Interplay between methylenetetrahydrofolate reductase gene polymorphism 677C→T and serum folate levels in determining hyperhomocysteinemia in heart transplant recipients

CLINICAL HEART TRANSPLANTATION

Interplay Between Methylenetetrahydrofolate Reductase Gene Polymorphism 677C3 T and Serum Folate Levels in Determining Hyperhomocysteinemia In Heart Transplant Recipients Luciano Potena, MD,a Francesco Grigioni, MD,a Mariagabriella Viggiani, MD,b Gaia Magnani, MD, PhD,a Simona Sorbello, MD,a Elena Falchetti, MD,a Simonetta Sassi, PhD,c Vilma Mantovani, MD,b Letizia Bacchi-Reggiani, BSc,a Carlo Magelli, MD,a and Angelo Branzi, MDa Background: Homocysteine metabolism is often impaired in heart transplant recipients, and increased total homocysteine plasma levels may constitute a risk factor for the development of heart allograft vascular disease. Although 677C3 T transition in methylenetetrahydrofolate reductase (MTHFR) is associated with increased homocysteine levels in the general population, it is unclear whether MTHFR polymorphism influences homocysteine metabolism after heart transplant. Methods: Homocysteine, serum folate, renal function, concentrations of cyclosporine and its metabolites, and MTHFR genotype were determined in 57 heart transplant recipients (age, 55 ⫾ 11 yr; 21% women; time from transplant, 48 ⫾ 42 months). Results: Forty nine percent of the study population presented with hyperhomocysteinemia. Homocysteine was 17.1 ⫾ 5.9 ␮mol/liter, 19.4 ⫾ 4.9 ␮mol/liter, and 26.3 ⫾ 14.2 ␮mol/liter for genotypes CC, CT, and TT, respectively (p ⫽ 0.028, Kruskal-Wallis test). At multivariate analysis, MTHFR genotype was independently associated with homocysteine (p ⫽ 0.005). When the study population was divided into 2 groups accordingly to serum folate levels (above/below the median value of 6.1 ng/ ml), MTHFR genotype remained a significant predictor of homocysteine only in patients with low serum folate (p ⫽ 0.048). Conclusions: This study demonstrates that hyperhomocysteinemia is frequent in heart transplant recipients and that the 677C3 T transition in the MTHFR gene independently and unfavorably influences homocysteine metabolism in this group of From the aInstitute of Cardiovascular Diseases, bLaboratory of Biology and Genetics, and cInstitute of Angiology and Coagulation Diseases, Academic Hospital S. Orsola-Malpighi, University of Bologna, Bologna, Italy. Submitted May 24, 2001; accepted July 26, 2001. Reprint requests: Carlo Magelli, MD, Institute of Cardiovascular Diseases, Pad. 21, Academic Hospital S.Orsola-Malpighi, Via

Massarenti, 9, 40138 Bologna. Italy. Telephone: ⫹39-051349858. Fax: ⫹39-051-344859. E-mail: [email protected] Copyright © 2001 by the International Society for Heart and Lung Transplantation. 1053-2498/01/$–see front matter PII S1053-2498(01)00350-3

1245

1246

Potena et al.

The Journal of Heart and Lung Transplantation December 2001

patients. Adequate folate intake may overcome genetic predisposition to hyperhomocysteinemia. J Heart Lung Transplant 2001;20:1245–1251.

H

eart transplantation (HT) is a valid therapeutic option reserved for patients with end-stage congestive heart failure. Unfortunately, long-term posttransplant outcome is still affected by heart allograft vascular disease (AVD).1 This accelerated form of atherosclerosis is caused by immunological and nonimmunologic mechanisms that lead to intimal hyperplasia and ultimately to progressive lumen loss in graft coronary arteries, with poor post-transplant prognosis.2– 4 Elevated plasma homocysteine (Hcy), a thiolcontaining intermediate of the methionine metabolic pathway, is an independent risk factor for

atherosclerosis in the general population.5–7 Recent studies have suggested that Hcy may even be involved in the pathogenesis of AVD.8 –10 Therefore, identification of the determinants of Hcy plasma levels after HT may permit novel therapeutic strategies to be developed to prevent hyperhomocysteinemia, to reduce the incidence of AVD, and to improve post-transplant prognosis. Nutritional habits, age, gender, several pathologic conditions, and certain drugs may influence Hcy plasma levels in the general population.6,7 Moreover, hyperhomocysteinemia may be caused by dysfunction of the folate-dependent enzyme methyl-

FIGURE 1 Overview of homocysteine and folate metabolic pathways. MTHFR lower

catalytic power impairs transformation of 5-10 methylen THF to 5⬘-methyl THF, necessary for the catabolism of Hcy to methionine, through methionine synthase. MTHFR, methylenetetrahydrofolate reductase; THF, tetrahydrofolate (adapted from: Potena et al. Familial susceptibility to coronary artery disease: homocysteine and genetic polymorphism of methylenetetrahydrofolate reductase. Ital Heart J Suppl 2001; 2(7):748 –53).

The Journal of Heart and Lung Transplantation Volume 20, Number 12

enetetrahydrofolate reductase (MTHFR). This MTHFR reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor involved in the remethylation of Hcy to methionine (Figure 1). A common C3 T transition in nucleotide 677 causes an alanine-to-valine substitution in the MTHFR sequence, leading to a thermolabile isoform that predisposes to hyperhomocysteinemia.11,12 Therefore, the compounded effects of MTHFR polymorphism and environmental factors ultimately influence Hcy plasma levels in the general population.13–15 In particular, MTHFR polymorphisms may constitute a genetic risk factor for coronary artery disease.16,17 However, prospective studies on populations living in countries with high nutritional intake have not revealed any adverse effect of MTHFR polymorphism on cardiovascular risk18,19 (such an effect seems more likely in “genetically vulnerable” populations with particular nutritional profiles.20,21 Heart transplant recipients are at high risk of graft atherosclerosis development, and their Hcy metabolism is frequently impaired.8,9,22 Thus, the effect of MTHFR polymorphism on Hcy and its interaction with environmental variables in HT patients may be a relevant issue. However, only one specific study has been performed on HT recipients, and this revealed no association between genetic predisposition and Hcy plasma levels or AVD.10 The present study was designed to further investigate whether 677C3 T mutation of MTHFR influences Hcy plasma levels in HT recipients, taking into account the possible influence of the interplay with environmental determinants of Hcy, such as folate status, renal function, time from HT, and immunosuppressive therapy.

Potena et al.

1247

samples using a spin column method (QiAmp DNA Blood Mini Kit, QIAGEN GmbH; Hilden, Germany). Briefly, 50 ng of human genomic DNA was amplified with 8 pmol each of the forward primer 5⬘-TTT GAG GCT GAC CTG AAG CAC TTG AAG GAG-3⬘ and the reverse primer 5⬘-GAG TGG TAG CCC TGG ATG GGA AAG ATC CCG-3⬘. Thirty-five cycles (95°C for 60 seconds, 60°C for 90 seconds, and 72°C for 60 seconds) were used to amplify the 173 base pair (bp) product. The 20-␮l polymerase chain reaction mixture contained 10 mmol/liter Tris-HCl, 50 mmol/liter KCl, 1,25 mmol/liter MgCl2, 5% dimethylsulfoxide, 200 ␮mol/ liter each nucleotide, and 0.8 U of Taq Gold DNA polymerase (PE Applied Biosystems; Foster City, CA). Because the C3 T bp substitution creates a HinfI restriction site, 10 units of HinfI restriction enzyme (Roche Diagnostics S.p.A.; Monza, Italy) and 2.5 ␮l of buffer were added to the polymerase chain reaction product and incubated at 37°C for 2 hours. Digestion products were visualized after electrophoresis on a 4% agarose gel with ethidium bromide. Wild types (i.e., 677CC) produced a singlet band at 173 bp. Heterozygotes (677CT) produced 173, 125, and 48 bp fragments. Homozygous mutants (677TT) produced 125 and 48 bp fragments.

Other Laboratory Assays

Between March and June 2000, a total of 87 HT recipients attended routine follow-up visits in our outpatient department. All these patients were receiving a standardized immunosuppressive therapeutic protocol based on cyclosporine, azathioprine, and prednisone.23 For the purposes of this study, we considered only the 57 patients who had not received any form of folate supplementation or methotrexate therapy in the previous 6 months.6

In all patients, total plasma Hcy was measured from venous blood samples obtained after an overnight fast. The blood specimens were immediately placed in ice and centrifuged within 2 hours. Plasma was eventually stored at ⫺70°C. The assay was based on high-performance liquid chromatography, as previously described.25 Intra-day (and inter-day) variability of the analytical method— calculated as the standard deviation of 6 assays from the same blood sample—was 1.5% (4.0%), 1.4% (2.9%), and 1.3% (3.9%) for values of Hcy of 6.5 ␮mol/liter, 15.5 ␮mol/liter, and 32.5 ␮mol/liter, respectively. The lowest value of Hcy detectable was 1 ␮mol/liter and the quantitative limit was 0.5 ␮mol/liter. Serum folate was determined using a chemoluminescencebased commercial kit (ACS-100 from Chiron Diagnostic; East Walpole, MA). Serum creatinine and whole blood cyclosporine and metabolites were assayed by standard laboratory methods.

Detection of MTHFR Polymorphism

Homocysteine Reference Values

The genotyping protocol used in all cases for the detection of the MTHFR 677C3 T polymorphism was adapted from the technique described by Schneider et al.24 DNA was isolated from blood

To define reference values for Hcy plasma concentration, we examined 147 apparently healthy subjects living in the same area as the patient population (44% women; age range, 14 to 94 years, mean

METHODS

1248

Potena et al.

The Journal of Heart and Lung Transplantation December 2001

TABLE I MTHFR genotypes with respect to patients clinical and biologic characteristics

Age (yr) Gender (men/women) Time from transplant (months) Cyclosporinemia (ng/ml) Cyclosporine metabolites (ng/ml) Serum creatinine (mg/dl) Serum folate (ng/dl) Plasma homocysteine (␮mol/liter)

Overall (n ⴝ 57)

Genotype CC (n ⴝ 18)

Genotype CT (n ⴝ 24)

Genotype TT (n ⴝ 15)

p

55 ⫾ 11 45/12 48 ⫾ 42 208.7 ⫾ 27.1 570.4 ⫾ 103.1 1.56 ⫾ 0.51 6.3 ⫾ 2.7 20.7 ⫾ 10.4

47 ⫾ 11 13/5 56 ⫾ 43 217.9 ⫾ 28.5 558.3 ⫾ 124.8 1.52 ⫾ 0.41 6.9 ⫾ 2.3 17.1 ⫾ 5.9

55 ⫾ 14 18/6 60 ⫾ 44 202.4 ⫾ 28.6 600.6 ⫾ 166.2 1.68 ⫾ 0.39 6.6 ⫾ 2.9 19.4 ⫾ 4.9

58 ⫾ 7 14/1 65 ⫾ 41 202.7 ⫾ 34.6 572.1 ⫾ 92.1 1.61 ⫾ 0.42 5.8 ⫾ 2.5 26.3 ⫾ 14.2

0.064 0.275 0.642 0.351 0.915 0.399 0.173 0.028

p values refer to Kruskal-Wallis test applied to the 3 different genotype groups. MTHFR, methylenetetrahydrofolate reductase

serum folate concentration, 5.09 ng/ml). The upper reference value was defined as the 90th percentile of Hcy values, specified for sex and age.15 References values turned out to be 16.4 ␮mol/ liter for men aged ⱕ45 and 20.3 ␮mol/liter for men aged ⬎45 years; 10.3 ␮mol/liter for women aged ⱕ45 years, and 14.6 ␮mol/liter for women aged ⬎45 years.

Statistical Analysis SPSS威 software packaging was used to perform statistical analysis. Continuous variables are expressed as mean ⫾ SD. Because most variables followed a skewed distribution, differences among groups were tested using Kruskal-Wallis, chi-square, or Mann-Whitney test as appropriate. Multiple linear regression analysis was performed to determine variables independently linked to Hcy levels. p ⬍ 0.05 was considered statistically significant.

among the 3 sub-groups (27% in CC, 54% in CT, and 66% in TT; p ⫽ 0.061). At univariate analysis, Hcy concentrations correlated with age (R ⫽ 0.302, p ⫽ 0.012), time from HT (R ⫽ 0.213, p ⫽ 0.058), serum creatinine (R ⫽ 0.205, p ⫽ 0.065), and serum folate (R ⫽ ⫺0.408, p ⫽ 0.001), but not with cyclosporine (R ⫽ ⫺0.20; p ⫽ 0.272) and cyclosporine metabolite concentrations (R ⫽ ⫺0.017, p ⫽ 0.913). Homocysteine concentration was lower in women than in men (15.7 ⫾ 3.3 vs 22.1 ⫾ 11.3 ␮mol/liter; p ⫽ 0.002). At stepwise multivariate linear regression analysis, serum folate, age, time from HT, and sex were independently associated with Hcy plasma levels (Table II). When 677 MTHFR polymorphism was factored in to the model, including all of the independent predictors of Hcy, the genotype also was independently associated with Hcy plasma levels (␤-coefficient, 0.349;

RESULTS Some relevant clinical and biologic characteristics of the study population can be seen in Table I. According to the reference values reported above, 28/57 (49%) patients presented with hyperhomocysteinemia. The distribution of the MTHFR genotypes follows: 18 (32%) patients were wild-type homozygous (CC), 24 (42%) were heterozygous (CT), and 15 (26%) mutant homozygous (TT). As can be seen from Table I, the Kruskal-Wallis test showed that Hcy levels differed significantly among the 3 genotypic patient sub-groups (17.1 ⫾ 5.9 ␮mol/liter for CC vs 19.4 ⫾ 4.9 ␮mol/liter for CT vs 26.3 ⫾ 14.2 ␮mol/liter for TT; p ⫽ 0.023). Moreover, as can be seen from Figure 2, presence of pathologic levels of Hcy showed a similar pattern

FIGURE 2 Hyperhomocysteinemia increases with respect to MTHFR genotypes (p ⫽ 0.06 at chi-square test). Hcy, homocystenemia; MTHFR, methylenetetrahydrofolate reductase.

The Journal of Heart and Lung Transplantation Volume 20, Number 12

Potena et al.

1249

TABLE II Environmental predictors of homocysteinemia at multiple linear regression analysis ␤-coefficient Age (per year) Male gender Time from transplant (per month) Serum Folate (per ng/ml)

0.403 0.229 0.374 ⫺0.318

B [95% confidence intervals], 4.165 [1.34 – 6.99]; p ⫽ 0.005). To discriminate the different effects of 677 MTHFR polymorphism and folic acid on Hcy plasma levels in HT recipients, the study population was divided into 2 groups with respect to folate serum levels, using the median value (6.1 ng/ml) as the cut-off point. As can be seen from Figure 3, when the Hcy levels in the 3 genotypic sub-groups were stratified in this way, a significant effect of MTHFR polymorphism was observed at the lower serum folate levels (p ⫽ 0.048, Kruskal-Wallis test) but not at the higher levels (p ⫽ 0.894). Thus, the mutant genotypes appeared to affect Hcy concentration only in patients with serum folate levels ⬍ 6.1 ng/ml.

DISCUSSION The determinants of Hcy metabolism in HT recipients and particularly the relationships among genetic and environmental factors are currently unknown. The one study so far performed10 failed to find any association between genetic predisposition and Hcy plasma levels in HT recipients. However,

FIGURE 3 Hcy plasma concentration with respect to MTHFR genotypes, stratified by low and high serum folate levels; MTHFR mutation determinate homocysteinemia increase only in patients with low folate serum concentration. (*p ⫽ 0.048 at KruskalWallis test). Hyc, homocysteinemia; MTHFR, methylenetetrahydrofolate reductase.

B [95% confidence intervals] 0.337 5.021 0.080 ⫺1.083

[0.136 to 0.537] [0.127 to 9.914] [0.028 to 0.132] [⫺1.846 to ⫺0.319]

p 0.001 0.045 0.003 0.006

that study did not take into account the possible interplay with serum folate levels. To our knowledge, our study provides the first evidence of a genetic element in the onset of hyperhomocysteinemia in HT patients. In particular, our results strongly suggest that (1) hyperhomocysteinemia is frequent in HT recipients and results from an interaction among genetic and environmental variables; (2) the transition 677C3 T in the MTHFR gene independently and unfavorably influences Hcy metabolism in this group of patients; (3) an adequate folate status might overcome genetic predisposition to Hcy after HT. Because definition of Hcy reference values in the literature has been heterogeneous and often arbitrary, the reported prevalence of hyperhomocysteinemia in HT recipients has ranged widely (from 48% to 87%).8,9,22,27 In this study, we matched the Hcy levels of our HT recipients with those of a sample of healthy subjects living in the same geographic area,26 presumably with rather similar nutritional habits and genetic features. Almost 50% of the HT recipients had Hcy plasma levels higher than our upper reference values, derived from the 90th percentile, specified for sex and age, as recommended by Selhub et al.15 This finding provides further confirmation of the high prevalence of Hcy metabolism disorders in HT recipients. Among the non-genetic factors we studied, low serum folate, older age, longer time from HT, and male gender were all independently associated with increased Hcy plasma levels.9,22,23,27–29 The ability of male gender and of folate status to influence Hcy has been previously reported.9,15 However, the independent association between follow-up time and Hcy requires some interpretation. Although, some authors have found a direct association between time from HT and raised Hcy levels,28 others have not,27,29 a discrepancy that can be only partially explained by differences in study design. In our study, the association between time from HT and raised Hcy was independent of all the other known determinants of homocysteinemia (i.e., serum folate and renal function). This finding appears to indicate

1250

Potena et al.

a time-dependant impairment of Hcy metabolism per se, linked to the HT condition. Immunosuppressive therapy has been indicated as a possible determinant of progressive impairment of Hcy metabolism.27,29 Although experimental data exist in support of this hypothesis,30 –32 clinical studies have once again generated conflicting results.23,27,29 In our study, no association emerged between immunosuppressive therapy and Hcy. Nevertheless, this could merely reflect difficulties in quantifying the burden of immunosuppression. Homozygosity for MTHFR mutant allele was high (26%) in our series of patients. However, this incidence is fully in keeping with previous data regarding the Italian population.33 Our data suggest that 677C3 T transition is an independent predictor of increased Hcy even in heterozygous patients, in keeping with the concept that the effect of the mutant allele in MTHFR activity is codominant.14 In our study, the adverse influence of the 677C3 T mutation was independent of folate concentration. Nevertheless, its deleterious effect on Hcy levels was overcome in patients who had adequate folate intake. Thus, although the burden of MTHFR mutant isoforms on Hcy metabolism seems to be augmented in the presence of environmental variables that predispose to hyperhomocysteinemia (e.g., low folate status, renal insufficiency, HT), it may be possible to circumvent this problem with nutritional measures such as folate supplementation. In conclusion, this observational study provides the first evidence that in HT recipients, MTHFR polymorphism is a major determinant of Hcy plasma levels. With their frequently high prevalence of hyperhomocysteinemia, HT recipients could constitute a sub-population in which environmental variables elicit a genotypically specific phenotype associated with increased risk for AVD. However, our study suggests that adequate folate assumption may overcome genetic predisposition to hyperhomocysteinemia among HT patients. Randomized prospective studies are now warranted to analyze the effects of MTHFR polymorphism and folate supplementation on hyperhomocysteinemia and AVD incidence after HT. The authors thank Paola Marchesini, Sandra Sassi, and Gianna Canu, the highly experienced nurses of our Heart Transplantation Center, who offered their skilled collaboration in collecting data for this study. We are also grateful to Robin M.T. Cooke for editing the manuscript and for helpful suggestions regarding its organization.

The Journal of Heart and Lung Transplantation December 2001 This study was supported by a grant from the foundation of Cassa di Risparmio di Bologna.

REFERENCES 1. Hosenpud J, Bennet L, Keck B, Fiol B, Novick R. The Registry of the International Society for Heart and Lung Transplantation: fourteenth official report. J Heart Lung Transplant 1997;16:691–712. 2. Johnson D, Gao S, Schroeder J, DeCampli W, Bilingham M. The spectrum of coronary artery pathologic findings in human cardiac allografts. J Heart Lung Transplant 1989;8: 349 –59. 3. Billingham M. Pathology and etiology of chronic rejection of the heart. Clin Transplant 1994;8:289 –92. 4. Weis M, Scheidt W. Cardiac allograft vasculopathy: a review. Circulation 1997;96:2069 –77. 5. Malinow M. Hyperhomocyst(e)inaemia: a common and easily reversible risk factor for occlusive atherosclerosis. Circulation 1990;81:2004 – 6. 6. Mayer E, Jacobsen D, Robinsom K. Homocysteine and coronary atherosclerosis. J Am Coll Cardiol 1996;27:517–27. 7. Malinow M, Bostom A; Kraus R. Homocysteine, diet and cardiovascular disease. Circulation 1999;99:178 – 82. 8. Ambrosi P, Garc¸on D, Riberi A, Habib G. Association of mild hyperhomocysteinemia with cardiac graft vascular disease. Atherosclerosis 1998; 138:347–50. 9. Gupta A, Moustapha A, Jacobsen D, Goosemastic M. High homocysteinemia, low folate and low vitamin B6 concentration: prevalent risk factors for vascular disease in heart transplant recipients. Transplantation 1998;65:544 –50. 10. Pethig K, Hoffmann A, Heublein B, TImke A, Gross G, Haverich A. Cardiac allograft vascular disease after orthotopic heart transplantation: methylenetetrahydrofolate reductase gene polymorphism C677T does not account for rapidly progressive forms. Transplantation 2000;69:449 –5. 11. Kang S, Zhou J, Wong P, Kowalisyn J, Strokosch G. Intermediate homocysteinemia: a thermolabile variant of methylenetetrahydrofolate reductase. Am J Hum Genet 1988;43: 414 –21. 12. Frosst P, Blom H, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Am J Hum Genet 1995;58: 35– 41. 13. Jaques P, Bostom A, Williams R, et al. Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 1996;93:7–9. 14. Girelli D, Friso S, Trebetti E, et al. Methylenetetrahydrofolate reductase C677T mutation, plasma homocysteine and folate in subjects from Northern Italy with or without angiographically doumented severe coronary atherosclerotic disease: evidence for an important genetic– environmental interaction. Blood 1998;91:4158 – 63. 15. Selhub J, Jacques P, Rosenberg I, et al. Serum total homocysteine concentration in the third national health and nutrition examination survey (1991–94): population reference ranges and contribution of vitamin status to high serum concentration. Ann Intern Med 1999;131:331–339. 16. Kang S, Passen E, Ruggie N, Wong P, Sora H. Thermolabile defect of methylenetetrahydrofolate reductase in coronary artery disease. Circulation 1993;88:1463–9. 17. Kluijtmans L, Kastelein J, Lindemans J, et al. Thermolabile

The Journal of Heart and Lung Transplantation Volume 20, Number 12

18.

19.

20.

21.

22.

23.

24.

25.

methylenetetrahydrofolate reductase in coronary artery disease. Circulation 1997;96:2573–7. Folsom A, Nieto F, McGovern P, et al. Prospective study of coronary artery disease incidence in relation to fasting total homocysteinemia, related genetic polymorphisms, and B vitamins. Circulation 1998;98:204 –10. van Bockxmeer F, Mamotte C, Vasikaran S, Taylor R. Methylenetetrahydrofolate reductase gene and coronary artery disease. Circulation 1997;95:21–3. Jee S, Beaty T, Suh I, Yoon Y, Appel L. The methylenetetrahydrofolate reductase gene is associated with increased cardiovascular risk in Japan, but not in other populations. Atherosclerosis 2000;153:161– 8. Zheng Y, Tong J, Do X, Pu X, Zhou B. Prevalence of methylenetetrahydrofolate reductase C677T and its association with arterial and venous thrombosis in the Chinese population. Br J Haematol 2000;109:870 – 4. Ambrosi P, Barlatier A, Habib G, Garc¸on D. Hyperhomocysteinemia in heart transplanted recipients. Eur Heart J 1994;15:1191– 6. Potena L, Grigioni F, Magnani G, et al. Increasing plasma homocysteine during follow-up in heart transplant recipients: effect of folate and renal function. Ital Heart J 2000;1:344 – 8. Schneider J, Rees D, Liu Y, Clegg J. Worldwide distribution of a common methylenetetrahydrofolate reductase mutation. Am J Hum Genet 1998;62:1258 – 60. Araki A, Sako Y. Determination of free and total homocysteine in human plasma by high performance liquid chromatography with fluorescence detection. J Chromatogr 1987; 422:43–52.

Potena et al.

1251

26. Sassi S, Campani B, Palareti G, Legnani G, Gironi G, Coccheri S. Plasma homocysteine level in a cohort of apparently healthy subjects in Northern Italy. Relation to age, sex, and nutritional status. Thromb Haemost 1999;82:773 (abstract). 27. Cook R, Tupper J, Parker S. Effect of immunosuppressive therapy, serum creatinine and time after transplant on plasma homocysteine in patients following heart transplantation. J Heart Lung Transplant 1999;18:420 – 4. 28. Berger P, Jones J, Olson L, et al. Increase in total plasma homocysteine concentration after cardiac transplantation. Mayo Clin Proc 1995;70:125–30. 29. Cole D, Ross H, Evrowsky J, et al. Correlation between total homocysteine and cyclosporine concentrations in cardiac transplant recipients. Clin Chemistry 1998;44: 2307–12. 30. Arnadottir M, Hultberg B, Vladov V; Nisson-Ehle P, Thysell H. Hyperhomocysteinemia in cyclosporine treated renal transplant recipients. Transplant 1996;61:509 –12. 31. Mc Nally P, Feehally J. Pathophysiology of Cyclosporin A nephrotoxocity: experimental and clinical observations. Nephrol Dial Transplant 1992;7:791– 804. 32. Chung G, Walker S, Vadher B, Murphy F, Leaver N, Banner N. Effect of Sandimmune cyclosporine on renal blood flow and renal function in heart transplant recipients. Transplant Proc 1998;30:1147– 8. 33. Sacchi E, Tagliabue L, Duca F, Mannucci P. High frequency of the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene in Northern Italy. Thromb Haemost 1997 (letter);78:963.