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Discussionseveral factors, and fully denatured DNA, freshly prepared bisulfite solution, low pH for thesulfonation and deamination steps, high pH for the desulfonation reaction, and a free radicalscavenger to minimize oxidative damages are essential (described in [140]). These factors areusually well taken care of in the many commercially available kits, including the EpiTectBisulfite Kit from QIAGEN which was used in the present thesis. This kit results in at least99% conversion and the repeated denaturation steps during the bisulfite incubation increasesthe reaction efficiency and significantly reduces the overall time consumption. The protocolis additionally shortened and standardized by clean-up using a QiaCube automated pipettingsystem.Methylation specific PCR (MSP)With MSP, first described in 1996, came the possibility to analyze many genes in largersample series[141]. The method relies on primer binding specificity to bisulfite convertedDNA as one primer pair specifically amplifies methylated DNA and one amplifiesunmethylated DNA. Primer design is therefore of crucial importance for a successful andreliable MSP result. Inclusion of multiple CpG sites improves the discrimination ofmethylated and unmethylated sequences. Non-CpG cytosines should additionally beincluded in the primers to avoid amplification of any unsuccessfully converted DNA.Without these cytosines, the methylated primers could also amplify unconverted,unmethylated DNA, yielding false positives.For all MSP primer sets included in papers Ia, Ib and II (expect for CRABP1) a minimum oftwo CpG sites are included in both the sense and antisense primer, ensuring gooddiscrimination between methylated and unmethylated template. For CRABP1 a single CpGsite was included in the sense primer. However, since this was located in the very 3’ end ofthe primer, high specificity was ensured. This was evident from validation analyses where themethylated CRABP1 primer set was challenged with an unmethylated bisulfite template fromnormal blood of a healthy person. The reaction produced no product. This was also the casefor the rest of the MSP primer pairs used in the present thesis. Additionally, none of theprimers amplified unconverted DNA, ensuring that the methylation status from the MSPanalyses was not over-estimated.44
DiscussionSince MSP only gives information on the methylation status of the gene in question based onthe CpG-sites covered by the primers, it is important that these specific sites arerepresentative for the gene promoter. In cases where you are looking for promotermethylation that affects the gene transcription, CpG sites in the close proximity to thetranscription start point will usually be most suitable. Hypermethylation of such promoterregions have in general shown a strong association with loss of or reduced gene expression.In contrast, hypermethylation within the body of the gene may be associated with an activetranscriptional state[142]. In the case of MLH1 only a few bases are correlated withtranscriptional repression, while the other CpG sites play a marginal role[143]. Even thougha similar result was seen for the MAL gene (see bislufite sequencing page 46), both casesrepresent exceptions to the general link between promoter methylation and transcription. Atthe same time they underline the importance of good study design and careful technicalvalidation.Scoring of MSP resultsThe MSP products were separated with agarose gel electrophoresis, stained with ethidiumbromide and visualized with UV. Samples with equal or stronger band intensity than thepositive control in the methylation specific reaction were denoted strongly methylated (++),while samples with less intense bands than the positive control were categorized as weaklymethylated. Samples with very weak band intensity and those with no visible PCR product inthe methylation-specific reaction were regarded as unmethylated. We considered carcinomaswith strong band intensities (++) as methylation positive for the gene promoter in question,while the benign lesions and normal mucosa are scored as positive also when weaklymethylated (+ and ++). The rationale for this is based on the clonal expansion theory (page12). In a carcinoma, tumorigenic alterations have already accumulated. Hence, only genesmethylated in the majority of tumor cells (++) would be functionally/biologically interesting.In contrast, benign precursors may need additional alterations and/or time in order todevelop a malignant potential. Therefore, methylation changes present only in a smallfraction of the adenoma cells may provide these cells with a future growth advantage leadingto a selection and clonal expansion. By scoring the normal samples and early lesions with a45
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 40 and 41: Results in Briefinstability, and se
Page 42 and 43: Results in BriefUnivariate survival
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
Page 91: Paper IbGuro E Lind, Terje Ahlquist
Page 94 and 95:
Journal of Translational Medicine 2
Page 96 and 97:
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Page 100 and 101:
Page 102 and 103:
Page 105:
Paper IITerje Ahlquist, Guro E Lind
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BackgroundMost cases of colorectal
Page 110 and 111:
ADAMTS1 CDKN2A CRABP1 HOXA9 MAL MGM
Page 112 and 113:
pseudogene, leading to a high rate
Page 114 and 115:
strands. Proc Natl Acad Sci U S A 1
Page 116 and 117:
concomitant absence of transcript a
Page 119 and 120:
Volume 10 Number 7 July 2008 pp. 68
Page 121 and 122:
682 RAS Signaling in Colorectal Car
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Page 127:
Table W2. Detailed Somatic Events o
Page 131 and 132:
Identification of RCC2 as a prognos
Page 133 and 134:
INTRODUCTIONMicrosatellite instabil
Page 135 and 136:
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
Page 147 and 148:
Multivariate analysesA multivariate
Page 149 and 150:
When comparing our findings of muta
Page 151 and 152:
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
Page 163 and 164:
Critical Reviews TM in Oncogenesis,
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TARGET GENES OF MSI COLORECTAL CANC
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