3.4.4202210G > A Mutation of the Prothrombin Gene (Factor II Leiden)

The 20210G > A mutation increases the efÞciency of prothrombin mRNA processing and mRNA stability, which in turn increases plasma prothrombin concentrations Ð a procoagulant effect. It is the second most common genetic defect causing thrombophilia, comprising 18% of cases of inherited thrombophilia.5,42,59 The prevalence of the heterozygous 20210G > A genotype in Europeans without venous thrombosis varies from 0.7% to 4.0%, but in patients with systemic venous thrombosis, it is from 8.0% to 16.0%.21,33,38,39 The prevalence of the mutation is higher in southern European whites than northern European whites, and much lower in non-whites.11 With such a low percentage, it would be difÞcult to assemble a large enough sample of patients with RVO to show an association if one were to exist.33

Heterozygosity for the 20210G > A mutation can increase plasma prothrombin levels from 25% to 30%, but some individuals have normal prothrombin levels.38,63 Case reports and series have suggested a link between the 20210G > A mutation and RVO.63 As shown in Table 3.7, in two studies of pooled RVOs, three studies of CRVOs, and three studies of BRVOs, there was no statistically signiÞcant difference in the frequency of the mutation (i.e., in percentage of heterozygous patients) among affected patients compared to controls. Exogenous risk factors for RVO such as oral contraceptive use along with a genetic substrate of the prothrombin 20210A mutation raise the risk of thrombosis.11 The consensus view advises against routine screening for the prothrombin 20210A mutation.6,7

3.4.5Plasminogen Activator Inhibitor-1

An increase in plasminogen activator inhibitor-1 (PAI-1) is theoretically prothrombotic, since the 4G allele of the plasminogen activator inhibitor-1

gene is associated with hypoÞbrinolysis.30 In a study of 44 pooled RVOs (42 CRVOs and 2 BRVOs) and 83 controls, there was a suggestion of a higher prevalence of this mutation among patients with RVOs. Sixty-four percent of the pooled RVO patients had the 4G allele compared to 48% of controls (P = 0.015). More studies are needed before this suggested association can be accepted.

Ophthalmic reports often do not separate primary and secondary abnormalities of PAI-1. There was a statistically signiÞcantly higher mean factor PAI-1 level in patients with CRVO compared to population-based controls in a study in England.86 In a separate study, elevated PAI-1 was higher (12.2 U/ml) in cases than in controls (6.3 U/ml) (P = 0.013).30 It is uncertain whether these results reßect primary disease among those with elevated levels. Routine screening for PAI-1 is not recommended.

3.4.6 Protein C

Protein C deÞciency has rarely been found in patients with RVO.80 Protein C is a vitamin K-dependent serine protease inhibitor of coagulation. When clotting is initiated at a site of vascular injury, activated factor V is a necessary cofactor in the production of thrombin. However, thrombin binds to thrombomodulin, forming a complex that cleaves protein C to form activated protein C (APC).53 APC inhibits coagulation by degrading activated factors V and VIII; therefore, reduced protein C activity is prothrombotic.49,75 The prevalence of reduced protein C activity in the general population is estimated to be 0.2Ð0.5%.49,77

Some cases of protein C deÞciency are primary, although ophthalmic association studies have not separated these cases from acquired cases. Primary deÞciency of protein C occurs as an autosomal recessive condition. Homozygous protein C deÞciency causes levels of protein C to be less than 1% of normal and is associated with neonatal thrombotic events and early death. Heterozygous protein C mutations producing

deÞciency are estimated to occur in 0.006% of persons and comprise 5.7% of cases of inherited thrombophilia.5,47,59 The prevalence of protein C deÞciency in systemic venous thrombosis is reported to be 3.1%.33 With such low prevalences, it is difÞcult to assemble a large enough sample of patients with RVO in which to be able to show an association if one were to exist.33 One study took the reverse approach and examined a family with known protein C deÞciency and a history of other systemic thromboses. Of 12 family members Þtting this description, fundus examination showed no evidence of RVO.82

Tourville performed a meta-analysis of six studies examining a possible association of protein C deÞciency and CRVO and nine studies examining a possible association of protein C deÞciency and pooled RVO cases through 2006.77 A total of 290 patients were examined in the CRVO studies and 568 patients in the pooled RVO studies. No studies involved a pure sample of BRVO and protein C deÞciency. All six CRVO studies found no association. Of the pooled RVO studies, three found an association and six did not. Since 2006, two additional case-control studies of pooled RVOs including 355 patients have also failed to Þnd an association with protein C deÞciency.3,66 The consensus is that screening for protein C deÞciency in RVO is unnecessary.77

3.4.7 Protein S

Protein S is a vitamin K-dependent glycoprotein that acts as a nonenzymatic cofactor with activated protein C to inhibit coagulation. Therefore, a deÞciency of protein S is prothrombotic.75 The prevalence of protein S deÞciency in the general population is estimated to be 0.14Ð1.3%, but in systemic venous thrombosis, it is reported to be 1.2%.49,77 In addition, total protein S and free protein S must be distinguished. A deÞciency of free protein S has been associated with CRVO in patients below the age of 50 with no deÞciency of total protein S.49

Cases of primary protein S deÞciency represent only a portion of the total cases of protein S

deÞciency, but ophthalmic reports do not generally distinguish the primary from the acquired subgroups. The gene for protein S is located on chromosome 3, and primary deÞciency is caused by autosomal dominant mutations. With such low prevalence of protein S deÞciency, it is hard to assemble a sample of patients with RVO large enough to show an association.33 In a meta-anal- ysis of seven studies published before 2006, with 307 total patients looking for an association of protein S deÞciency and CRVO, six studies found no association and one study without a control group suggested an association.77 Similarly, of eight studies of 566 pooled RVO patients, no association was found in six and an association was found in two studies. The two positive studies were from the same group and have been criticized for methodological problems of noncomparable controls and high rates of positive associations for many factors examined.77 Since the meta-analysis, two additional case-control studies with 355 pooled RVO patients found no association with protein S deÞciency.3,66 As with protein C, the consensus is that screening for protein S deÞciency in RVO is unnecessary.77

3.4.8 Fibrinogen

Fibrinogen is a glycoprotein with three paired protein chains (Aa, Bb, and gg) that is converted to Þbrin through the action of thrombin. Fibrin stabilizes thrombus, so an increased Þbrinogen level is procoagulant. A Þbrinogen b gene polymorphism involving a G-to-A substitution at position −455 in the 5¢ promoter region, when present on both alleles, is associated with higher plasma Þbrinogen levels than the more common homozygous G genotype. Although suspected of association with BRVO, no association has been found.84 In a study of 294 patients with BRVO, the b-455AA genotype was found in 5.1% of BRVOs and 7.1% of the controls (P = 0.730).84

Ophthalmic reports do not generally separate primary from acquired cases of abnormal Þbrinogen levels. The importance of elevated Þbrinogen in RVO is unclear. Elevated Þbrinogen

has been reported in some case-control studies of patients with pooled RVO compared to controls,1,68,76,78 but not in others.28,41,78,86 In one study, ischemic but not nonischemic RVOs had elevated Þbrinogen concentrations.78 Paradoxically, reduced Þbrinogen has also been reported in CRVO patients without known risk factors when compared to controls.4 In the EDCCS, an elevated Þbrinogen was associated with an increased risk of HCRVO.73 The inconsistency of results suggests that Þbrinogen levels have minor importance if any in most cases of RVO, so routine screening for Þbrinogen concentration in patients with RVO is not recommended.

3.4.9 Factor XII

Factor XII increases Þbrinolysis by promoting the conversion of plasminogen to plasmin. It also promotes coagulation by involvement in the Þrst step of the intrinsic coagulation cascade, so it has both prothrombotic and antithrombotic functions. Nevertheless, the overall effect of a deÞciency of factor XII is procoagulant.

The factor XII gene polymorphism characterized by a C-to-T substitution at nucleotide 46 is associated with lower plasma XII levels and has been investigated as a risk factor for BRVO. No association was found.84 In a study of 294 patients with BRVO, the −46TT genotype was found in 7.5% of BRVOs and 7.1% of controls (P = 0.177).84

Not all reports separate primary from acquired cases of factor XII deÞciency. In a case-control study of 150 pooled RVO cases and 135 controls, 9.35% of cases had low factor XII compared to 0.7% of controls (P = 0.009).45 The results were dependent on age. Prevalence of factor XII deÞciency was not different from controls among patients greater than age 45, but only among patients 45 years of age or less.45 This study pooled 94 patients with CRVO and 56 patients with BRVO, and the results were similar in the RVO subgroups to those in the pooled group.45 Routine screening for factor XII concentration in patients with RVO is not recommended.

3.4.10 Antithrombin Deficiency

Antithrombin (AT), also called antithrombin III in the older literature, is a plasma protein that inactivates thrombin, IXa, Xa, XIIa, activated protein C, and kallikrein.47 Heparin increases its anticoagulant potency 1,000-fold. Functional tests for antithrombin are necessary rather than antigenic tests, which do not distinguish functional from dysfunctional protein.80

As with many coagulopathies, AT deÞciency can be primary (genetic) or secondary (acquired in association with other conditions).69 Secondary causes of antithrombin deÞciency include atherosclerosis, cancer, liver disease, pregnancy, oral contraceptive use, and disseminated intravascular coagulation.69 There is little information in the literature regarding acquired AT deÞciency and RVO. Most reports measure AT levels, ignore the contribution of nongenetic confounding factors, and consider the results to reßect primary AT deÞciency.

Reduced antithrombin levels are prothrombotic.8 However, some studies report abnormalities in antithrombin, which could include elevated levels that would not normally be expected to predispose patients to RVO. For example, levels of antithrombin were checked in 51 patients with either CRVO or HCRVO in an uncontrolled study.35 Abnormalities were found in 16%, but whether the abnormalities were reductions or elevations was not reported.35 Similarly, abnormalities without stated direction were found in 17% of an uncontrolled series of 30 patients with BRVO.35 No action was recommended based on these test results.35

In the EDCCS, an elevated antithrombin was paradoxically associated with an increased risk of CRVO.76 Again, enigmatically, a higher mean AT level was found in patients with CRVO than in population-based controls.86 On the other hand, in a case-control study of 42 patients with chronic pooled RVOs, the mean AT concentration was lower than that of the controls.78 To attempt to explain the paradox, some have suggested that elevated AT occurs as a compensatory response to elevated procoagulant factors, but this suggestion has not been tested.86

Primary AT deÞciency is inherited as an autosomal dominant condition and is characterized by recurrent thromboembolism and disseminated intravascular coagulation.69 At least 220 mutations of the SERPINC 1 gene found on chromosome 1 cause primary AT deÞciency. The prevalence of primary AT deÞciency in the general population has been reported to be 0.02Ð 0.18%.48,75,77 The small prevalence makes it difÞcult to assemble a large enough sample size to demonstrate any association with RVO. Although case reports of CRVO in patients with AT deÞciency exist, a genetic association with RVO has not been demonstrated. In a meta-anal- ysis of eight studies with 357 total patients looking for an association of AT deÞciency and CRVO, seven studies found no association and one study without a control group suggested an association.77 Similarly, of six studies with 390 pooled RVOs, no association was found in Þve of the studies, although an association was found in one study that lacked a control group. The consensus is that screening for AT deÞciency in RVO is unnecessary.77

3.4.11Glucose-6-Phosphate Dehydrogenase Deficiency

Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme found in the cytoplasm that regulates production of nicotinamide adenosine dinucleotide phosphate (NADPH), therefore protecting the cell against oxidative damage. The gene for G6PD is found on the X chromosome, and 300 mutations are known. G6PD deÞciency is protective against malaria infection, and geographical prevalence of G6PD mutations and G6PD deÞciency correlates with historical areas of endemic malaria infection. Because it was found that G6PD deÞciency protects against ischemic heart disease and stroke, its relationship with RVO was investigated in a case-control study from Sardinia where G6PD deÞciency is found in 10Ð15% of the population.64 G6PD levels were determined in 448 patients with RVO and 896 age and gender-matched controls, with selective

G6PD genotyping done in nine RVO cases and 19 controls having G6PD deÞciency. G6PD deÞciency was found in 4.7% of RVO patients and 11.9% of controls (P < 0.005).64 The G6PD gene Mediterranean mutation was found in 67.9% of those genotyped.64 Routine screening of patients with RVO for G6PD deÞciency is not recommended.

3.4.12Other Negative Genetic Association Studies

A number of genetic mutations suspected of a relationship to RVO and studied by multiple independent investigators have been reviewed in previous sections. In no case has the conclusion been reached that an association exists, nor that screening should be pursued routinely. Many other gene polymorphisms have been investigated for association with retinal vein occlusions and have also been found not associated, but these have lacked independent, conÞrmatory studies, unlike the mutations reviewed previously. Before accepting a negative result as valid, the reader is cautioned to consider the power of the study to detect an association. That depends on the size of the study and the magnitude of any purported association.54

The following gene polymorphisms were investigated in a study of 398 Austrian Caucasian patients with BRVO, 315 patients with CRVO, and 355 hospital-based control subjects without RVO: interleukin (IL) 1b −511C>T, interleukin 1 receptor antagonist (IL1RN) 1018T > C, interleukin 4 (IL4) −584C > T, interleukin 6 (IL6) −174G > C, interleukin 10 (IL10) −592C > A, interleukin 18 (IL18) 183A > G, tumor necrosis factor (TNF)-a −308G > A, monocyte chemoattractant protein (MCP)-1/CCL2 −2518A > G, interleukin 8 (IL*) −251A > T, and RANTES (CCL5) −403G > A.54,74 None of these mutations was found to be associated with BRVO or CRVO. In the BRVO study, the statistical power to detect an OR greater than or equal to 1.55 was 0.80 or greater for each of the polymorphisms listed.74