<i>SIX6</i> variants identified by sequencing POAG cases and controls.

<p>Coordinates are based on the Hg19 reference; MAF = minor allele frequency;</p><p>AA = amino acid; GERP = Genomic Evolutionary Rate Profiling.</p

<i>In vivo</i> zebrafish morpholino complementation assay showing the effect of <i>SIX6</i> nonsynonymous variants.

<p>Zebrafish embryos were microinjected with a translation blocking morpholino designed to target <i>six6a</i>. Total eye size (µm²) was measured 3 days post fertilization. Compared to the uninjected controls, morphants showed a significant reduction in eye size. Zebrafish were co-injected with the morpholino and a human <i>SIX6</i> allele (Glu93Gln, Glu129Lys, Asn141His, Leu205Arg, Thr212Met, or Ser242IIe). Results of each allele were compared to the <i>SIX6</i> non-risk allele (Ref). P-values are provided below the mean of each treatment.</p

OCT measurements from POAG cases homozygous for rs33912345.

<p>SD = standard deviation; OCT = optical coherence tomography.</p

<i>In vitro</i> luciferase assay results showing the effect of <i>SIX6</i> enhancer variants.

<p><i>SIX6</i> enhancer alleles were tested using a dual luciferase assay and the ratio of the experimental luciferase: control luciferase was calculated (DLR ratio). All vectors were co-transfected with NeuroD and E47. In this context, the <i>SIX6</i> enhancer is functioning to increase expression compared to the empty vector (pGL4.23), driven by a minimal promoter. Compared to the reference enhancer (Ref), one variant (Chr14:60974449_G) significantly increases the enhancer's activity.</p

Functional evaluation of <i>SIX6</i> variants on the volume of the optic nerve.

<p>Representative whole mount images of acetylated-tubulin expression in the heads of zebrafish embryos injected with a control or <i>six6a</i> morpholino, rescued by co-injection with human non-risk SIX6 transcript or a transcript containing the Leu205Arg hypomorphic variant (A). Acetylated-tubulin staining is restricted primarily to axon tracts and can be used to visualize the optic nerve. Relative to the control morphants, volumetric regions of interest (ROI) along the optic nerve in <i>six6a</i> morphants were reduced significantly. Co-injection of human variants revealed a hypomorphic (Leu205Arg, Asn141His) or benign (Glu93Gln) role of the variants on the optic nerve (B). Sample size for all injection paradigms ranged from 7–9 and p-values are plotted for each comparison (*** p<0.001; ** p<0.01). No significant changes in the volume of other axonal tracts in the head (marked by an asterisk) were detected. Standard error of the mean is shown and white scale bars = 20 um.</p

Morpholino knockdown of <i>six6a</i>.

<p>Zebrafish were microinjected with a <i>six6a</i> translation blocking morpholino. Lateral images, taken 3 days post fertilization (3 dpf), of a wild-type zebrafish (left) and a morpholino injected zebrafish (right) are shown, highlighting the small eye phenotype (dashed circle).</p

Results from the <i>in vivo</i> zebrafish <i>six6a</i> morpholino complementation assay.

<p>N = sample size; SD = standard deviation.</p