Complex disease and long-range regulation: Interpreting the GWAS using a Dual Colour Transgenesis Strategy in Zebrafish

Complex disease A disease caused by many factors difficult to identify Cancer Diabetes Obesity Depression Multiple sclerosis Crohn’s disease and many more.. Arguments that the inheritance of a disorder is polygenic usually derive from the observation that, while the disorder might aggregate in families, it does not tend to segregate in ways that are consistent with simple Mendelian inheritance.  Difficulties in Confounding, similar symptoms pooled nd generisation proble Why is it difficult? Strange inheritance patterns- sporadic cases Classification of disorders based on symptoms may be problematic Environmental and stochastic factors

Genome-Wide Association Studies Trying to resilve the genetic component of the complex diseases, scientinsts thought a very logical approach: Compare the DNA of one group of people with a disease to another group that doesn’t have the disease, identify the DNA region specific to the disease group, and then find the specific gene and mutations that lead to the disease. Gwas good for finding the major contributing genes

The majority of the GWAS signals is found in the non-coding genome Gwas good for finding the major contributing genes but the majority of signals is found in the non-coding genome.  Examples of four different common diseases, showing the number of associated single nucleotide polymorphisms (SNPs) that lie in putative enhancers, promoters and exons [6-8]. Putative enhancer elements were defined by chromatin features in each of the four indicated cell types. Olivia Corradin and Peter C Scacheri, Genome Med, 2014

Why is it difficult to identify the causative variants in the cis-regulatory elements and the physiological context? Linkage Disequilibrium Blocks GWAS arrays contain only index SNPs that represent SNPs in the same linkage disequilibrium (LD) block. Hence, the causative SNP may not be the one indicated in the GWAS GWAS follow a simple design: compare allele frequencies for hundreds of thousands of common variants spread across the genome between large samples of disease cases and controls [10]. If a variant predisposes to the disease, even slightly, this should be apparent as an increased allelic frequency in cases versus controls, given a large enough sample size. This design has been applied to many different complex disorders, with varying degrees of success. It is important to note that these studies use a sample of single nucleotide polymorphisms (SNPs) to tag variation across the genome (on the basis of blocks of low recombination or linkage disequilibrium (LD)). If a single SNP shows an association with the disease, this does not necessarily mean that that SNP itself is involved, the association could be due to any of the other variants in LD with it. As we will see, these can include tagged rare variants [27]. The goal of these studies is thus not necessarily to identify causal alleles but to point to loci that might harbor them. Enhancer variation may have a very subtle effect on the transcription of a target gene but in a a specific and tight developmental window this effect may be crucial Nature of Enhancer Function Long distance from target genes, Tissue and stage specific-transcription factor stoichiometry Effect of non-coding variation difficult to predict

Functional Characterisation of non-coding variation using a Dual -Colour Strategy in Zebrafish- Overview 1) Gateway cloning 2 different haplotypes (e.g. obesity risk haplotype and non-risk haplotype) upstream of different reporter genes to destination vector with TOL2 recombination sites

Functional Characterisation of non-coding variation using a Dual -Colour Strategy in Zebrafish- Overview 2) Injections 1-2 cell embryos, Tol2 transposase mRNA

Functional Characterisation of non-coding variation using a Dual -Colour Strategy in Zebrafish- Overview 3) Outcrosses Observing the different patterns of reporter activation

Functional Characterisation of non-coding variation using a Dual -Colour Strategy in Zebrafish- Overview 4) Knock down candidate enhancer-binding proteins Observe changes in reporter activation pattern

A proof of principle study- SHH enhancer

A proof of principle: Identifying the target gene mRNA in-situ hybridization for candidate target gene Compare patterns

A proof of principle: Identifying the enhancer-binding factor Morpholino knock-down

Advantages of the model Zebrafish Transparent – easy observation Vertebrate – conserved developmental procedures Conserved transcription factor networks – works also when the enhancer is not conserved in Zebrafish

Advantages of the strategy Quick – 3-4 months Robust Cost effective In vivo GWAS point to the right direction