Discovery and functional characterization of glaucoma susceptibility variations

Abstract

Glaucoma, the major cause of visual blindness throughout the world, is characterized by optic nerve degeneration and visual defects. Primary open angle glaucoma (POAG

MIM 137760) is the most common type of glaucoma that generally accompanies elevated intraocular pressure (IOP). One subtype of POAG where the patient who has an IOP within the normal range of < 22 mmHg, is classified as normal tension glaucoma (NTG). Although the exact mechanism of glaucoma has not been completely established, it is most likely a genetically heterogeneous disorder resulting from the interaction of multiple genes and environmental factors. Identifying the unknown genetic factors to contribute the POAG development is necessary to understand the pathogenesis of glaucoma and consequently discover the therapeutic target. In this study, novel POAG associated variants and gene were discovered using single nucleotide polymorphisms (SNP) based case-control association study which is very effective tools to identify the common modest-risk variants of complex diseases. To elucidate the genetic association in POAG etiology, the functional study was performed using animal model of glaucoma. In addition, replication study was also conducted using Korean population to validate previously discovered POAG associated gene through genome wide association study (GWAS) in other population. Transferrin (TF) was regarded as promising candidate gene for POAG because the genomic position was mapped on the human chromosome 3q22.1, which was previously identified as a putative POAG locus. Transferrin expression was increased in POAG patients and it has protected various cells from several pathologic conditions implicated in glaucoma pathogenesis. In addition, it was reported that the cellular uptake of transferrin was blocked by mutation of OPTN known to one of glaucoma causing genes. To determine the genetic association of transferrin with POAG, eighteen tag SNPs were selected in 3kb either side of TF gene, and genotyped in 348 POAG patients and 932 controls of Korean ancestry. Two intronic SNPs, rs8177215 (OR=1.6, p=0.00061) and rs1880669 (OR=0.71, p=0.00072), were significantly and independently associated with POAG susceptibility. Especially, individuals carrying two risk alleles (AA) of rs8177215 had 2.9 fold increased odds of having POAG (OR=2.9, p=0.00023 in recessive model). There was no preferential association with POAG according to IOP level or gender. The imputation was performed to infer the alleles of ungenotyped SNP probably significantly associated with POAG. Total six imputed SNPs were significant association with POAG susceptibility (0.0025 ≤ p ≤ 0.000045). The functional prediction for all POAG associated SNPs in TF revealed that one imputed SNP (rs1799852) located in 48bp from 5’end of TF exon 7 had a function as an exon splicing enhancer (ESE). The POAG risk allele (T) of rs1799852 was expected to disrupt binding of splicing factor. As a matter of fact that two minor allele (TT) increase 2.9 fold risk of POAG, decreased expression of transferrin resulted from aberrant splicing may increase the susceptibility of POAG. In addition, the risk allele of most POAG associated SNPs were known to decrease the serum transferrin level in human clinical studies. To validate the POAG association with TF and influence of transferrin amount in glaucomatous injury, the role of transferrin was examined in acute high IOP induced retina ischemia reperfusion model in SD rats. Transferrin level was significantly increased at 12 hs and 24 hs after retinal ischemia reperfusion injury by 4.1±0.5 and 4.5±0.6 fold respectively compared with control retina (p<0.01). The expression of ceruloplasmin, which is required for iron binding activity of transferrin, was also increased after 12 hs and 24 hs by 2.6±0.6 and 3.0±0.8 fold respectively (p<0.05). Intraperitoneally injected apotransferrin was well delivered to retina and significantly decreased apoptotic cell in GCL region (13.8±6.4 ) compared with vehicle treated retina (36.1±11.7) at 6 hs after ischemia (p<0.001). In addition, daily administration of apotransferrin enhanced RGC survival by an approximately 20% compared with vehicle treated ischemic retina at 6 days after ischemia. In conclusion, transferrin level determinant TF SNPs were associated with POAG susceptibility. Systemic apotransferrin administration, which mimicked the high concentration of transferrin level by genotype of human, significantly enhanced RGC survival. This study implicates that transferrin amount is important in pathologic condition of glaucoma development. The human CAV1-CAV2 locus had been associated with susceptibility to primary open-angle glaucoma in four studies using Caucasian, Chinese, and Pakistani populations, although not in several other studies using non-Korean populations. In this study with Korean participants, the CAV1-CAV2 locus was investigated for associations with susceptibility to primary open-angle glaucoma accompanied by elevated intraocular pressure (IOP), namely high-tension glaucoma (HTG), as well as with IOP elevation, which is a strong risk factor for glaucoma. Two single-nucleotide polymorphisms (SNPs) were genotyped in 1,161 Korean participants including 229 HTG patients and 932 healthy controls and statistically examined for association with HTG susceptibility and IOP. One SNP was rs4236601 G>A, which had been reported in the original study and the other SNP was rs17588172 T>G, which was perfectly correlated ($r^2$=1) with another reported SNP rs1052990. The expression quantitative trait loci (eQTL) analysis was performed using the Genevar data. Both SNPs were associated with HTG susceptibility, but rs4236601 association disappeared when adjusted for rs17588172 genotype and not vice versa. The minor allele G of rs17588172 was associated significantly with 1.5-fold increased susceptibility to HTG (p=0.0069) and marginally with IOP elevation (p=0.043) versus the major allele T. This minor allele was additionally associated with decreased CAV1 and CAV2 mRNA in skin and adipose according to the Genevar eQTL analysis. In conclusion, the minor allele G of rs17588172 in CAV1-CAV2 locus is associated with decreased expression of CAV1 and CAV2 in some tissues, marginally with IOP elevation and consequently with increased susceptibility to HTG.