Community Research and Development Information Service - CORDIS

Periodic Report Summary 1 - FINDING CRC GENES (Identification of new colorectal cancer susceptibility genes)

- Summary description of the project objectives
The overall aim of this proposed project is to discover new colorectal cancer (CRC) susceptibility genes to better understand the colorectal carcinogenesis process and, at the same time, improve the molecular diagnosis of families affected by a colorectal neoplasia.This general aim can be divided in two specific and complementary aims:
Aim 1: Our first aim will be to identify new highly penetrant CRC susceptibility genes and variants using an exome-wide approach. Recent studies have demonstrated the feasibility of whole-exome sequencing (WES) as a powerful discovery tool for germline mutations in other familial cancers. By accurately selecting the families to be studied we expect to increase the gene identification rate. To this aim, we will restrict our WES analysis to highly enriched familial cases from two populations with an already demonstrated founder character (Québec –Canada– and Catalonia –Spain–).
Aim 2: Given the recent evidences of the involvement of two spindle-assembly checkpoint (SAC) genes (BUB1B and BUB1) to hereditary colorectal carcinogenesis, our second aim is to identify new moderately penetrant CRC susceptibility genes and/or variants that are involved in the SAC pathway. From previous evidences we hypothesise that diminished expression of SAC genes may lead to carcinogenesis. To test subtle expression changes we will measure the allele-specific expression in the SAC genes in a case-control study design using RNAseq, thus we will be able to test expression imbalances not only in the SAC pathway but also in the whole transcriptome.
- A description of the work performed since the beginning of the project. Description of the main results achieved so far.
Aim 1. For the first aim of this project, we have contacted and successfully enrolled into the study 9 families that fall into one of the following clinical criteria: (a) unexplained polyposis; (b) familial colorectal cancer of unknown etiology; (c) young cases of colorectal cancer before age 25; and (c) other interesting phenotypes. For all of them we have been able to collect samples from a significant number of individuals, both from the germline (blood or saliva) and from the tumor (FFPE samples), which have been submitted to WES. For some of these families we have already found putatively interesting variants that could be further studied. Other families are still being processed. Additionally, regarding the polyposis category, several families have been enrolled but not submitted to WES because further molecular diagnosis was necessary to discard mutations in the well-known APC gene. To this end, we have designed a new HaloPlex test for the APC gene including promoter, introns and exons that should help us improve the molecular test on polyposis families. Five families have been submitted to this HaloPlex test. Following our interest on novel CRC founder effects we have discovered and described a novel founder MSH6 mutation in the French Canadian population. On the same vein, we have designed an approach to find new CRC founder effects in the AJ population combining a Fluidigm chip with next generation sequencing.
Aim 2. Here we aim to discover alterations in gene expression that could be related to colorectal carcinogenesis. Although our initial target was the spindle-assembly checkpoint (SAC) pathway, for methodological and strategical reasons, we decided to extend our analysis to the whole transcriptome by using RNAseq. We had been able to collect lymphoblastoid cell line (LCL) pellets from 389 cases (CRCindividuals with positive family history) and 410 controls. As a proof-of-concept batch, we have submitted 96 of the cases for RNAseq. These 96 cases were selected considering the clinical and genotyping information available. As the control group, we decided to use publicly available data from 344 LCL controls (Lappalainen et al. Nature, 2013). From these samples we have obtained the RNAseq results and, in collaboration with Dr. TomiPastinen, started the bioinformatics analysis. RNAseq technology permits us to look into several levels of analysis. Firstly, and as our initial interest, we have looked into the allelic imbalances, both recurrently found in several samples or found as isolated events. Secondly, we will look into changes on the total RNA expression, again both as recurrent or isolated events. Thirdly, we are interested in detecting aberrant isoforms due to deleterious splicing events. Finally, we will try to get variant calling information through the RNAseq data of the cases in order to detect putative causative mutations.
Additionally, related to our initial interested with the SAC pathway, we have found two mutations in the CDC20 gene, present in two cancer families. The validation of the two missense mutations that we are performing comprises functional tests on yeast, HeLa cells and primary skin fibroblasts from two mutation carriers. To our knowledge, this is the first time that CDC20 gene is related to carcinogenesis.
- Expected final results and their potential impact and use.
CRC is one of the most important causes of morbidity and mortality in occidental countries. An early diagnosis of CRC as well as clinical surveillance of those who have an increased risk to develop a CRC leads to an important decrease of CRC morbidity and mortality. Genetic diagnosis represents a powerful tool to detect at risk patients, and although some genes have been demonstrated to cause CRC, they still explain a small proportion of cases. On these bases, a better understanding of the CRC genetic contribution is necessary to achieve a meaningful impact on the disease. By finding new cancer susceptibility genes and variants we will decrease the number of families that remain with an unknown genetic cause of their familial cancer after attending genetic counselling and diagnostics units.

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