Hrev_master Veins and Lymphatics 2012; volume 1:e8 [Veins and Lymphatics 2012; 1:e8] [page 31] Familial hyperhomocysteinemia, age and peripheral vascular diseases - an Italian study Sandro Michelini,1 Marco Cardone,1 Adriano Micci,1 Francesco Cappellino,1 Alessandro Fiorentino,1 Vincenzo Sainato,1 Maria Mora,1 Rachele Todisco,1 Maddalena Todini,1 Serena Michelini,2 Guido Valle3 1Hospital San Giovanni Battista – ACISMOM, Rome; 2La Sapienza University, 2nd School of Medicine, Rome; 3Nuclear Medicine Unit, Scientific Institute “Casa Sollievo della Sofferenza”, San Giovanni Rotondo (FG), Italy Abstract Hyperhomocysteinemia is a widely recog- nized, although not yet entirely understood, risk factor for cardiovascular disease. Particularly, the complex relationships between age, hyper- homocysteinemia, predisposing genetic factors and peripheral vascular diseases have not been fully evaluated. Our contribution to this issue is a retrospective analysis of a large series of patients with peripheral arterial, venous and lymphatic disease, and of their blood relatives, with special reference to homocysteine plasma levels, age and methylenetetrahydrofolate reductase (MTHFR) polymorphisms. Serum homocysteine was measured in 477 patients (286 males, 191 females, age range 19-78 years) with various vascular clinical conditions: post- phlebitic syndrome (46) recurrent venous ulcers (78), arterial diseases (101) primary lym- phoedema (87), secondary lymphoedema (161) and outlet thoracic syndrome (4), and in 50 nor- mal controls. A MTHFR study for polymorphisms was carried on in the subjects with homocys- teine values exceeding 15 �mol/L. Serum homo- cysteine determination and MTHFR polymor- phism studies were performed also in 1430 healthy blood related relatives (mainly siblings, descendents and sibling descendents) of the subjects with hyperhomocysteinemia and MTHFR polymorphisms. We found MTHFR poly- morphisms in 20% of controls and in 69.3%, 69.5% and 53.8% of hyperhomocysteinemic sub- jects with arterial diseases, postphlebitic syn- drome and venous ulcers, respectively. As expected, the percentage of hyperhomocys- teinemia in patients with secondary lym- phoedema and with thoracic outlet syndrome did not show significant differences compared to the control group. A MTHFR polymorphism was found in 116 out of the 214 hyperhomocys- teinemic patients, i.e., in the 54% of the overall patient population with hyperhomocysteinemia (214 patients). Interestingly 750 (52%) out of the 1430 blood relatives of the 116 patients with hyperhomocysteinemia and MTHFR polymor- phisms showed at least one polymorphism in MTHFR gene. In this latter group of 750 healthy blood-related relatives bearing a MTHFR poly- morphism the finding of hyperhomocysteine- mia increased according to the age class from 1.6% in the age range <40 years up to 54.9% in the age range >60 years. The present study demonstrate that patients with peripheral arte- rial disease, post-phlebitic syndrome, venous ulcers and primary lymphoedema show a signif- icantly higher incidence of hyperhomocysteine- mia compared to controls, and adds further evi- dence to the causative role of hyperhomocys- teinemia in the development of both arterial and venous disease. Moreover our data indicate a possible causative role of hyperhomocysteine- mia in primary lymphoedema. In more than 50% of our hyperhomocysteinemic patients a polymorphism of MTHFR (C677T and/or A1298C) was detected. In subjects with these polymorphisms the frequency of hyperhomocys- teinemia increases with age. We observed a quite similar frequency of the two polymor- phisms in the studied population and therefore claim for the need to study both C677T and A1298C mutations in hyperhomocysteinemic patients. Introduction The possible role of homocysteine high lev- els as a risk factor for vascular disease was described for the first time more than forty years ago,1,2 and in the following decades a large amount of evidence has demonstrated that even mild increases in homocysteinemia are associated with an increased risk of cardio- vascular diseases3,4 including venous throm- boembolic disease.5 Hyperhomocysteinemia is frequently associated with MTHFR polymor- phisms C677T and A1298C. MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofo- late to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methion- ine. Figure 1 summarizes homocysteine metabolism.6-13 Figure 2 shows the pathophysi- ologic mechanism(s) of hyperhomocysteine- mia-induced vascular damage.14-18 In the last few years, however, the causative role of hyperhomocysteinemia in cardiovascu- lar disease has been questioned mainly based on the reports that failed to demonstrate a clin- ical benefit after lowering homocysteinemia levels with vitamins B6, B12 and folate.19-21 Particularly, it has been pointed out that treatment with homocysteine lowering agents, i.e. folic acid and B6 and B12 vitamins, over prolonged times, does not reduce the inci- dence of cardiovascular adverse effects,19 although it has been reported that folate, vita- min B12 intake is associated with a reduction of the risk of ischemic stroke.22,23 Therefore further studies have been advocated to address this issue.24 In order to understand the relationships between homocysteinemia and vascular dis- ease we made a retrospective analysis of the patients who were referred to vascular disease rehabilitation program of our institute for peripheral arterial, venous and lymphatic dis- orders, paying special attention to homocys- teine blood levels, MTHFR polymorphisms and age. The influence of MTHFR polymorphisms on homocysteine blood levels was also studied in the available healthy blood related relatives of hyperhomocysteinemic subjects with MTHFR polymorphisms. Materials and Methods A general description of the study is reported in Figure 3. Briefly, serum concentration of homocysteine was measured in 477 consecutive patients (286 males, 191 females, mean age 56.5 years, age range 19-78 years) with various vascular clinical conditions: post-phlebitic syn- drome (46) recurrent venous ulcers (78), peripheral arterial diseases (101) primary lym- phoedema (87), secondary lymphoedema (161) outlet thoracic syndrome (4) admitted to the rehabilitation program of our Hospital. Serum homocysteine determination was also per- formed in 50 normal control subjects (29 males and 21 females, mean age 55 years). The quantitative determination of the Correspondence: Sandro Michelini, Hospital “San Giovanni Battista” - ACISMOM, via Luigi Ercole Morselli 13, 00148 Rome, Italy. Tel. +393358410881. E-mail: sandro.michelini@fastwebnet.it Key words: homocysteine, methylenetetrahydro- folate reductase, thrombosis risk factor. Acknowledgments: the authors gratefully thank Prof. Rossella Antonelli (Endocrinology Unit, ASL RM/C, Hospital Sant’Eugenio, Rome) for her kind suggestions and comments on the text. Received for publication: 22 September 2012. Revision received: 5 December 2012. Accepted for publication: 6 December 2012. This work is licensed under a Creative Commons Attribution 3.0 License (by-nc 3.0). ©Copyright S Michelini et al., 2012 Licensee PAGEPress, Italy Veins and Lymphatics 2012; 1:e8 doi:10.4081/vl.2012.e8 No n- co mm er cia l u se on ly Article [page 32] [Veins and Lymphatics 2012; 1:e8] homocysteine serum levels was performed with an automated latex enhanced immunoas- say (HemosIL, Homocysteine - 0020007800, Instrumental Laboratory SpA, Milano, Italy). When the plasma homocysteine values exceeded 15 �mol/L patients underwent MTHFR study for C677T and/or A1298C poly- morphisms. MTHFR polymorphism analysis was performed after genetic amplification on venous blood EDTA treated samples by molecu- lar genetic real time techniques in an associat- ed laboratory (BIOS, Rome, Italy). Homocysteine plasma determination and MTHFR polymorphism studies were performed also on 1430 healthy blood related relatives (mainly, siblings, descendents and sibling descendents) of the subjects with hyperhomo- cysteinemia and MTHFR polymorphisms. Results Patients Hyperhomocysteinemia was found in 20% of controls and in 69.3%, 69.5% and 53.8 % of sub- jects with arterial diseases, postphlebitic syn- drome and venous ulcers, respectively (Table 1). As expected, the percentage of hyperhomocys- teinemia in patients with secondary lymphoede- ma and with thoracic outlet syndrome did not show significant differences compared to the control group (Table 1). At least one MTHFR polymorphism was found in 116 out of the 214 hyperhomocysteinemic patients i.e. in the 54% of the overall patient population with hyperho- mocysteinemia (Table 2). Blood related relatives of hyperhomocysteinemic patients bearing a methylenetetrahydrofolate reductase polymorphism In 750 (52%) out of the 1430 blood relatives of the 116 patients with hyperhomocysteinemia and MTHFR polymorphisms at least one poly- morphism in MTHFR gene was found. In this group of 750 asymptomatic subjects bearing a MTHFR polymorphism the frequency of the find- ing of hyperhomocysteinemia increased accord- ing to the age class from 1.6% in the age range <40 years up to 54.9% in the age range >60 years (Table 3). C677T polymorphism-associated hyperhomocysteinemia did not significantly dif- fer from A1298C-induced hyperhomocysteine- mia either in frequency or in hyperhomocys- Figure 1. Homocysteine metabolism. Homocysteine is a sulphydryl amino acid derived from the intracellular demethylation of methio- nine. Homocysteine, when activated, yields a methyl group to different receivers (including creatine, steroid hormones, purine bases of DNA and RNA) and then it can be converted into homocysteine. Homocysteine may be, in turn, transformed irreversibly into cystathio- nine and then cysteine, or, in the absence of dietary methionine, remethylated to methionine. A series of enzymes and cofactors regu- late these pathways. Homocysteine is produced through two possible pathways: remethylation or trans-sulphuration. The remethyla- tion process converts back homocysteine to methionine (utilizing folate, vitamin B12 or trimethylglycine). The trans-sulphuration process utilizes vitamin B6, pyridoxal-5-phosphate, and catabolizes the homocysteine excess into metabolites that can be excreted from the organism. A mildly failure of the remethylation pathway (often due to reduced levels of folate, vitamin B12 or genetic defects) can increase significantly the homocysteine plasma levels. A mild failure in the trans-sulphuration pathway (caused by genetic defect or indadeguate levels of vitamin B6) can only increase slightly the homocysteine plasma concentration. There are several causes of hyper- homocysteinemia, some create a deficiency of the enzyme co-factors, and others reduce the activity of enzymes, involved in its metabo- lism. The deficiencies of vitamin B12, B6 or folic acid may be due to an inadequate diet, intake of drugs like methotrexate, nitroxide and levodopa or conditions involving hormonal changes like pregnancy and hypothyroidism. No n- co mm er cia l u se on ly Article [Veins and Lymphatics 2012; 1:e8] [page 33] Figure 2. Hyperhomocysteinemia mechanisms of endothelial damage and thrombosis. Hyperhomocysteinemia induces an oxidative stress through both direct and indirect effects. Particularly important are the inhibition of glutathione peroxidase, the reduction of cys- teine blood concentration, which results in depressed glutathione levels, the overexpression of NADPH and the reduced availability of nitric oxide. In its turn the depression of nitric oxide activity activates MMP-2 and MMP-9 with consequent increased aggregation of platelets and increased interaction between platelets and endothelium. Moreover MMP activation increases the production of endostatin from collagen and of angiostatin from plasminogen with consequent vascular damage. Thrombophilia is also sustained by the hyperho- mocysteinemia-enhaced activation of TF, of TAFI and of factor XIII. All these data explain why abnormally elevated homocysteine blood levels result in endothelial damage and in a consequent increase of the risk of both arterial and venous thrombosis. TF, tissue factor; TAFI, thrombin activable fibrinolysis inhibitor; MMP-2, matrix metalloproteinase 2; MMP-9, matrix metalloproteinase 9. No n- co mm er cia l u se on ly Article [page 34] [Veins and Lymphatics 2012; 1:e8] teine induced levels (Table 4). Moreover, the association of the two polymorphisms in the same subject did not result either in an earlier appearance of hyperhomocysteinemia or in higher homocysteinemia levels. From a clinical point of view our findings indicate a quite simi- lar frequency of the two polymorphisms in the studied population and therefore the need to study both C677T and A1298C mutations in hyperhomocysteinemic patients (Table 4). Discussion and Conclusions Despite some uncertainity due to the limited number of control subjects the present study shows a far higher incidence of hyperhomocys- teinemia in patients with peripheral artery dis- ease, post-phlebitic syndrome, venous ulcers and primary lymphoedema compared to con- trols, therefore adding further evidence to the causative role of hyperhomocysteinemia in the development of both peripheral thrombotic arte- rial and venous disease and of primary lym- phoedema. The patients with primary lym- phoedema recruited in the present study had no evidence of other vascular disease. In more than 50% of our hyperhomocys- teinemic patients a polymorphism of MTHFR (C677T and/or A1298C) was detected. It has been also observed that in subjects with MTHFR C677T and/or A1298C polymorphism the frequency of hyperhomocysteinemia increases with age. This latter finding is espe- cially unexpected taking into account that genetic disorders usually, although not always, produce clinically evident disorders in the first decades of life. This could possibly be explained by an age related reduction of the defences against oxidative stress. Another interesting observation is that C677T polymorphism-associated hyperhomo- cysteinemia did not significantly differ from A1298C-induced hyperhomocysteinemia either in frequency or in severity. Moreover, surpris- ingly, the association of the two polymorphisms in the same subject is not associated either with an earlier appearance of hyperhomocysteine- mia or with higher homocysteinemia levels. The very similar frequency of the C677T and A1298C polymorphisms in the patient popula- tion implies the need to study both C677T and A1298C mutations in hyperhomocysteinemic patients. Particularly, it is known that in south- ern Europe, and especially in Italy, the preva- lence of C677T homozygous polymorphism is quite higher compared with northern and cen- tral Europe.25 Finally, at authors’ knowledge this is the first report indicating a possible causative role of hyperhomocysteinemia in primary lym- phoedema. Table 1. Homocysteine plasmatic values according to the underlying disease (data on 477 patients) and in the control group (50 subjects). Test results Groups Arterial Post-phlebitic Venous leg Primary Secondary Thoracic outlet Control diseases syndrome ulcers lymphoedema lymphoedema syndrome subjects 101 46 78 87 161 4 50 5 to 15 mmol/L Normal levels 31 14 36 61 117 4 40 15 to 30 mmol/L Moderate 44 17 30 19 28 0 9 hyperhomocysteinemia 30 to 100 mmol/L Intermediate 17 10 9 7 15 0 1 hyperhomocysteinemia >100 mmol/L Severe 9 5 3 0 1 0 0 hyperhomocysteinemia Hyperhomocysteinemic - 69.3 69.5 53.8 29.9 27.3 0 20 subjects (%) Hyperhomocysteinemic subjects were significantly (P<0.001 at Chi-square test) more frequent in the groups of subjects with arterial diseases, postphlebitic syndrome, venous ulcers and primary lymphoedema compared to controls. Table 2. Patients bearing A1298C and/or C677T polymorphisms (n=116), methylenetetrahydrofolate reductase polymorphism, homo- cysteinemia level and associated clinical disorders. Mutation Moderate Intermediate Severe Arteriopathy Thrombo-phlebitis Venous hyperhomo- hyperhomo- hyperhomo- leg ulcers cysteinemia cysteinemia cysteinemia C677T eterozygous 18 subjects 15 3 1 11 3 4 C677T homozygous 16 subjects 2 6 4 8 2 6 A1298C homozygous 28 subjects 17 10 1 19 5 4 A1298C homozygous 16 subjects 6 11 2 7 3 6 C677T eterozygous A1298C eterozygous 9 6 4 8 6 5 5 subjects C677T eterozygous+A1298C homozygous 6 3 3 5 5 2 19 subjects C677T homozygous+A1298C eterozygous 0 0 2 1 1 0 12 subjects C677T homozygous+A1298C homozygous 2 2 1 1 3 1 2 subjects The first three columns refer to the degree of hyperhomocysteinemia. The last three columns illustrate the associated vascular disease. No n- co mm er cia l u se on ly Article [Veins and Lymphatics 2012; 1:e8] [page 35] Table 3. Age related finding of hyperhomocysteinemia in asymptomatic methylenetetrahydrofolate reductase polymorphism bearers. Age No. subjects C677T C677T A1298C A1298C No. subjects with % subjects with (years) eterozygotic homozygotic eterozygotic homozygotic hyperhomo- hyperhomo- mutation mutation mutation mutation cysteinemia cysteinemia ≤39 83 24 16 28 19 2 1.66 40-49 254 66 48 89 55 34 13.4 50-59 211 56 45 61 56 79 37.4 ≥60 202 53 42 63 55 111 54.9 The frequency of hyperhomocysteinemic subjects among age groups resulted always highly significant (P<0.001 at Chi-square test). Notice that in 38 cases a double polymorphism was found. Table 4. Incidence of methylenetetrahydrofolate reductase (MTHFR) polymorphisms in the 750 blood relatives of the patients with MTHFR mutation and relative incidence and level of related hyperhomocysteinemia. Mutation Moderate hyperhomocysteinemia Intermediate hyperhomocysteinemia Severe hyperhomocysteinemia C677T eterozygotic 199 subjects 20 23 8 C677T homozygotic 151 subjects 22 27 9 A1298C eterozygotic 241 subjects 23 25 9 A1298C homozygotic 185 subjects 28 26 6 Notice that 95 out of 226 (42%) bearers of a MTHFR polymorphism presented, at a carefully made clinical interview, a history for peripheral vascular disease. Figure 3. Study design, patients and subjects. MTHFR, methylenetetrahydrofolate reductase. No n- co mm er cia l u se on ly Article [page 36] [Veins and Lymphatics 2012; 1:e8] References 1. Gibson JB, Carson NAJ, Neill DW. Pathological findings in homocystinuria. J Clin Path 1964;17:427-37. 2. McCully KS. Vascular pathology of homo- cysteinemia: implications for the patho- genesis of arteriosclerosis. Am J Pathol 1969;56:111-28. 3. Graham IM, O’Callaghan P. Vitamins, homocysteine and cardiovascular risk. Cardiovasc Drugs Ther 2002;16:383-9. 4. 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