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MTHFR C677T variant on homocysteine
THE C677T THERMOLABILE
VARIANT OF METHYLENE TETRAHYDROFOLATE REDUCTASE ON HOMOCYSTEINE,
FOLATE AND VITAMIN B12 IN A HEMODIALYSIS CENTER Abstract Homocysteine is a risk factor for cardiovascular disease. Mutations in a key enzyme in homocysteine metabolism, methylenetetrahydrofolate reductase, may contribute to hyperhomocysteinemia and alter folate and cobalamin levels. After starting hemodialysis, 10 mg oral folate daily and 500 µg intravenous methylcobalamin once weekly were prescribed to 27 hemodialysis patients (time on hemodialysis > 12 months) and two groups were defined: Group A normal; Group B heterozygous. Initial, third and twelfth month measurements of homocysteine, serum folate and vitamin B12 levels were collected and analyzed. Heterozygous state of methylenetetrahydrofolate reductase prevalence was 48% and homozygozity 4%. Hyperhomocysteinemia was present in both groups. Cobalamin final levels were significantly lower in Group B compared to Group A. Homocysteine, serum folate and cobalamin levels at third and twelfth month were significantly different from baseline levels but non-different between them in both groups. In Group B, vitamin B12 at third month was significantly higher than initial, but final measurements were not different from baseline determinations. In conclusion, the heterozygous prevalence of the enzyme in hemodialysis patients is similar to that reported in the general population; hyperhomocysteinemia is frequent in hemodialysis patients and final levels in heterozygous patients are significantly higher than in normal patients. Cobalamin levels are lower in the heterozygous group. After one year of treatment, homocysteine tends to increase, suggesting a secondary resistance phenomenon to vitamin supplementation in heterozygous patients Key words: homocysteine, MTHFR, folate, folic acid,
cobalamin, vitamin B12, hemodialysis
La variante termolábil C677T de la enzima metileno-tetrahidrofolato-reductasa
sobre homocisteína, folatos y vitamina B12 en hemodializados crónicos.
La homocisteína es un factor de riesgo de enfermedad
cardiovascular. Mutaciones en la enzima metilenetetrahidrofolato
reductasa pueden contribuir a la hiperhomocisteinemia alterando los
niveles séricos de folato y cobalamina. Luego del ingreso a
hemodiálisis, se prescribió 10 mg diarios de ácido fólico y 500 µg
semanales intravenosos de metilcobalamina a veintisiete pacientes en
hemodiálisis (tiempo en hemodiálisis > 12 meses) y se definieron dos
grupos: Grupo A normal para la enzima; Grupo B heterocigota.
Mediciones iniciales (prediálisis) de homocisteína, folato sérico y
vitamina B12, al tercer y duodécimo mes fueron recolectados. El estado
heterocigota de la enzima tuvo una prevalencia del 48% y el homocigota
un 4%. Los valores iniciales de homocisteína estaban elevados en ambos
grupos. Los niveles finales de cobalamina fueron significativamente
más bajos en el grupo B. Tanto la homocisteína como el folato y la
cobalamina al mes tres y doce fueron significativamente diferentes a
los iniciales pero no diferentes entre sí. En el grupo B, la vitamina
B12 al tercer mes fue significativamente más elevada que la inicial,
pero las determinaciones finales no fueron diferentes a las basales.
En conclusión, la prevalencia heterocigota de la enzima en los
pacientes en hemodiálisis es similar a la de la población general; la
hiperhomocisteinemia es frecuente en los pacientes hemodializados y
los niveles al año son significativamente más altos en los
heterocigotas, si bien dentro de parámetros normales. Los niveles de
cobalamina son más bajos en el grupo heterocigota. Al año de
tratamiento, la homocisteína mostró una tendencia a elevarse,
sugiriendo la existencia de una resistencia secundaria al suplemento
vitamínico en los pacientes heterocigotas. Palabras clave: homocisteína, MTHFR, folato, ácido fólico, cobalamina, vitamina B12, hemodiálisis
Postal address: Dr. Hernán Trimarchi, Hospital Británico, Perdriel 74, 1280 Buenos Aires, Argentina. Fax: (54-11) 4304 3393 email: htrimarchi@hotmail.com
Received: 13-VI-2001 Accepted: 18-XII-2001
Homocysteine (Hcy), an intermediary amino acid formed by the conversion of methionine to cysteine, is an independent risk factor for atherosclerotic vascular disease and recurrent venous thromboembolism, two frequent complications of end-stage renal disease patients1-6. Homocysteine is metabolized by transsulfuration (vitamin B6 acts as a cofactor) and mainly by remethylation (vitamin B12 is the cofactor). In the remethylation pathway, Hcy is remethylated to methionine in a reaction catalyzed by methionine synthase; the methyl group comes from the active form of folic acid methyltetrahydrofolate, which therefore acts as a cosubstrate7, 8. Elevations in plasma Hcy can be caused by a variety of disorders: genetic defects, nutritional deficiencies in the vitamin cofactors, and other causes such as renal failure, liver disease or drugs8. Among the genetic defects, the most common cause of genetic hyperhomocys-teinemia is due to a thermolabile variant of methylene-tetrathydrofolate reductase (MTHFR) with reduced enzy-matic activity9, 10, with a prevalence of the homozygous state estimated between 5 to 14 percent in the general population11-13 and similar to the 10 percent reported in hemodialysis (HD) patients14. The enzyme MTHFR is required for the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydro-folate, thus generating the active folate derivative required for the remethylation of Hcy to methionine15, 16. A variant of this enzyme with decreased activity contains an alanine-to-valine substitution at amino acid 677 (MTHFR 677C®T). It is well established that such mutation of the gene coding for 5,10 methylenetetrahydrofolate reductase may predispose hyperhomocysteinemia12. We decided to determine the
prevalence of the variant states of MTHFR in a HD center in Buenos
Aires and assess the impact of this mutation on Hcy, serum folic acid
(sFA) and vitamin B12 (vitB12) levels when compared with patients
lacking this mutation in stable chronic HD patients. Measurements of
these variables were made before the first HD was performed in each
patient (baseline, To) and at the third (T3) and twelfth (T12) month
postdialysis. Material and Methods Study design MTHFR status has recently
been determined in March 2001 in all patients who were hemodialyzed
thrice weekly at the Hospital Británico during the year 2000 and for
at least 12 months. Patients who were dialyzed for more than one year
and died at the time of MTHFR determination, had frozen serum stored
which was later processed for such purpose. Baseline levels, third
month (T3) and one year (T12) reported measurements of Hcy, sFA and
vitB12 were retrospectively recollected and analyzed. Patient characteristics A total of 27 chronic
hemodialysis patients were included in this study. Patients were free
from malignancy, end-stage chronic heart failure, active liver or
thyroid disease, uncontrolled diabetes mellitus and malnourishment and
had serum albumin > 3 g/dl and hematocrits > 32%. Patients were
consequently divided into three groups according to the MTHFR status (Table
1). Thirteen patients (48%) were normal for the enzyme (Group A); in
this group 8 patients (62%) were male, age 59.4±4.6 years and time on
HD was 25.2±6.4 months. Causes of end-stage renal disease were:
Diabetes mellitus in 2, glomerulonephritis in 5, polycystic kidney
disease in 4 and ischaemic nephropathy in 2. Group B consisted of
thirteen patients (48%) who were heterozygous; in this group 7
patients (54%) were male, age 64.8±3.5 years, time on hemodialysis was
13.1±1.4 months. Causes of end-stage renal disease were: Diabetes
mellitus in 3, glomerulonephritis in 6, polycystic kidneys in 1
patient and ischaemic nephropathy in 3. Group C (4%) included only one
patient who was homozygous, male gender, 40 years old and had been on
HD for 20 months. He had polycystic kidney disease as the cause of
renal failure. Group C was excluded from group comparisons due to
small size (n=1). Hemodialysis was performed in a high-flux manner
with bicarbonate bath, mean Qd:500 ml/minute and mean Qb:350±50 ml/minute;
biocompatible membranes were used: polysulphone F80® (Fresenius
Germany), cellulose triacetate FB210® (Nipro, Japan) and CT190G® (Baxter,
USA). Each HD session averaged 3.5±0.5 hours thrice weekly. Biochemical measurements Homocysteine (normal:10 ± 5
µmol/l) was measured by fluorescent polarization immunoassay, while
sFA (normal: >10 ng/ml) and vitB12 (normal: 200-900 pg/ml) blood
levels were determined by radioimmunoassay. All levels were measured
predialysis in fasting conditions; baseline levels (To) correspond to
those measured at the first HD performed in the patient, while
subsequent measurements belong to the third month (T3) and the twelfth
month (T12) of dialysis. DNA extraction and mutation
detection DNA extraction was
performed as originally described17 from an entire blood sample kept
at -20°C. Screening for the MTHFR 677C®T substitution was performed by
amplification of a 198-bpDNA fragment and followed by Hinf I digestion,
as originally described10. Usual medications
prescribed All patients received
erythropoietin (2000-4000 U subcuta-neously thrice a week)
postdialysis and intravenous iron saccharate to reach a transferrin
saturation between 20 and 50%. Most patients are on multivitamins in
the predialysis period, but are routinely started on folic acid (10 mg/day
orally) and iv methylcobalamin (500 µg/once weekly postdialysis) when
admitted to the HD unit at the Hospital Británico. Statistical analyses Results are expressed as the mean ± standard error of the mean (SEM), unless especified otherwise. Mann-Whitney U test was used for differences between groups of cuantitative variables. Chi square or Fisher test was used for qualitative variable comparitions; finally, Wilcoxon signed ranks test was used to compare intragroup results.
Results Intergroup results Results are depicted in
Table 2 No differences were found with respect to initial Hcy, initial sFA, or serum vitB12 baseline levels; after three months of HD, no significant differences were found between both groups. Finally, after one year of treatment Hcy levels were significantly higher in Group B with respect to Group A. No differences were found regarding SFA levels. Noteworthy, despite continuous therapy, vitamin B12 blood levels were significantly lower in Group B with respect to Group A, although levels were well above normal refe-rence values. Additionally, regarding
thromboembolic events, no significant differences were found between
both groups. In group A, 4 thromboses of arteriovenous accesses were
diagnosed during the study (30.7%) versus 6 events in group B (46.2%);
in this group 5 thromboses occurred in the arteriovenous accesses and
1 patient had pulmonary thromboembolism. No differences between both
populations were observed with respect to clotting complications of
the extracorporeal circuit. Finally, 1 patient from group A (7.7%)
died due to hypovolemic shock and 2 from group B (15%) of myocardial
infarction and chronic heart disease. These differences were non
significant. Intragroup results Results are shown in Table
3 Group A: Hcy, sFA and vitB12 blood levels were significantly different from their corresponding initial levels, but T3 and T12 measurements were non-different between them. Group B: Significant
reductions were observed in Hcy To-T3 and To-T12 blood levels, but
were not statistically different between them, although T12
measurements were higher than T3. Significant rises in To-T3 and To-T12
SFA levels were observed. Finally, vitamin B12 T3 concentrations were
statistically higher than baseline; T12 were lower than T3 but lacked
significant statistical difference, and non-different from initial
ones. Discussion Our results show that the prevalence of the heterozygous variant of MTHFR in a HD center in Buenos Aires was 48%, similar to the 42.8% reported in a previous study from 418 healthy blood donors in Argentina18, demonstrat-ing that this mutation is not associated with renal failure and is not a risk factor to develop end-stage renal disease. To our knowledge, no reported data exist about the prevalence of the thermolabile variant of MTHFR in a HD center in Argentina. Despite both normal and heterozygous patients presented decreased significantly Hcy levels after three months of vitamin supplementation and were non-different between them, Hcy levels in the heterozygous group were significantly higher than in the normal group after one year of treatment, although these final levels were statistically lower than baseline ones. Moreover, albeit T12 levels were non-different from T3, they showed a climbing trend, which may be explained by a secondary resistance of the enzyme to a constant dose of folic acid. These results confirm previous ones which show that such mutation predisposes to higher Hcy levels9, 10, and such resistance is observed at constant doses of vitamin therapy, being folate the most important vitamin involved in Hcy metabolism11, 15. This MTHFR malfunction can partially contribute to the hypomethylation phenomenon described in uremia, by which Hcy levels remain high19, 20. One possibility to overcome such enzymatic derangement could be to assess Hcy levels after higher doses of folate supplementation (> 20 mg/day in patients on 10 mg/day) in heterozygous patients. In our center, we have already increased the dose of intravenous methylcobalamin from 500 µg once a week to 500 µg thrice weekly maintainig constant folate daily doses of 10 mg, and no significant reduction in Hcy levels was obtained after six months of therapy. Whether MTHFR heterozygous people are exposed to a higher risk of atherosclerotic complications (coronary heart disease, stroke, etc) or thromboembolic events is to be determined. In our study, these differences were statistically non-significant probably due to the small number of patients included. Likewise, we cannot conclude from this study that T12 Hcy levels in Group B (normal but significantly higher than in Group A) are related to additional cardiovascular or thromboembolic risks. Curiously, initial Hcy levels were high in all patients despite baseline sFA and vitamin B12 blood levels were normal (Table 2), demonstrating that well above or supra-physiological concentrations of both vitamins must be achieved to lower Hcy significantly. (Normal folate and vitB12 levels could be due to previous multivitamin supplementation even at low doses: average 1 mg oral folate and 200 µg oral cobalamin preparations). With respect to intragroup comparisons, folate plus vitamin B12 supplementation rapidly and efficiently decreased Hcy in both groups (T3 vs To). In Group B, T12 vitamin B12 levels were non different from initial ones, again showing that the heterozygous population of renal patients is unable to maintain vitB12 levels in the rising pattern that people without MTHFR mutations show after intravenous methylcobalamin supplementation. We have not found any data in the literature reporting any association between MTHFR heterozygozity and low-normal vitB12 concentrations, albeit in a recent report homozygous subjects carrying the MTHFR C 677T variant have higher folate and vitamin B12 requirements21. Noteworthy and anecdotically, in our study six patients from Group B but no patient from Group A were Helicobacter pylori positive, a recently reported possible cause of cobalamin deficiency22. All six patients lacked antiparietal cell and intrinsic factor antibodies. Again, no association between MTHFR mutations and lower vitB12 levels have been reported previously. Whether MTHFR heterozygozity predisposes to Helicobacter pylori superinfection through folic acid deficiency and vitB12 malabsorption has not been assessed. In conclusion, heterozygous MTHFR prevalence in HD patients is similar to that reported in the general population; plasma Hcy in heterozygous patients is significantly higher than in normal MTHFR patients; after one year of therapy with 10 mg daily oral folic acid and 500 µg weekly intravenous methylcobalamin, a secondary resistance phenomenon to vitamin supplementation in MTHFR heterozygous patients is observed, by which Hcy tend to increase. This pilot study includes a small group of patients so that all results must be analyzed with caution.
References 1. Bostom A, Culleton B. Hyperhomocysteinemia in chronic renal disease. J Am Soc Nephrol 1991; 10: 981-90. 2. Foley R, Parfrey P, Sarnak M. Clinical epidemiology of cardiovascular disease in chronic renal disease. Am J Kidney Dis 1998; 112S-9S. 3. Robinson K, Gupta A, Dennis V, et al. Hyperhomocys-teinemia confers an independent increased risk of atherosclerosis in end-stage renal disease and is closely linked to plasma folate and pyridoxine concentrations. Circulation 1996; 94: 2743-8. 4. Moustapha A, Naso A, Nahlawi M,et al. Prospective study of hyperhomocysteinemia as an adverse cardiovascular risk factor in end-stage renal disease. Circulation 1998; 97: 138-41. 5. Clarke R, Daly L, Robinson K, et al. Hyperhomocys-teinemia: An independent risk factor for vascular disease. N Engl J Med 1991; 324: 1149-55. 6. Shemin D, Lapane K, Bausserman L, et al. Plasma total homocysteine levels and hemodialysis access thrombosis: a prospective study. J Am Soc Nephrol 1999; 10: 1095-9. 7. Teferri A, Pruthi R. The biochemical basis of cobalamin deficiency. Mayo Clin Proc 1994; 69: 181-6. 8. Moghadasian M, McManus B, Frohlich J. Homocyst(e)ine and coronary artery disease. Arch Inter Med 1997; 157: 2299-308. 9. Kang S, Wong P, Susmano A, et al. Thremolabile methy-lenetetrahydrofolate reductase: an inherited risk factor for coronary artery disease. Am J Hum Genet 1991; 48: 536-45. 10. Frosst P, Blom H, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase (letter) Nat Genet 1995; 10: 111-3. 11. Gallagher P, Meleady R, Shields D, et al. Homocysteine and risk of premature coronary heart disease. Evidence for a common gene mutation. Circulation 1996; 94: 2154-8. 12. Guttormsen A, Ueland P, Nesthus I, et al. Determinants and vitamin responsiveness of intermediate hyperhomo-cysteinemia (or=40 micromol/liter). The Hordaland Homocysteine Study. J Clin Invest 1996; 98: 2174-83. 13. Klujitmans L, Kastelein J, Lindemans J, et al. Thermolabile methylenetetrahydrofolate reductase in coronary artery disease. Circulation 1997; 96: 2573-7. 14. Van Guldener C, Stam F, Stehouwer D. Homocysteine metabolism in renal failure. Kidney Int 2001; 59 Suppl 78: 234S-237S. 15. Fowler B. The folate cycle and disease in humans. Kidney Int 2001; 59 Suppl 78: 221S-9S. 16. Födinger M, Wagner O, Hörl W, et al. Recent insghts into the molecular genetics of the homocysteine metabolism. Kidney Int 2001; 59 Suppl78: 238S-242S. 17. Lahiri D, Nurnberger J. A rapid non-enzimatic method for the preparation of HMW DNA from blod for RELP studies. Nucleic Acid Research 1991; 19: 5444-9. 18. Genoud V, Castañon M, Annichino-Bizzacchi J, et al. Prevalence of three prothrombotic polymorphisms: Factor V G1691A, Factor II G20210A and methylenetetra-hydrofolate reductase (MTHFR) C 677T in Argentina. Thromb Res 2000: 100; 127-31. 19. Perna A, Ingrosso D, Galletti P, et al. Membrane protein damage and methylation reactions in chronic renal failure. Kidney Int 1996: 50: 358-66. 20. Perna A, Ingrosso D, Castaldo P, et al. Homocysteine and transmethylations in uremia. Kidney Int 2001; 59 Suppl 78: 230S-3S. 21. D‘Angelo A, Coppola A, Madonna P, et al. The role of vitamin B12 in fasting hyperhomocysteinemia and its interaction with the homzygous 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. 22. Kaptan K, Beyan C, Ugur
Ural A, et al. Helicobacter pylori. Is it a novel causative agent in
vitamin B12 deficiency?. Arch Inter Med 2000; 160: 1349-53. Table 1.– Patient
characteristics Group Male Age Time on HD DM GN PKD Isch Nephr CHD Deaths % (years) (months) % % % % A n=13 62 59.4±4.6 25.2±6.4 15 39 31 15 1 1 B n=13 54 64.8±3.5 13.1±1.4 23 46 8 23 3 2 C* n=1 100 40.4 20 0 0 100
0 0 0 Abbreviations: HD, hemodialysis; DM, diabetes mellitus; GN, glomerulonephritis; PKD, polycystic kidney disease; Isch Nephr, ischemic nephropathy; CHD, chronic heart disease; Symbols: *: excluded from study; %, percent Table 2.– Intergroup
results Group Homo cyst eine Serum Folic Acid Vita min B12 normal 10±5 µmol/l normal >10 ng/ml normal 200-900 pg/ml To T3 T12 To T3 T12 To T3
T12 A 21.7±1.5 12.8±1.1 12.7±0.9ª 22.9±6.9 349.9±93.7 372.1±93.8 1673±495 2756±569 3643±846b B 23.2±2.9 16.1±2.0 16.3±0.9ª 30.2±16.5 178.8±39.6 235.8±52.2 1507±92 2358±587 1505±293b C* 64 11 23 2.4 321 295 481
1094 2316 Symbols: To: baseline levels; T3: three months postdialysis; T12: twelve months postdialysis; * : excluded a P=0.014 b P=0.039 Table 3.– Intragroup
differences Measurement Period Group A
Group B Homocysteine (µmol/l) To-T3 21.7±5.3 vs 12.8±3.9; P=0.002 23.1±10.7 vs 16.1±7.3; P=0.009 Homocysteine (µmol/l) To-T12 21.7±5.3 vs 12.7±3.3; P=0.003 23.1±10.7 vs 16.3±3.5; P=0.039 Serum Folic Acid (ng/ml) To-T3 22.9±32.4 vs 349.9±337.9; P=0.001 30.2±59.5 vs 178.8±142.9; P=0.001 Serum Folic Acid (ng/ml) To-T12 22.9±32.4 vs 372.1±338.2; P=0.001 30.2±59.5 vs 235.8±188.3; P=0.002 Vitamin B12 (pg/ml) To-T3 1673±1785 vs 2755±2050; P=0.016 1507±1742 vs 2357±2117; P=0.033 Vitamin B12 (pg/ml) To-T12
1673±1785 vs 3643±3051; P=0.011 1507±1742 vs 1505±1057; p=ns Results are expressed as
the mean ± SD |
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