The role of SIX6 in primary open-angle glaucoma. M. Ulmer1, Y. Liu2, E. Oh2, Y. Liu1, L. Pasquale3, J. Wiggs3, A. Ashley-Koch1, R. Allingham4, M. Hauser1,4, NEIGHBORHOOD Consortium 1) Center for Human Genetics, Duke University, Durham, NC; 2) Center for Human Disease Modeling, Duke University, Durham, North Carolina; 3) Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts; 4) Department of Ophthalmology, Duke University School of Medicine, Durham, North Carolina.

   Purpose: To follow up a genome-wide association by identifying sequence variants within and near the SIX6 gene, to assess their association with primary open angle glaucoma (POAG), and to perform functional testing. Methods: Meta-analyses of the NEIGHBOR and GLAUGEN datasets (>6500 samples) found significant association between POAG disease status and the SIX6 locus on chromosome 14q23 (top SNP rs10483727, p-value = 3.9 X 10-11, OR= 1.32). SIX6 is a homeobox gene that plays a role in ocular development; it has been associated with quantitative optic nerve endophenotypes and haploinsufficiency in humans causes bilateral anophthalmia. To identify potential causal variants, we sequenced a well described enhancer element, the promoter, and the exons of the SIX6 gene in 262 POAG cases and 279 controls. Variants identified are currently being functionally tested using morpholino antisense oligonucleotide technology in developing zebrafish and dual luciferase assays. Results: The common, nonsynonymous variant, rs33912345 (His141Asn), was identified by sequencing and is significantly associated with POAG (p-value = 4.2 X 10-10, OR = 1.27). This variant is in strong LD with rs10483727 (r2 = 0.95) first identified in the NEIGHBOR/GLAUGEN meta-analysis. We identified five additional nonsynonymous variants within the SIX6 gene, (Glu93Gln, Glu129Lys, Leu205Arg, Thr212Met, and Ser242Ile). Morpholino knockdown of SIX6 in the zebrafish results in reduced eye size. This phenotype can be rescued by the wild-type human SIX6 cDNA, but at least 3 variants identified by sequencing prevent full functional rescue (His141Asn, Thr212Met, Ser242Ile; all p < 0.0001). Within the SIX6 enhancer, 5 variants were identified, including 4 novel variants. These are currently being tested to determine their effects on SIX6 expression levels using dual luciferase assays. Conclusions: SIX6 plays a pivotal role in retinal development including direct regulation of retinal ganglion cells, the cell type whose apoptotic death leads to blindness in POAG patients. Through direct in vivo testing, we have demonstrated that rs33912345 (His141Asn) has reduced functional activity and may well be the primary driver of the genome-wide association signal in this locus. We have also identified rare protein coding variants that affect gene function in vivo, as well as variants in the regulatory regions of the SIX6 gene that may be functional through altering gene dosage.

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