Discovery and Functional Annotation of SIX6 Variants in Primary Open-Angle Glaucoma

Abstract

Glaucoma is a leading cause of blindness worldwide. Primary open-angle glaucoma (POAG) is the most common subtype and is a complex trait with multigenic inheritance. Genome-wide association studies have previously identified a significant association between POAG and the SIX6 locus (rs10483727, odds ratio (OR) = 1.32, p = 3.87×10−11). SIX6 plays a role in ocular development and has been associated with the morphology of the optic nerve. We sequenced the SIX6 coding and regulatory regions in 262 POAG cases and 256 controls and identified six nonsynonymous coding variants, including five rare and one common variant, Asn141His (rs33912345), which was associated significantly with POAG (OR = 1.27, p = 4.2×10−10) in the NEIGHBOR/GLAUGEN datasets. These variants were tested in an in vivo Danio rerio (zebrafish) complementation assay to evaluate ocular metrics such as eye size and optic nerve structure. Five variants, found primarily in POAG cases, were hypomorphic or null, while the sixth variant, found only in controls, was benign. One variant in the SIX6 enhancer increased expression of SIX6 and disrupted its regulation. Finally, to our knowledge for the first time, we have identified a clinical feature in POAG patients that appears to be dependent upon SIX6 genotype: patients who are homozygous for the SIX6 risk allele (His141) have a statistically thinner retinal nerve fiber layer than patients homozygous for the SIX6 non-risk allele (Asn141). Our results, in combination with previous SIX6 work, lead us to hypothesize that SIX6 risk variants disrupt the development of the neural retina, leading to a reduced number of retinal ganglion cells, thereby increasing the risk of glaucoma-associated vision loss.

Quantitative Trait Locus Analysis of SIX1-SIX6 With Retinal Nerve Fiber Layer Thickness in Individuals of European Descent

“…Our findings, along with prior studies, 43, 44 support the importance of SIX1-SIX6 in quantitative trait measures such as RNFL. Future studies to understand the genetic contribution of these additional markers identified in the SIX1-SIX6 region, either individually or in combination, on RNFL thickness would be interesting and meaningful.…”

“…43, 44 Ulmer et al, 43 examining the sequence variants of the coding and regulatory regions of SIX6 in POAG , identified 6 non-synonymous SNPs, of which only 1 SNP (rs33912345) was a common variant. This SNP is in high linkage disequilibrium with our lead SNP of rs10483727 (r 2 = 1.00; D′ = 1.00 using SNAP 45 ).…”

Exome Sequence Analysis Suggests that Genetic Burden Contributes to Phenotypic Variability and Complex Neuropathy

“…To assess the specificity of our in vivo model we also tested for genetic interaction between GDAP1 , a bona fide CMT driver, and 3 genes that have not been associated previously with peripheral neuropathy. Two of those are expressed in the CNS and cause other neuropathologies ( SIX6 : optic nerve atrophy [Carnes et al, 2014]; RP1L1 : retinal degeneration and cerebellar disorganization [Davidson et al, 2013]), and the third is expressed ubiquitously and causes VACTERL ( ANKRD6 ; unpublished data). We injected sub-effective doses of each of the tested genes alone and also in pair-wise combinations (Supplementary Figure 6).…”

Association of SIX1/SIX6 locus polymorphisms with regional circumpapillary retinal nerve fibre layer thickness: The Nagahama study

“…To date, genetic studies on cpRNFLT have shown consistent contributions of the single nucleotide polymorphisms (SNPs) rs33912345 and rs10483727 located within the SIX1/SIX6 loci to cpRNFL thinning in the superior and inferior sectors, but not in the temporal, nasal, and global sectors 30–32 . The significance of the SIX1/SIX6 locus in glaucoma was initially discovered by GWAS for VCDR and primary open-angle glaucoma (POAG), and subsequent studies confirmed the association of polymorphisms in this region with glaucoma onset.…”