We have been working with Bristol University for just over 4 years now. Professor Paul Martin, Professor Kate Nobes, Dr Beck Richardson and Dr Chrissie Hammond are our main collaborators.
The first stage of our research is to work out which genes we wanted to focus on. We used data from genome wide association studies, or GWAS, to do this.
GWAS studies search the whole genome for small variations called single nucleotide polymorphisms, or SNPs for short. GWAS studies look into whether SNPs occur more frequently in people with a particular phenotype, such as a disease, than in people without the phenotype. Each study looks at many thousands of SNPs in thousands of people’s genomes at the same time.
To generate this data, large numbers of cases and controls are genotyped using microarrays. This level of analysis can be carried out by cohort studies such as the Bristol-based ALSPAC cohort. ALSPAC is a study that recruited 14,000 mothers during pregnancy in the early 1990s and has collected huge amounts of phenotypic and genetic information from them and their families for the last 25 years.
In one example we used GWAS studies to look for particular SNPs that were associated with osteoarthritis, including this study carried out by the arcOGEN consortium. This study looked at over 7000 patients and over 11000 controls, including samples from the ALSPAC study, to identify SNPS associated with osteoarthritis. The results are represented in a Manhattan Plot, named after its resemblance to the Manhattan skyline. Each dot represents a single SNP with its chromosome position on the x axis and its association with osteoarthritis on the y axis. Any dots above this line of significance indicate that the SNP could be associated with osteoarthritis. SNPs are located in or close to genes, indicating which genes we should look at.
These genes were identified from GWAS studies in humans, but we’re using zebrafish as our model organism. We then had to find zebrafish orthologues using the ZFin database, which generated gnl3, pbrm1 and stab1 as our genes of interest.
Every year we plan to use GWAS to find different genes of interest. The genes of interest are chosen by us, we also get involved in primer design for the purposes of running a PCR. PCR and gel electrophoresis are the techniques that we use to find out more about the expression of our chosen genes, and more specifically when those particular genes are switched on. We have plans this year to introduce some immunohistochemistry to further explore where in the zebrafish our genes of interest are expressed.