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Title: Discovery and Functional Annotation of SIX6 Variants in Primary Open-Angle Glaucoma}
Article:
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.
Introduction:
Primary open-angle glaucoma( POAG) is the most common type of glaucoma, a group of diseases that affect approximately 60 million people worldwide and is a leading cause of blindness[1]. Glaucoma is characterized by the progressive death of retinal ganglion cells, leading to optic nerve atrophy and loss of vision. POAG is a complex inherited disorder for which an increasing number of genetic associations have been described, each contributing modestly to disease burden[2],[3]. A recent POAG genome-wide association study found a significant genetic association( rs10483727, odds ratio( OR) = 1. 32, p = 3. 87×10−11) at the SIX1/SIX6 locus[4]. Variants in the SIX1/SIX6 locus were first associated with quantitative optic nerve parameters in controls, including vertical cup-disc ratio( VCDR), which is used clinically to diagnose and monitor POAG progression[5],[6]. Several studies have independently confirmed the association of the SIX1/SIX6 locus with both VCDR and POAG[7]–[9]. The human SIX gene family consists of six members( SIX1–SIX6), all of which contain two shared protein domains; a DNA binding homeobox domain and a SIX domain, which binds downstream effector molecules[10],[11]. Members of this conserved gene family were originally identified through homology to the Drosophila melanogaster( Drosophila) sine oculis( so) gene, which is required for proper eye development[10],[11] and are thought to function as transcription factors, regulating key developmental steps through a complex regulatory network. During embryonic development, SIX1 is expressed broadly in multiple tissues, including the otic vesicle and the limb mesenchyme. However, expression of SIX6 is restricted to regions of the retina and the pituitary[10],[12]. Drosophila with null so alleles have restricted retinal development, while morpholino knockdown of six6b in Danio rerio( zebrafish) embryos was recently shown to result in a small eye phenotype[10],[13]. In humans, a large deletion on chromosome 14q22. 3-q23 that includes SIX6 causes bilateral anophthalmia, the absence of both eyes, demonstrating the importance of SIX gene family members in ocular development and human disease[10],[11],[14]–[16]. In this study, we have extended the current understanding of the molecular contributions of SIX6 to POAG risk. First, we identified potential POAG risk alleles by sequencing the SIX6 gene in a case-control dataset; we found both common and rare coding changes within SIX6 in POAG cases, as well as sequence variants in the SIX6 enhancer. We then used the zebrafish system to demonstrate that these human coding variants have functional consequences in eye development. This analysis of 2–3 day old zebrafish embryos is not intended to fully recapitulate the glaucomatous phenotype; however, it provides in vivo data about the functional effects of human genetic variation on the human SIX6 protein in the context of eye development. We next used luciferase reporter assays, through which we show that a sequence variant found in the SIX6 enhancer of POAG patients may increase SIX6 expression. Finally, we demonstrate that POAG cases homozygous for the SIX6 risk allele rs33912345 have a significantly thinner retinal nerve fiber layer, suggesting a glaucomatous pathogenic mechanism driven by SIX6 dysfunction.
Discussion:
The SIX1/SIX6 locus has been shown to be associated significantly with POAG in several independent studies; however, the causal variant( s) driving this association have remained unknown[4],[6]–[9],[13], as has the direction of effect of these common alleles on protein function. We have demonstrated through several lines of evidence—the tissue specificity of the SIX gene family described in the literature; the identification of SIX6 missense variants in our POAG dataset; and the results of the in vivo and in vitro assays— that SIX6 is the most likely POAG susceptibility gene in this region. We identified both common and rare coding variants that alter the function of the SIX6 protein. We also identified a novel variant within the SIX6 enhancer that appears to disrupt the regulation of SIX6 expression, suggesting both regulatory and coding variants may influence POAG susceptibility at this locus. Finally, we identified hitherto unknown clinical features in POAG patients that may be dependent upon SIX6 genotype: patients homozygous for the SIX6 risk allele have a significantly thinner retinal nerve fiber layer than patients homozygous for the SIX6 non-risk allele. The common variant, rs33912345( Asn141His), which we showed has significantly reduced function in an in vivo model, was associated with POAG in our dataset and is in strong linkage disequilibrium with the originally identified GWAS SNP, rs10483727[4]. This conserved amino acid is located within the alpha helix structure of the DNA homeobox domain of SIX6. Interestingly, the ancestral allele( C, His) is associated with POAG risk. The allele frequency of this variant differs markedly among populations( C allele frequency: YRI( 0. 99), ARF( 0. 96), ASN( 0. 76), and CEU( 0. 42); from 1000 Genomes release 14)[20]. This locus is associated significantly with an increase in vertical cup-disc ratio( VCDR) in population controls, indicating that it may be involved in the development of the optic nerve. VCDR is also a clinical measure used to track disease progression in POAG patients[5],[6],[9],[13],[21]. We note that African populations have larger VCDR and an increase in overall POAG prevalence[22]–[24] compared to CEU populations; in a recent study, the prevalence of POAG in Ghana exceeded 17%, the highest rate observed anywhere in the world[25]. While rs33912345 is not associated with POAG risk in a West African POAG case/control dataset from Ghana, the frequency of the ancestral( risk) allele is 99% in both cases and controls[26]. We hypothesize that differences in the structure of the optic nerve and the higher risk of POAG in individuals with African ancestry may, in part, arise from the fact that essentially all individuals in this population are homozygous for the rs33912345 ancestral risk allele. Given the association of the SIX6 locus with neural retinal measurements such as VCDR, it is reasonable to hypothesize causal variants may function by inducing quantitative changes in cell populations in the retina. There is extensive evidence that SIX6 regulates early retinal progenitor cell proliferation during eye development[18],[19],[27]. Li et al. showed that Six6−/− mice display varying degrees of retinal hypoplasia that is due to a decrease in retinal ganglion cell proliferation arising from an early exit from the cell cycle during development, and results in a 20% decrease in the number of retinal ganglion cells by P35[18]. This is consistent with functional studies of XOptx2, the Xenopus ortholog of SIX6[19]. We have shown a reduction in eye size and in the volume of the optic nerve upon MO knockdown of zebrafish six6a, and we were able to rescue these phenotypes with co-injection of the human SIX6 non-risk allele, demonstrating that the zebrafish six6a gene is the likely functional ortholog to human SIX6. We identified five alleles that could not rescue the small eye and optic nerve phenotypes, and we observed a reduction in retinal nerve fiber layer thickness in POAG patients homozygous for the His141 SIX6 risk allele. Taken together with previously published findings, our results suggest that risk variants in human SIX6 increase POAG susceptibility by negatively affecting retinal ganglion cell development, likely leading to a reduction in the number of retinal ganglion cells in adulthood. Given that retinal ganglion cells are lost during the normal aging process, we speculate that this rate of loss could be increased by the presence of additional POAG risk alleles or other risk factors such as increased intraocular pressure[28]. The development of glaucomatous optic neuropathy and associated visual field loss would thus be hastened by a reduction in the initial number of retinal ganglion cells that an individual possesses. Future work will test the possibility that SIX6 variants also alter the rate of RGC death in the adult. In summary, we have identified multiple common and rare SIX6 sequence variants in POAG cases, and used in vivo and in vitro assays to demonstrate that these variants have functional consequences on SIX6 expression and protein function. While other risk factors may be required for the onset of POAG, our data suggest that attenuation of SIX6 protein function increases an individual's susceptibility to developing the disease via changes to retinal development. Additional work is needed, possibly through the use of transgenic animal model studies, to fully understand the role of SIX6 in POAG.
Primary open angle glaucoma is a blinding disease for which there is currently no cure , only treatments that may slow its progress . To help understand the mechanisms of this disease and to design more effective treatments , we identified previously a locus , SIX6 , that increases the risk of glaucoma . This gene is involved in early eye development and helps to form the retina . In this paper , we test specific sequence variants in SIX6 that are found in glaucoma patients . We show that these variants have a reduced function that interferes with their ability to direct proper formation of the retina . One variant in particular is common , and may be the main reason that this gene is important in the glaucoma disease process . Patients who have two copies of this sequence variant show a change in the structure of their eye consistent with fewer neurons that carry the visual signal to the brain . These neurons typically die as people age , and people who begin life with fewer visual neurons may have an increased risk of glaucoma . Additional research in this topic may lead to new treatments that preserve sight .