Genetic dissection of Irinotecan toxicity and lymphocyte tumor infiltration
Meijer, Gustaaf Eduard
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The aim of this project was twofold (1) genetic mapping of the genes that cause Irinotecan induced toxicity and (2) identification of the genes that regulate tumour lymphocyte infiltration. In addition, a project was started in order to test the feasibility of a novel alternative for mouse genotyping. Irinotecan is a first line drug used in the treatment of metastatic colorectal cancer. However, Irinotecan has a narrow therapeutic index due to major drug toxicities being diarrhea, neutropenia and extreme weight loss. The exact genetic basis for Irinotecan toxicity is not fully elucidated even after a decade of research. To date, the only established genetic variant is UGT1A1*28 however this does not predict toxicity in all patients because of currently unknown genes involved. Previous research in our lab using the recombinant congenic strain mouse model a genetic locus on chromosome 1 termed Adri1 has been mapped. Through linkage analysis we have narrowed down the location of the genes to a smaller upper middle region of the locus. The preliminary results suggested that the genes are located in this region and is now undergoing further testing in order to validate these findings. For the second project we investigated the genetic basis for differences in tumour lymphocyte infiltration. The ability of patient's immune system to respond to a tumour through lymphocyte infiltration has an impact on the prognosis. Genetic mapping using the recombinant congenic strain our lab has previously mapped four genetic loci termed Lynf loci (Lynf1, Lynf2, Lynf3, Lynf4). The focus of this project was the Lynf4 locus which fine mapping revealed to be a pseudo-gene. Our current hypothesis is that this pseudo-gene is a regulatory element that influences adjacent candidate genes that regulate tumour lymphocyte infiltration. We selected and tested 4 out of 50 candidate genes because these genes are in closest proximity to Lynf4. The genes we tested did not seem to show genotype dependent changes in gene expression levels. However, we the expression analysis needs to be repeated and the remaining candidate genes tested before we can validate our hypothesis. In addition to gene mapping another study was conducted aimed at streamlining the process of mouse genotyping. A major rate limiting factor for mouse genotyping is the sample analysis using gel electrophoresis. A recent publication (Thomson et al. 2012), described an alternative for gel electrophoresis for genotyping using high resolution meting curve (HRM). This technique used sequence length polymorphisms that produce PCR products that differ in melting temperature. Melting curve analysis of the PCR products using Real-Time PCR instrumentation allows for allele discrimination excluding the need for gel electrophoresis. We tested several micro-satellite markers however only one marker produced melting curves that differ as such that the alleles could be distinguished. Furthermore, clear identification of the genotype was mostly restricted to heterozygote samples. Second, we tested an insertion marker and genotyping was successful after repeated validation. Due to lack of advanced software that allow for high resolution meting curve analysis we could not implement this technique for micro-satellite marker based genotyping.