**2. Methods**

#### **2.1. Subjects**

The present study was undertaken to evaluate the association of APOE allele and genotype in Saudi primary glaucoma and pseudoexfoliation syndrome patients. A total of 200 unrelated Saudi patients with primary glaucoma [primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG)] and 51 pseudoexfoliation syndrome (PEX) were recruited from ophthalmology clinic of the Riyadh Military Hospital, Saudi Arabia. The glaucoma patient group consisted of 100 males and 100 females, with age at diagnosis ranging from 30 to 78 years (mean ± SD: 58±14.4). The control group consisted of 200 unrelated subjects, with 160 males and 40 females, ages ranging from 20 to 58 years (mean ± SD: 45±11.6). The diagnosis of PG was based on clinical observations:

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 on some other day.

#### **2.2. Visual fields**

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

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

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

The present study was undertaken to evaluate the association of APOE allele and genotype in Saudi primary glaucoma and pseudoexfoliation syndrome patients. A total of 200 unrelated Saudi patients with primary glaucoma [primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG)] and 51 pseudoexfoliation syndrome (PEX) were recruited from ophthalmology clinic of the Riyadh Military Hospital, Saudi Arabia. The glaucoma patient group consisted of 100 males and 100 females, with age at diagnosis ranging from 30 to 78 years (mean ± SD: 58±14.4). The control group consisted of 200 unrelated subjects, with 160 males and 40 females, ages ranging from 20 to 58 years (mean ± SD: 45±11.6). The diagnosis

allele frequencies of ε2, ε3, and ε4 [52-58].

132 Glaucoma - Basic and Clinical Aspects

in Saudi population.

of PG was based on clinical observations:

**2. Methods**

**2.1. Subjects**

that this will be a useful approach for glaucoma and PEX as well.

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%; falsepositive and false-negative < 33%).

### **2.3. Diagnostic definitions**

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 of media opacities.

Blindness was defined as a best-corrected logMAR visual acuity of < 2/40 (log MAR 1.3) and/or constriction of the VF to <10° from fixation in the better eye [71]. Hyperopia was defined as spherical equivalent > 0.50diopter(D)inaphakic eye [72].Diabetesmellituswasdetectedbased oncurrentuseofantidiabeticmedicationand/orrandombloodsugarlevel>200mg/dl[73].Thus the primary Glaucoma patients were separated in two groups (POAG and PACG) as follows:

Ethics Committee of the Hospital, and written informed consent was obtained from all study

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

The genotypes of the APOE polymorphisms were determined using APOE StripAssayTM kit based on polymerase chain reaction (PCR) and reverse-hybridization technique (ViennaLab Labordiagnostika GmbH, Vienna, Austria). The procedure included three steps: (1) DNA isolation, (2) PCR amplification using biotinylated primers, (3) hybridization of amplification product to a test strip containing allele-specific oligonucleotide probes immobilizd as an array of parallel lines. Bound biotinylated sequences were detected using streptavidin- alkaline phosphatase and color substrates. To cross-check the results the genotypes of the APOE polymorphisms were also determined by PCR and restriction fragment length polymorphism (RFLP) technique. Primers were designed on the basis of the sequence data for APOE available in the GenBank to amplify the coding sequence of APOE. PCR was performed using PuRe Taq

*Forward primer:* 5- GAC GCG GGC ACG GCT GTC CAA GGA GCT GCA GGC

*Backward primer:* 5 - AGG CCA CGC TCG ACG CCC TCG CGG GCC CCG GCC

Genomic DNA was extracted from whole blood using a commercial kit (Qiamp; Qiagen, Hiden, Germany). The 200–300 ng of genomic DNA was used as a template in 25 *μ*l reaction. Genomic DNA was amplified for 40 cycles. Each cycle consisted of: 94 °C for 30 sec, 68 °C for 10 sec, 72 °C for 1 min; PCR products obtained were separated by electrophoresis on 1.5% agarose gel in TAE buffer, visualized by ethidium bromide fluorescence. Fragments with the expected size were cut from the gel, purified using GFX PCR DNA Gel band purification kit (Amersham, USA). Purified DNA was digested with *Cfo* I (Hha I) enzyme, separated by agarose gel electrophoresis to identify the genotype. On the basis of size and number of various fragments generated, APOE genotypes were determined as ε2/ε2 with 144 bp and 96 bp, ε3/ε3 with 144 bp and 48 bp, ε4/ ε4 with 72 bp and 48 bp, ε2/ε3 with 144 bp, 96 bp and 48 bp, ε3/ ε4 with 144 bp, 72 bp and 48 bp, and ε2/ε4 with 144 bp, 96 bp, 72 bp and 48 bp fragments. The prevalence of various genotypes in patients and controls was determined. Complete

matching of results was obtained following both of the above mentioned procedures.

Frequencies of various alleles and genotypes for each polymorphism were compared between patients and controls and analyzed by Fisher's exact test and the *P*-values < 0.05 were consid‐ ered as significant. The strength of the association of disease with respect to a particular allele/ genotype is expressed by odd ratio interpreted as *relative risk* (RR) according to the method of Woolf as outlined by Schallreuter *et al* [74]. The RR was calculated only for those alleles and

TGG TAC ACT-3

GAC GCA GGC CCG GCT GGA CGC GGA CAT GGA GGA-3

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135

Ready-To-Go PCR Beads (Amersham, USA) with following primers:

participants.

**2.4. Genotyping**

**2.5. Statistical analysis**

POAG: Anterior chamber angles open and appearing normal by gonioscopy, typical features of glaucomatous optic disc as defined earlier, and visual field defects corresponding to the optic disc changes.

PACG: At least two of the criteria mentioned: glaucomatous optic disc damage or glaucoma‐ tous visual field defects in combination with anterior chamber angle partly or totally closed, appositional angle closure or synechiae in angle, absence of signs of secondary angle closure (e.g., uveitis, lens related glaucoma; microspherophakia; evidence of neovascularization in the angle and associated retinal ischemia or congenital angle anomalies). Patients with signs of intracranial disease that would cause optic nerve atrophy in x-ray computerized tomography or magnetic resonance imaging were excluded.

Diagnosis of PEX among Saudi patients visiting Primary Care Clinics of Riyadh Military Hospital was undertaken by a team of ophthalmologists. Patients visiting primary care clinic were offered free eye examination to exclude the presence of PEX. Consent was obtained from the patients after describing them the features of PEX syndrome. Patients who suffered ocular trauma or with active eye condition, and/or has undergone ocular surgery were excluded from this study.

All patients were subjected to interviews and initial evaluation was performed by the oph‐ thalmic assistant (OA). Demographic data were collected, complaints of the eye and family history of eye problems were recorded. Visual acuity was recorded. After the preliminary examination and interview all patients were examined by an ophthalmologist for identifying the factors for PEX syndrome by the external eye examination: PEX flakes on pupil margin (undilated examination), Iris transillumination defects, evaluation of anterior chamber depth by Van Herick's technique, measurement of intraocular pressure, poor pupil dilation, and examination of the crystalline lens surface after papillary dilation for the presence of PEX material. After identification of PEX, the patients were short listed and further rechecking and confirmation of PEX syndrome was performed by (1) slit lamp examination of the anterior segment which included flakes on the pupillary margin, iris transillumination defects, flare in the A/C and corneal edema, (2) measurement of intraocular pressure (IOP) with Goldman tanometer (3) gonioscopy to record angle depth PEX flakes and/or hyperpigmentation on the trabecular meshwork which was followed by examination after dilation which included Poor pupillary dilation, flakes on the anterior lens capsule, posterior synechiae, lens opacity, phacodenesis and lens subluxation, bilaterality and symmetry and optic nerve head cupping. Out of 51 confirmed cases of PEX 25 were males and 26 females. The average age of PEX positive males and females patients was 70.43±9.62 years and 65.56±7.45 years respectively.

Venous blood was collected from the confirmed PEX and PG patients as well as healthy controls, stored at -20ºC before extraction of DNA. The study protocol was approved by the Ethics Committee of the Hospital, and written informed consent was obtained from all study participants.

#### **2.4. Genotyping**

Blindness was defined as a best-corrected logMAR visual acuity of < 2/40 (log MAR 1.3) and/or constriction of the VF to <10° from fixation in the better eye [71]. Hyperopia was defined as spherical equivalent > 0.50diopter(D)inaphakic eye [72].Diabetesmellituswasdetectedbased oncurrentuseofantidiabeticmedicationand/orrandombloodsugarlevel>200mg/dl[73].Thus the primary Glaucoma patients were separated in two groups (POAG and PACG) as follows:

POAG: Anterior chamber angles open and appearing normal by gonioscopy, typical features of glaucomatous optic disc as defined earlier, and visual field defects corresponding to the

PACG: At least two of the criteria mentioned: glaucomatous optic disc damage or glaucoma‐ tous visual field defects in combination with anterior chamber angle partly or totally closed, appositional angle closure or synechiae in angle, absence of signs of secondary angle closure (e.g., uveitis, lens related glaucoma; microspherophakia; evidence of neovascularization in the angle and associated retinal ischemia or congenital angle anomalies). Patients with signs of intracranial disease that would cause optic nerve atrophy in x-ray computerized tomography

Diagnosis of PEX among Saudi patients visiting Primary Care Clinics of Riyadh Military Hospital was undertaken by a team of ophthalmologists. Patients visiting primary care clinic were offered free eye examination to exclude the presence of PEX. Consent was obtained from the patients after describing them the features of PEX syndrome. Patients who suffered ocular trauma or with active eye condition, and/or has undergone ocular surgery were excluded from

All patients were subjected to interviews and initial evaluation was performed by the oph‐ thalmic assistant (OA). Demographic data were collected, complaints of the eye and family history of eye problems were recorded. Visual acuity was recorded. After the preliminary examination and interview all patients were examined by an ophthalmologist for identifying the factors for PEX syndrome by the external eye examination: PEX flakes on pupil margin (undilated examination), Iris transillumination defects, evaluation of anterior chamber depth by Van Herick's technique, measurement of intraocular pressure, poor pupil dilation, and examination of the crystalline lens surface after papillary dilation for the presence of PEX material. After identification of PEX, the patients were short listed and further rechecking and confirmation of PEX syndrome was performed by (1) slit lamp examination of the anterior segment which included flakes on the pupillary margin, iris transillumination defects, flare in the A/C and corneal edema, (2) measurement of intraocular pressure (IOP) with Goldman tanometer (3) gonioscopy to record angle depth PEX flakes and/or hyperpigmentation on the trabecular meshwork which was followed by examination after dilation which included Poor pupillary dilation, flakes on the anterior lens capsule, posterior synechiae, lens opacity, phacodenesis and lens subluxation, bilaterality and symmetry and optic nerve head cupping. Out of 51 confirmed cases of PEX 25 were males and 26 females. The average age of PEX positive males and females patients was 70.43±9.62 years and 65.56±7.45 years respectively.

Venous blood was collected from the confirmed PEX and PG patients as well as healthy controls, stored at -20ºC before extraction of DNA. The study protocol was approved by the

optic disc changes.

134 Glaucoma - Basic and Clinical Aspects

this study.

or magnetic resonance imaging were excluded.

The genotypes of the APOE polymorphisms were determined using APOE StripAssayTM kit based on polymerase chain reaction (PCR) and reverse-hybridization technique (ViennaLab Labordiagnostika GmbH, Vienna, Austria). The procedure included three steps: (1) DNA isolation, (2) PCR amplification using biotinylated primers, (3) hybridization of amplification product to a test strip containing allele-specific oligonucleotide probes immobilizd as an array of parallel lines. Bound biotinylated sequences were detected using streptavidin- alkaline phosphatase and color substrates. To cross-check the results the genotypes of the APOE polymorphisms were also determined by PCR and restriction fragment length polymorphism (RFLP) technique. Primers were designed on the basis of the sequence data for APOE available in the GenBank to amplify the coding sequence of APOE. PCR was performed using PuRe Taq Ready-To-Go PCR Beads (Amersham, USA) with following primers:


Genomic DNA was extracted from whole blood using a commercial kit (Qiamp; Qiagen, Hiden, Germany). The 200–300 ng of genomic DNA was used as a template in 25 *μ*l reaction. Genomic DNA was amplified for 40 cycles. Each cycle consisted of: 94 °C for 30 sec, 68 °C for 10 sec, 72 °C for 1 min; PCR products obtained were separated by electrophoresis on 1.5% agarose gel in TAE buffer, visualized by ethidium bromide fluorescence. Fragments with the expected size were cut from the gel, purified using GFX PCR DNA Gel band purification kit (Amersham, USA). Purified DNA was digested with *Cfo* I (Hha I) enzyme, separated by agarose gel electrophoresis to identify the genotype. On the basis of size and number of various fragments generated, APOE genotypes were determined as ε2/ε2 with 144 bp and 96 bp, ε3/ε3 with 144 bp and 48 bp, ε4/ ε4 with 72 bp and 48 bp, ε2/ε3 with 144 bp, 96 bp and 48 bp, ε3/ ε4 with 144 bp, 72 bp and 48 bp, and ε2/ε4 with 144 bp, 96 bp, 72 bp and 48 bp fragments. The prevalence of various genotypes in patients and controls was determined. Complete matching of results was obtained following both of the above mentioned procedures.

#### **2.5. Statistical analysis**

Frequencies of various alleles and genotypes for each polymorphism were compared between patients and controls and analyzed by Fisher's exact test and the *P*-values < 0.05 were consid‐ ered as significant. The strength of the association of disease with respect to a particular allele/ genotype is expressed by odd ratio interpreted as *relative risk* (RR) according to the method of Woolf as outlined by Schallreuter *et al* [74]. The RR was calculated only for those alleles and genotype which were increased or decreased in patients as compared to normal Saudis. The RR was calculated for all the subjects using the formula given below:

**Allele**

**Genotype**

populations (Table 3).

**Glaucoma (N=400) Control (N=400)**

**Number Frequency (%) Number Frequency(%)**

ε4 49 12.25 17 4.25 0.0001‡ 3.145 0.506\* ε3 346 86.50 383 95.75 0.0001‡ 0.284 0.544 ε2 5 1.25 0 0.0 0.030‡ - -

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

Our study on various genotypes of APOE also showed variations in patient and control groups (Table 2). The prevalence of ε3/ε3, ε3/ε4, ε4/ε4, ε2/ε3, and ε2/ε4 was 75.5, 20.5, 1.5,1.5 and 1.0%

ε3/ε3 151 75.50 183 91.50 0.0001‡ 0.286 0.530 ε3/ε4 41 20.50 17 8.50 0.0006‡ 2.775 0.491\*

ε4/ε4 3 1.50 0 0.0 0.1240 - ε2/ε3 3 1.50 0 0.0 0.1240 - ε2/ε4 2 1.00 0 0.0 0.2493 - ε2/ε2 0 0.0 0 0.0 - - -

N, number of subjects; RR, relative risk; EF, etiological fraction; PF, preventive fraction; ‡, statistically significant

Though the frequency of ε3/ε3 genotype was higher in both the test and control Saudi population, the statistical analysis of data showed strongly significant difference in ε3/ ε3 genotype frequencies between patients and controls (P=0.0001, RR=0.286, PF=0.53). The difference in the frequencies of the second common genotype (ε3/ ε4) was also statistically significant between the two groups (*P*=0.0006) being more in glaucoma patients. Genotypes ε4/ε4, ε2/ε3 were found only in 1.5% and ε2/ε4 in 1% of patients while being completely absent in the controls (*P*=0.124). The genotypes ε2/ε2 was absent in both patient and control groups. These results indicated that allele ε4 and genotype ε3/ ε4 are associated with glaucoma and can be a risk factor while allele ε3 and genotype ε3/ ε3 may be protective in Saudis. The frequencies of various genotypes and alleles were not significantly different in male and female patients clearly indicating that gender plays no role in genotype/ allele distributions among

N, number of alleles; RR, relative risk; EF, etiological fraction; PF, preventive fraction; ‡, statistically significant

**Table 1.** Distribution of APOE allele frequencies in glaucoma patients and matched control subjects.

**Glaucoma (N=200) Control (N=200)**

**Number Frequency (%) Number Frequency (%)**

in patients and 91.5, 8.5, 0,0 and 0 % in control group respectively.

**Table 2.** Distribution of APOE genotypes in glaucoma patients and matched controls

*P-value* **RR EF\*/PF**

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137

*P-value* **RR EF\*/PF**

RR = (a) × (d)/(b) × (c)

a = number of patients with expression of allele or genotype

b = number of patients without expression of allele or genotype

c = number of controls with expression of allele or genotype

d = number of controls without expression of allele or genotype.

*Etiologic Fraction* (EF): The EF indicates the hypothetical genetic component of the disease. Values 0.0-0.99 are of significance. It was calculated for positive association (RR>1) using the following formula [75].

EF = (RR-1)f/RR, where f = a/a+c

*Preventive Fraction* (PF): The PF indicates the hypothetical protective effect of one specific antigen for the disease. It was calculated for negative association only where RR<1 using following formula [75].

PF = (1-RR)f/RR (1-f) + f, where f = a/a+c

Values <1.0 indicated the protective effect of the genotype/ allele against the manifestation of disease.
