No association between global leukocyte DNA methylation and homocysteine levels in schizophrenia patients

Available online at www.sciencedirect.com

Schizophrenia Research 101 (2008) 50 – 57 www.elsevier.com/locate/schres

No association between global leukocyte DNA methylation and homocysteine levels in schizophrenia patients A. Bromberg, J. Levine, B. Nemetz, R.H. Belmaker, G. Agam ⁎ Psychiatry Research Unit, Faculty of Health Sciences, Stanley Research Center, Ben-Gurion University of the Negev & Beersheva Mental Health Center, Beersheva, Israel Received 18 September 2007; received in revised form 30 December 2007; accepted 4 January 2008 Available online 13 February 2008

Abstract Meta-analysis recently suggested that a 5 μM increase in homocysteine is associated with a 70% higher risk for schizophrenia. Elevated homocysteine is reported to alter macromolecule methylation. We studied whether elevated plasma homocysteine levels in schizophrenia are associated with altered leukocyte global DNA methylation. DNAwas extracted from peripheral blood leukocytes of 28 schizophrenia patients vs. 26 matched healthy controls. Percent of global genome DNA methylation was measured using the cytosine-extension method. Homocysteine levels were higher in schizophrenia patients than in controls. No difference in global DNA methylation between schizophrenia patients and control subjects was found (74.0% ± 14.8 vs. 69.4 ± 22.0, p = 0.31). A significant interaction between diagnosis and smoking on DNA methylation was obtained (F = 6.8, df = 1,47, p = 0.032). Although leukocytes may be a useful cell model to evaluate epigenetic changes such as global DNA methylation in brain, future studies should compare global DNA methylation in peripheral tissue vs. brain in laboratory animals. © 2008 Elsevier B.V. All rights reserved. Keywords: Leukocyte; DNA methylation; Schizophrenia; Homocysteine

1. Introduction Schizophrenia is a complex psychiatric disorder hypothesized to involve an interaction of multiple susceptibility genes (Lewis et al., 2005), environmental factors (Cannon et al., 2002) and epigenetic effects (Grayson et al., 2005). DNA methylation, the covalent binding of a methyl group to the 5-carbon position of the cytosine residues within CpG dinucleotides, affects eukaryotic ⁎ Corresponding author. Faculty of Health Sciences, Ben-Gurion University of the Negev, PO Box 4600 Beer-Sheva 84170, Israel. Tel.: +972 8 6401737; fax: +972 8 6401740. E-mail address: [email protected] (G. Agam). 0920-9964/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2008.01.009

gene regulation (Robertson and Wolffe, 2000; Jones and Takai, 2001) and cellular differentiation (Bird and Wolffe, 1999). DNA methylation status is dynamic and responds to physiological and pathological conditions such as development, cell differentiation (Chen et al., 2003), aging (Rampersaud et al., 2000) and cancer (Robertson and Wolffe, 2000). The methyl donor is S-adenosylmethionine (SAM), produced from the dietary amino acid methionine. After the donation of the methyl group SAM is converted to S-adenosylhomocysteine (SAH) which is hydrolyzed to homocysteine. This reaction is reversible; high intracellular homocysteine levels shift the equilibrium between SAH and homocysteine back to SAH production (Hoffman et al., 1980). SAH inhibits the

A. Bromberg et al. / Schizophrenia Research 101 (2008) 50–57

activity of most SAM-dependent methyltransferases, including DNA methyltransferases (Hoffman et al., 1980), therefore elevated homocysteine and SAH concentrations may cause DNA hypomethylation. Such negative correlation between plasma SAH or homocysteine levels and global DNA methylation was reported in some (Yi et al., 2000; Castro et al., 2003) but not all (Choi et al., 2003; Bonsch et al., 2004; Fux et al., 2005) studies. Others, as well as our group, reported elevated plasma homocysteine levels in schizophrenia patients, particularly in young males (Regland et al., 1995; Levine et al., 2002; Applebaum et al., 2004; Nevo et al., 2006). Moreover, a recent population-based birth cohort of schizophrenia and schizoaffective patients vs. matched controls found that elevated maternal third-trimester serum homocysteine levels was associated with over two-fold increase in schizophrenia risk in the offspring (Brown et al., 2007). These studies may be rebated to classical reports that orally loaded methionine, the precursor of homocysteine, exacerbates psychotic symptoms in about 40% of schizophrenia patients (Cohen et al., 1974). A recent meta-analysis of eight cross-sectional case-control studies suggested that a 5 μM increase in homocysteine levels is associated with a 70% higher risk for schizophrenia (Muntjewerff et al., 2006). Moderately elevated plasma homocysteine levels in the range of 7.3–24.4 μM were found to be associated with human brain atrophy (Sachdev et al., 2002), with in vitro neurotoxicity via NMDA receptors dependent on glycine levels (Lipton et al., 1997). High homocysteine levels (up to 250 μM in vitro) were found to be involved in neuronal DNA damage and apoptosis (Kruman et al., 2000). Homocysteine was reported to accelerate oxidative damage of endothelial cells (Lentz, 2005). Since NMDA receptorinvolved neurotoxicity (Lau and Zukin, 2007), elevated brain cell apoptosis (Glantz et al., 2006) and hypoxia (Murray, 1994) have been reported to contribute to the pathophysiology of schizophrenia, each of the effects of homocysteine described above, or any combination of them, may be involved in the etiology of the disorder. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in SAM's de novo synthesis. Its 677T allele, associated with elevated plasma homocysteine (Frosst et al., 1995) and with neuropsychiatric disorders including schizophrenia (Regland et all., 1997; Joober et al., 2000; Sazci et al., 2003), was recently found to be associated with global DNA hypomethylation (Matsubayashi et al., 2005; Graziano et al., 2006). DNA-methyltransferse1 (DNMT1), one of the enzymes that catalyze DNA methylation, was found to be upregulated in GABAergic neurons in prefrontal cortex of schizophrenia patients and bipolar patients with psychosis (Veldic et al., 2005).

51

In accordance with Bleich et al.'s (Bleich et al., 2007) recent comment the present study aimed to assess a hypothesis of the mechanism by which elevated plasma homocysteine levels are involved in the etiology of schizophrenia. Namely, we used leukocyte-derived DNA to study whether elevated plasma homocysteine levels in schizophrenia are associated with altered global DNA methylation in schizophrenia patients vs. healthy control subjects. 2. Methods 2.1. Subjects Twenty eight familialy unrelated schizophrenia patients [aged 39 ± 13.7 (S.D.); 18F,10M] from the BeerSheva Mental Health Center and 26 age- and sex-matched familialy unrelated healthy controls [42 ± 10.0; 16F,10M] were recruited. Patients were diagnosed by two independent psychiatrists according to DSM-IV criteria. All patients were being treated with antipsychotic medications. All participants were interviewed for demographic and lifestyle data, including age, gender and smoking habits. For the schizophrenia patients, illness-related data as duration of illness, general and antipsychotic medication was provided by their attendant psychiatrists. Subjects suffering from chronic non-psychiatric diseases associated with altered folate and homocysteine levels, i.e. Alzheimer's disease, diabetes type II, cardiovascular disease or renal failure were excluded. Subjects treated chronically with medications that may alter DNA methylation, e.g. valproate, were excluded. The study was approved by the Helsinki Committee (institutional review board) of Ben Gurion University. All subjects provided written informed consent. 2.2. Plasma homocysteine levels Three ml blood samples were obtained in ice-cooled EDTA tubes. Plasma was separated by centrifugation at 250 g for 15 min and stored at − 20 °C. Total homocysteine levels were measured by high-performance liquid chromatography (HPLC) with fluorescence detection following labeling of homocysteine with monobromobimane according to a modification of the method of Araki and Sako (Araki and Sako, 1987). 2.3. Global genome DNA methylation Genomic DNA was extracted from white blood cells of 3 ml blood samples using the MasterPure™ DNA Purification Kit (Epicentre, Madison, Wisconsin, USA).

52

A. Bromberg et al. / Schizophrenia Research 101 (2008) 50–57

Global genome DNA methylation was measured using a modification of the radiolabeled [3H]dCTP-extension assay (Pogribny et al., 1999). Briefly, DNA samples (2 μg each) were digested overnight with 40 units of methylation-insensitive restriction endonuclease MspI (New England Biolabs, Ipswich, MA, USA) or with 40 units of HpaII (New England Biolabs, Ipswich, MA, USA) (methylation-sensitive isoschizomer). A third DNA aliquot was incubated without restriction enzymes (background control). Single nucleotide extension reaction was performed in a 50 μl reaction mixture (0.5 μg of DNA, 1× PCR buffer II, 1.0 mM MgCl2, 0.5 units of DNA polymerase (Fisher Biotec, Australia), 0.1 μl of [3H]dCTP (S.A. 57.4 Ci/mmol; Amersham Biosciences, Little Chalfont, UK) at 56 °C for 1 h, then placed on ice. Triplicate 10 μl aliquots from each reaction were applied on Whatman DE-81 ion-exchange filters (Whatman, Middlesex, UK) and washed ×4 with 0.5 M Naphosphate buffer pH 7.0 at room temperature. The filters were dried and counted. [3H]dCTP incorporation into DNA was expressed as mean dpm/min/μg DNA after subtraction of the dpm incorporated into undigested samples (background). The percent of global genome DNA methylation was calculated as [1 − (dpm incorporated following HpaII/dpm incorporated following MspI)] × 100%. 2.4. Statistical analysis Differences between means analyzed by the Student's t-test or ANOVA, Pearson's correlation coefficients and their statistical significance and power analysis were obtained using the software Statistica (StatSoft Inc., Tulsa, USA).

2.5. Calculation of chlorpromazine equivalents of antipsychotic treatment Antipsychotic dose was converted into chlorpromazine equivalents using conversion tables in Bazire (Bazire, 2003) and according to Woods (Woods, 2003). 3. Results Mean plasma homocysteine levels were about 1.5 fold higher (15.8 μM ± 10.5 vs. 10.6 ± 2.7, p = 0.016) in schizophrenia patients (n = 28) vs. gender- and age-matched control subjects (n = 26) (Fig. 1), contributed by a trend of 1.8 fold higher levels in males (20.7 μM ± 15.1 vs.11.5 ± 2.3, n = 10 in each diagnostic group, p = 0.061) and 1.3 fold higher levels in females (13.0 μM ± 5.6 vs. 9.9 ± 2.5, n = 18 patients vs. 16 controls, p = 0.054). No differences were found in global leukocyte DNA methylation between schizophrenia patients and controls (74.0% ± 14.8 vs. 69.4 ± 22.0, p = 0.31), or between males and females (76.3% ± 18.0 vs. 69.7 ± 17.9, p = 0.19) (Fig. 2). Power analysis considering our S.D.s shows that our sample size would have been sufficient to detect a 20% difference in global DNA methylation at a power of 80%. To reach statistical significance of the 7% difference we have found, a sample size of over 220 subjects in each group would have been needed. However, since previous studies suggested that gender (Kawakami et al., 2006; Shimabukuro et al., 2006) and smoking (Lin et al., 2007; Smith et al., 2007) affect global DNA methylation we performed a three-way ANOVA of global DNA methylation with diagnosis, smoking and gender as independent factors. A significant interaction between diagnosis and smoking on DNA methylation was obtained (F = 6.8,

Fig. 1. Elevated plasma homocysteine levels in schizophrenia. Horizontal lines denote the means; vertical lines denote S.D. Homocysteine was measured by HPLC. ⁎Student's t-test, p = 0.016.

A. Bromberg et al. / Schizophrenia Research 101 (2008) 50–57

53

Fig. 2. Leukocyte global DNA methylation in schizophrenia determined by the [3H]cytosine-extension assay. No significant difference was obtained, Student's t-test, p = 0.31.

df = 1,47, p = 0.032). LSD post-hoc comparisons revealed that within non-smoking subjects leukocyte global DNA methylation in female schizophrenia patients (n = 9) was higher than in control females (n = 9) (76.2% ± 5.7 vs. 55.8% ± 6.0, p = 0.018). Leukocyte global DNA methylation in control non-smoking females (n = 9) was lower than in control non-smoking males (n = 8) (55.8% ± 6.0 vs. 72.9 ± 5.7, p = 0.045) (Fig. 3). Since gender and smoking have also been reported as determinants of plasma homocysteine levels (Nygard et al., 1998; Refsum et al., 2006), we also performed three-way ANOVA of plasma homocysteine levels with diagnosis, smoking and gender as independent factors. A significant main effect of diagnosis was found (F = 5.41, df = 1,47, p = 0.024), but no interactions. Homocysteine levels in non-smoking schizophrenia

females were over 50% higher than in non-smoking control females (14.4 μM ±6.1 vs. 9.1 ± 2.1, p = 0.03). Pearson's correlation parameters between DNA methylation and homocysteine levels in non-smoking females were r = 0.41, p = 0.085. Homocysteine levels and leukocyte global DNA methylation did not differ between smoking and nonsmoking subjects (homocysteine levels: 14.4 μM ± 10.6 vs. 12.3 ± 5.2, p = 0.34; global DNA methylation: 73.8 % ± 13.6 vs. 71.1 ± 21.3, p = 0.59), nor was there a difference in males (homocysteine levels: 19.2 μM ± 15.2 vs. 12.9 ± 7.5, p = 0.21; global DNA methylation: 72.9 % ± 14.7 vs. 75.0 ±20.8, p = 0.71) or females (homocysteine levels: 11.2 μM ± 4.2 vs. 11.9 ± 5.1, p = 0.68; global DNA methylation: 71.1 % ±12.6 vs. 68.6±21.8, p = 0.70).

Fig. 3. Leukocyte global DNA methylation in non-smoking subjects. Horizontal lines denote the means; vertical lines denote S.D. Post-hoc: ⁎p = 0.045, ⁎⁎p = 0.018.

54

A. Bromberg et al. / Schizophrenia Research 101 (2008) 50–57

Table 1 Correlation of leukocyte global DNA methylation and plasma homocysteine levels with age (A) and with chlorpromazine equivalents of antipsychotic treatment (B)

A Global DNA methylation (%) Plasma homocysteine (μM)

Whole cohort (n = 54)

Males (n = 20)

Females (n = 34)

Schizophrenia patients (n = 28)

Control subjects (n = 26)

r = 0.15, p = 0.29 r = 0.03, p = 0.84

r = 0.03, p = 0.88 r = 0.08, p = 0.73

r = 0.2, p = 0.27 r = 0.4, p = 0.29

r = 0.61, p = 0.76 r = 0.07, p = 0.72

r = 0.38, p = 0.048⁎ r = 0.10, p = 0.62

B

Global DNA methylation (%) Plasma homocysteine (μM)

All schizophrenia patients (n = 28)

Schizophrenia males (n = 10)

Schizophrenia females (n = 17)

r = 0.26, p = 0.24 r = 0.24, p = 0.29

r = 0.54, p = 0.10 r = 0.16, p = 0.65

r = 0.15, p = 0.69 r = 0.12, p = 0.75

⁎ Indicates statistical significance.

Correlation of leukocyte global DNA methylation with age is summarized in Table 1A. Leukocyte global DNA methylation correlated with age only in the control group (r = 0.38, p = 0.048), contributed by the control females: r = 0.50, n = 16, p = 0.05; males: r = 0.7, n = 10, p = 0.82. This result is not significant after correction for multiple testing. Homocysteine levels and leukocyte global DNA methylation did not correlate with chlorpromazine equivalents of antipsychotic treatment in the patient group (homocysteine levels: r = 0.24, n = 26, p = 0.29; leukocyte global DNA methylation: r = 0.26, n = 26, p = 0.24) (Table 1B), nor was there correlation in male (homocysteine levels: r = 0.16, n = 10, p = 0.65; global DNA methylation: r = 0.54, n = 10, p = 0.10), or female patients (homocysteine levels: r = 0.12, n = 18, p = 0.75; global DNA methylation: r = 0.15, n = 18, p = 0.69). Neither homocysteine levels nor leukocyte global DNA methylation correlated with the duration of illness either when the whole patient group was analyzed (homocysteine levels: r = 0.07, n = 26, p = 0.75; leukocyte global DNA methylation: r = 0.10, n = 26, p = 0.61), nor when analyzed by gender (males: homocysteine levels, r = 0.39, n = 10, p = 0.30; global DNA methylation, r = 0.32, n = 10, p = 0.40; females: homocysteine levels, r = 0.38, n = 18, p = 0.13; global DNA methylation, r = 0.03, n = 18, p = 0.90). 4. Discussion The results of the current study confirm previous findings of elevated plasma homocysteine levels in schizophrenia patients (Levine et al., 2002; Muntjewerff et al., 2006). In the previous studies elevated homocysteine levels were found mainly in male schizophrenia patients (Regland et al., 1997; Levine et al., 2002; Applebaum et al., 2004). Similarly, in the cohort of the

present study a stronger trend of elevation of plasma homocysteine was found in male schizophrenia patients. Since homocysteine is a crucial metabolite of one carbon metabolism, and since several studies reported an association between elevated plasma homocysteine and leukocyte global DNA hypomethylation in healthy young women (James et al., 2002), in patients with vascular disease (Castro et al., 2003) and in male schizophrenia patients (Shimabukuro et al., 2007), we hypothesized altered leukocyte DNA methylation in schizophrenia patients. The present study did not find a difference in leukocyte global DNA methylation between schizophrenia patients and healthy controls despite the elevated plasma homocysteine levels. Other studies in patients with chronic alcoholism (Bonsch et al., 2004), in healthy males (Fux et al., 2005) and in rats (Choi et al., 2003; Davis and Uthus, 2003) also failed to find an association between homocysteine levels and global DNA methylation. A lack of difference in global DNA methylation between patients and controls does not rule out possible differences in methylation at specific functionally relevant sites potentially involved in schizophrenia. Smoking and gender may be confounding factors when measuring global DNA methylation. The reports in the literature dealing with this issue are controversial. Some studies found that smoking is associated with global DNA hypomethylation (Smith et al., 2007; Ting Hsiung et al., 2007) apparently mediated by smoking lowering blood folate, associated with an increase in homocysteine (Refsum et al., 2006). Other studies found hypermethylation (Piyathilake et al., 2001; Lin et al., 2007). The relevance of these studies for the present work may be limited, since they dealt with cancer cells or cancer patients. Gender effect on global DNA methylation has also been found. Namely, using alternative methodology, Shimabukuro et al. (Shimabukuro et al., 2007) found

A. Bromberg et al. / Schizophrenia Research 101 (2008) 50–57

higher leukocyte global DNA methylation in healthy males vs. females similarly to the finding of the present study. Differently from the present report this group observed a tendency toward lower DNA methylation in schizophrenia patients than in controls in males but not in females. Kawakami et al. (2006) found higher DNA methylation of the tumor suppressor genes ERalpha and MYOD in colonic mucosa cells of healthy female subjects. In the present study concomitant analysis of the effect of diagnosis, smoking and gender on leukocyte global DNA methylation revealed a significant interaction between diagnosis and smoking. Namely, smoking decreased global DNA methylation in the patients and increased it in the controls. Hence, significantly higher leukocyte global DNA methylation between schizophrenia females vs. control females was obtained only in nonsmoking subjects, suggesting that smoking is indeed a confounding factor in DNA methylation. Neither plasma homocysteine levels nor leukocyte global DNA methylation differed between smoking and non-smoking subjects suggesting that smoking does not necessarily affect leukocyte global DNA methylation via lowering folate and elevating homocysteine levels. A direct effect of nicotine on DNA methylation is a possibility (Soma et al., 2006). An additional confounding factor may be antipsychotic medication. Shimabukuro et al. found that haloperidol treatment decreased leukocyte global DNA methylation in male rats, but unexpectedly, increased it in females. In brain haloperidol treatment resulted in a decrease in global DNA methylation in female, but not male rats (Shimabukuro et al., 2006). These effects may reflect the involvement of estrogen in DNA methylation (Yokomori et al., 1995; Friso et al., 2007). The field of epigenetics and the methodologies developed to assess DNA methylation are relatively new and limited (Mill and Petronis, 2007). The methodology used in the present study provides quantitative information of the whole genome. Since about 70% of global DNA methylation exists in CpG islands of gene promoters, an indication of the degree of DNA methylation in these sites is obtained. The assay we used, based on restriction by methylation-sensitive/insensitive enzymes, has some technical limitations i.e. restriction enzymes do not cleave all potential methylation sites in the genome and restriction sites may be altered by mutations or polymorphisms. Moreover, the extent of global DNA methylation differs between tissues (Caudill et al., 2001; Davis and Uthus, 2003; Lund et al., 2004), and drugs affect global DNA methylation in a tissuespecific manner (Shimabukuro et al., 2006). Therefore, although leukocytes may be a useful cell model to eva-

55

luate epigenetic changes such as global DNA methylation in brain (Mill and Petronis, 2007), future studies should compare global DNA methylation in peripheral tissue vs. brain in laboratory animals. Role of funding source None. Contributors A Bromberg: extracted DNA from leukocytes, established and carried out the assays of DNA methylation and wrote the manuscript's first draft J Levine: screened patients and collected blood samples B Nemetz: screened patients and collected blood samples RH Belmaker: served as a clinical and conceptual advisor G Agam: supervised the lab work and the preparation of the manuscript and edited the final version All authors contributed to and have approved the final manuscript. Conflict of interest All authors declare that they have no conflicts of interest. Acknowledgment We are grateful to Dr. Yuly Bersudsky for his scholar statistical advice.

References Applebaum, J., Shimon, H., Sela, B.A., Belmaker, R.H., Levine, J., 2004. Homocysteine levels in newly admitted schizophrenic patients. J. Psychiatr. Res. 38, 413–416. Araki, A., Sako, Y., 1987. Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection. J. Chromatogr. 422, 43–52. Bazire, 2003. Psychotropic Drug Directory. Quay Books, Salisbury. Bird, A.P., Wolffe, A.P., 1999. Methylation-induced repression—belts, braces, and chromatin. Cell 99, 451–454. Bleich, S., Frieling, H., Hillemacher, T., 2007. Elevated prenatal homocysteine levels and the risk of schizophrenia. Arch. Gen. Psychiatry 64, 980–981. Bonsch, D., Lenz, B., Reulbach, U., Kornhuber, J., Bleich, S., 2004. Homocysteine associated genomic DNA hypermethylation in patients with chronic alcoholism. J. Neural Transm. 111, 1611–1616. Brown, A.S., Bottiglieri, T., Schaefer, C.A., Quesenberry Jr., C.P., Liu, L., Bresnahan, M., Susser, E.S., 2007. Elevated prenatal homocysteine levels as a risk factor for schizophrenia. Arch. Gen. Psychiatry 64, 31–39. Cannon, M., Jones, P.B., Murray, R.M., 2002. Obstetric complications and schizophrenia: historical and meta-analytic review. Am. J. Psychiatry 159, 1080–1092. Castro, R., Rivera, I., Struys, E.A., Jansen, E.E., Ravasco, P., Camilo, M.E., Blom, H.J., Jakobs, C., Tavares de Almeida, I., 2003. Increased homocysteine and S-adenosylhomocysteine concentrations and DNA hypomethylation in vascular disease. Clin. Chem. 49, 1292–1296. Caudill, M.A., Wang, J.C., Melnyk, S., Pogribny, I.P., Jernigan, S., Collins, M.D., Santos-Guzman, J., Swendseid, M.E., Cogger, E.A., James, S.J., 2001. Intracellular S-adenosylhomocysteine concentrations predict global DNA hypomethylation in tissues of methyldeficient cystathionine beta-synthase heterozygous mice. J. Nutr. 131, 2811–2818.

56

A. Bromberg et al. / Schizophrenia Research 101 (2008) 50–57

Chen, T., Ueda, Y., Dodge, J.E., Wang, Z., Li, E., 2003. Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol. Cell. Biol. 23, 5594–5605. Choi, S.W., Friso, S., Dolnikowski, G.G., Bagley, P.J., Edmondson, A.N., Smith, D.E., Mason, J.B., 2003. Biochemical and molecular aberrations in the rat colon due to folate depletion are age-specific. J. Nutr. 133, 1206–1212. Cohen, S.M., Nichols, A., Wyatt, R., Pollin, W., 1974. The administration of methionine to chronic schizophrenic patients: a review of ten studies. Biol. Psychiatry. 8, 209–225. Davis, C.D., Uthus, E.O., 2003. Dietary folate and selenium affect dimethylhydrazine-induced aberrant crypt formation, global DNA methylation and one-carbon metabolism in rats. J. Nutr. 133, 2907–2914. Friso, S., Lamon-Fava, S., Jang, H., Schaefer, E.J., Corrocher, R., Choi, S.W., 2007. Oestrogen replacement therapy reduces total plasma homocysteine and enhances genomic DNA methylation in postmenopausal women. Br. J. Nutr. 97, 617–621. Frosst, P., Blom, H.J., Milos, R., Goyette, P., Sheppard, C.A., Matthews, R.G., Boers, G.J., den Heijer, M., Kluijtmans, L.A., van den Heuvel, L.P., et al., 1995. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat. Genet. 10, 111–113. Fux, R., Kloor, D., Hermes, M., Rock, T., Proksch, B., Grenz, A., Delabar, U., Bucheler, R., Igel, S., Morike, K., Gleiter, C.H., Osswald, H., 2005. Effect of acute hyperhomocysteinemia on methylation potential of erythrocytes and on DNA methylation of lymphocytes in healthy male volunteers. Am. J. Physiol., Renal. Physiol. 289, F786–F792. Glantz, L.A., Gilmore, J.H., Lieberman, J.A., Jarskog, L.F., 2006. Apoptotic mechanisms and the synaptic pathology of schizophrenia. Schizophr. Res. 81, 47–63. Grayson, D.R., Jia, X., Chen, Y., Sharma, R.P., Mitchell, C.P., Guidotti, A., Costa, E., 2005. Reelin promoter hypermethylation in schizophrenia. Proc. Natl. Acad. Sci. U. S. A. 102, 9341–9346. Graziano, F., Kawakami, K., Ruzzo, A., Watanabe, G., Santini, D., Pizzagalli, F., Bisonni, R., Mari, D., Floriani, I., Catalano, V., Silva, R., Tonini, G., Torri, V., Giustini, L., Magnani, M., 2006. Methylenetetrahydrofolate reductase 677C/T gene polymorphism, gastric cancer susceptibility and genomic DNA hypomethylation in an at-risk Italian population. Int. J. Cancer 118, 628–632. Hoffman, D.R., Marion, D.W., Cornatzer, W.E., Duerre, J.A., 1980. S-Adenosylmethionine and S-adenosylhomocystein metabolism in isolated rat liver. Effects of L-methionine, L-homocystein, and adenosine. J. Biol. Chem. 255, 10822–10827. James, S.J., Melnyk, S., Pogribna, M., Pogribny, I.P., Caudill, M.A., 2002. Elevation in S-adenosylhomocysteine and DNA hypomethylation: potential epigenetic mechanism for homocysteinerelated pathology. J. Nutr. 132, 2361S–2366S. Jones, P.A., Takai, D., 2001. The role of DNA methylation in mammalian epigenetics. Science 293, 1068–1070. Joober, R., Benkelfat, C., Lal, S., Bloom, D., Labelle, A., Lalonde, P., Turecki, G., Rozen, R., Rouleau, G.A., 2000. Association between the methylenetetrahydrofolate reductase 677C−NT missense mutation and schizophrenia. Mol. Psychiatry 5, 323–326. Kawakami, K., Ruszkiewicz, A., Bennett, G., Moore, J., Grieu, F., Watanabe, G., Iacopetta, B., 2006. DNA hypermethylation in the normal colonic mucosa of patients with colorectal cancer. Br. J. Cancer 94, 593–598. Kruman II, Culmsee, C., Chan, S.L., Kruman, Y., Guo, Z., Penix, L., Mattson, M.P., 2000. Homocysteine elicits a DNA damage response

in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J. Neurosci. 20, 6920–6926. Lau, C.G., Zukin, R.S., 2007. NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders. Nat. Rev. Neurosci. 8, 413–426. Lentz, S.R., 2005. Mechanisms of homocysteine-induced atherothrombosis. J. Thromb. Haemost. 3, 1646–1654. Levine, J., Stahl, Z., Sela, B.A., Gavendo, S., Ruderman, V., Belmaker, R.H., 2002. Elevated homocysteine levels in young male patients with schizophrenia. Am. J. Psychiatry 159, 1790–1792. Lewis, S.J., Zammit, S., Gunnell, D., Smith, G.D., 2005. A meta-analysis of the MTHFR C677T polymorphism and schizophrenia risk. Am. J. Med. Genet., B, Neuropsychiatr. Genet. 135, 2–4. Lin, R.K., Hsu, H.S., Chang, J.W., Chen, C.Y., Chen, J.T., Wang, Y.C., 2007. Alteration of DNA methyltransferases contributes to 5'CpG methylation and poor prognosis in lung cancer. Lung Cancer 55, 205–213. Lipton, S.A., Kim, W.K., Choi, Y.B., Kumar, S., D'Emilia, D.M., Rayudu, P.V., Arnelle, D.R., Stamler, J.S., 1997. Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor. Proc. Natl. Acad. Sci. U. S. A. 94, 5923–5928. Lund, G., Andersson, L., Lauria, M., Lindholm, M., Fraga, M.F., VillarGarea, A., Ballestar, E., Esteller, M., Zaina, S., 2004. DNA methylation polymorphisms precede any histological sign of atherosclerosis in mice lacking apolipoprotein E. J. Biol. Chem. 279, 29147–29154. Matsubayashi, H., Skinner, H.G., Iacobuzio-Donahue, C., Abe, T., Sato, N., Riall, T.S., Yeo, C.J., Kern, S.E., Goggins, M., 2005. Pancreaticobiliary cancers with deficient methylenetetrahydrofolate reductase genotypes. Clin. Gastroenterol. Hepatol. 3, 752–760. Mill, J., Petronis, A., 2007. Molecular studies of major depressive disorder: the epigenetic perspective. Mol. Psychiatry 12, 799–814. Muntjewerff, J.W., Kahn, R.S., Blom, H.J., den Heijer, M., 2006. Homocysteine, methylenetetrahydrofolate reductase and risk of schizophrenia: a meta-analysis. Mol. Psychiatry 11, 143–149. Murray, R.M., 1994. Neurodevelopmental schizophrenia: the rediscovery of dementia praecox. Br. J. Psychiatry. 6–12 Suppl. Nevo, G.A., Meged, S., Sela, B.A., Hanoch-Levi, A., Hershko, R., Weizman, A., 2006. Homocysteine levels in adolescent schizophrenia patients. Eur. Neuropsychopharmacol. 16, 588–591. Nygard, O., Refsum, H., Ueland, P.M., Vollset, S.E., 1998. Major lifestyle determinants of plasma total homocysteine distribution: the Hordaland homocysteine study. Am. J. Clin. Nutr. 67, 263–270. Piyathilake, C.J., Frost, A.R., Bell, W.C., Oelschlager, D., Weiss, H., Johanning, G.L., Niveleau, A., Heimburger, D.C., Grizzle, W.E., 2001. Altered global methylation of DNA: an epigenetic difference in susceptibility for lung cancer is associated with its progression. Hum. Pathol. 32, 856–862. Pogribny, I., Yi, P., James, S.J., 1999. A sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands. Biochem. Biophys. Res. Commun. 262, 624–628. Rampersaud, G.C., Kauwell, G.P., Hutson, A.D., Cerda, J.J., Bailey, L.B., 2000. Genomic DNA methylation decreases in response to moderate folate depletion in elderly women. Am. J. Clin. Nutr. 72, 998–1003. Refsum, H., Nurk, E., Smith, A.D., Ueland, P.M., Gjesdal, C.G., Bjelland, I., Tverdal, A., Tell, G.S., Nygard, O., Vollset, S.E., 2006. The Hordaland homocysteine study: a community-based study of homocysteine, its determinants, and associations with disease. J. Nutr. 136, 1731S–1740S. Regland, B., Johansson, B.V., Grenfeldt, B., Hjelmgren, L.T., Medhus, M., 1995. Homocysteinemia is a common feature of schizophrenia. J. Neural. Transm., Gen. Sect. 100, 165–169.

A. Bromberg et al. / Schizophrenia Research 101 (2008) 50–57 Regland, B., Germgard, T., Gottfries, C.G., Grenfeldt, B., KochSchmidt, A.C., 1997. Homozygous thermolabile methylenetetrahydrofolate reductase in schizophrenia-like psychosis. J. Neural. Transm. 104, 931–941. Robertson, K.D., Wolffe, A.P., 2000. DNA methylation in health and disease. Nat. Rev. Genet. 1, 11–19. Sachdev, P.S., Valenzuela, M., Wang, X.L., Looi, J.C., Brodaty, H., 2002. Relationship between plasma homocysteine levels and brain atrophy in healthy elderly individuals. Neurology 58, 1539–1541. Sazci, A., Ergul, E., Guzelhan, Y., Kaya, G., Kara, I., 2003. Methylenetetrahydrofolate reductase gene polymorphisms in patients with schizophrenia. Brain Res. Mol. Brain Res. 117, 104–107. Shimabukuro, M., Jinno, Y., Fuke, C., Okazaki, Y., 2006. Haloperidol treatment induces tissue- and sex-specific changes in DNA methylation: a control study using rats. Behav. Brain Funct. 2, 37. Shimabukuro, M., Sasaki, T., Imamura, A., Tsujita, T., Fuke, C., Umekage, T., Tochigi, M., Hiramatsu, K., Miyazaki, T., Oda, T., Sugimoto, J., Jinno, Y., Okazaki, Y., 2007. Global hypomethylation of peripheral leukocyte DNA in male patients with schizophrenia: a potential link between epigenetics and schizophrenia. J. Psychiatr. Res. 41, 1042–1046. Smith, I.M., Mydlarz, W.K., Mithani, S.K., Califano, J.A., 2007. DNA global hypomethylation in squamous cell head and neck cancer

57

associated with smoking, alcohol consumption and stage. Int. J. Cancer 121, 1724–1728. Soma, T., Kaganoi, J., Kawabe, A., Kondo, K., Imamura, M., Shimada, Y., 2006. Nicotine induces the fragile histidine triad methylation in human esophageal squamous epithelial cells. Int. J. Cancer 119, 1023–1027. Ting Hsiung, D., Marsit, C.J., Houseman, E.A., Eddy, K., Furniss, C.S., McClean, M.D., Kelsey, K.T., 2007. Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma. Cancer Epidemiol. Biomark. Prev. 16, 108–114. Veldic, M., Guidotti, A., Maloku, E., Davis, J.M., Costa, E., 2005. In psychosis, cortical interneurons overexpress DNA-methyltransferase 1. Proc. Natl. Acad. Sci. U. S. A. 102, 2152–2157. Woods, 2003. Chlorpromazine equivalent doses for the newer atypical antipsychotics. J. Clin. Psychiatry 64, 663–667. Yi, P., Melnyk, S., Pogribna, M., Pogribny, I.P., Hine, R.J., James, S.J., 2000. Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation. J. Biol. Chem. 275, 29318–29323. Yokomori, N., Moore, R., Negishi, M., 1995. Sexually dimorphic DNA demethylation in the promoter of the Slp (sex-limited protein) gene in mouse liver. Proc. Natl. Acad. Sci. U. S. A. 92, 1302–1306.