Development of a novel mouse model for the colorectal cancer risk locus at Xp22.2
View/ Open
McBride2016.pdf (4.369Mb)
Date
29/11/2016Item status
Restricted AccessEmbargo end date
31/12/2100Author
McBride, Andrew Niall
Metadata
Abstract
Colorectal cancer (CRC) is the third most common cancer globally with around 1.3
million cases diagnosed annually. In cases of inherited CRC where none of the rare,
high-risk mutations associated with familial syndromes are observed it has been
theorised that the heritable risk is due to common, low-risk variants in the genome.
Identifying these variants of modest or small effect size has become possible due to
the use of large genome-wide association studies (GWAS). Through a meta-analysis
of five previous GWAS, Dunlop et al. identified three novel susceptibility loci at 6p21,
11q13.4 and Xp22.2. The aims of this study were to further characterise the Xp22.2
risk locus and investigate the function of the putative risk gene SHROOM2.
The variant originally identified in the Dunlop et al. study as showing an association
with CRC risk rs5934683 is located between SHROOM2 and GPR143. Genome–wide
expression analysis has shown that the variant is an eQTL (expression quantitative
locus), affecting expression of SHROOM2, but not GPR143. An important caveat to
this analysis was that the commercial array used to measure gene expression does not
detect all predicted GPR143 transcripts. Hence it was important to understand whether
GPR143 might be involved at the locus and whether there was altered expression in
normal colonic mucosa. I have analysed the expression of GPR143 and shown that it
is poorly expressed in normal mucosa and that expression of the alternative transcripts
is rare. This provided further evidence for the gene of interest at Xp22.2 being
SHROOM2 and thus became the focus for further investigation.
In order to understand SHROOM2 function a knockout mouse was generated allowing
studies of gene function beyond the previously used in vitro systems. Embryonic stem
cells containing a Shroom2 knockout first allele were obtained from the International
Knockout Mouse Consortium and a novel mouse line established. This mouse line was
found to be an incomplete knockout and a Cre recombination strategy was employed
to remove the critical exon and create a true null allele for Shroom2. This model was
validated as being a true knockout of Shroom2 at both the RNA and protein level and
the model subjected to initial phenotyping focusing on tissues where the gene has
previously been identified as expressed. To investigate the role of Shroom2 as a CRC susceptibility gene preliminary data has
been gathered from crosses to the ApcMin/+ CRC model, and analysis of the intestines
of the Shroom2KO line has been undertaken. Two spontaneously occurring anorectal
adenomas have been identified in Shroom2 null mice, and an additional mid-colonic
polyp phenotype identified when crossed onto the ApcMin/+ background. Additionally,
embryonic fibroblasts have been used in growth and wound healing assays to
determine what effect total loss of Shroom2 has at a cellular level. Proteomics analysis
to identify significantly altered pathways associated with Shroom2 loss has also been
carried out and has highlighted a number of interesting targets for further investigation.
In summary, a novel Shroom2 knockout mouse model has been developed to
investigate the CRC susceptibility locus identified at Xp22.2. Preliminary data from
this mouse model appears to confirm SHROOM2 as having a role in tumour
development in the large intestine.