
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On the assumption that DNA repair a
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In order to ensure that gene mutati
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Figure 2. Mutation frequency differ
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and TAF1B (0.50), ACVR2A and ASTE1
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Multivariate analysesA multivariate
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When comparing our findings of muta
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The test series included a low numb
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entering M-phase remains to be seen
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12. Duval A, Reperant M, Hamelin R
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34. Martineau-Thuillier S, Andreass
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AppendicesAppendix I:List of abbrev
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Critical Reviews TM in Oncogenesis,
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TARGET GENES OF MSI COLORECTAL CANC
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