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Novel genetic and epigenetic alterations in ... - Ous-research.no

Novel genetic and epigenetic alterations in ... - Ous-research.no Novel genetic and epigenetic alterations in ... - Ous-research.no

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Discussionthan 96% DHPLC do not stand back in sensitivity for any of these high-throughputmethods. In order to be time efficient and not spend time on method optimization,collaboration with a national diagnostic centre for NF1-related diseases in Rome, Italy, wasinitiated. Here a lab protocol for NF1 analysis was well established, analyzing both sequencealterations and copy number changes[153;154]. The DHPLC primers were generallypositioned approximately 50 to 60 bp away from the intron–exon boundary to allow thedetection of splicing defects while minimizing intronic polymorphisms. Melting temperatureof all fragments was optimized to yield as high sensitivity as possible. In addition, a largenumbers of normal control samples were analyzed and recorded, making it easy todistinguish between mutations and common polymorphisms.Direct sequencingDye terminator sequencing has up until now been considered the golden standard inmutation analysis as it describes any sequence variant with a high accuracy[18]. Still, alimitation with direct sequencing is the detection level. It has been estimated that thismethod can detect and quantify minor sequence variants mutations present in as little as10% of a virus population[155], although other studies claim that this number is as high as30%[18]. The detection level can vary somewhat from sequence to sequence, and althoughwe are able to see base changes at lower cut-offs in a designed sensitivity test using titrationsof known mutations, our experience is that the sensitivity level is approximately 15% forunknown mutations. This means that when running several unknown samples, sequencechanges will be scored when present at this level or higher. Mutations in genes present in asmaller fraction (than 15%) of the sample (e.g. by-stander genes, see page 61-62) will bescored as wild type. In such cases a cloning strategy or another more sensitive method couldbe used, but one might discuss whether it is interesting, in a biologic perspective, to detectmutations that falls below the detection threshold. Such mutations will per definition only bepresent in a few of the tumor cells as discussed on pages 61-62, and are not expected toprovide the tumor with a selective advantage.In addition to the inherent nature of random template selection in PCR assays, the basecallingalgorithm contributes to this relatively poor sensitivity. Although improvements in50

Discussionthe sequencing technology make it possible to sequence large number of samples, thethroughput of direct sequencing is still relatively poor as the data analysis is labor-intensive.Automated sequence scanning softwares are developed, but as with the sensitivity level, thebase calling algorithm makes automated mutation calling difficult when the signal to noiseratio is suboptimal. However, with a good signal-to noise ratio, such programs can be verylabor-saving when looking at distinct mutation hot-spots such as the V600E in BRAF. Theprogram can quickly zoom in on the codon and call possible mutations, and it is easy for theinvestigator to quickly confirm or refute the results. Hence, genes with well defined mutationregions are more easily adapted to direct sequencing. In the remaining cases manual readingof electropherograms (which requires skills in addition to time) is still preferable.Here, direct sequencing has been used for analyzing BRAF and KRAS in a tumor seriesevaluated to contain an average of 84% tumor cells[156]. Both of these genes contain knownmutation hot-spots, and should be easily picked up by an automated analysis. Both genesadditionally provide a strong selective advantage for the tumor when mutated and theiroccurrence is therefore expected to be well above the detection limit of 15%.Fragment analysisMSI is also called the mutator phenotype (page 30)[91]. Since the MSI-inflicted indels mostoften are confined to microsatellite regions, a diagnostic approach is applicable. Directsequencing could be an option, but due to the previously mentioned specificity issue as wellas the workload, other methods such as fragment analysis could be more suitable. This is asensitive and high throughput method to describe PCR-products when performed withfluorescent primers in a capillary electrophoresis system[157]. Fragment analysis was usedwhen exploring the microsatellite-containing regions of each of 41 genes in paper IV as wellas the MSI-analysis. Using this method, the presence and identity of the mutation was easilydetected within the same run (Figure 13).51

Discussionthe sequenc<strong>in</strong>g tech<strong>no</strong>logy make it possible to sequence large number of samples, thethroughput of direct sequenc<strong>in</strong>g is still relatively poor as the data analysis is labor-<strong>in</strong>tensive.Automated sequence scann<strong>in</strong>g softwares are developed, but as with the sensitivity level, thebase call<strong>in</strong>g algorithm makes automated mutation call<strong>in</strong>g difficult when the signal to <strong>no</strong>iseratio is suboptimal. However, with a good signal-to <strong>no</strong>ise ratio, such programs can be verylabor-sav<strong>in</strong>g when look<strong>in</strong>g at dist<strong>in</strong>ct mutation hot-spots such as the V600E <strong>in</strong> BRAF. Theprogram can quickly zoom <strong>in</strong> on the codon <strong>and</strong> call possible mutations, <strong>and</strong> it is easy for the<strong>in</strong>vestigator to quickly confirm or refute the results. Hence, genes with well def<strong>in</strong>ed mutationregions are more easily adapted to direct sequenc<strong>in</strong>g. In the rema<strong>in</strong><strong>in</strong>g cases manual read<strong>in</strong>gof electropherograms (which requires skills <strong>in</strong> addition to time) is still preferable.Here, direct sequenc<strong>in</strong>g has been used for analyz<strong>in</strong>g BRAF <strong>and</strong> KRAS <strong>in</strong> a tumor seriesevaluated to conta<strong>in</strong> an average of 84% tumor cells[156]. Both of these genes conta<strong>in</strong> k<strong>no</strong>wnmutation hot-spots, <strong>and</strong> should be easily picked up by an automated analysis. Both genesadditionally provide a strong selective advantage for the tumor when mutated <strong>and</strong> theiroccurrence is therefore expected to be well above the detection limit of 15%.Fragment analysisMSI is also called the mutator phe<strong>no</strong>type (page 30)[91]. S<strong>in</strong>ce the MSI-<strong>in</strong>flicted <strong>in</strong>dels mostoften are conf<strong>in</strong>ed to microsatellite regions, a diag<strong>no</strong>stic approach is applicable. Directsequenc<strong>in</strong>g could be an option, but due to the previously mentioned specificity issue as wellas the workload, other methods such as fragment analysis could be more suitable. This is asensitive <strong>and</strong> high throughput method to describe PCR-products when performed withfluorescent primers <strong>in</strong> a capillary electrophoresis system[157]. Fragment analysis was usedwhen explor<strong>in</strong>g the microsatellite-conta<strong>in</strong><strong>in</strong>g regions of each of 41 genes <strong>in</strong> paper IV as wellas the MSI-analysis. Us<strong>in</strong>g this method, the presence <strong>and</strong> identity of the mutation was easilydetected with<strong>in</strong> the same run (Figure 13).51

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