Contribution of the Cystathionine β-Synthase Gene (844ins68) Polymorphism to the Risk of Early-onset Venous and Arterial Occlusive Disease and of Fasting Hyperhomocysteinemia

Summary

The frequency of the heterozygous 844ins68 mutation of the cystathionine β-synthase (CBS) gene and of its association with the homozygous C677T transition of the methylenetetrahydrofolate reductase (MTHFR) gene, plasma fasting tHcy, folate and vitamin B₁₂ levels were evaluated in 309 consecutive patients with objectively diagnosed early-onset venous (n = 200) or arterial thromboembolic disease (n = 109) recruited over 25 months in Milan (North Italy) and Naples (South Italy). The above gene polymorphisms were also evaluated in a population of 787 unmatched controls, 204 of whom – similar to patients for age- and sex-distribution – had fasting tHcy, vitamins and activated protein C resistance measured in their plasma.

Moderate fasting hyperhomocysteinemia was detected in 15.5% of patients and in 5.9% of 204 controls (Mantel-Haenszel OR after stratification for type of occlusive disease and gender: 2.88; 1.48–5.32). The frequencies of the 677TT mutation of the MTHFR gene and of the heterozygous 844ins68 insertion of the CBS gene were not significantly different in the patient (19.4% and 6.9%) and the control population (16.5% and 7.8%), but the association of the two gene polymorphisms – found in 3.9% of patients and in 1.1% of controls – was significantly associated with an increased risk of venous or arterial occlusive diseases (RR = 3.63; 1.48–8.91). The MTHFR 677TT mutation (RR: 6.92; 3.86–12.4) and its association with the 844ins68 insertion (RR: 21.9; 8.35–57.4), but not the isolated insertion (RR: 0.71), were more frequent in patients and controls with fasting hyperhomocysteinemia than in normohomocysteinemic subjects, irrespective of the type of occlusive disease (venous or arterial). When adjusted for determinants of hyperhomocysteinemia in the patient and the control populations (generalized linear model), fasting tHcy levels were significantly higher in subjects with association of the two gene abnormalities (24.2 ± 3.8 µmol/L) than in subjects with the MTHFR 677TT mutation only (14.0 ± 5.8 µmol/L, p = 0.004). Activated protein C resistance was significantly more prevalent in venous patients (9.9%) than in controls (3.9%, OR = 2.69; 1.08–6.88). Six of 21 venous patients with APCresistance also had hyperhomocysteinemia (RR = 5.04; 0.68–37.6), but isolated fasting hyperhomocysteinemia retained statistical significance for the association with venous occlusive disease (RR = 2.84; 1.34–6.01).

Heterozygosity for the 844ins68 mutation of the CBS gene is not per se a risk factor for premature arterial and/or venous occlusive diseases. However, when detected in combination with thermolabile MTHFR, it increases by almost 4-fold the risk of occlusive diseases (arterial and/or venous), by increasing the risk and the degree of fasting hyperhomocysteinemia.

Key words

Vascular occlusive disease - thrombosis - APC-resistance - hyperhomocysteinemia - methylenetetrahydrofolate reductase - cystathionine β-synthase

• References

• 1 D’Angelo A, Selhub J. Homocysteine and thrombotic disease. Blood 1997; 90: 1-11.
• 2 Boers GHJ. Hyperhomocysteinemia as a risk factor for arterial and venous disease. A review of evidence and relevance. Thromb Haemost 1997; 78: 520-2.
• 3 Refsum H, Ueland PM, Nygård O, Vollset SE. Homocysteine and cardiovascular disease. 1998; 49: 31-62.
• 4 Brattström L. Common mutation in the methylenetetrahydrofolate reductase gene offers no support for mild hyperhomocysteinemia being a causal risk factor for cardiovascular disease. Circulation 1997; 96: 3805-6.
• 5 Cattaneo M. Hyperhomocysteinemia, atherosclerosis and thrombosis. Thromb Haemost 1999; 81: 165-76.
• 6 Evans RW, Shaten BJ, Hempel JD, Cutler JA, Kuller LH. for the MRFIT Research Group. Homocysteine and risk of cardiovascular disease in the Multiple Risk Factor Intervention Trial. Arterioscler Thromb Vasc Biol 1997; 17: 1947-53.
• 7 Folsom AR, Nieto FJ, McGovern PG, Tsai MY, Malinow MR, Eckfeldt JH, Hess DL, Davis CE. Prospective study of coronary heart disease incidence in relation to fasting homocysteine, related genetic polymorphisms, and B vitamins. The Atherosclerosis Risk in Communities (ARIC) Study. Circulation 1998; 98: 204-10.
• 8 Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJH, den Heijer M, Kluijtmans LAJ, van den Heuvel LP, Rozen R. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nature Genet 1995; 10: 111-3.
• 9 Guttormsen AB, Ueland PM, Nesthus I, Nygard O, Schneede J, Vollset SE, Refsum H. Determinants and vitamin responsiveness of intermediate HHcy (≥ 40 µmol/liter). J Clin Invest 1996; 98: 2174-83.
• 10 Gudnason V, Stansbie D, Scott J, Bowron A, Nicaud V, Humphries S. on behalf of the EARS group. C677T (thermolabile alanine/valine) polymorphism in methylenetetrahydrofolate reductase (MTHFR): its frequency and impact on plasma homocysteine concentration in different European populations. Atherosclerosis 1998; 136: 347-54.
• 11 Kluijtmans LAJ, Kastelein JJP, Lindemans J, Boers GHL, Heil SG, Bruschke AVG, Jukema JW, van den Heuvel LPWJ, Trijbels FJM, Boerma GJM, Verheugt FWA, Willems F, Blom HJ. Thermolabile methylenetetrahydrofolate reductase in coronary artery disease. Circulation 1997; 96: 2573-7.
• 12 Kluijtmans LAJ, den Heijer M, Reitsma PH, Heil SG, Blom HJ, Rosendaal FR. Thermolabile methylenetetrahydrofolate reductase and factor V Leiden in the risk of deep vein thrombosis. Thromb Haemost 1998; 79: 254-8.
• 13 D’Angelo A, Beltrametti C. Venous disease. In “Homocysteine in Health and Disease”. Carmel R, Jacobsen DW. eds. New York: Cambridge University Press; 2000. in press.
• 14 Kang S-S, Zhou J, Wong PWK, Kowalysin J, Strokosch G. Intermediate homocysteinemia: a thermolabile variant of methylenetetrahydrofolate reductase. Am J Hum Genet 1988; 43: 414-21.
• 15 Rozen R. Genetic predisposition to hyperhomocysteinemia: deficiency of methylenetetrahydrofolate reductase (MTHFR). Thromb Haemost 1997; 78: 523-6.
• 16 D’Angelo A, Coppola A, Madonna P, Fermo I, Pagano A, Mazzola G, De Stefano V, Vigano’ D’Angelo S, Galli L, Mancini FP, Cerbone AM, Di Minno G. The role of vitamin B12 in fasting hyperhomocysteinemia and its interaction with the homozygous C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene. A case-control study of patients with early-onset thrombotic events. Thromb Haemost 2000; 83: 563-70.
• 17 Ridker PM, Hennekens CH, Selhub J, Miletich JP, Malinow MR, Stampfer MJ. Interrelationship of hyperhomocyst(e)inemia. Factor V Leiden, and risks of future venous thrombosis. Circulation 1997; 95: 1777-82.
• 18 Petri M, Roubennoff R, Dallal GE, Nadeau MR, Selhub J, Rosenberg IH. Plasma homocysteine as a risk factor for atherothrombotic events in systemic lupus erythematosus. Lancet 1996; 348: 1120-4.
• 19 Falcon CR, Cattaneo M, Panzeri D, Martinelli I, Mannucci PM. High prevalence of hyperhomocysteinemia in patients with juvenile venous thrombosis. Arterioscl Thromb 1994; 14: 1080-3.
• 20 Fermo I, Vigano’ D’Angelo S, Paroni R, Mazzola G, Calori G, D’Angelo A. Prevalence of moderate hyperhomocysteinemia in patients with earlyonset venous and arterial thrombosis. Ann Intern Med 1995; 123: 747-53.
• 21 Leclerc D, Campeau E, Goyette P, Adjalla CE, Christensen B, Ross M, Eydoux P, Rosenblatt DS, Rozen R, Gravel RA. Human methionine synthase: cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders. Hum Mol Genet 1996; 05: 1867-74.
• 22 Tsai MY, Bignell M, Schwichtenberg K, Hanson NQ. High prevalence of a mutation in the cysthationine β synthase gene. Am J Hum Genet 1996; 59: 1262-7.
• 23 Sperandeo MP, de Franchis R, Andria G, Sebastio G. A 68bp insertion found in a homocystinuric patient is a common variant and is skipped by alternative splicing of the cysthationine β synthase mRNA. Am J Hum Genet 1996; 59: 1391-3.
• 24 Fermo I, Arcelloni C, Mazzola G, D’Angelo A, Paroni R. High-performance liquid chromatographic method for measuring total plasma homocysteine levels. J Chromatography B 1998; 719: 31-6.
• 25 Sambrook J, Fritsch EF, Maniatis T. In: Molecular Cloning. A Laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press; New York: 1989
• 26 De Franchis R, Mancini FP, D’Angelo A, Sebastio G, Fermo I, De Stefano V, Margaglione M, Mazzola G, Di Minno G, Andria G. C667T mutation of the 5.10-methylenetetrahydrofolate reductase gene and hyperhomocysteinaemia in thrombotic vascular disease. Am J Hum Genet 1996; 59: 262-4.
• 27 Sebastio G, Sperandeo MT, Panico M, de Franchis R, Kraus JP, Andria G. The molecular basis of homocystinuria due to cysthationine β synthase deficiency in Italian families and report of four novel mutations. Am J Hum Genet 1995; 56: 1324-33.
• 28 Mazzola G, Vigano’D’Angelo S, Bernardi F, Palareti G, Simioni P, D’Angelo A. Measurement of APC response in factor V+VIII depleted plasma: improved sensitivity and specificity over conventional assays. Thromb Haemost 1995; 73: 1364 (#1776).
• 29 De Franchis R, Buoniconti A, Mandato C, Pepe A, Sperandeo MP, Del Gado R, Capra V, Salvaggio E, Andria G, Mastroiacovo P. The C677T mutation of the 5,10-methylenetetrahydrofolate reductase gene is a moderate risk factor for spina bifida in Italy. J Med Genet 1998; 35: 1009-13.
• 30 Cattaneo M, Tsai MY, Bucciarelli P, Taioli E, Zighetti ML, Bignell M, Mannucci PM. A common mutation in the methylenetetrahydrofolate reductase gene (C677T) increases the risk for deep vein thrombosis in patients with mutant factor V (Factor V: Q506). Arterioscl Thromb Vasc Biol 1997; 17: 1662-6.
• 31 Margaglione M, D’Andrea G, d’Addedda M, Giuliani N, Cappucci G, Iannaccone L, Vecchione G, Grandone E, Brancaccio V, Di Minno G. The methylenetetrahydrofolate reductase TT677 genotype is associated with venous thrombosis independently of the coexistence of the FV Leiden and the prothrombin A20210 mutation. Thromb Haemost 1998; 79: 907-11.
• 32 Salomon O, Steinberg DM, Zivelin A, Gitel S, Dardik R, Rosenberg N, Berliner S, Inbal A, Many A, Lubetski A, Varon D, Martinowitz U, Seligsohn U. Single and combined prothrombotic factors in patients with idiopathic venous thromboembolism. Prevalence and risk assessment. Arterioscl Thromb Vasc Biol 1999; 19: 511-8.
• 33 Kluijtmans LAJ, Boers GHJ, Trijbels FJM, van Lith-Zanders HMA, van den Heuvel LPWJ, Blom HJ. A common 844ins68 insertion variant in the cysthationine β synthase gene. Biochem Mol Ned 1997; 62: 23-25.
• 34 Giusti B, Comeglio P, Attanasio M, Gori AM, Brunelli T, Prisco D, Pepe G, Gensini F, Abbate R. Different distribution of the double mutant “T833C/68bp insertion” in cysthationine β synthase gene in Northern and Southern Italian populations. Thromb Haemost 1997; 78: 1293.
• 35 Ramsbottom D, Scott JM, Molloy A, Weir DG, Kirke PN, Mills JL. Are common mutations of cystathionine β-synthase involved in the aetiology of neural tube defects?. Clin Genet 1997; 51: 39-42.
• 36 Franco RF, Trip MD, ten Cate H, Prins MH, Kastelein JJP, Reitsma PH. The prevalence of the 68bp insertion in the cysthationine β synthase gene in patients with premature arterial vascular disease: correlation with homocysteine levels and vitamin status. Blood 1997; 90 (Suppl. 01) 153a.
• 37 Kluijtmans LAJ, Verhoef P, Boers GJH, den Heijer M, van den Heuvel LPWJ, Trijbels JMF, Blom HJ. Cysthationine β synthase and mild hyperhomocysteinemia: an association study. The Netherlands J Med 1998; 52 (Suppl): S34.
• 38 Tsai MY, Bignell MK, Schwichtenberg K, Yang F, Hanson NQ. Genetic causes of mild hyperhomocysteinemia in patients with premature coronary artery diseases. The Netherlands J Med 1998; 52 (Suppl): S48.
• 39 Orendac M, Muskova B, Richterova F, Zvarova J, Stefek M, Zaykova E, Stribnye J, Hyanek J, Kraus JP, Kozich V. Mutation C677T in the MTHFR gene and polymorphism 844ins68bp in the CBS gene: risk factors for peripheral arterial occlusive disease?. The Netherlands J Med 1998; 52 (Suppl): S47.
• 40 Orendac M, Muskova B, Richterova E, Zvarova J, Stefek M, Zaykova E, Kraus JP, Stribrny J, Hyanek J, Kozich V. Is the common 844ins68 polymorphism in the cystathionine β-synthase gene associated with atherosclerosis?. J Inher Metab Dis 1999; 22: 674-5.
• 41 Selhub J, Miller JW. The pathogenesis of homocysteinemia: interruption of the coordinate regulation by S-adenosylmethionine of the remethylation and transsulfuration of homocysteine. Am J Clin Nutr 1991; 55: 131-8.
• 42 Andria G, Buoniconti A, Sperandeo MP, de Franchis R, D’Angelo A, Sebastio G, Guttormsen AB, Refsum H, Ueland PM. Intermediate hyperhomocysteinemia and the 844ins68 mutation of the cystathionine β-synthase gene. Am J Hum Genet. 1998; 63 (Suppl): A207 (#1186).
• 43 Guastadnes M, Rüdiger N, Rasmussen K, Ingerslev J. Intermediate and severe hyperhomocysteinemia with thrombosis: a study of genetic determinants. Thromb Haemost 2000; 83: 554-8.
• 44 Robinson K, Arheart K, Refsum H, Brattstrom L, Boers G, Ueland P, Rubba P, Palma-Reis R, Meleady R, Daly L, Witteman J, Graham I. for the European COMAC Group. Low circulating folate and vitamin B6 concentrations. Risk factors for stroke, peripheral vascular disease, and coronary heart disease. Circulation 1998; 97: 437-43.
• 45 Mazzola G, Fermo I, Crippa L, Paroni R, Tomassini L, Vigano’ D’Angelo S, D’Angelo A. Changes in homocysteine levels 2, 6, and 8 hours after methionine load: dependence of Δ post-methionine load tHcy on fasting tHcy and gender. Thromb Res. 1998; 91 (Suppl. 01) S100.