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MSI detection by next generation sequencing in Lynch syndrome patients -

(2011) Knopperts, Alain

Introduction: Colorectal carcinomas are the second most common cancer in the Netherlands. Approximately 5% of all colorectal cancers are hereditary, with Lynch syndrome (LS) as the most common form. Besides colorectal cancers LS patients can also have cancers in other organs, for example colorectal, endometrial, ovary carcinomas. The one major hallmark of LS related tumours is a malfunction of the DNA Mismatch repair system (MMR). MMR involves several proteins, and activation of the system is initiated by the recognition of mismatches in DNA. The most important proteins of the MMR system are MSH2, MSH6, MLH1 and PMS2. These proteins initiate MMR and assemble the machinery for adequate repair of the DNA. The malfunctioning of the MMR system is caused by a germline mutation in one of the MMR genes, followed by a somatic mutation of the wildtype allele of the same gene. As consequence of the failure of this MMR system, mutations will not be repaired properly and therefore mutations will accumulate. The most sensitive spots for mutations to occur are parts of DNA which contain repetitive sequences. Mutation in repetitive sequences are called microsatellite instability (MSI). MSI is a hallmark of Lynch syndrome. It is still not yet clear whether MSI is present only in tumours of mutation carriers or whether MSI can already be detected before tumours are diagnosed in for instance peripheral blood lymphocytes (PBLs). Indeed, some studies suggested that slightly elevated levels of MSI are detectable in PBLs as a consequence of heterozygous germline mutations. However, some other studies suggest that MSI will occur anyway as a consequence of aging. In this study we try to confirm these hypotheses using a highly sensitive method to analyze MSI. This method is called next-generation sequencing.
Materials and Methods: In this study we selected 39 control DNA samples (DNA isolated out of PBLs), DNA samples of 42 presymptomatic carriers (DNA also isolated from PBLs) and 9 DNA samples isolated from tumours to determine if elevated MSI indeed is detectable in PBLs in MMR germline mutation carriers and in these age-matched controls. Tests were performed with five different mononucleotide markers. We also selected samples with different ages whether indeed a correlation with age does exist. These samples are analyzed with the conventional MSI test method and with a new far more sensitive method called next generation sequencing (NGS).
Results: The MSI profiles of tumour DNAs as seen with the conventional MSI detection method and with the new NGS method, were identical. This makes clear that NGS is capable to detect MSI properly. Subsequent analysis of the carriers and the age-matched control group showed that there was no significant difference in MSI between the carrier group and the control group. For some markers there was a slight elevation of MSI detectable, however, these results were not significant. Also, we could not confirm a correlation between age and MSI.
Conculsion: Next generation sequencing is a valid method to detect MSI in tumour tissue. We did not see a difference in MSI profile of DNA out of PBLs of MMR germline mutation carriers when compare to age matched controls nor did we see an age effect. Therefore we could not confirm the results found in previous studies.

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