An Updated Review on the Genetics of Primary Open Angle Glaucoma

Epidemiological studies suggest that by 2020 the prevalence of primary open angle glaucoma (POAG) is estimated to increase to 76.0 million, and to 111.8 million by 2040 globally due to the population aging. The prevalence of POAG is the highest among those of African descent, followed by Asians, and the lowest in Europeans. POAG is a genetically complex trait with a substantial fraction exhibiting a significant heritability. Less than 10% of POAG cases in the general population are caused by specific gene mutations and the remaining cases are polygenic. Quantitative traits related to POAG pathogenesis such as intra-ocular pressure (IOP), vertical cup/disc ratio (VCDR), optic disc area, and central corneal thickness (CCT) are highly heritable, and likely to be influenced at least in part by genes and show substantial variation in human populations. Recent genome-wide association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs) at different loci including CAV1/CAV2, TMCO1, CDKN2B-AS1, CDC7-TGFBR3, SIX1/SIX6, GAS7 and ATOH7 to be associated with POAG and its related quantitative traits (endophenotypes). The chapter provides a brief overview on the different GWAS and SNP association studies and their correlation with various clinical parameters important for POAG in the population worldwide, including the Middle East.


Introduction
Glaucoma is a chronic and progressive group of optic neuropathies affecting more than 60 million people globally [1]. It is associated with death of retinal ganglion cells resulting in characteristic cupping or degeneration of the optic nerve head and loss of peripheral vision [2]. Primary open angle glaucoma (POAG) is one of the most common types of glaucoma which is clinically characterized by an open and normal anterior iridocorneal chamber angle [2]. POAG can either occur with increased intraocular pressure (IOP) or normal IOP, the latter being referred to as normal-tension glaucoma (NTG).
Although there are many postulated mechanisms of retinal ganglion cell damage, the exact etiology of POAG still remains obscure. The well-recognized risk factors associated with POAG include elevated IOP, age, family history, gender, ethnicity, central corneal thickness, and myopia. A recent large prospective study indicated that POAG with early paracentral visual field loss displays distinct as well common risk factor profiles as compared to those with peripheral vision loss [3]. Raised IOP is the most important and the only modifiable risk factor in the development and progression of POAG. Several large population-based studies in the past have confirmed that the reduction of IOP reduces the progression of glaucoma in patients with or without elevated IOP [4][5][6][7][8].
Similarly, findings of the meta-analysis from the Eye Diseases Prevalence Research Group have shown that the occurrence of glaucoma increases with increasing age among all ethnicities (Europeans, Blacks, and Hispanics) [7,9]. Age was also reported to be associated with POAG in patients with ocular hypertension in two large population-based studies [5,8]. Family history is another important risk factor in the development of glaucoma [7,[10][11][12]. A positive family history of POAG significantly increases the odds (varying from five to 10 times) for the development of POAG [13]. In the Melbourne [14] and Rotterdam studies [11], males showed a trend towards increased risk of POAG which was absent in the Barbados Eye Study [7] and the Beaver Dam Eye Study [10]. Similarly, the Eye Disease Prevalence Research Group [9] reported no gender-related association of glaucoma among the European, African American, and Hispanic subjects. However, a recent systematic review of 3497 POAG cases out of 146,882 participants with gender-specific data showed that the age-adjusted prevalence is higher in men compared to women, and that this finding remains consistent across all ethnic groups provides very strong evidence for the association of POAG with gender [15]. Several studies have shown POAG to be more prevalent with rapid and severe disease progression in people of African-Caribbean as compared to European descent, Hispanics, and Asians [9,16]. Central corneal thickness (CCT) has also been reported to be associated with POAG, particularly in the ocular hypertension patients [17,18]. Although the precise mechanism(s) are still unclear, this may be in part due to the effect of corneal thickness on IOP measurement, and increased susceptibility to optic nerve damage [19,20]. In addition, studies have shown that individuals with thicker corneas are less responsive to topical ocular hypotensive medications [21]. Myopia is also considered to be an important risk factor for POAG as it can increase susceptibility of myopic nerves to glaucomatous damage [22]. Moderate-to-high levels of myopia conferred two-to three-fold increased risk in the Australian [23], US Caucasian [24], and the Chinese populations [25]. Other predisposing factors for POAG include adult-onset diabetes and hypertension. Although there are conflicting reports regarding the risk of POAG in individuals with diabetes [26,27], a recent systematic review and meta-analysis of 13 studies, which included six population-based cohorts and seven case-control studies, showed increased risk of POAG (relative risk of 1.4 and 1. 49, respectively) in individuals with diabetes [28]. Multiple epidemiological studies have also reported a role of hypertension as a risk factor for POAG [23,29]. Treatment of hypertensive patients with beta-blockers results in nocturnal hypotension and is a potential risk factor for glaucomatous optic neuropathy [30]. The mechanism(s) by which hypertension induces optic nerve damage are still unclear.
POAG is a genetically complex trait with a substantial fraction exhibiting a significant heritability. Genetic linkage studies of large affected families have so far identified at least 20 chromosomal loci (GLC1A-P) that are linked to POAG. The causative genes that are capable of causing POAG with minimal influence from other gene(s) or the environment and that have been consistently implicated so far include myocilin (MYOC), optineurin (OPTN), WD repeat domain 36 (WDR36), ankyrin repeat and SOCS-box containing 10 (ASB10), Cytochrome P450 family 1, subtype B, polypeptide 1 (CYP1B1), and neurotrophin 4 (NTF4) as reviewed elsewhere [31,32]. Twin studies and family-based studies have discovered a number of genes. However, these disease-causing genes account for <10% of POAG cases in the general population. It is therefore likely that the hereditary aspect of many of the remaining cases of POAG is due to the combined effects of several genes (polygenic) and that gene-environment interactions are important. Quantitative endophenotype traits related to POAG pathogenesis such as IOP, vertical cup-to-disc ratio (VCDR), and CCT [10,33,34] are highly heritable, likely to be influenced at least in part by genes, and are highly polymorphic. Recent advances in genomic technologies and genome-wide association studies (GWAS) have greatly accelerated the discovery and understanding of genes and genomic regions associated with POAG and influencing the quantitative endophenotype traits related to POAG pathogenesis, which will be the main focus of this chapter.

Epidemiology of POAG
Recent epidemiological studies suggest that, in 2013, almost 64.3 million people (aged between 40 and 80 years) were affected by glaucoma globally, and this number is expected to increase to 76.0 million by 2020 and to 111.8 million by 2040 due to the population aging [35]. POAG accounts for a major three-quarters (74%) of all glaucoma cases [1]. Another recent meta-analysis estimated the global number of POAG cases in 2015 at 57.5 million, rising to 65.5 million by 2020 [15]. Almost half (47%) of these will those of Asian descent, while a quarter (24%) will be European [1]. The risk and subtypes of glaucoma are known to vary among races and countries [36]. A meta-analysis conducted by the Eye Disease Prevalence Research Group showed that, in the United States, African Americans have a higher POAG prevalence than Caucasians. The prevalence of POAG in individuals ě40 years old was observed to be 1.86%, including 1.57 million Caucasian and 398,000 African American subjects. In 2020, this number is estimated to rise up to 3.36 million due to the population aging [9]. In all the age groups, there was an increased prevalence of glaucoma in individuals of African descent compared with European-derived individuals [37]. Similarly, a recent meta-analysis of 81 22, 1.41). It is clearly evident that individuals of African descent are associated with increased risk (estimated incidence is two to five times higher) of developing glaucoma compared with individuals of European descent. The reasons for this increased risk of glaucoma among individuals of African descent are still not clear. The Barbados Eye Study reported a prevalence of 7% in Africans, suggesting an influence of ancestral factors [7]. Several other factors that may also be influential could be physiological or anatomical differences in the optic disc or corneas, environmental factors, social differences or genetics [26].

Genotype-Phenotype Association in POAG
Association studies using the candidate-gene approach and GWASs have been particularly useful tools in identifying genetic factors, each of which may have a relatively small effect but contributes to a large number of cases. Unlike the candidate-gene approach, GWAS is an unbiased (without bias to known protein functionality gene) genome-wide approach that compares the genotypic profile of single nucleotide polymorphisms (SNPs) throughout the genome in cases (affected) and controls (unaffected), thus identifying genomic region(s) associated with a disease or trait of interest. The large population sample required in GWASs to achieve a genome-wide statistical significance (p-value of less than 5ˆ10´8) has been greatly facilitated by the formation of the International Consortia. However, since GWAS can rarely identify functional or causal variant(s), further in-depth genotyping and functional testing in addition to replication studies in independent cohorts of different population groups are considered a standard requirement to conclusively validate genes or genomic regions identified from GWAS. Using this powerful approach (GWAS), recent genetic studies have identified genes or genetic variants with modest effect to be associated with POAG and related quantitative traits (Table 1). These studies have provided better insights into the genetic basis of POAG and improved our understanding of the underlying pathophysiology of the disease.

GWAS and POAG
Nakano et al. described the first GWAS in the Japanese POAG population with patients predominantly having NTG [38]. This was a two-stage GWAS involving a discovery cohort and a replication cohort. The study reported significant loci on chromosomes 1, 10 and 12 that included genes such as ZP4, PLXDC2 and TMCT2 (DKFZp762A217), respectively. However, none of the SNPs achieved a genome-wide significance (p < 5ˆ10´8) even in the combined analysis and, therefore, they await further evaluation in additional cohorts. Meguro et al. reported the first genome-wide significant (p = 2.5ˆ10´9, odds ratio (OR) = 2.80) association for SNP rs3213787 in SRBD1 in the Japanese NTG population [39]. Two other studies have replicated this finding in a Japanese NTG and high-tension glaucoma (HTG) cohort [89] and a US Caucasian POAG cohort [110], but not in the African-Caribbean cohort [66].
GWASs have been able to identify certain common variants that are of significance to the understanding of POAG pathogenesis. These include SNPs near CAV1 and CAV2 in an Icelandic cohort [40], in TMCO1 and CDKN2B-AS1 in an Australian cohort [41], in CDKN2B-AS1, SIX1/SIX6, and the 8q22 locus in Europeans [42], in GAS7 and TMCO1 in US Caucasians [49], and in CDKN2B-AS1, CDC7/TGFBR3 and FNDC3B in Asian, African and European cohorts [48].
The caveolin genes have been postulated to influence transforming growth factor-beta (TGF-β) or nitric oxide signaling pathways involved in POAG pathogenesis. The locus on chromosome 7q31 has been studies in US Caucasians, Africans, and the Saudi Arabian population with inconsistent results [40,66,[71][72][73]100]. A recent meta-analysis of five studies, including 5774 POAG cases and 40,598 healthy controls, suggested that SNP rs4236601 is associated with POAG risk in Caucasian and Asian populations but not in African and Saudi populations [111]. Australian GWAS identified two loci, TMCO1 (1q24) and CDKN2B-AS1 (9p21), to be associated with advanced glaucoma. The association of the TMCO1 locus with POAG has been replicated in another GWAS for a Caucasian cohort [49], and associated with increase in IOP as well [49,93]; the carriers of risk alleles for SNP rs4656461 have been reported to be associated with a younger age at diagnosis [92]. The ciliary body, trabecular meshwork and retina show abundant TMCO1 expression. However, its precise role in POAG pathogenesis is unclear. So far, there are no published reports of association studies at the TMCO1 locus in the Middle East population.
Since the identification of the association between the CDKN2B/CDKN2B-AS1 locus and POAG in the Australian cohort, several GWASs have replicated this association in the US Caucasian [42], Japanese [43][44][45], Asian, African, and European populations [48], providing strong evidence for the association of this locus with POAG. In addition, many studies have reported a positive association of SNPs in CDKN2B in several other populations using a candidate-gene approach [66,67,69,70,74]. These SNPs are located in an anti-sense non-protein coding gene, CDKN2BAS, within the CDKN2A/B gene cluster. CDKN2B is a tumor suppressor gene and, with its suggested role in the TGF-β pathway, may play a critical role in glaucoma pathogenesis [112,113]. Interestingly, carriers of the CDKN2B-AS1 risk alleles are associated with larger VCDR [53,54] and low IOP as compared to the wild-type carriers [74]. On the basis of these findings, it has been suggested that the CDKN2B/CDKN2B-AS1 locus of 9p21 may possibly predispose a person to glaucomatous optic neuropathy in a mechanism that may not be dependent on IOP and highlights the importance of the chromosome 9p21 susceptibility locus as a risk factor in the development of POAG [114].
Recently, Li et al. performed a GWAS on 3504 POAG cases and 9746 controls. The positive significant findings of this phase were then replicated in 9173 POAG cases and 26,780 controls across 18 different collections of Asian, African, and European populations including a replication cohort from our center in Saudi Arabia [48]. The study confirmed and provided strong evidence of an association at the CDKN2B-AS1 locus (rs2157719, OR = 0.71, p = 2.81ˆ10´3 3 ), and also identified SNP rs1192415 in the CDC7-TGFBR3 gene (1p22) showing significant association with POAG (OR = 1.13, p = 1.60ˆ10´8) in the Asian, African and European populations, as well as SNP rs4894796 in FNDC3B (3q25.31) showing a significant association in Asians only (OR = 0.89, p = 7.93ˆ10´8).
Interestingly, these results were found to be non-significant in the Saudi replication cohort, indicating that the genetic cause for POAG in the Saudi population may be different than those from Asian, African and European descent.
GWAS studies by Wiggs et al. and Osman et al. in the Caucasian POAG and Japanese POAG cases, respectively, have demonstrated a strong association of SNP rs10483727 located in the in the intergenic region between the SIX1 and SIX6 locus (14q23) [42,43]. SIX6 has been shown to express in the developing and adult human retina [115]. Moreover, the association of SNP rs10483727 in the SIX1/SIX6 region has also been replicated in other Caucasian POAG cohorts [67,69,70,88] but not in the African-Caribbean subjects [66]. After the association of the CDKN2B-AS1 region on chromosome 9p21, the second most consistent association with POAG has been observed in the SIX1/SIX6 locus and so it would be interesting to know if this locus is associated with POAG in the Saudi or other Middle Eastern populations. However, currently there are no published reports of association of SIX1/SIX6 locus with POAG in the middle-east population.
These variants were significantly associated with glaucoma and the related functional visual field loss that could make them future study targets for glaucoma patients in the Middle East.

GWAS and Quantitative Endophenotype Traits
The genetic evaluation of quantitative endophenotype traits is often very useful in complex multifactorial diseases to understand the contribution of specific traits to the overall disease phenotype. A similar strategy has been successfully used in POAG to understand the contribution of proposed endophenotypes including IOP, VCDR, optic disc area and CCT to the overall disease process. GWASs have been performed to examine the genetic components of these endophenotypes in POAG and the normal population. van Koolwijk and colleagues performed a GWAS for IOP in POAG patients of European descent and identified SNPs rs11656696 and rs7555523, located in GAS7 and TMCO1, respectively, suggesting a role for these two genes in IOP regulation [49]. Other loci found to be associated with IOP so far include FNDC3B, ABCA1, ABO, 11p11.2, ARHGEF12 [50,52]. Another three loci, FAR2, GGA3, and PKDREJ, did not reach a genome-wide significance level (p < 10´5) [51]. Three independent GWASs have evaluated the association of optic disc parameters (VCDR and optic disc area) in the normal general population. The loci associated included ATOH7, CDC7/TGFBR3 and SALL1, CARD10 for the optic disc area, and CDKN2B, SIX1, SCYL1/LTBP3, CHEK2, and DCLK1, in addition to ATOH7, for the VCDR [53][54][55][56]. An exome sequencing also reported the SIX6 locus to influence VCDR (p = 7.74ˆ10´7) [57]. A subsequent meta-analysis of the Rotterdam study with the Twin UK study [54] demonstrated a strong association of ATOH7, CDKN2B, and SIX1 in POAG with borderline association for CDC7/TGFBR3 and SALL4 (both p = 0.04). CARD10 was not found to be associated with African-Caribbean POAG cases [66], whereas CHEK2 was reported to be associated with VCDR and HTG among the Japanese [67] but not in Europeans [70]. Moreover, multiple studies have provided strong evidence of association of ATOH7 [66-69] CDKN2B(-AS1) [66,67,69,70,74] and SIX1/SIX6 [69,70] with POAG. CCT is an important risk factor for POAG in individuals with increased IOP, and over 26 loci have been reported [116]. GWASs have identified several loci associated with CCT in the normal general population (Asian and European descent) and POAG cases (US Caucasians). These loci include ZNF469, COL5A1, AKAP13, AVGR8, and COL8A2 [58][59][60]. The ZNF469 and COL5A1 loci have been found to be associated with CCT in both the Caucasian and Asian cohorts [58,59].
The possible role of these newly discovered loci associated with POAG and its endophenotypes in understanding the pathophysiology of POAG has been elegantly reviewed by Iglesias et al. elsewhere [117]. The review integrates current knowledge in POAG from human and experimental data and dissects the contribution of the newly discovered genetic loci with the known molecular and biological processes, including extracellular matrix remodeling; TGF-β and tumor necrosis factor α (TNF-α) signaling; and the vascular tone pathway, that have been implicated in the pathogenesis of POAG.

Final Remarks
There is significant progress in understanding the genetic basis of POAG, largely due to the application of GWAS methodology in different populations. In recent years, GWASs have identified several loci associated with POAG including CAV1/CAV2, TMCO1, CDKN2B-AS1, CDC7-TGFBR3, SIX1/SIX6, GAS7 and ATOH7.
The association between the CDKN2B(-AS1) locus on chromosome 9p21 and POAG has been extensively established across different populations and represents a major genetic risk factor for POAG. Studies involving the SIX1/SIX6 and the ATOH7 loci affecting the optic disc parameters and POAG itself have also been reproducible. Other loci seem to be more ethnicity-specific. CAV1/CAV2 and CDC7-TGFBR3 loci do not seem to contribute to POAG in the Middle East and the role of other newly discovered loci is yet to be established. Moreover, the GSTT1/GSTM1 genotypes were found to be strongly associated with POAG in the Middle Eastern population and more studies may be needed to examine the role of oxidative stress and anti-oxidant pathways in this population.
Based on the current and new genes identified in glaucoma, it may be possible to develop an algorithm of SNP risk scores to assess the future risk of POAG in patients, which could be clinically useful. However, despite the tremendous progress, the genetic basis of POAG is still not completely understood and further investigations are needed to identify novel genes and pathways contributing to glaucoma that may help define disease-specific targets and facilitate the development of diagnostic and therapeutic strategies. Appears to contribute to glaucomatous optic neuropathy as a non-IOP-related genetic factor; exact mechanism is not known

ELOVL5
ELOVL Fatty Acid Elongase 5 Catalytic activity Appears to contribute to glaucomatous optic neuropathy as a non-IOP-related genetic factor; exact mechanism is not known    May be through lowering the plasma level of vitamin C. Low level of vitamin C was found in POAG patients carrying mutation in this gene. Exact link between low vitamin C level and POAG is not determined Since the advent of GWAS studies, more and more genes and SNPs have been discovered in association of POAG. However, the usefulness (in term of clinical application and developing therapeutic modalities) of those discoveries is still limited. It will take multiple genotype-phenotype studies in various centers and multiethnic groups before establishing the applicability of those SNPs and/or genes to POAG or POAG clinical indices. As for the development of new therapeutic agents, the process will be lengthy and may take several years before effective therapeutic modalities for POAG are available. The whole process from discovering new genetic markers (SNPs) or genes to developing new therapeutic agents may take several steps and many years. Those steps are: (i) Discover those genes and/or SNPs associated with POAG, which is underway thanks to new emerging technologies in molecular genetics such as exome sequencing and GWAS technologies. This may take up to 10 years to complete; (ii) Establish the association of various SNPs and genes with POAG in various ethnicities, larger cohorts, and in multiple centers. This is important as initial discovery studies are conducted on specific ethnicities and in smaller cohorts; (iii) Conduct functional studies in order to understand how those genes and/or SNPs contribute to POAG pathogenesis; (iv) Develop therapeutic agents based on our understanding of the function of the genes associated with POAG. This step is the longest and expected to take at least 10-15 years. This should not hold us back or make us think less of genetic studies as those may prove to be the only way to improve our current understanding of the etiology of glaucoma and facilitate the development of diagnostic and therapeutic strategies.
Author Contributions: Khaled Abu-Amero and Altaf A. Kondkar reviewed the literature and participated equally in writing the manuscript. Kakarla V. Chalam, supervised the whole process, revised the clinical information reported All three authors approved the final version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.