RØYRVIK ET AL.gene must be validated by functional suppressor studies <strong>in</strong> <strong>in</strong> vivo or <strong>in</strong> vitromodels. These criteria have been criticized as excessively narrow: 160 (1) Inactivationevents are <strong>no</strong>t the only functionally important types of mutation, for example,AXIN2 <strong>and</strong> TCF4 are ack<strong>no</strong>wledged target genes but the mutations to which theyare subject are <strong>no</strong>t expected to be <strong>in</strong>activat<strong>in</strong>g. (2) Biallelic <strong>in</strong>activation need <strong>no</strong>tbe a requirement <strong>in</strong> the case of haplo<strong>in</strong>sufficiency (see below), c.f. the dearth ofbiallelic TAF1B <strong>in</strong>activation. 44 To be truly useful, 3 <strong>and</strong> 4 would have to entailcomplete k<strong>no</strong>wledge both of all possible growth suppressor pathways <strong>and</strong> ofevery gene or pathway <strong>in</strong>volved mismatch repair proficient cancers, <strong>and</strong> 4 <strong>in</strong>cludesthe assumption that the molecular pathway to MSS <strong>and</strong> MSI tumors areessentially equivalent, which is <strong>no</strong>t necessarily the case. Nor do all ack<strong>no</strong>wledgedtarget genes participate <strong>in</strong> growth suppressor pathways, most <strong>no</strong>tablyMSH3/6. 159 Regard<strong>in</strong>g criterion 5, such studies are lack<strong>in</strong>g for most target genes,<strong>and</strong> furthermore, it is unsuitable for several types of potential target genes. Thetransformed phe<strong>no</strong>type, for example, will <strong>no</strong>t be reversed <strong>in</strong> the event of re<strong>in</strong>troductio<strong>no</strong>f a wild-type mutator target gene to a system, 160 <strong>and</strong> a gene with <strong>no</strong>k<strong>no</strong>wn functional significance <strong>in</strong> tumor progression may yet be of prog<strong>no</strong>sticcl<strong>in</strong>ical significance. Among the genes which have been subject to functionalstudies are AXIN2, BAX, E2F4, RIZ, TCF4, <strong>and</strong> TGFRII. 45,68,111,156,161,162Duval <strong>and</strong> Hamel<strong>in</strong> proposed a fourfold, functional classification of affectedgenes <strong>in</strong>to survivor genes, hibernator genes, cooperator genes <strong>and</strong> transformatorgenes. 159Survivor genes encode vital products <strong>and</strong> whose <strong>in</strong>activation should exert anegative selection pressure. Hibernator genes are <strong>no</strong>nvital <strong>and</strong> downregulated,<strong>and</strong> should have a mutation rate <strong>in</strong> the background range. Cooperator genes designatesets of genes with the same term<strong>in</strong>al effect, e.g., promotion of apoptosis,which have a synergistic effect without any one gene requir<strong>in</strong>g a high mutationfrequency. Transformator genes are those which, upon mutation, <strong>in</strong>dependentlyconfer a selection advantage to the cells concerned, <strong>and</strong> therefore should have thehighest mutation frequency. These categories are thought to be mutated <strong>in</strong> a preferentialorder, with the transformator TGFRII be<strong>in</strong>g among the earliest. 49,16352The statistical regression model presented by Woerner et al. takes <strong>in</strong>to considerationthe fact that longer repetitive tracts are more mutable (see below), i.e.the background rate for them is higher, <strong>and</strong> a gene with a mutation frequencyabove the 95% prediction <strong>in</strong>terval for any given repeat length is considered a realtarget gene. TGFRII, BAX, TCF4, MSH3, ACVR2, PTHL3, HT001, <strong>and</strong>SLC23A1 are, by this method, considered genu<strong>in</strong>e positive targets for MSI colorectalcancer, while the authors ack<strong>no</strong>wledge the <strong>in</strong>applicability of the model asregards target pathways, c.f. cooperator genes above.Due to the difficulties of implement<strong>in</strong>g clear-cut qualitative criteria—functional aspects of s<strong>in</strong>gle <strong>genetic</strong> products <strong>and</strong> their <strong>in</strong>teractions are often <strong>in</strong>sufficientlyelucidated, likewise signal<strong>in</strong>g pathways <strong>and</strong> cascades—it has beenmost common to use the unmanipulated mutation frequency as the primary or240
TARGET GENES OF MSI COLORECTAL CANCEReven sole criterion for target gene detection, <strong>and</strong> to treat any <strong>in</strong>volvement of afrequently mutated gene <strong>in</strong>, e.g., apoptosis or cell cycle control as a bonus. A<strong>no</strong>therpotentially complicat<strong>in</strong>g factor is that, however detrimental frameshiftsusually are, mutations <strong>in</strong> the repetitive tracts of target genes do <strong>no</strong>t <strong>in</strong>variablycause complete <strong>in</strong>activation. This appears to be the case for AXIN2, where themutated product is more stable than wild type <strong>and</strong> may have a dom<strong>in</strong>ant negativeeffect, 68 <strong>and</strong> also for the mutated isoform of TCF4. 162 Both mutated gene productsencourage <strong>in</strong>appropriate WNT signal<strong>in</strong>g activation, which is cancerpromot<strong>in</strong>g.164The very fact that so many different mutational constellations exist suggeststhat the are few, if any, truly key genes for carc<strong>in</strong>ogenesis among the target genes,TGFRII be<strong>in</strong>g the only one to have been accorded such status. 44,55,159 Rather, thecumulative effect of many different <strong>and</strong> <strong>in</strong>terchangeable mutations may drivetumorigenesis, with very few of the total be<strong>in</strong>g decisive <strong>in</strong> themselves. 159 Nor isit likely that all the relevant microsatellite-conta<strong>in</strong><strong>in</strong>g genes have as yet beentested for mutations <strong>in</strong> MSI tumors, or even necessarily characterized <strong>in</strong> anyDNA sequence database. Several studies have used ge<strong>no</strong>me-wide sequence databasesearches for genes conta<strong>in</strong><strong>in</strong>g cMNRs as a basis for potential target gene selection.44,53,55,105,119 Such a search * currently yields well over 1000 prote<strong>in</strong>-cod<strong>in</strong>ggenes conta<strong>in</strong><strong>in</strong>g the most promis<strong>in</strong>g (N)8 repeats, the figure ris<strong>in</strong>g more thantenfold when the range is exp<strong>and</strong>ed to <strong>in</strong>clude (N)6-7. Even allow<strong>in</strong>g for unpublishednegative data, it may be seen from the number of genes <strong>in</strong> Table I thatfundamental target genes may still lurk <strong>in</strong> the unplumbed depths of the humange<strong>no</strong>me.VII. PERSPECTIVESIn addition to the mutational frequency <strong>and</strong> association studies of small numbersof target genes, future work will hopefully determ<strong>in</strong>e the prog<strong>no</strong>stic values ofdist<strong>in</strong>ct comb<strong>in</strong>ations of target genes—the prog<strong>no</strong>stic value of any target genemutations is at present unclear. None of the studies exam<strong>in</strong><strong>in</strong>g the matter consistentlycorroborate each other, despite be<strong>in</strong>g almost solely concerned with BAX<strong>and</strong> TGFRII mutations <strong>and</strong> their <strong>in</strong>ferred effect on patient survival ranges froma poor prog<strong>no</strong>sis through prog<strong>no</strong>stically irrelevant to improved survival.108,154,161,166,167* Scann<strong>in</strong>g for mo<strong>no</strong>repeats <strong>in</strong> human genes. The 41,030 cod<strong>in</strong>g sequences <strong>in</strong> the transcripts of 20,484 humanprote<strong>in</strong>-cod<strong>in</strong>g genes were downloaded us<strong>in</strong>g the BioMart service at www.biomart.org 165 on March 13, 2007. APerl script was written to scan the sequences for repeats. For each gene, only the longest cod<strong>in</strong>g sequence wasconsidered. The script identified all mo<strong>no</strong>nucleotide repeats of length six <strong>and</strong> over, <strong>and</strong> also produced summary<strong>in</strong>formation about the repeats <strong>in</strong> each gene (longest repeat, number of repeats, sum of length of repeats).241
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Novel genetic and epigenetic altera
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TABLE OF CONTENTSACKNOWLEDGEMENTS .
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ACKNOWLEDGEMENTSThe present work ha
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Prefacetechnology[3]. This new tech
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SummaryThe subgroup of carcinomas w
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Introduction“Epigenetic inheritan
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Introductionamino acid change it is
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Introductionmethylation during embr
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IntroductionDNA is most of the time
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IntroductionFigure 5. DNA methylati
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IntroductionFigure 6. Incidence rat
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IntroductionFigure 8. Tumor staging
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Introductioninasmuch as 80% of colo
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IntroductionInstabilities involved
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Introductionthere seems to be a fid
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Introductionsevere alterations are
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Introductionpopulation-wide screeni
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IntroductionFigure 12. Present and
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RESULTS IN BRIEFPaper Ia. “DNA hy
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Results in Briefinstability, and se
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Results in BriefUnivariate survival
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Discussionseveral factors, and full
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Discussionlow threshold, we increas
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DiscussionIt may seem like unnecess
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Discussionthan 96% DHPLC do not sta
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DiscussionFigure 13. Mutation detec
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DiscussionClinical impact of molecu
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Discussionmarkers with a very high
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Discussionchromosomes in metaphase[
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DiscussionThese examples underline
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Discussiongenes. One is based on mu
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CONCLUSIONSWe have identified novel
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Future PerspectivesMolecular risk a
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REFERENCES1. Breasted J (1930) The
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References29. Deng G, Chen A, Pong
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References57. Al-Sukhni W, Aronson
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References84. Kunkel TA (1993) Nucl
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ReferencesLeggett B, Levine J, Kim
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References133. Lind GE, Thorstensen
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References156. Meling GI, Lothe RA,
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ReferencesT, Song X, Day RH, Sledzi
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References196. Honda S, Haruta M, S
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ORIGINAL ARTICLESAPPENDIXAppendix I
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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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Journal of Translational Medicine 2
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Journal of Translational Medicine 2
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Journal of Translational Medicine 2
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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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