Can Heterozygosity of MTHFR and Hyperhomocysteinemia be Risk Factors for Both Retinal/Vitreal Hemorrhages in Retinal Vasculitis and Retinal Vein Occlusion?

Retinal vein occlusion (RVO) is one of the most common vascular diseases of the eye and a frequent cause of severe visual loss. It is multifactorial in origin with both local factors and systemic diseases being of etiological importance. Many thrombophilic conditions have recently been identified and studies looking at their potential role in RVO have been undertaken. The aim of this study was to investigate the role of methylenetetrahydrofolate reductase (MTHFR) heterozigosity associated with normal homocysteine level in blood as risk factor for thromboembolic retinal/vitreal manifestations in patients with retinal vasculitis (RV) and RVO where the other risk factors (such as hypertension; atherosclerosis; diabetes; antiphospholipid syndrome) are excluded. Second question was if anticoagulation treatment should be applied together with systemic corticosteroids and/ immunosuppressive therapy? Results obtained from the examined group showed that heterozigosity for MTHFR C677T gene is of statistical significance and correlates with raised values of homocysteine (correlation coefficient = 0.23). At the same time increased levels of homocysteine correlate (correlation coefficient = 0.01) with appearance of RVO. At r = 0.67, if there is no RV present, existence of a single mutated allele for MTHFR C677T gene does not correlate with RVO. There is however significant correlation between the mutation for MTHFR and hemorrhagic type of RV associated with RVO and/ with isolated hemorrhagic vasculitis without RVO (correlation coefficient = 0.33; correlation coefficient = 0.27; respectively).


Introduction
Retinal vasculitis (RV) is an inflammation of retinal blood vessels, which can be isolated or associated with systemic vasculitis.
Muffing, narrowing and occlusion of blood vessels lead to retinal ischemia and are basic clinical signs noted on the retina [1].In absence of retinal ischemia and independently of its etiology, some cases of retinal vasculitis are followed by pronounced retinal and vitreal hemorrhages and/ hemophthalmos.In these cases pathophysiological mechanisms can be based on thrombophilia and/ antiphospholipid syndrome if diabetes, hypertension and atherosclerosis are excluded.
Following diabetic retinopathy, retinal vein occlusion (RVO) is the second most common retinal vascular occlusive disease that can affect people of all ages.According to Hayreh at al. and Mitchel et al.RVO appears to be a condition with a multiple etiology and there is an increased incidence with age (patients are predominantly over 65) [2,3].
RVO may increase venous pressure and lead to retinal capillary decompensation with macular edema and have as consequence decrease/loss of visual efficiency.Exact pathogenesis of RVO is unclear.Primary mechanisms of blood vessel occlusion are degenerative changes of vessel walls, abnormal perivascular changes as well as various hematological factors.Two out of three Virchow's classical factors that play a role in thrombogenesis (stasis, vessel damage and hypercoagulability) have been frequently reported in patients with RVO.Over the past couple of years, especially in case of younger patients, physicians' have been highly interested in thrombophilic risk factors, such as antithrombin deficiency; protein C and S deficiency; hyperhomocysteinaemia; factor V Leiden and II mutations and antiphospholipid antibodies associated with venous thromboembolisms (VTE) [4][5][6][7].

Polymorphism
(C677T) in methylenetetrahydrofolate reductase (MTHFR) gene has been reported to affect homocysteine levels.In particular, elevated levels of homocysteine are generally accepted to be a risk factor for systemic vascular disease and venous thrombosis.In MTHFR studies, the difference in homocysteine concentration arisen from a single gene mutation is allocated at random [8,9].
Homocysteine is formed in metabolic demethylation process from methionine and it contains a sulphydryl amino group."Total plasma (or serum) homocysteine" (tHcy) is a combination of free thiol; disulphide-bound to plasma proteins and a combination of homocysteine bound to itself (dimer homocysteine) or with other thiols (bound with cysteine thus forming homocysteine-cysteine mix disulphide) [10].An abnormal tHcy is defined by an arbitrary cut-off in distribution of concentration found in the "normal population", similarly to hypertension and hypercholesterolemia. Homocysteine is metabolized by remethylation of transsulphuration.One of two methionineconserving remethylation pathways metabolizes homocysteine if protein intake is low.Methionine synthase, N 5 ,N 10 methylenetetrahydrofolate and N 5 ,N 10 methylenetetrahydrofolate reductase have an important role in metabolic process of homocysteine in the liver.Similar process occurs in other tissue types.Additionally, vitamin B12 (Cobalamin) is an essential cofactor for methionine synthase [4].
The most common genetic cause of severe hyperhomocysteinemia and classic homocystinuria (congenital homocystinuria) is homozygous deficiency of cystathionine β-synthase (CβS).Heterozygosity is often associated with normal basal tHcy, and it remains unclear whether heterozigosity is associated with additional risk of vascular events.Homozygous deficiency of MTHFR, both deficiency and impaired activity of methionine synthase, are rare cause of severe hyperhomocysteinaemia [11].Single point mutation in the coding region of MTHFR gene (C-to-T substitution at nucleotide 677) is the most common enzyme defect which occurs with moderately raised tHcy levels.This in turn is associated with thermolabile MTHFR variant which is only half as active [12].Activated circulating immune complexes against phospholipid membranes and plasma proteins can lead to arterial and venous thromboembolisms.RVO can be one of the first manifestations of antiphospholipid syndrome [1].

Aim
The  In our sample, results obtained from the examined group showed that occurrence of heterozigosity for MTHFR C677T gene is of statistical significance (Figure 3) and correlates with raised values of homocysteine (correlation coefficient = 0.23).At the same time increased levels of homocysteine correlate (correlation coefficient = 0.01) with appearance of RVO.At r = 0.67, if there is no RV present, existence of a single mutated allele for MTHFR C677T gene does not correlate with RVO.There is however significant correlation between the mutation for MTHFR and hemorrhagic type of RV associated with RVO and/ with isolated hemorrhagic vasculitis without RVO (correlation coefficient = 0.33; correlation coefficient = 0.27; respectively) (   mentioned anticoagulant therapy, these patents also received corticosteroid and immunosuppressive agents (Figure 5).

% of patients
Following treatment, retinal hemorrhages and edema were reduced and macular edema disappeared all together.Gene polymorphisms affecting hemostasis may also play a role in pathogenesis of RVO and studies to determine prevalence of genetic polymorphisms of various factors implicated in hypercoagulability among patients with RVO have been performed [13].
Thrombophilia plays an important role in the pathogenesis of RVO in patients who have no acquired risk factors.Screening for thrombophilia in patients where these factors have been excluded, seem to be somewhat controversial in that they leave a question concerning the association of RVO and hereditary thrombophilial abnormalities unanswered.Various coagulation disorders induced by genetic mutations (antithrombin III gene mutations, protein C and protein S deficiencies, factor V Leiden mutation, or MTHFR mutations) are often associated with an increased risk for both systemic and retinal vein occlusion [14][15][16][17][18][19][20][21].
In the year 1976, Wilcken presented a first case controlled study which suggested that the higher than normal levels of homocysteine which can otherwise be found in a ''normal'' population seem to be a predisposing factor for RVO.Some studies which have been performed at a later date ______________________________________________________________________________________________________________ _____________ Jelena Paovic, Predrag Paovic, Vojislav Sredovic, Zoran Dimcic (2015), Research in Immunology: An International Journal, DOI: 10.5171/2015.246396 have also confirmed it to be a risk factor for venous thrombosis [22,23,24].On the other hand, our results show that RVO and hemorrhagic retinal manifestations can occur in patients with retinal vasculitis at normal levels of homocysteine.Although the exact mechanisms by which hyperhomocysteinaemia causes thrombosis are unknown, it has been shown to be toxic to endothelial cells, to impair thrombomodulin expression and to reduce protein C activation via vascular endothelial cell activator [25- Predrag Paovic, Vojislav Sredovic, Zoran Dimcic (2015), Research in Immunology: An International Journal, DOI: 10.5171/2015.246396

Figure 5 :
Figure 5: Retinal vein occlusion follow up patient which was treated with anticoagulant and corticosteroid therapy (a -macular edema, optical coherent tomography, bangiography, -initial examination, 2 -follow up)