our preliminary observations that we initially found puzzling: that the levels of tolerated load did not significantly vary depending on the functional annotation of regulated genes (not shown) and that candidate Drosophila enhancers with seemingly very deleterious mutations at Bin, Tin and Twi binding sites were still able to drive reporter gene expression in vitro (Figure S7 in Additional file 1). It is clear, however, that this phenomenon requires further investigation, perhaps drawing more input from the biology of individual TFs. Finally, it is worth noting that a number of disease-causing mutations are located in regulatory regions, and presumably are either not buffered or inappropriately buffered. A well-studied example of this is the regulatory mutations in Pax6 regulatory regions associated with neurodevelopmental abnormalities [59]. In addition, the majority of genome-wide association studies do not implicate a protein-coding variant [20]. To fully understand these diseases we must gain a more complete knowledge of how variation impacts regulatory function.
