**The Role of Apolipoprotein E Gene Polymorphisms in Primary Glaucoma and Pseudoexfoliation Syndrome**

Najwa Mohammed Al- Dabbagh, Sulaiman Al-Saleh, Nourah Al-Dohayan, Misbahul Arfin, Mohammad Tariq and Abdulrahman Al-Asmari

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54614

**1. Introduction**

Primary glaucoma (PG) is one of the most common eye diseases which may potentially result in bilateral blindness. Glaucoma affects 70 million people and is the second leading cause of blindness worldwide. It is estimated that by the year 2020, this number would rise to around 79.6 million [1]. The prevalence of glaucoma varies widely across the different ethnic groups [2-8] and is significantly higher in blacks (4.7%) as compared to the white (1.3%) population [9]. The prevalence of both primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) is higher in western region of Saudi Arabia as compared to other Asian countries [10]. To date no national study has been undertaken to determine the exact preva‐ lence of glaucoma in Saudi Arabia, though it is one of the major causes of blindness in this country.

The glaucomas are a group of relatively common optic neuropathies in which pathological loss of retinal ganglion cells cause progressive loss of sight and associated alteration in the retinal nerve fiber layer and optic nerve head. Recent studies clearly suggest that abnormalities in structure and function of retinal nerve fiber layer (RNFL) are proportional to the loss of retinal ganglion cells in glaucoma [11]. Studies on two independent patients' populations also confirmed a close association between RNFL thickness and several visual parameters [12]. The retina is a light capturing tissue consisting of more than fifty different types of cells each performing unique function that ultimately provide the visual centers in the brain the information to achieve image formation and visual perception. Photo production require the

© 2013 Al- Dabbagh et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

retina to have a high metabolic rate, multiple and complex membrane structures [13,14]. The photo receptor outer segments are enriched in polyunsaturated fatty acids including highly light sensitive docosahexenoic acid [15]. Recent experimental study suggests a clear role of fatty acids and cholesterol in optic nerve head blood flow and retinal nerve fibers structures. Retina has a unique mechanism for lipid uptake of low density lipoproteins which provides blood-borne lipids to all the cellular layers of retina [16,17]. Moreover, to keep its steady state lipid composition retina has the ability to synthesize cholesterol [18]. Defects in lipid metab‐ olism in neural retina result in detrimental consequences on its structure and function. Published data clearly suggest the crucial role of lipids and lipoproteins in the pathophsiology of glaucoma [19]. Evidence from population and family studies supports heredity of glaucoma to be a complex trait. It is a genetically heterogeneous disorder attributed to the effects of individual causative mutations as well as interactions of multiple genes with a variety of environmental factors [20].

ciliary body stroma, sclera, and cornea, in eyes with PEX. Besides its presence in the eye the PEX material is found in many other parts of the body such as the eyes, skin, heart, lungs, liver,

The Role of Apolipoprotein E Gene Polymorphisms in Primary Glaucoma and Pseudoexfoliation Syndrome

http://dx.doi.org/10.5772/54614

131

PEX is a heterogeneous group of disorders with both Mendelian and multifactorial traits. Even within individual families, there can be large variations in the phenotypic presentation of gene mutations. Therefore, multifactorial etiologies must be involved in PEX development. This can include polygenic and environmental factors [35]. Some genes may act as susceptibility factors that allow other genes or environmental influences to produce PEX. Further, familial aggre‐ gation and the increased frequency of PEX in relatives of affected subjects compared with relatives of unaffected subjects [36,37] suggest an underlying genetic component [38]. The main problems with studies on the genetic background of PEX have been the asymptomatic nature of PEX and late age of onset which make it difficult to collect multi-generation families with several affected individuals for linkage and association studies. A wide variety of inheritance models have been suggested depending on the study material [39] and, of these, the autosomal dominant mode of inheritance with incomplete penetrance has received the most support [40,41]. However, most of these studies investigating PEX inheritance have been based on small pedigrees making hypotheses about the inheritance model uncertain. Thorleifsson et al. [42] explained the genetic aetiology of PEX in virtually all instances. In Iceland and Sweden, the high-risk haplotype is very common with a frequency that averages about 50% in the general population; approximately 25% are homozygous (two copies) for the haplotype with the

Apolipoprotein E (APOE) is the major apolipoprotein in the central nervous system, which plays important role in the uptake and redistribution of cholesterol within neuronal network [43]. Immunologically, APOE is present in many cerebral and systemic amyloidoses; such as late-onset Alzheimer's disease, Down's syndrome, and prion disorders. It is thought that APOE can promote the aggregation of amyloidogenic proteins into the β-pleated sheet conformation that is typical of all amyloid deposits, and is directly involved in the amyloid deposition and fibril formation [44,45]. This widespread association of APOE with biochemi‐ cally diverse amyloids has led scientists to postulate a more general role for it in the process

APOE is synthesized by Muller cells (the predominant glial cells of the retina) and released into the vitreous and then transported into the optic nerve through anterograde rapid transport where it has an important role in axonal nutrition [46]. It has been suggested that APOE plays a role in neuronal survival following ischemia and other chemical insults and particular APOE isoform may be related to neuronal degeneration in glaucoma [47]. APOE, is a 34-kDa glycosylated protein, composed of 299 amino acids encoded by a four exon polymorphic gene on chromosome 19q13.2. The gene encoding APOE has three polymorphic variants in human designated as ε2, ε3, and ε4. These variants differ from one another by the presence of either C or T nucleotide at codons 112 and 158. These three alleles encode different APOE isoforms which vary significantly in structure and function including receptor binding capacity and lipid metabolism [48]. As each individual human being carries two allelic copies in a gene, six possible genotypes (ε2/ε2, ε3/ε3, ε2/ε3, ε3/ε4, ε2/ε4, and ε4/ε4) are formed by different

kidney, gall bladder, blood vessels, optic nerves, and meninges [26,33,34].

highest risk.

of amyloid formation.

Pseudoexfoliation syndrome (PEX) is another common and clinically significant systemic condition and represents a complex, multifactorial, late-onset disease of worldwide signifi‐ cance with an estimated prevalence ranging from 10% to 20% of the general population [21]. It is clinically diagnosed by observation of whitish flake-like deposits of PEX material on anterior segment structures, particularly on the anterior lens surface and the pupillary border of the iris. Despite its worldwide distribution, there is a clear tendency for PEX syndrome to cluster geographically and in certain racial or ethnic subgroups. For example, there is a high prevalence of PEX syndrome in Nordic, Baltic, Mediterranean, and Arabian populations, where it affects up to 30% of individuals over age 60. The reported mean age of PEX patients ranges from 69 to 75years, and most epidemiological surveys demonstrate an increasing prevalence with increasing age. There is a significantly higher frequency and severity of optic nerve damage at the time of diagnosis, worse visual field damage, poor response to medica‐ tions, more severe clinical course, and more frequent necessity for surgical intervention.

PEX is characterized by the pathological production and accumulation of an abnormal fibrillar extracellular material in the surface lining of the anterior and posterior chambers of the eye. The characteristic fibrillar PEX material is composed of microfibrillar subunits surrounded by an amorphous matrix. The material has a complex glycoprotein/proteoglycan structure composed of a protein core surrounded by glycosaminoglycan [22,23].

The fibrillar portion has been characterized as amyloid laminin, oxytalan, and various elastic tissue and basement membrane components [24-26]. Numerous studies showed positive reactions of PEX material to Congo red, showing its intense fluorescence with thioflavin T and S, and positive immunofluorescence with antiserum to amyloid, affinity for ruthenium red, positive histochemical tests for tyrosine and tryptophan [27-30]. However some other studies failed to demonstrate a positive reaction with Congo red in exfoliative deposits [24,27]. Hypothetically, amyloid might deposit in the vicinity of PEX material fibers because of the affinity they both have for elastic tissues. Moreover amyloid in the skin accumulates close to elastic fibers [31]. It has been suggested that the amyloid component normally present on elastic fibers may serve as a ligand for the amyloid–elastic fiber association [32]. Meratoja and Tarkkanen [30] showed amyloid positive material in sites atypical for PEX disease, such as the ciliary body stroma, sclera, and cornea, in eyes with PEX. Besides its presence in the eye the PEX material is found in many other parts of the body such as the eyes, skin, heart, lungs, liver, kidney, gall bladder, blood vessels, optic nerves, and meninges [26,33,34].

retina to have a high metabolic rate, multiple and complex membrane structures [13,14]. The photo receptor outer segments are enriched in polyunsaturated fatty acids including highly light sensitive docosahexenoic acid [15]. Recent experimental study suggests a clear role of fatty acids and cholesterol in optic nerve head blood flow and retinal nerve fibers structures. Retina has a unique mechanism for lipid uptake of low density lipoproteins which provides blood-borne lipids to all the cellular layers of retina [16,17]. Moreover, to keep its steady state lipid composition retina has the ability to synthesize cholesterol [18]. Defects in lipid metab‐ olism in neural retina result in detrimental consequences on its structure and function. Published data clearly suggest the crucial role of lipids and lipoproteins in the pathophsiology of glaucoma [19]. Evidence from population and family studies supports heredity of glaucoma to be a complex trait. It is a genetically heterogeneous disorder attributed to the effects of individual causative mutations as well as interactions of multiple genes with a variety of

Pseudoexfoliation syndrome (PEX) is another common and clinically significant systemic condition and represents a complex, multifactorial, late-onset disease of worldwide signifi‐ cance with an estimated prevalence ranging from 10% to 20% of the general population [21]. It is clinically diagnosed by observation of whitish flake-like deposits of PEX material on anterior segment structures, particularly on the anterior lens surface and the pupillary border of the iris. Despite its worldwide distribution, there is a clear tendency for PEX syndrome to cluster geographically and in certain racial or ethnic subgroups. For example, there is a high prevalence of PEX syndrome in Nordic, Baltic, Mediterranean, and Arabian populations, where it affects up to 30% of individuals over age 60. The reported mean age of PEX patients ranges from 69 to 75years, and most epidemiological surveys demonstrate an increasing prevalence with increasing age. There is a significantly higher frequency and severity of optic nerve damage at the time of diagnosis, worse visual field damage, poor response to medica‐ tions, more severe clinical course, and more frequent necessity for surgical intervention.

PEX is characterized by the pathological production and accumulation of an abnormal fibrillar extracellular material in the surface lining of the anterior and posterior chambers of the eye. The characteristic fibrillar PEX material is composed of microfibrillar subunits surrounded by an amorphous matrix. The material has a complex glycoprotein/proteoglycan structure

The fibrillar portion has been characterized as amyloid laminin, oxytalan, and various elastic tissue and basement membrane components [24-26]. Numerous studies showed positive reactions of PEX material to Congo red, showing its intense fluorescence with thioflavin T and S, and positive immunofluorescence with antiserum to amyloid, affinity for ruthenium red, positive histochemical tests for tyrosine and tryptophan [27-30]. However some other studies failed to demonstrate a positive reaction with Congo red in exfoliative deposits [24,27]. Hypothetically, amyloid might deposit in the vicinity of PEX material fibers because of the affinity they both have for elastic tissues. Moreover amyloid in the skin accumulates close to elastic fibers [31]. It has been suggested that the amyloid component normally present on elastic fibers may serve as a ligand for the amyloid–elastic fiber association [32]. Meratoja and Tarkkanen [30] showed amyloid positive material in sites atypical for PEX disease, such as the

composed of a protein core surrounded by glycosaminoglycan [22,23].

environmental factors [20].

130 Glaucoma - Basic and Clinical Aspects

PEX is a heterogeneous group of disorders with both Mendelian and multifactorial traits. Even within individual families, there can be large variations in the phenotypic presentation of gene mutations. Therefore, multifactorial etiologies must be involved in PEX development. This can include polygenic and environmental factors [35]. Some genes may act as susceptibility factors that allow other genes or environmental influences to produce PEX. Further, familial aggre‐ gation and the increased frequency of PEX in relatives of affected subjects compared with relatives of unaffected subjects [36,37] suggest an underlying genetic component [38]. The main problems with studies on the genetic background of PEX have been the asymptomatic nature of PEX and late age of onset which make it difficult to collect multi-generation families with several affected individuals for linkage and association studies. A wide variety of inheritance models have been suggested depending on the study material [39] and, of these, the autosomal dominant mode of inheritance with incomplete penetrance has received the most support [40,41]. However, most of these studies investigating PEX inheritance have been based on small pedigrees making hypotheses about the inheritance model uncertain. Thorleifsson et al. [42] explained the genetic aetiology of PEX in virtually all instances. In Iceland and Sweden, the high-risk haplotype is very common with a frequency that averages about 50% in the general population; approximately 25% are homozygous (two copies) for the haplotype with the highest risk.

Apolipoprotein E (APOE) is the major apolipoprotein in the central nervous system, which plays important role in the uptake and redistribution of cholesterol within neuronal network [43]. Immunologically, APOE is present in many cerebral and systemic amyloidoses; such as late-onset Alzheimer's disease, Down's syndrome, and prion disorders. It is thought that APOE can promote the aggregation of amyloidogenic proteins into the β-pleated sheet conformation that is typical of all amyloid deposits, and is directly involved in the amyloid deposition and fibril formation [44,45]. This widespread association of APOE with biochemi‐ cally diverse amyloids has led scientists to postulate a more general role for it in the process of amyloid formation.

APOE is synthesized by Muller cells (the predominant glial cells of the retina) and released into the vitreous and then transported into the optic nerve through anterograde rapid transport where it has an important role in axonal nutrition [46]. It has been suggested that APOE plays a role in neuronal survival following ischemia and other chemical insults and particular APOE isoform may be related to neuronal degeneration in glaucoma [47]. APOE, is a 34-kDa glycosylated protein, composed of 299 amino acids encoded by a four exon polymorphic gene on chromosome 19q13.2. The gene encoding APOE has three polymorphic variants in human designated as ε2, ε3, and ε4. These variants differ from one another by the presence of either C or T nucleotide at codons 112 and 158. These three alleles encode different APOE isoforms which vary significantly in structure and function including receptor binding capacity and lipid metabolism [48]. As each individual human being carries two allelic copies in a gene, six possible genotypes (ε2/ε2, ε3/ε3, ε2/ε3, ε3/ε4, ε2/ε4, and ε4/ε4) are formed by different combinations of these three alleles. The frequency of these genotypes differ significantly among different ethnic groups, however, APOE ε3/ε3 is the most predominant genotype and ε3 the most common allele in majority of populations [49-51]. The ε3 allele is considered to be the ancestral allele; and ε2 and ε4 are considered as variants, on the basis of single point mutations. Global studies on the APOE locus have shown highly significant variations in the allele frequencies of ε2, ε3, and ε4 [52-58].

A comprehensive eye examination was done that included best-corrected visual acuity (BCVA) measurements using logarithm of the minimum angle of resolution (logMAR) 4-m charts (Light House Low Vision Products, New York, NY), applanation tonometry, gonioscopy, dilated fundus examination, optic disc photography, and visual field (VF) examination. On gonioscopy, an angle was considered occludable if the pigmented trabecular meshwork was not visible in >180° of angle in dim illumination. Laser iridotomy was performed in subjects with occludable angles after consent was obtained, and they had the rest of the examination

The Role of Apolipoprotein E Gene Polymorphisms in Primary Glaucoma and Pseudoexfoliation Syndrome

http://dx.doi.org/10.5772/54614

133

Automated VFs were performed for all the subjects with BCVA of 4/16 (logMAR 0.6) or better, using frequency-doubling perimetry (Carl Zeiss Meditec, Inc., Dublin, CA). All eligible subjects underwent C-20-1 screening (if the results were unreliable or abnormal, the test was repeated) and the N-30 threshold test. The reliability criteria were no fixation or false-positive errors for the C-20-1 screening test and < 20% fixation errors and <33% false-positive and falsenegative errors for the threshold N-30 test. Visual fields with no depressed points to any level of sensitivity were considered to be normal. A provisional diagnosis of suspected glaucoma was made when the subject had one or more of the following conditions: intraocular pressure (IOP) ≥ 21 mmHg in either eye; vertical cup-to-disc ratio (VCDR) ≥ 0.7 in either eye or CDR asymmetry ≥ 0.2; and focal thinning, notching, or a splinter hemorrhage. All these subjects were asked to perform a threshold VF test using the Swedish interactive threshold algorithm Standard 30-2 program (model 750, Carl Zeiss Meditec). A glaucomatous field defect was diagnosed using a single reliable threshold VF examination of the central 30° (Swedish interactive threshold algorithm Standard 30-2). The field was considered to be abnormal if the glaucoma Hemi-field test results were outside normal limits and ≥3 abnormal contiguous nonedge points (except the nasal horizontal meridian) were depressed to *P*< 5% [69]. Reliability criteria were as recommended by the instrument's algorithm (fixation losses <20%; false-

The distribution of VCDR and IOP was obtained from those subjects with reliable and normal supra-threshold VF testing using frequency-doubling perimetry. Cases of glaucoma were defined using the International Society of Geographical and Epidemiologic Ophthalmology classification [70]. Glaucoma was classified according to 3 levels of evidence. In category 1, diagnosis was based on structural and functional evidence. It required CDR or CDR asym‐ metry ≥ 97.5th percentile for the normal population or a neuroretinal rim width reduced to ≥ 0.1 CDR (between 11- and 1-o'clock or 5- and 7-o'clock) with a definite VF defect consistent with glaucoma using the Swedish interactive threshold algorithm 30-2. Category 2 was based on advanced structural damage with unproved field loss. This included those subjects in whom VFs could not be determined or were unreliable, with CDR or CDR asymmetry ≥ 99.5th percentile for the normal population. Lastly, category 3 consisted of persons with an IOP ≥ 99.5th percentile for the normal population, whose optic discs could not be examined because

on some other day.

**2.2. Visual fields**

positive and false-negative < 33%).

**2.3. Diagnostic definitions**

of media opacities.

The complex genetic contributions to glaucoma and PEX have been attributed to the effects of individual causative mutations as well as interactions of multiple genes with a variety of environmental factors. However, most of the identified genes do not appear to have a major role in the complex phenotype. Recent whole genome–association studies have successfully identified a number of single nucleotide polymorphisms as genetic factors conferring sus‐ ceptibility to complex diseases, such as age-related macular degeneration, and it is expected that this will be a useful approach for glaucoma and PEX as well.

Earlier studies clearly point towards a possible association between APOE alleles and glau‐ coma. However, the results of these studies are contradictory. Some investigators suggested positive association [47,59,60] while others have shown no link at all [61-63]. Moreover, earlier studies were mainly restricted to white populations from Australia [47], United Kingdom [62,63] and Sweden [61] with only few reports from other ethnic groups restricted to Chinese and Japanese [59,60,64,65]. Similarly APOE polymorphism and the presence of ε 2 alleles have been reported to be significantly associated with the development of PEX in Turkish patients [66]. However, APOE genotypes and PEX seems to differ among study populations and no significant differences in allele and genotype frequencies between PEX and control were observed in European patients from Norway [67] and Germany [68]. Moreover, the informa‐ tion about the association of APOE alleles with glaucoma and PEX in Arabs is very limited. Therefore, this study on underlying genetics in these complex disorders will help analyze the genetic aspect of PEX and glaucoma in Saudi patients. In this study, we evaluated the possible association of alleles/genotypes of APOE with primary glaucoma (POAG and PACG) and PEX in Saudi population.
