Figure 4

Two intergenic case-only variants disrupt a repressor element and change gene expression in vitro . (a) The two case-only variants are just 20 bases apart in a 2.5 Mb gene desert between DSC3 and CDH2 on canine chromosome 7. (b) The syntenic region of human chromosome 18 shows markers of DNase hypersensitivity, transcription factor binding, repressor binding, histone methylation [30], and mammalian constraint [31]. (c) Both variants, SNP55 and SNP35, alter bases that are highly constrained across mammals, SNP55 to an A and SNP35 to a T. Compositions of the four bases for each position in the 29 mammals comparison [27] are shown with different grey scale and height of bars (the higher the more conserved in multiple species). (d) The wild-type regulatory sequence represses luciferase reporter expression in SK-N-BE(2) neuroblastoma cells. Both variants significantly change the extent of repression relative to wild type, with SNP35-G more repressive and SNP55-A less repressive. The firefly luciferase expression in the test plasmids were normalized against the co-transfected Renilla luciferase expression in pGL4.73. The P-value of the significance of the change relative to wild type is shown above each bar, with vertical lines showing standard error of the mean. (e) An electrophoretic mobility shift assay testing the wild-type alleles (lanes 1 to 4) and the OCD-risk alleles (lanes 5 to 8) of SNP35 (top gel) and SNP55 (bottom gel) show that nuclear protein binding (red arrow) to the SNP35 locus is disrupted by the risk allele. Nuclear extract was derived from SK-N-BE(2) cells. We used a 200-fold molar excess of competitor where appropriate.