**3. Results**

Out of 200 PG patients 134 were diagnosed as having POAG and 66 as having PACG. Diagnosis of POAG was based on category 1 in 20 subjects (14.93 %) and category 2 in 114 subjects (85.07 %). Between category 1 and category 2 there was no significant different in age, IOP and gender distribution. One subject was blind in both eyes and 1 subject had unilateral blindness due to POAG. There were 66 subjects with PACG. Diagnosis was based on category 1 in 16 subjects (24.24%), category 2 in 46 subjects (69.70%), and category 3 in 4 subjects (6.06%). Three subjects (4.55 %) bilaterally, 4 (6.06 %) were unilaterally blind due to PACG.

The results of frequency of APOE alleles and genotypes in the PG patients and the control subjects are summarized in Tables 1, 2,3,4,5 and 6. The frequency of the ε3 alleles was signif‐ icantly lower in the glaucoma patients (86.5 %) compared to the control subjects (95.75 %, *P*=0.0001, RR=0.284, PF=0.544). On the other hand the frequencies of the ε4 allele was signifi‐ cantly higher in the glaucoma patients as compared to controls (12.25% vs 4.25%, *P*=0.0001, RR=3.145, EF=0.506). The allele ε2 was present only in 5 patients while totally absent in control groups (Table 1).


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.

genotype which were increased or decreased in patients as compared to normal Saudis. The

*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

*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

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

Out of 200 PG patients 134 were diagnosed as having POAG and 66 as having PACG. Diagnosis of POAG was based on category 1 in 20 subjects (14.93 %) and category 2 in 114 subjects (85.07 %). Between category 1 and category 2 there was no significant different in age, IOP and gender distribution. One subject was blind in both eyes and 1 subject had unilateral blindness due to POAG. There were 66 subjects with PACG. Diagnosis was based on category 1 in 16 subjects (24.24%), category 2 in 46 subjects (69.70%), and category 3 in 4 subjects (6.06%). Three subjects

The results of frequency of APOE alleles and genotypes in the PG patients and the control subjects are summarized in Tables 1, 2,3,4,5 and 6. The frequency of the ε3 alleles was signif‐ icantly lower in the glaucoma patients (86.5 %) compared to the control subjects (95.75 %, *P*=0.0001, RR=0.284, PF=0.544). On the other hand the frequencies of the ε4 allele was signifi‐ cantly higher in the glaucoma patients as compared to controls (12.25% vs 4.25%, *P*=0.0001, RR=3.145, EF=0.506). The allele ε2 was present only in 5 patients while totally absent in control

(4.55 %) bilaterally, 4 (6.06 %) were unilaterally blind due to PACG.

RR was calculated for all the subjects using the formula given below:

a = number of patients with expression of allele or genotype

c = number of controls with expression of allele or genotype

b = number of patients without expression of allele or genotype

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

RR = (a) × (d)/(b) × (c)

136 Glaucoma - Basic and Clinical Aspects

following formula [75].

following formula [75].

disease.

**3. Results**

groups (Table 1).

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

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

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


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

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

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 populations (Table 3).


**Genotype/Allele Open angle glaucoma (134)**

**Genotype/Allele Angle closure glaucoma (66)**

**N (%)**

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

**Table 6.** Distribution of APOE genotype/ allele frequencies in patients with PACG and matched controls

Over all prevalence of PEX in our study was 3.03%. Unilateral PEX was noted in 38% while bilateral PEX in 62% of the PEX patients (Figures.1 & 2). However, there was no significant

be protective.

**N (%)**

**Controls (200)**

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

ε3/ε4 30 (22.39) 17 (8.50) 0.0006‡ 3.105 0.432*\** ε4/ε4 3 (2.24) 00 0.063 - ε3/ε3 98 (73.13) 183 (91.50) 0.0001‡ 0.252 0.507 ε2/ε3 2 ( 1.49 ) 00 0.160 - ε2/ε4 1 ( 0.75 ) 00 0.401 - ε4 37 ( 13.81) 17(4.25) 0.0001‡ 3.608 0.495*\**

ε3 228 (85.07 ) 383 (95.75) 0.0001‡ 0.253 0.524

The frequency of allele ε3 and ε3/ε3 genotype was significantly higher in controls (*P*=0.0001). Similarly, the frequency of various genotypes of APOE differ between PACG and controls but the differences were not statistically significant except for ε3/ε3(*P*=0.022) (Table 6). However, the frequency of allele ε4 was higher in PACG whereas ε3 in controls indicating that the allele ε4 is also significantly associated with PACG in Saudis while genotype ε3/ε3 and allele ε3 may

> **Controls (200) N (%)**

ε3/ε4 11 ( 16.66 ) 17 (8.50) 0.067 2.152 0.210\* ε4/ε4 0 00 - - ε2/ε3 1 (1.52) 00 0.248 - ε2/ε4 1 (1.52) 00 0.248 - ε3/ε3 53 ( 80.30 ) 183 (91.50) 0.022‡ 0.378 0.269 ε4 12 ( 9.09 ) 17(4.25) 0.045‡ 2.252 0.229\* ε2 2 ( 1.52) 00 0.061 - ε3 118(89.39 ) 383 (95.75) 0.010‡ 0.374 0.282

ε2 3 ( 1.12 ) 00 0.064 -

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

**Table 5.** Distribution of APOE genotype/ allele frequencies in patients with POAG and matched controls

**N (%)** *p-value RR EF\*/PF*

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139

*P-value RR EF\*/PF*

**Table 3.** Distribution of APOE genotypes and alleles in male and female glaucoma patients

Though the distribution of APOE genotypes and alleles was not significantly different in two types of glaucoma (Table 4) however when compared with controls separately, significant difference was found in the frequencies of genotypes ε3/ε4, ε3/ε3 and alleles ε4 and ε3 in POAG and controls.


**Table 4.** Comparison of APOE genotype/ allele frequencies in patients with POAG and PACG

The frequency of genotype ε3/ ε4 and ε 4 allele was significantly more (*P*= 0.0006 and 0.0001 respectively) in POAG patients as compared to controls (Table 5).

The Role of Apolipoprotein E Gene Polymorphisms in Primary Glaucoma and Pseudoexfoliation Syndrome http://dx.doi.org/10.5772/54614 139


**Genotype/Allele**

138 Glaucoma - Basic and Clinical Aspects

N, number of subjects

and controls.

N, number of subjects

**Male (N=100) Female (N=100)**

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

ε3/ε3 71 71.00 80 80.00 0.143 ε3/ε4 26 26.00 15 15.00 0.079 ε4/ε4 0 0.0 3 3.00 0.123 ε2/ε3 2 2.00 1 1.00 0.623 ε2/ε4 1 1.00 1 1.00 0.999

ε3 170 85.00 176 88.00 0.385 ε4 27 13.50 22 11.00 0.451 ε2 3 1.50 2 1.00 0.685

Though the distribution of APOE genotypes and alleles was not significantly different in two types of glaucoma (Table 4) however when compared with controls separately, significant difference was found in the frequencies of genotypes ε3/ε4, ε3/ε3 and alleles ε4 and ε3 in POAG

ε3/ε4 30 (22.39) 11 (16.66) 0.456 ε4/ε4 3 (2.24) 00 0.552 ε3/ε3 98 (73.13) 53 (80.30) 0.298 ε2/ε3 2 (1.49) 1 (1.52) 1.000 ε2/ε4 1 (0.75) 1 (1.52) 0.552 ε4 37 (13.81) 12 (9.09) 0.197 ε2 3 (1.12) 2 (1.52) 0.666 ε3 228 (85.07) 118(89.39) 0.277

The frequency of genotype ε3/ ε4 and ε 4 allele was significantly more (*P*= 0.0006 and 0.0001

**Angle closure glaucoma (66) N (%)**

**Table 3.** Distribution of APOE genotypes and alleles in male and female glaucoma patients

**N (%)**

**Table 4.** Comparison of APOE genotype/ allele frequencies in patients with POAG and PACG

respectively) in POAG patients as compared to controls (Table 5).

**Genotype/Allele Open angle glaucoma (134)**

*P***-value**

*P-value*

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

**Table 5.** Distribution of APOE genotype/ allele frequencies in patients with POAG and matched controls

The frequency of allele ε3 and ε3/ε3 genotype was significantly higher in controls (*P*=0.0001). Similarly, the frequency of various genotypes of APOE differ between PACG and controls but the differences were not statistically significant except for ε3/ε3(*P*=0.022) (Table 6). However, the frequency of allele ε4 was higher in PACG whereas ε3 in controls indicating that the allele ε4 is also significantly associated with PACG in Saudis while genotype ε3/ε3 and allele ε3 may be protective.


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

**Table 6.** Distribution of APOE genotype/ allele frequencies in patients with PACG and matched controls

Over all prevalence of PEX in our study was 3.03%. Unilateral PEX was noted in 38% while bilateral PEX in 62% of the PEX patients (Figures.1 & 2). However, there was no significant difference in the prevalence of PEX in male and female. Prevalence distribution of PEX with the age in Saudi population is summarized in (Table 7). The prevalence of PEX varied from 0.50% to 25% in various age groups. The majority of the patients screened was in the age group of 50-60 years followed by those from <50 years¸ 61-70 years, 71-80 years and 81-100 years groups. The prevalence of PEX increased with progressing of age.


**Allele**

groups (Table 9).

among populations.

**Pseudoexfoliation (N=102) Control (N=400)**

**Table 8.** Distribution of APOE allele frequencies in PEX patients and matched controls

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

**Figure 2.** Shows deposition of PEX material more peripherally indicating wide pupillary excursion

ε4 15 14.70 17 4.25 0.0004‡ 3.884 0.347 ε3 84 82.35 383 95.75 0.0001‡ 0.207 0.373 ε2 3 2.94 0 0.0 0.0081‡ - -

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

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

Our study on various genotypes of APOE also showed variations in PEX patient and control groups (Table 9). The prevalence of ε3/ε3, ε3/ε4, ε4/ε4, ε2/ε3 and ε2/ε4 was 70.58, 21.56, 1.96, 1.96 and 3.92% in patients and 91.5, 8.5, 0, 0, and 0 % in control group respectively. Though the frequency of ε3/ε3 genotype was high in both the test and control Saudi population, the statistical analysis of data showed significant difference in ε3/ ε3 genotype frequencies between patients and controls, being more in controls than patients (*P*=0.0002, RR=0.222, PF=0. 363). The difference in the frequencies of the second common genotype ε3/ ε4 was also statistically significant between the two groups and was found to be increased in PEX patient group (*P*=0.012, RR=2.96, EF=0.259). Genotypes ε4/ε4, ε2/ε3 and ε2/ε4 were found only in patients while being completely absent in the controls. The genotype ε2/ε2, was absent in both the

These results indicated that alleles ε4 and ε2 and genotype ε3/ ε4 and ε2/ε4 were associated with PEX 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 almost similar in male and female patients clearly indicating that gender plays no role in genotype/ allele distributions

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

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141

**Table 7.** Age specific prevalence of PEX in Saudi patients

**Figure 1.** Showing massive PEX material in the papillary area forming a membrane like deposit

The results of frequency of APOE alleles and genotypes in the PEX patients and the control subjects are summarized in Tables 8 and 9. The frequency of the ε3 alleles was significantly lower in the PEX patients (82.35 %) compared to the control subjects (95.75 %, *P*=0.0001, RR=0.207, PF=0.373). On the other hand the frequencies of the ε2 and ε4 allele were significantly higher in the PEX patients as compared to controls (2.94% vs 0.00%, *P*=0.0081 and 14.70% vs 4.25%, *P*=0.0004, RR=3.884, EF=0.347 respectively). The allele ε2 was absent in control group (Table 8).

The Role of Apolipoprotein E Gene Polymorphisms in Primary Glaucoma and Pseudoexfoliation Syndrome http://dx.doi.org/10.5772/54614 141

**Figure 2.** Shows deposition of PEX material more peripherally indicating wide pupillary excursion

difference in the prevalence of PEX in male and female. Prevalence distribution of PEX with the age in Saudi population is summarized in (Table 7). The prevalence of PEX varied from 0.50% to 25% in various age groups. The majority of the patients screened was in the age group of 50-60 years followed by those from <50 years¸ 61-70 years, 71-80 years and 81-100 years

**Age group (years) Patients screened (N) PEX positive patients (N) Frequency of PEX (%)** <50 600 3 0.50 51-60 850 27 3.17 61-70 200 16 8.00 71-80 30 4 13.33 81-100 4 1 25 Total 1684 51 3.03

groups. The prevalence of PEX increased with progressing of age.

**Figure 1.** Showing massive PEX material in the papillary area forming a membrane like deposit

The results of frequency of APOE alleles and genotypes in the PEX patients and the control subjects are summarized in Tables 8 and 9. The frequency of the ε3 alleles was significantly lower in the PEX patients (82.35 %) compared to the control subjects (95.75 %, *P*=0.0001, RR=0.207, PF=0.373). On the other hand the frequencies of the ε2 and ε4 allele were significantly higher in the PEX patients as compared to controls (2.94% vs 0.00%, *P*=0.0081 and 14.70% vs 4.25%, *P*=0.0004, RR=3.884, EF=0.347 respectively). The allele ε2 was absent in control group

N, number of patients

140 Glaucoma - Basic and Clinical Aspects

(Table 8).

**Table 7.** Age specific prevalence of PEX in Saudi patients


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

**Table 8.** Distribution of APOE allele frequencies in PEX patients and matched controls

Our study on various genotypes of APOE also showed variations in PEX patient and control groups (Table 9). The prevalence of ε3/ε3, ε3/ε4, ε4/ε4, ε2/ε3 and ε2/ε4 was 70.58, 21.56, 1.96, 1.96 and 3.92% in patients and 91.5, 8.5, 0, 0, and 0 % in control group respectively. Though the frequency of ε3/ε3 genotype was high in both the test and control Saudi population, the statistical analysis of data showed significant difference in ε3/ ε3 genotype frequencies between patients and controls, being more in controls than patients (*P*=0.0002, RR=0.222, PF=0. 363). The difference in the frequencies of the second common genotype ε3/ ε4 was also statistically significant between the two groups and was found to be increased in PEX patient group (*P*=0.012, RR=2.96, EF=0.259). Genotypes ε4/ε4, ε2/ε3 and ε2/ε4 were found only in patients while being completely absent in the controls. The genotype ε2/ε2, was absent in both the groups (Table 9).

These results indicated that alleles ε4 and ε2 and genotype ε3/ ε4 and ε2/ε4 were associated with PEX 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 almost similar in male and female patients clearly indicating that gender plays no role in genotype/ allele distributions among populations.


found a weak association between APOE ε4 and retinal microvascular degeneration. Contrary to these findings a decrease risk of NTG in Chinese [59,60] and POAG in Japanese with ε4 allele [64] has been reported, whereas some investigators reported no link between APOE polymor‐

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143

Besides glaucoma, APOE ε4 allele has been identified as a genetic susceptibility factor for a variety of neurodegenerative disorders in diverse ethnic populations [83-86 ]. APOE ε4 allele has also been associated with early age-at-onset of AD in a dose dependent manner [87,88]. Interestingly, a high incidence of glaucoma in AD patients clearly suggests a close association between ophthalmic and neurodegenerative disorders [89,90]. It has been hypothesized that the cellular mechanisms involved in the degeneration of optic nerve cells in glaucoma are quite similar to the neurodegenerative changes in AD [47,91,92]. APOE allele ε4 is also strongly linked with increased risk of Parkinson's disease, schizophrenia and coronary artery disease [93-99]. Possession of the ε4 allele is also associated with a retarded recovery after traumatic head injury [100,101]. The exact mechanism by which APOE ε4 exerts its deleterious effect is far from clear. However, APOE alleles has been reported to modulate the biological functions of APOE in part by altering the binding of the different lipoprotein lipid classes [93]. Individ‐ uals carrying the ε4 allele have higher plasma and neuronal levels of cholesterol as compared to individuals with ε2 or ε3. APOE immunoreactivity has been localized to basal laminar deposits and soft drusen in age related macular degeneration [102]. APOE has also been localized to the Müller cells (specialized retinal glia) [46,102] and this protein may be increased in Müller cells in glaucomatous eyes [103], indicating that this glial cell may have a role in the

On the other hand, earlier genetic studies support the concept that APOE would directly be involved in the amyloid deposition and fibril formation; and they suggest a close association between one of the main isoforms of APOE encoded by the ε4 allele and both familial and sporadic late-onset Alzheimer's disease (AD) [44,45]. In addition, deposits in various amy‐ loidoses and prion diseases such as Down's syndrome, cystatin C-related Icelandic-type hereditary amyloid angiopathy, Creutzfeldt-Jakob disease, Lewy body dementia, dementia in Parkinson's disease include both biochemically and immunohistochemically detectable

The higher frequency of ε3/ ε3 in controls as compared to the patients indicated a protective effect of ε3/ ε3 on development of glaucoma in Saudis. Though the genotypes ε4/ε4, ε2/ ε3 and ε2/ε4 were only found in glaucoma patients and completely absent in normal Saudi population however, the differences were statistically insignificant. The genotypes ε2/ε2, was absent in both patients and control group. Earlier studies on APOE polymorphism in general healthy population also showed absence of genotypes containing ε2 allele among Saudis [51,108] as

This study showed that prevalence of PEX in Saudi Population was 3.03%. No significant difference was found in prevalence of PEX between male and female whereas the rate of prevalence varied in different age group. the prevalence of PEX increased with progressing of age. Earlier investigators from Saudi Arabia using a very small hospital based study reported overall prevalence of PEX as 9.3% [110]. PEX occurs worldwide, although reported prevalence

phism and glaucoma [61,62].

retinal response to glaucomatous injury.

amounts of APOE [104-107].

well as Native Americans [109].

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

**Table 9.** Distribution of APOE genotype frequencies in PEX patients and matched controls

### **4. Discussion**

The result of this study showed a very high frequency (95.75%) of allele ε3, very low frequency (4.25%) of ε4 and absence of allele ε2 in control population. Global studies on APOE locus have shown highly significant variations in allele frequencies among various populations. Studies from various geographical locations and ethnicities have reported a wide range of frequencies of ε2 (0-12%), ε3 (75-90 %) and ε4 (6-20%) [52-58]. The differences in the APOE genotype/allele frequencies in different populations may be attributed to environmental factors as well as genetic differences. The ε3 allele is the most frequent in all the human groups, especially in populations with a long established agricultural economy, whereas APOE ε4 allele remains higher in populations where the economy of foraging still exists or food supply is/was scarce and sporadically available [76]. Data on APOE allele frequencies collected from literature showed that the APOE allele distributions were different between North and South Europe. Additionally, compared to northern European countries, Mediterranean countries such as Italy, Turkey and Greece had lower frequencies of APOE- ε2 and ε4 alleles [77-79].

Results of present study revealed significant differences in the frequencies of ε3 and ε4 alleles in glaucoma patient as compared to control groups (Table 1). Allele ε3 being more common in controls while ε4 was predominant in glaucoma patients suggesting that the inheritance of the ε4 allele might be a risk factor whereas ε3 might exert a protective effect for glaucoma in Saudi population. Neuroprotective effect of ε3 is also evident from several earlier studies. APOE has an isoform specific effect on neuronal growth with ε3 stimulating neuronal elongation and neurite outgrowth on dorsal root ganglion [80]. In individuals with acute cerebral ischemia, such as an intracerebral hemorrhage, the ε3 allele confers a much higher survival and functional recovery whereas ε4 leads higher rate of disability and mortality [81]. Our results clearly suggest that presence of ε4 is associated with high risk of both POAG and PACG. Vickers *et al* [47] also reported an association between the ε4 allele and NTG in the Tasmanian population. Recently, Yaun *et al* [65] reported that the ε4 may be a latent risk factor in developing primary glaucoma in Chinese population. On the other hand Liew *et al* [82] found a weak association between APOE ε4 and retinal microvascular degeneration. Contrary to these findings a decrease risk of NTG in Chinese [59,60] and POAG in Japanese with ε4 allele [64] has been reported, whereas some investigators reported no link between APOE polymor‐ phism and glaucoma [61,62].

Besides glaucoma, APOE ε4 allele has been identified as a genetic susceptibility factor for a variety of neurodegenerative disorders in diverse ethnic populations [83-86 ]. APOE ε4 allele has also been associated with early age-at-onset of AD in a dose dependent manner [87,88]. Interestingly, a high incidence of glaucoma in AD patients clearly suggests a close association between ophthalmic and neurodegenerative disorders [89,90]. It has been hypothesized that the cellular mechanisms involved in the degeneration of optic nerve cells in glaucoma are quite similar to the neurodegenerative changes in AD [47,91,92]. APOE allele ε4 is also strongly linked with increased risk of Parkinson's disease, schizophrenia and coronary artery disease [93-99]. Possession of the ε4 allele is also associated with a retarded recovery after traumatic head injury [100,101]. The exact mechanism by which APOE ε4 exerts its deleterious effect is far from clear. However, APOE alleles has been reported to modulate the biological functions of APOE in part by altering the binding of the different lipoprotein lipid classes [93]. Individ‐ uals carrying the ε4 allele have higher plasma and neuronal levels of cholesterol as compared to individuals with ε2 or ε3. APOE immunoreactivity has been localized to basal laminar deposits and soft drusen in age related macular degeneration [102]. APOE has also been localized to the Müller cells (specialized retinal glia) [46,102] and this protein may be increased in Müller cells in glaucomatous eyes [103], indicating that this glial cell may have a role in the retinal response to glaucomatous injury.

**4. Discussion**

**Genotype**

142 Glaucoma - Basic and Clinical Aspects

The result of this study showed a very high frequency (95.75%) of allele ε3, very low frequency (4.25%) of ε4 and absence of allele ε2 in control population. Global studies on APOE locus have shown highly significant variations in allele frequencies among various populations. Studies from various geographical locations and ethnicities have reported a wide range of frequencies of ε2 (0-12%), ε3 (75-90 %) and ε4 (6-20%) [52-58]. The differences in the APOE genotype/allele frequencies in different populations may be attributed to environmental factors as well as genetic differences. The ε3 allele is the most frequent in all the human groups, especially in populations with a long established agricultural economy, whereas APOE ε4 allele remains higher in populations where the economy of foraging still exists or food supply is/was scarce and sporadically available [76]. Data on APOE allele frequencies collected from literature showed that the APOE allele distributions were different between North and South Europe. Additionally, compared to northern European countries, Mediterranean countries such as

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

**Table 9.** Distribution of APOE genotype frequencies in PEX patients and matched controls

**Pseudoexfoliation (N=51) Control (N=200)**

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

ε3/ε3 36 70.58 183 91.50 0.0002‡ 0.222 0.363 ε3/ε4 11 21.56 17 8.50 0.012‡ 2.960 \*0.259 ε4/ε4 1 1.96 0 0.0 0.203 - ε2/ε2 0 0 0 0.0 - - ε2/ε3 1 1.96 0 0.0 0.203 - ε2/ε4 2 3.92 0 0.0 0.040‡ - -

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

Italy, Turkey and Greece had lower frequencies of APOE- ε2 and ε4 alleles [77-79].

Results of present study revealed significant differences in the frequencies of ε3 and ε4 alleles in glaucoma patient as compared to control groups (Table 1). Allele ε3 being more common in controls while ε4 was predominant in glaucoma patients suggesting that the inheritance of the ε4 allele might be a risk factor whereas ε3 might exert a protective effect for glaucoma in Saudi population. Neuroprotective effect of ε3 is also evident from several earlier studies. APOE has an isoform specific effect on neuronal growth with ε3 stimulating neuronal elongation and neurite outgrowth on dorsal root ganglion [80]. In individuals with acute cerebral ischemia, such as an intracerebral hemorrhage, the ε3 allele confers a much higher survival and functional recovery whereas ε4 leads higher rate of disability and mortality [81]. Our results clearly suggest that presence of ε4 is associated with high risk of both POAG and PACG. Vickers *et al* [47] also reported an association between the ε4 allele and NTG in the Tasmanian population. Recently, Yaun *et al* [65] reported that the ε4 may be a latent risk factor in developing primary glaucoma in Chinese population. On the other hand Liew *et al* [82] On the other hand, earlier genetic studies support the concept that APOE would directly be involved in the amyloid deposition and fibril formation; and they suggest a close association between one of the main isoforms of APOE encoded by the ε4 allele and both familial and sporadic late-onset Alzheimer's disease (AD) [44,45]. In addition, deposits in various amy‐ loidoses and prion diseases such as Down's syndrome, cystatin C-related Icelandic-type hereditary amyloid angiopathy, Creutzfeldt-Jakob disease, Lewy body dementia, dementia in Parkinson's disease include both biochemically and immunohistochemically detectable amounts of APOE [104-107].

The higher frequency of ε3/ ε3 in controls as compared to the patients indicated a protective effect of ε3/ ε3 on development of glaucoma in Saudis. Though the genotypes ε4/ε4, ε2/ ε3 and ε2/ε4 were only found in glaucoma patients and completely absent in normal Saudi population however, the differences were statistically insignificant. The genotypes ε2/ε2, was absent in both patients and control group. Earlier studies on APOE polymorphism in general healthy population also showed absence of genotypes containing ε2 allele among Saudis [51,108] as well as Native Americans [109].

This study showed that prevalence of PEX in Saudi Population was 3.03%. No significant difference was found in prevalence of PEX between male and female whereas the rate of prevalence varied in different age group. the prevalence of PEX increased with progressing of age. Earlier investigators from Saudi Arabia using a very small hospital based study reported overall prevalence of PEX as 9.3% [110]. PEX occurs worldwide, although reported prevalence

rates vary extensively with geographical location, as well as with ethnicity [21,111]. The prevalence of PEX varies significantly among Asians. The prevalence of PEX has been report‐ ed to be 3.01% and 6.28% in two different age groups in Southern Indian population [112], 6.45 % in Pakistani population [113], 3.4% in Japanese [114], 0.4% in Chinese [115] and 0.2 to 0.7% in Chinese Singaporeans [116]. In Scandinavia, the prevalence among persons over age 60 varies from over 20% in Finland to about 25% in Iceland. Aasved [117] found prevalence of 6.3%, 4.0%, and 4.7% in persons over age 60 in Norway, England, and Germany, respectively. Forsius [118] studiedprevalence inpatients over age 60 years invariedgroups andfoundprevalence ranging from 0% in Greenland Eskimos to 21% in Icelanders. Lantukh and Piatin [119] found a low prevalence in native Siberian Tchutchee, but a much higher rate among immigrants to the area indicating ethnic variations. Similarly in New Mexico, Spanish-American men are nearly six times as likely to develop PEX than are non-Spanish-Americans [120].

protective for PEX (*P*=0.0002) similar to PG in Saudi patients. In addition, the control group had a significantly higher frequency of the ε3 allele (95.75%) than the PEX group (82.35%), showing that this allele had a protective effect for developing the disease (P=0.0001). This is in agreement with Yilmaz et al [66] who reported a protective role of APOE ε3 allele in patients with exfoliation syndrome in Turkish population. However there are reports indicating no association of APOE genotypes and PEX in Germans or Italians [68] and Norwegians [67].

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In the literature, ε 4 allele has been shown to be risky for developing amyloidoses in AD [44,45,104,106,107]. Yilmaz et al [66] suggested PEX to belong to the amyloidosis group depending on the deposition of amyloid or amyloid-like material throughout the body. As stated earlier inheritance of the ε4 allele has also been associated with elevated risk to Alz‐ heimer's disease. In this regard, it is interesting that visual deficits have been reported in Alzheimer's disease cases. However, there are conflicting reports as to whether visual field loss observed in a relatively high proportion of Alzheimer's disease cases is associated with retinal or central damage [127-129]. It has also been noted that both Alzheimer's disease and Parkinson's disease cases have increased glaucomatous retinal changes [90]. In the light of the these findings, there may be similar cellular processes involving APOE related to neuronal damage. It has been argued that both Alzheimer's disease and glaucoma/PEX are ultimately axon damaging conditions and it is how nerve cells respond to this injury that leads to overall neuronal degeneration and the clinical picture of progressive loss of function [130]. Müller cells that express particular APOE isoforms may thus have an important role in regulating the response of retinal ganglion cells to injury. However, it cannot be ruled out that APOE may be acting centrally to promote β-amyloid fibril formation in structures such as the lateral geniculate nucleus [131] and that these plaques are causing damage to retinal axons and visual pathways. In this regard, it would be intriguing to determine whether glaucoma and PEX cases

The result of this study suggests that APOE alleles may influence the risk of glaucoma and PEX. The inheritance of the ε4 allele is associated with elevated risk of POAG, PACG and PEX and ε3 may exert protection for both type of glaucoma as well as PEX. Genotypes containing allele ε2 (ε2/ε3, ε2/ε4) were found only in small number of patients (3POAG, 2 PACG and 1PEX) whereas altogether absent in Saudi normal population so it is difficult to derive any conclusion. Further studies involving larger number of patients from different race/tribes of Saudi Arabia are warranted to reach any definite conclusion as the APOE allele frequencies from same population (Turkish) reported by different authors are not uniform [66,132,133]. These differences in the distribution of APOE allele and genotype in single population in different studies have been attributed to geographical/ racial differences and/ or variations in

Though the inheritance of the ε4 allele seems to be associated with elevated risk of primary glaucoma and PEX in our Saudi population. However, it will be important to replicate these results in populations from other geographical locations of Saudi Arabia. The significance of inheritance of these APOE allelic isoforms has yet to be established, as is the case for the potential role of this protein in many other neurodegenerative conditions, but it may be linked with associated hypertension, formation of central β-amyloid deposits or a more general role

may have a higher incidence of Alzheimer-type dementia.

genotyping methodology.

The prevalence of PEX may also vary within the same country in similar environments and over short distances as found in present study. Similarly, in France the prevalence in over age 70 years varies from 3.6% in Toulon to 20.6% in Brest [121]. Ringvold et al [122] also found rates of 10.2%, 19.6%, and 21.0% in three closely situated municipalities in central Norway. The reasons underlying true variations, both from one population to another and within more or less homogeneous populations, remain to be explained. Geographic distribution patterns may perhaps be explained either by regional gene pools or by environmental influences. Persons living at lower latitudes (Greece, Saudi Arabia, and Iran) appear to develop PEX at younger ages [123]. Exposure to sunlight (ultraviolet radiation) may or may not be implicated. Forsius and Lukka [124] found no PEX in Eskimos versus 20% among Lapps living at the same latitude.

Similar to our observations, the prevalence of PEX increases with age in most of the studies [112,114,117]. Forsius [125] found PEX incidence to double every decade after age 50. These variations in prevalence rates may consequently be caused, to varying degrees, by genuine differences in genetic, ethnic and environmental factors and by methodological differences in age and sex distribution, diagnostic criteria, experience of the examiners in diagnosing the syndrome and the thoroughness of their examination [126].

This study also indicated that allele ε4 was associated with PEX and can be a risk factor while allele ε3 may be protective for PEX similar to PG in Saudi patients. Allele ε2 was found in only 2.94% of the PEX while totally absent in controls. Contrary to our results, Yilmaz et al [66] reported a close association of ε2 allele with PEX in Turkish population. According to them PEX have significantly higher frequency of ε2 allele (50%). In their study the frequency of genotypes carrying ε2 allele was also significantly higher in PEX. They have suggested that especially when ε2 allele is heterozygous, the possibility of developing PEX increases which could be an indicator for pathogenicity when this allele frequency is over 30% in the PEX group. In our study ε2/ ε3 and ε2/ ε4 genotypes are found only in 1 and 2 cases respectively. As the genotype frequencies are low in these groups, it is difficult to make general conclusion on statistically insignificant data.

On the other hand our results for APOE polymorphism in PEX indicated that genotype ε3/ ε4 was also associated with PEX (*P*=0.012) and can be a risk factor while genotype ε3/ ε3 may be protective for PEX (*P*=0.0002) similar to PG in Saudi patients. In addition, the control group had a significantly higher frequency of the ε3 allele (95.75%) than the PEX group (82.35%), showing that this allele had a protective effect for developing the disease (P=0.0001). This is in agreement with Yilmaz et al [66] who reported a protective role of APOE ε3 allele in patients with exfoliation syndrome in Turkish population. However there are reports indicating no association of APOE genotypes and PEX in Germans or Italians [68] and Norwegians [67].

rates vary extensively with geographical location, as well as with ethnicity [21,111]. The prevalence of PEX varies significantly among Asians. The prevalence of PEX has been report‐ ed to be 3.01% and 6.28% in two different age groups in Southern Indian population [112], 6.45 % in Pakistani population [113], 3.4% in Japanese [114], 0.4% in Chinese [115] and 0.2 to 0.7% in Chinese Singaporeans [116]. In Scandinavia, the prevalence among persons over age 60 varies from over 20% in Finland to about 25% in Iceland. Aasved [117] found prevalence of 6.3%, 4.0%, and 4.7% in persons over age 60 in Norway, England, and Germany, respectively. Forsius [118] studiedprevalence inpatients over age 60 years invariedgroups andfoundprevalence ranging from 0% in Greenland Eskimos to 21% in Icelanders. Lantukh and Piatin [119] found a low prevalence in native Siberian Tchutchee, but a much higher rate among immigrants to the area indicating ethnic variations. Similarly in New Mexico, Spanish-American men are nearly six

The prevalence of PEX may also vary within the same country in similar environments and over short distances as found in present study. Similarly, in France the prevalence in over age 70 years varies from 3.6% in Toulon to 20.6% in Brest [121]. Ringvold et al [122] also found rates of 10.2%, 19.6%, and 21.0% in three closely situated municipalities in central Norway. The reasons underlying true variations, both from one population to another and within more or less homogeneous populations, remain to be explained. Geographic distribution patterns may perhaps be explained either by regional gene pools or by environmental influences. Persons living at lower latitudes (Greece, Saudi Arabia, and Iran) appear to develop PEX at younger ages [123]. Exposure to sunlight (ultraviolet radiation) may or may not be implicated. Forsius and Lukka [124] found no PEX in Eskimos versus 20% among Lapps living at the same

Similar to our observations, the prevalence of PEX increases with age in most of the studies [112,114,117]. Forsius [125] found PEX incidence to double every decade after age 50. These variations in prevalence rates may consequently be caused, to varying degrees, by genuine differences in genetic, ethnic and environmental factors and by methodological differences in age and sex distribution, diagnostic criteria, experience of the examiners in diagnosing the

This study also indicated that allele ε4 was associated with PEX and can be a risk factor while allele ε3 may be protective for PEX similar to PG in Saudi patients. Allele ε2 was found in only 2.94% of the PEX while totally absent in controls. Contrary to our results, Yilmaz et al [66] reported a close association of ε2 allele with PEX in Turkish population. According to them PEX have significantly higher frequency of ε2 allele (50%). In their study the frequency of genotypes carrying ε2 allele was also significantly higher in PEX. They have suggested that especially when ε2 allele is heterozygous, the possibility of developing PEX increases which could be an indicator for pathogenicity when this allele frequency is over 30% in the PEX group. In our study ε2/ ε3 and ε2/ ε4 genotypes are found only in 1 and 2 cases respectively. As the genotype frequencies are low in these groups, it is difficult to make general conclusion on

On the other hand our results for APOE polymorphism in PEX indicated that genotype ε3/ ε4 was also associated with PEX (*P*=0.012) and can be a risk factor while genotype ε3/ ε3 may be

times as likely to develop PEX than are non-Spanish-Americans [120].

syndrome and the thoroughness of their examination [126].

latitude.

144 Glaucoma - Basic and Clinical Aspects

statistically insignificant data.

In the literature, ε 4 allele has been shown to be risky for developing amyloidoses in AD [44,45,104,106,107]. Yilmaz et al [66] suggested PEX to belong to the amyloidosis group depending on the deposition of amyloid or amyloid-like material throughout the body. As stated earlier inheritance of the ε4 allele has also been associated with elevated risk to Alz‐ heimer's disease. In this regard, it is interesting that visual deficits have been reported in Alzheimer's disease cases. However, there are conflicting reports as to whether visual field loss observed in a relatively high proportion of Alzheimer's disease cases is associated with retinal or central damage [127-129]. It has also been noted that both Alzheimer's disease and Parkinson's disease cases have increased glaucomatous retinal changes [90]. In the light of the these findings, there may be similar cellular processes involving APOE related to neuronal damage. It has been argued that both Alzheimer's disease and glaucoma/PEX are ultimately axon damaging conditions and it is how nerve cells respond to this injury that leads to overall neuronal degeneration and the clinical picture of progressive loss of function [130]. Müller cells that express particular APOE isoforms may thus have an important role in regulating the response of retinal ganglion cells to injury. However, it cannot be ruled out that APOE may be acting centrally to promote β-amyloid fibril formation in structures such as the lateral geniculate nucleus [131] and that these plaques are causing damage to retinal axons and visual pathways. In this regard, it would be intriguing to determine whether glaucoma and PEX cases may have a higher incidence of Alzheimer-type dementia.

The result of this study suggests that APOE alleles may influence the risk of glaucoma and PEX. The inheritance of the ε4 allele is associated with elevated risk of POAG, PACG and PEX and ε3 may exert protection for both type of glaucoma as well as PEX. Genotypes containing allele ε2 (ε2/ε3, ε2/ε4) were found only in small number of patients (3POAG, 2 PACG and 1PEX) whereas altogether absent in Saudi normal population so it is difficult to derive any conclusion. Further studies involving larger number of patients from different race/tribes of Saudi Arabia are warranted to reach any definite conclusion as the APOE allele frequencies from same population (Turkish) reported by different authors are not uniform [66,132,133]. These differences in the distribution of APOE allele and genotype in single population in different studies have been attributed to geographical/ racial differences and/ or variations in genotyping methodology.

Though the inheritance of the ε4 allele seems to be associated with elevated risk of primary glaucoma and PEX in our Saudi population. However, it will be important to replicate these results in populations from other geographical locations of Saudi Arabia. The significance of inheritance of these APOE allelic isoforms has yet to be established, as is the case for the potential role of this protein in many other neurodegenerative conditions, but it may be linked with associated hypertension, formation of central β-amyloid deposits or a more general role in the regulation of lipids following axonal injury. However, our results together with similar data elucidated a potential overlap between the degenerative pathways underlying glaucoma/ PEX and Alzheimer-type dementia and brain injury.

District, Guangzhou. Investigative Ophthalmology & Visual Science 2006;47:

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

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