Genetics and Glaucoma: the state of the art

Glaucoma is the second leading cause of irreversible blindness worldwide. Although genetic background contributes differently to rare early-onset glaucoma (before age 40) or common adult-onset glaucoma, it is now considered an important factor in all major forms of the disease. Genetic and genomic studies, including GWAS, are contributing to identifying novel loci associated with glaucoma or to endophenotypes across ancestries to enrich the knowledge about glaucoma genetic susceptibility. Moreover, new high-throughput functional genomics contributes to defining the relevance of genetic results in the biological pathways and processes involved in glaucoma pathogenesis. Such studies are expected to advance significantly our understanding of glaucoma’s genetic basis and provide new druggable targets to treat glaucoma. This review gives an overview of the role of genetics in the pathogenesis or risk of glaucoma.


Introduction
Glaucoma is the second leading cause of blindness worldwide, and it is estimated that its incidence will increase in the next 20 years (1)(2)(3).The term glaucoma brings together a heterogeneous group of neurodegenerative diseases characterized by Retinal Ganglion Cell (RGC) degeneration as well as typical morphological changes in the Optic Nerve Head (ONH), i.e., the narrowing of the neuroretinal rim, the enlargement of the excavation, and the loss of prelaminar neural tissue (4,5).
In general, all glaucoma forms are categorized into primary and secondary forms, according to their etiology.Primary glaucoma is defined as an isolated, idiopathic disease of the anterior chamber of the eye and the optic nerve whereas the secondary one is characteristic of specific syndromic frameworks (e.g., nail patella, exfoliation syndrome, aniridia), or associated with predisposing events, including systemic diseases (e.g., diabetes) and pharmacological treatment.non-development of neural crest-derived tissues, which results in developmental anomaly at the angle of the anterior chamber (9).Finally, PACG is responsible for most cases of glaucoma-induced bilateral blindness in populations, such as Asians (10).In such glaucoma, the anatomic narrow-angle leads to trabecular meshwork (TM) obstruction and, consequently, to peripheral anterior synechiae, elevated intraocular pressure IOP, iris whirling or sectoral atrophy, and excessive pigment deposition on the trabecular surface (10)(11)(12)(13).
Glaucoma can occur at all ages exhibiting a classic (Mendelian) inheritance pattern (i.e., onset before the age of 40) (14,15) or a complex inheritance pattern [i.e., onset after the age of (16,17)].Therefore, genetic background is considered a precondition that can directly cause or contribute to glaucoma.This present review aims to summarize the main achievement in genetic studies of glaucoma, mainly focusing on glaucoma-causing genes.

Methods
The data in this present comprehensive review were collected using two different searches: PubMed1 and the online database: genome aggregation database, 2 and the NHGRI-EBI Catalog of Human Genome-Wide Association Studies. 3The search of the references using PubMed identified a total of 157 hits with no time frame relative to keywords such as "Genetic of glaucoma" "POAG guidelines, " "JOAG management, " "MYOC mutations in glaucoma, " "CYP1B1 in glaucoma, " "PACG guidelines, " "clinical and genetic update of glaucoma, " "candidate genes involved in glaucoma." Thus, the combined information obtained from articles relevant to the subject matter and the online database has represented the basis for writing the review.

Primary open angle glaucoma
Primary open angle glaucoma is defined as a progressive chronic bilateral or asymmetric ocular disease (18).Clinical evidence in addition to visual field defects and the age of patients (i.e., > the 40s) includes structural abnormalities to the optic disk (OD) rim or parapapillary retinal nerve fiber layer (RNFL) (19).The severity of POAG damage can be distinguished as mild, moderate, and severe based on the presence or not of possible changes in the optic disk, RNFL damage, and degree of visual field abnormalities.
Although it is not an effective treatment for all patients, IOP management is the only clinically addressable risk factor to prevent or delay POAG progression (3).Primary open angle glaucoma guidelines (20) suggest the use of topical medications (e.g., Prostaglandin analogs, beta-blockers, alpha 2 adrenergic agonists, parasympathomimetic, and topical and oral carbonic anhydrase inhibitors) as the first line of treatment to reach the target pressure.However, since treatment regimens require daily applications to control IOP, noncompliance, undesirable adverse effects, and cost limit their effectiveness (21,22).Laser trabeculoplasty and glaucoma surgery (e.g., trabeculectomy, aqueous shunts, non-penetrating glaucoma surgery, micro-invasive glaucoma surgery, and so on…) are considered for further IOP reduction in eyes with inadequate initial responses (23).
Adult-onset POAG is a complex disease often caused by an intricate interplay at molecular levels between predisposing genes and evidence-based risk factors (e.g., ethnicity, gender, aging, IOP, central corneal thickness, migraine headache and peripheral vasospasm, myopia, systemic diseases, steroid responsiveness, positive history of glaucoma) (3,24,25).Primary open angle glaucoma may be inherited as a Mendelian trait, as in the case of causative mutations in genes such as the myocilin (MYOC), optineurin (OPTN), and TANK-binding kinase 1 (TBK1) (26,27), where other genes or environmental factors play little role (See below).
Generally, POAG is unsuitable for linkage studies because they require many living POAG-affected relatives and this, for obvious reasons, is not always possible.Thus, highlighting an inheritance pattern for POAG represents a real challenge compared to the childhood-onset glaucomas (24,(28)(29)(30)(31).
Genome-Wide Association Studies (GWAS) provide information about the common POAG genetic variants, i.e., Single Nucleotide Polymorphisms (SNPs), that are found among populations of diverse ancestry (Table 1).In this regard, recurring SNPs may constitute a quantifiable relationship between the genetic variability of POAG and its clinical manifestation (endophenotypes) (30,41,42).However, SNPs have limited predictive value in terms of risk assessment and prognosis of POAG due to its complex inheritance pattern (43).

Juvenile open angle glaucoma
Juvenile open angle glaucoma, as above mentioned, is an uncommon subset (0,38,100,000) of POAG that affects individuals during childhood or early adulthood.
In most cases, JOAG is characterized by severe IOP elevation (>40 mmHg) due to immaturity of the conventional outflow pathway, and thick corneas.However, the extent of clinical manifestations, including the IOP elevation, degree of goniodysgenesis, and the inheritance pattern, as well as the age at onset, are subject to individual variations.In this regard, four phenotype groups have been identified to classify affected individuals based on the age of onset, highest untreated IOP, gonioscopic findings, and iris features (52) (Table 2).
Juvenile open angle glaucoma patients are often asymptomatic until an advanced stage, therefore, a prompt diagnosis is not always possible.When JOAG becomes symptomatic results in blurred vision, eye pain, decreased visual acuity, tearing, blinking, and glare (55).As known, an early JOAG diagnosis increases the chance of preserving vision and preventing permanent visual field loss.Surgery (i.e., trabeculectomy, external trabeculectomy, goniotomy, drainage implants, gonioscopy-assisted transluminal trabeculectomy, and so on…) is often required since the affected individuals are refractory to the standard primary drug approaches to manage the IOP (e.g., β blockers, topical carbonic anhydrase inhibitor, prostaglandin analogs, α-adrenergic agonists) (56, 57).

Myocilin
The MYOC gene, also known as Trabecular meshwork Inducible Glucocorticoid Response (TIGR), maps to the GLC1A (Glaucoma 1A) locus at chromosome 1q21-q31.It consists of three exons: the first encodes for amino-terminal region of the protein containing a peptide signal sequence and a leucine zipper-like motif (i.e., where the myocilin-myocilin interactions take place) with periodic arginine and leucine repeats arranged along an α-helix; the second encodes for the protein central region; the third encodes for the carboxyl-terminal half of myocilin, the olfactomedin (OLF)-domain, which includes the majority of the identified disease causing variants (58).
Myocilin encodes the myocilin glycoprotein expressed in several ocular tissues, such as the cornea, iris, ciliary body, retinal epithelium, and TM.Myocilin mutations can be responsible for the improper function of the eye tissue where the protein is commonly highly expressed, e.g., TM (59).Moreover, myocilin is found also in non-ocular tissues, such as the heart tissue and skeletal muscle but there is no evidence of its involvement in systemic disease (60,61).
Myocilin in its wild-type (wt) state is a ubiquitous protein whose function in the eye has not been determined yet.One suggestion is that myocilin contributes to the maintenance of IOP homeostasis, although it has not been confirmed due to conflicting and contradictory data.While some studies report that myocilin up-regulation is responsible for IOP elevation (62, 63), other ones report that neither its overexpression nor its haploinsufficiency is the primary mechanism for glaucoma phenotype.(31,64,65).This latter evidence has challenged the pathological role of myocilin in promoting steroid-induced glaucoma (~40% of cases) after long-term glucocorticoid treatment (e.g., dexamethasone) (66).In this regard, previous studies on steroid-induced myocilin overexpression excluded its role in both ocular hypertension and endoplasmic reticulum (ER) stress (67,68).However, a myocilin stress-related function in TM has been suggested because stressful stimuli, such as hydrogen peroxide and elevated IOP, increase its expression (69,70).
The first evidence that showed a correlation between POAG and mutant myocilin date from 1997 (71).Linkage studies have shown that MYOC mutations, accounting for 8-34% of JOAG, 2-4% of adultonset POAG cases, and in part for NTG, are mainly inherited in an autosomal dominant manner with incomplete penetrance (72).In this regard, disease severity varies between individuals due to a strong genotype-phenotype correlation (73).A meta-analysis of the penetrance of MYOC mutation revealed that except for a few variants (e.g., Pro370Leu, Thr377Arg, Asp380Ala, and Asn480Lys) having a stable penetrance, the penetrance of MYOC mutations can be influenced by factors such as aging, and ethnicity (74).
Under physiological conditions, wt myocilin after intracellular endoproteolytic processing, produces two fragments, i.e., the 35kD fragment containing the C-terminal OLF-like domain and the 20kD fragment containing the N-terminal leucine zipper-like domain (75).However, only the 35kD fragment is co-secreted with non-processed protein leading to assume that it may play a role in the regulation of the myocilin extracellular activity (76,77).The best known-myocilininduced disease phenotype is characterized by an intracellular abnormal buildup of myocilin in TM (26,78,79), resulting in a toxic gain-of-function disease mechanism.Indeed, most of the myocilinassociated glaucoma mutations are missense clustered in the OLF domain (Table 3).These mutations by inducing defects in myocilin stability, promote the mutant myocilin retention into the endoplasmic reticulum (ER) of TM cells with toxic consequences, such as ER stress, cytotoxicity, and TM apoptosis, which in turn affect the IOP control (61,75,(91)(92)(93).However, also nonsense mutations are involved in about 5.7% of JOAG and adult-onset POAG cases.Notably, the nonsense mutation Gln368Stop is responsible for 40% of High Tension Glaucoma (HTG) (91,94,95).

Familial normal tension glaucoma
Normal-tension glaucoma (NTG) is an OAG characterized by an IOP within a statistically normal range (≤21 mmHg).The NTG pattern of damage in the optic disk, retinal nerve fiber layer, and visual field significantly differs from that found in POAG, suggesting that the optic neuropathy is due to other mechanisms rather than elevating IOP (96).In this regard, a higher sensitivity to normal pressure, vascular dysregulation, an abnormally high translaminar pressure gradient, and a low cerebrospinal fluid pressure in the optic nerve sheath compartment, have been proposed to contribute to NTG pathogenesis (97).
Familial NTG refers to an early onset (at 20-35 years old) bilateral disease with variable severity (99).Optineurin, MYOC, and TBK1 are indicated as NTG-causing genes, according to the Mendelian autosomal dominant inheritance pattern.
The optineurin-mediated autophagy-dysfunction by either overstimulation or by defects that block its function has attracted considerable attention due to its involvement in a variety of neurodegenerative diseases (glaucoma, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease), inflammatory diseases (Crohn's disease, acute liver failure, rheumatoid arthritis) cancer, and nephropathy (104).Notably, the autophagy-mediated RGC death in POAG, also known as autophagic cell death, seems to be promoted by an excess of catabolic activity induced by chronic IOP elevation (105) or alterations in OPTN and TBK1 (106).Under physiological conditions, OPTN to link structures targeted for elimination with proteins of an assembling autophagosome must be phosphorylated by TBK1.However, POAG-associated OPTN mutations may increase the autophagy activation by promoting OPTN phosphorylation via TBK1.Moreover, other OPTN functions may be altered by specific mutations (see below).On the other hand, the gain-of-function mutation and copy number variation (CNV) of the TBK1 gene have been proposed as possible Normal Tension Glaucoma (NTG, a POAG subtype) causing mechanism (107, 108).
Rezaie et al. ( 109) described four sequence alterations in OPTN involved in autosomal dominant adult-onset glaucoma cases, i.e., Glu50Lys, Arg545Gnl, 691_692insAG, and Met98Lys.Most of the affected families harboring these OPTN alterations had normal IOP, and only a small portion had high IOP.Among the OPTN polymorphisms, Glu50Lys, which is located within a putative bZIP motif, is the most recurrent likely disease-causing variant associated with hereditary NTG in Caucasian and Hispanic individuals.The Glu50Lys-induced functional alterations leading to RGC apoptosis are probably related to both autophagy and trafficking dysfunction and an increase in oxidative stress (110,111).
Arg545Gnl may be considered a disease-causing variant only in Asian populations because this role was not shown in other studies (112).691_692insAG variant was described as a dominant mutation responsible for familial POAG in Japan (109) and Eastern Europe (112).Met98Lys was initially proposed as an attributable risk factor for familial and sporadic POAG cases but due to conflicting results among diverse populations, it was not confirmed (113)(114)(115).
Interesting, semiquantitative, hierarchical evidence-based rules for locus interpretation (Sherloc), which are a refinement of the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines, are providing an accurate clinical prediction of the functional consequences of OPTN mutations (116).The algorithms used to predict the effect of sequence change suggest that variants affecting protein sequence (e.g., missense mutation), its function (e.g., splicing mutation), or its abundance (e.g., frameshift or nonsense mutations) are presumed more likely to be deleterious (Please visit the National Library of Medicine for more information). 4Further studies on larger populations are required to confirm the incrimination of OPTN in POAG, to discriminate OPTN mutations causing POAG from those acting only as risk factors, and to ascertain the rate of loss-of-function OPTN variants in different populations.

PGC
Primary congenital glaucoma is considered a non-syndromic glaucoma form characterized by a childhood onset with variable incidence across countries and ethnic groups, but it is most widespread in countries where consanguineous marriages abound (e.g., Middle East) (117,118).
This pediatric glaucoma occurs at birth or within the first 3 years of life, and the clinical outcomes vary depending on whether the neonatal/newborn-onset PGC (within a month after birth) or lateonset-PGC (after 2 years of age).Indeed, neonatal PGC has a poor visual prognosis compared to that diagnosis later, especially if accompanied by amblyopia (119, 120).Moreover, other elements strongly associated with poor long-term visual outcomes are unilateral disease, multiple surgeries, poor vision at diagnosis, and ocular co-morbidities.
The main mechanisms involved in PCG are related to trabecular dysgenesis due to mutations affecting normal TM development with or without iridodysgenesis and maldeveloped trabecular angle meshwork with or without dysgenesis of the Schlemm's canal (121)(122)(123).Moreover, histopathological findings of the anterior chamber angle of PCG eyes reveal an immature anatomic relationship involving iris, TM, and ciliary body (119,124,125).Clinical features include high IOP, an increase in horizontal corneal diameter, the presence of corneal edema, Haab's striae, the pupillary reflex, refraction, axial length, and keratometry (124).
High IOP and its fluctuations are managed through multiple or single surgical drainage procedures (e.g., goniotomy and trabeculotomy ab externo) aimed at targeting the abnormal trabecular angle.Topical hypotensive medications could be used post-op (126).
Genetically, PCG is considered heterogeneous.10-40% of affected individuals show several pathogenetic variants of CYP1B1 or LTBP2 inherited in an autosomal recessive manner and with varying degrees of penetrance (127).Moreover, also autosomal dominant and pseudodominant inheritance patterns have been described as well (117,128,129).
Human Cyp1b1 consists of three exons and two introns, where the coding region is located within the second and the third exons.It is expressed constitutively in many parenchymal and stromal tissues, including the liver, brain, kidney, prostate, breast, cervix, uterus, ovary, lymph nodes, heart, placenta, lung, skeletal muscle, and eye (85,151).
The role of CYP1B1 in PCG and JOAG is not clear, but it has been hypothesized that it plays a metabolic role in eye development and function, as well as cellular redox homeostasis maintenance (152)(153)(154).Therefore, the most accepted theory to explain the link between CYP1B1 mutation and PCG holds that mutated CYP1B1 is the leading cause of maladapted and dysfunctional TM due to defects in embryonic neural crest cell migration during development.This condition, in turn, leads to increased IOP, compression of the optic nerve, and blindness, if not treated surgically (155).Moreover, mutations in the Cyp1b1 gene, in addition to being strongly related to the pathophysiology of PCG, are associated with the most severe disease outcomes.Notably, a genotype-phenotype study in a large cohort of Moroccan PCG reports that both patients carrying CYP1B1 mutation and those with double CYP1B1 null alleles showed the most severe phenotype when compared with patients genetically negative (156).
Most of the CYP1B1 mutations responsible for a maldeveloped and dysfunctional TM are missense (Table 5), but how they affect disease phenotype and their frequency among different populations is still under investigation (152,155).Recently, "in silico" methods (supported by databases, network analysis, and machine learning approaches) were used to identify possible high-risk deleterious variations by predicting the functional importance (neutral or deleterious) of the altered amino acid substitution on protein stability and the resulting phenotypic characteristics (e.g., disruption of the structural conformation), considering the allele frequency of variants (157).Based on such approaches, three novel missense mutations, i.e., Leu487Pro, Leu177Arg and Trp434Arg, and a transversion mutation Asp374Glu were identified in consanguineous Pakistani families as impacting the CYP1B1 protein stability, structure, and function (158).The results obtained from computational tools, functional assays, segregation analysis, genetic studies, and epidemiological data could potentially be helpful for the understanding and management of PCG.

Primary angle closure glaucoma
Primary angle closure glaucoma (PACG) is characterized by at least 180° of iridotrabecular contact, resulting from mechanical obstruction of the TM in the anterior chamber angle and glaucomatous optic neuropathy due to elevated IOP.PACG pathogenesis can be explained as a pupillary block, which is the most common one, plateau iris or peripheral iris crowding, and multiple mechanism patterns, according to clinical phenotype, anatomic configurations, etiology and natural history (159).
From a clinical point of view, PACG is classified into acute, sub-acute, and chronic angle-closure glaucoma, based on the timing of onset, symptoms, and clinical findings.However, is not unusual to observe a combination of these clinical types in patients.Indeed, acute/subacute angle-closure glaucoma may progress to a chronic stage if it does not remit spontaneously or under medical intervention or vice-versa.
In acute glaucoma, the sudden elevation of IOP (usually more than 50 mmHg) leads to severe symptoms, including pressure-induced corneal edema (i.e., blurred vision and halos around lights), mid-dilated pupil, lens opacity (glaucomflecken), vascular congestion, headache, eye pain, and redness (160).The subacute presentation, which may occur before the acute attack or before peripheral anterior synechiae (in the case of chronic IOP elevation), can be asymptomatic or show mild prodromal symptoms, such as headache, blurred vision, and halos, coinciding with IOP spikes (161).Finally, in chronic angleclosure glaucoma, IOP is usually chronically raised and generally, no symptoms are experienced when vision loss occurs.The closure of the Diagnosis of PACG is associated with its natural course and is often delayed until an advanced phenotypic stage, i.e., when the patient has chronic visual loss or acute angle closure.Several risk factors, such as aging, female gender, Inuit, East Asian, and South Indians ethnicity, anatomical features (e.g., shallow anterior chamber depth, short axial length, thick and anteriorly positioned lens, plateau iris configuration, and hyperopia), family history (i.e., first-degree relatives), and environmental factors, are involved in promotion of the disease progression (165)(166)(167).

Conclusion
Although impressive advancements in glaucoma study and treatment are progressing, it still represents the second leading cause of blindness worldwide.Only a small portion of rare early-onset glaucomas (before age 40) show Mendelian inheritance, while the common adult-onset forms are characterized by a complex inheritance.
GWAS studies aim to identify either genetic variants associated with disease or endophenotypes across ancestries and important functional insights underlying biological mechanisms of glaucoma pathogenesis.GWAS results are clinically important to identify subjects with an increased risk of the disease and they can be the starting point for studying the contribution of protein-changing, coding sequence genetic variants.Thus, next-generation sequencing technologies coupled with high throughput functional genomics can enrich our understanding of the disease pathophysiological mechanisms by identifying novel disease-associated or causative genetic variants for predicting glaucoma risk.However, although gene-based screening can be useful for genetic counseling and treatment and surveillance plans in families with a history of earlyonset glaucoma, a limitation is a relatively low diagnostic yield after testing for the genes currently known (170).Moreover, gene-based screening for adult-onset glaucoma seems to be accurate when comprises a large number of risk alleles.
The study of the complex genetic background in glaucoma is in the making but in the future, the hope is that it will become a tool to assess personal risk for the disease and facilitate novel therapeutic strategies.

TABLE 1
List of some known SNPs identified in different glaucoma cohorts (source: GWAS Catalog).

TABLE 2
Four phenotypic groups of JOAG.

TABLE 3
List of some pathogenic/likely pathogenic glaucoma-associated myocilin missense mutations.