Molecular Psychiatry (2006) 11, 143–149 & 2006 Nature Publishing Group All rights reserved 1359-4184/06 $30.00 www.nature.com/mp ORIGINAL ARTICLE Homocysteine, methylenetetrahydrofolate reductase and risk of schizophrenia: a meta-analysis JW Muntjewerff1, RS Kahn2, HJ Blom3 and M den Heijer4 1GGz Nijmegen, Mental Health Institute Nijmegen, Nijmegen, The Netherlands; 2Department of Psychiatry, University Medical Center Utrecht, Utrecht, The Netherlands; 3Laboratory of Paediatrics and Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands and 4Department of Endocrinology and Epidemiology and Biostatistics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Elevated plasma homocysteine concentration has been suggested as a risk factor for schizophrenia, but the results of epidemiological studies have been inconsistent. The most extensively studied genetic variant in the homocysteine metabolism is the 677C4T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene, resulting in reduced enzyme activity and, subsequently, in elevated homocysteine. A meta-analysis of eight retrospective studies (812 cases and 2113 control subjects) was carried out to examine the association between homocysteine and schizophrenia. In addition, a meta-analysis of 10 studies (2265 cases and 2721 control subjects) on the homozygous (TT) genotype of the MTHFR 677C4T polymorphism was carried out to assess if this association is causal. A 5 lmol/l higher homocysteine level was associated with a 70% (95% confidence interval, CI: 27– 129) higher risk of schizophrenia. The TT genotype was associated with a 36% (95% CI: 7–72) higher risk of schizophrenia compared to the CC genotype. The performed meta-analyses showed no evidence of publication bias or excessive influence attributable to any given study. In conclusion, our study provides evidence for an association of homocysteine with schizophrenia. The elevated risk of schizophrenia associated with the homozygous genotype of the MTHFR 677C4T polymorphism provides support for causality between a disturbed homocysteine metabolism and risk of schizophrenia. Molecular Psychiatry (2006) 11, 143–149. doi:10.1038/sj.mp.4001746; published online 20 September 2005 Keywords: ; genetics; homocysteine; methylenetetrahydrofolate reductase; psychiatric disorder; neurodevelopment; schizophrenia

Introduction of schizophrenia. Evolving from the original trans- hypothesis postulating that an abnor- It is commonly accepted that both genetic and mally (toxic) methylated metabolite might be the environmental factors contribute to the aetiology cause of schizophrenia,6 it was suggested subse- and clinical phenotype of schizophrenia. Despite quently that disturbances in the single-carbon meta- numerous family, twin, and adoption studies in bolism itself might be causative.7 Methylation which evidence for hereditary and environmental processes are vital for normal cell functioning contributions to schizophrenia is presented,1–4 no because of their key role in protein, lipid and DNA underlying inherited mechanism has yet been identi- metabolism, gene expression, synthesis of neurotrans- fied. Although several putative susceptibility genes mitters and detoxification processes.8 Initial clinical for schizophrenia have been identified,5 the patho- evidence for an impaired methyl-carbon pathway in genic mechanisms remain to be resolved. Evidence of schizophrenic patients stems from many an association between schizophrenia and a specific administration studies ranging from the 1960s until aberrant metabolism could reveal new candidate the early 1990s. These studies showed that treatment genes and might provide more insight in the complex with high-daily doses of L-methionine resulted in aetiology of schizophrenia. exacerbation of schizophrenic symptoms.9,10 In addi- Dysfunctional single-carbon transfer reactions in- tion, as compared to controls the rate of whole body volving the methionine–homocysteine metabolism methylation was found to be decreased in patients have been proposed to be relevant in the aetiology with schizophrenia.11,12 It was suggested that these findings might reflect an enzymatic defect or a failure Correspondence: Dr JW Muntjewerff, GGz Nijmegen, Mental of a control mechanism of the single-carbon cycle Health Institute Nijmegen, PO Box 7049, 6503 GM Nijmegen, in schizophrenic patients.13 The finding of a defect The Netherlands; E-mail: [email protected] in methylation due to severe dysfunction of 5,10- Received 17 December 2004; revised 6 July 2005; accepted 9 methylenetetrahydrofolate reductase (MTHFR) result- August 2005; published online 20 September 2005 ing in in a subject with Homocysteine metabolism and schizophrenia: a meta-analysis JW Muntjewerff et al 144 schizophrenia-like symptoms14 was therefore of great phrenia for cases, (b) use of a case–control design and interest. Homocysteine is a sulphur containing ami- (c) publication in an English-language, peer-reviewed, no-acid derived from the demethylation of the indexed scientific journal. Meeting abstracts were not essential amino-acid methionine, which is the only allowed. We also included our recent published dietary precursor of homocysteine. The metabolism of data.22 homocysteine depends on several B-vitamins includ- ing folate, cobalamin, and riboflavin. Data extraction and synthesis MTHFR is the essential enzyme in the folate- For the meta-analysis of homocysteine and schizo- mediated single-carbon transfer reactions. A common phrenia, information was extracted from each study polymorphism exists in the catalytic domain of the on: the odds ratio (OR) and 95% confidence interval MTHFR enzyme. The gene is localized on chromo- (CI) as a risk estimate for the association between some 1p36.3,15 in which a C4T substitution at elevated homocysteine and schizophrenia and the position 677 results in a substitution of to number of cases and controls. If the OR and 95% CI .16 The reported prevalence of the homozygous were not provided, they were calculated from the 677C4T (TT) genotype is between 0 and 32% of the number of cases and controls above and below a population world-wide,17 ranging from 5 to 15% in particular homocysteine concentration as a cutoff Caucasian populations.18 The single amino-acid sub- point from the control distribution in each study stitution results in impaired flavin adenine dinucleo- using Woolf’s method.23 The homocysteine studies tide (FAD) binding, leading to loss of folate resulting, included in our meta-analysis reported different in its turn, in reduced activity of MTHFR.19 Subjects definitions of hyperhomocysteinemia by using differ- with the TT genotype have about 25% higher ent cutoff levels. Most studies provided the 90th homocysteine levels than those with the normal percentile of homocysteine levels in the control homozygous (CC) genotype,18 whereby the impact of group, while other studies reported the 95th or 75th the polymorphism varies according to folate or percentile. For pooling of risk estimates, it is essential riboflavin status.20,21 that these estimates are based on a single unit of Elucidation of an association between a genetic comparison. Previous studies on homocysteine and variant associated with elevated homocysteine levels homocysteine-related diseases reported linear dose– and schizophrenia might be informative about the effects relations.24–26 In line with these findings, we hypothesis that higher levels of homocysteine play a calculated the OR for a 5 mmol/l increase in homo- causal role in the aetiology of schizophrenia. Indivi- level, a standard reference increment. To dual case–control studies concerning homocysteine estimate the log OR for a 5 mmol/l increase in or the 677C4T polymorphism included too few cases homocysteine, information about the normal distribu- and controls to produce reliable evidence for an tion was used to calculate the expected mean level of association with schizophrenia. homocysteine in the groups being compared. For The aim of this study was to examine the associa- example, the mean value in the top fifth of a normal tion between homocysteine and schizophrenia by distribution is 1.4 standard deviation (s.d.) above the conducting a meta-analysis of case–control studies on mean, and the mean in the bottom fifth is 1.4 s.d. this topic. In order to assess whether such association below the mean. Hence, the OR comparing the top to is causal, we have also performed a meta-analysis of the bottom fifth is also the OR for a 2.8 s.d. difference all case–control observational studies with available in homocysteine levels. Wherever possible, the s.d. in data on the MTHFR 677C4T polymorphism and controls was used to estimate the difference in mmol/l schizophrenia. between the top and bottom fifth of a study, but if this was not reported, then a weighted average was used of the s.d. from the studies that did report Method it. Assuming the association was log–linear, it was Study identification then possible to calculate the OR for a unit change Eligible studies were identified by searching the in homocysteine and hence for a 5 mmol/l increase in electronic NLM MEDLINE database for relevant homocysteine. reports published up to and including December For the meta-analysis of the MTHFR 677C4T 2004 using the search terms (‘homocysteine’ or polymorphism and schizophrenia, data were col- ‘hyperhomocysteinemia’ or ‘MTHFR’) and (‘schizo- lected on the frequency of CC, CT and TT genotypes phrenia’). Additionally, the reference lists of original in cases and controls or calculated from the articles on this topic were scanned to identify articles allele frequencies. Subjects with the TT genotype missed by the computerized search. have approximately 2.7 mmol/l (25%) higher homo- cysteine levels compared to subjects with the CC Study selection genotype, while the mean difference of homocysteine Studies examining total serum or plasma homocys- concentration between the CT and CC genotype is teine levels or the 677C4T polymorphism in the only about 0.29 mmol/l.25Therefore, the TT genotype MTHFR gene were included in the meta-analysis. The was assigned as the risk genotype, because of the following additional criteria were used to select relatively strong contrast in homocysteine level studies for data extraction: (a) a diagnosis of schizo- between the TT and CC genotype. The study-specific

Molecular Psychiatry Homocysteine metabolism and schizophrenia: a meta-analysis JW Muntjewerff et al 145 ORs and 95% CIs for TT compared with CC genotype studies and for all studies when taken together. A were estimated using Woolf’s method.23 5 mmol/l increase in measured homocysteine was By combining the pooled risk estimate of the associated with an overall 70% (95% CI: 27–129) association between the TT genotype and schizo- increased risk of schizophrenia. Sequential removing phrenia (OR) with the published estimate of a mean and replacement of individual studies from the difference in plasma homocysteine between TT and calculation of the pooled OR produced risk estimates CC genotype (Dhcy), we derived an equivalent risk ranging from 1.58 to 1.94 with 95% CIs that always estimate for a 5 mmol/l increase in homocysteine encompassed 1.0. This finding indicates that the concentration of OR5/Dhcy (raising OR to the power of significance of the pooled OR was not excessively 5/Dhcy). Subsequently, we compared the derived and influenced by any single study. There was significant measured risk estimates. heterogeneity between the results of the studies Random effects model meta-analyses were per- included (w2 ¼ 17.949; df ¼ 7; P ¼ 0.012), suggesting formed according to the methods of DerSimonian the presence of some moderating variable. Several and Laird,27 and 95% CIs were constructed by using characteristics of the included homocysteine studies Woolf’s method.23 The significance of the pooled ORs are presented in Table 1, showing heterogeneity of was determined by the z-test. Heterogeneity (greater exclusion criteria and of age and sex distributions. In variation among study results than would be expected Figure 1 the distribution of the ORs of the individual through change) was assessed using standard w2 tests. homocysteine studies are ordered by the inverse of The influence of individual studies on the pooled the standard error of the risk estimate. It indicates that ORs was determined by sequentially removing each much of the evidence for an association of homo- study and recalculating the pooled OR and 95% CI. cysteine and schizophrenia comes from two larger The individual studies were ordered by the inverse of studies which deviate significantly from an OR of the standard error (s.e.) of the risk estimate to evaluate 1.0.32,34 The shape of the funnel also suggests that a publication bias.28 A symmetrical inverted funnel few smaller studies finding an inverse association implies absence of significant selection or publication may not have been published. However, the effect of bias. In addition, publication bias was tested by these studies on the overall risk estimate is likely to Egger’s test.28 If necessary, data were completed by be small. In addition, Egger’s regression asymmetry personal communication with the original authors. test showed no evidence of publication bias (a ¼À0.9; The type I error rate was set at 0.05. All statistical t ¼À0.73; P ¼ 0.49). analyses were conducted by using Stata 8.0. MTHFR and schizophrenia A total of nine published articles22,31,33,36–41 reported Results on the relationship between schizophrenia and the Homocysteine and schizophrenia MTHFR 677C4T polymorphism. One article pro- Data for the meta-analysis were obtained from eight vided data on two separate studies.40 Thus, data were published case–control studies22,29–35 with a total obtained from 10 studies involving 2265 cases and number of 812 cases and 2113 control subjects. Figure 2721 control subjects. All cases were diagnosed 1 shows the ORs and 95% CIs of schizophrenia according to standard diagnostic systems (DSM-III, associated with a 5 mmol/l increase in measured DSM-V or ICD-9). All studies used a standardized plasma total homocysteine, separately for individual method to determine the MTHFR 677C4T genotypes.

Study Cases/controls

Susser et al 30 17/24 Regland et al 29 20/20 Muntjewerff et al 33 35/104 Muntjewerff et al 22 62/432 Goff et al 35 91/248 Virgos et al 31 210/218 Applebaum et al 34 184/305 Levine et al 32 193/762

Combined 812/2113 1.70 (1.27-2.29) 0.5 1 2 Odds ratio Figure 1 Odds ratios (ORs) and 95% confidence intervals (CIs) of schizophrenia for a 5 mmol/l increase in plasma total homocysteine concentration for each study separately and combined (Studies are ordered by the inverse of the s.e. of each estimate. The size of the data markers is inversely proportional to the s.e. The horizontal lines represent the 95% CIs. The combined OR and 95% CI is indicated by the diamond).

Molecular Psychiatry Homocysteine metabolism and schizophrenia: a meta-analysis JW Muntjewerff et al 146 Table 1 Descriptive characteristics of eight case–control studies on the association of schizophrenia and homocysteine

Study Study area Percentage of Mean age (years) Diagnostic Exclusion criteria males instrument

Cases/controls Cases/controls

Regland et al.29 Sweden 55/55 32/32 DSM-III-R Not reported Susser et al.30 USA Not reported Not reported DSM-III-R Substance abuse Virgos et al.31 Spain 67/89 58/58 ICD-9 Levine et al.32 Israel 78/64 Not reported DSM-IV Cardiovascular disease and substance abuse Muntjewerff et al.33 The Netherlands 77/29 39/36 DSM-IV Neural tube defects, liver and renal dysfunction, use of folate antagonists or vitamin supplements Applebaum et al.34 Israel 69/50 Not reported DSM-IV Cardiovascular, renal and endocrinological diseases, and substance abuse Goff et al.35 USA 70/55 43/57 Not reported Renal dysfunction, alcohol abuse, and use of drugs affecting folate or homocysteine levels Muntjewerff et al.22 The Netherlands 76/43 42/51 DSM-IV Physical illness, relevant homocysteine levels modulating drugs, and use of vitamin supplements

Study Study area Cases/controls Muntjewerff et al 33 The Netherlands 34/97 Tan et al 41 Singapore 236/120 Joober et al 38 Canada 105/90 Sazci et al 39 Tur key 130/226 Virgos et al 31 Spain 210/218 Muntjewerff et al 22 The Netherlands 254/414 Yu et al, Chinese series40 China 230/251 Kunugi et al 37 Japan 343/258 Arinami et al 36 Japan 297/419 Yu et al, Scottish series40 Scottland 426/628

Combined 2265/2721 1.36 (1.07-1.72) 0.5 1 2 Odds ratio Figure 2 Odds ratios (ORs) and 95% confidence intervals (Cls) of schizophrenia for TT versus CC genotype of the MTHFR 677C4T polymorphism of each study seperately and combined (Studies are ordered by the inverse of the s.e. of each estimate. The size of the data markers is inversely proportional to the s.e. The horizontal lines represent the 95% CIs. The combined OR and 95% CI is indicated by the diamond).

Figure 2 shows the results of individual and pooled the results of individual studies (w2 ¼ 13.973; df ¼ 9; studies for the TT genotype. Overall, the TT genotype P ¼ 0.123). The absence of asymmetry in the distribu- was associated with a 36% (95% CI: 7–72) increased tion of the ORs of the individual studies as shown in risk of schizophrenia compared with the CC geno- Figure 2, and the outcome of the Egger regression type. Sequential removing and replacement of in- asymmetry test (a ¼À0.06; t ¼À0.04; P ¼ 0.97) reduce dividual studies from the calculation of the pooled the likelihood of publication bias. OR produced risk estimates ranging from 1.26 to 1.43 with 95% CIs that always encompassed 1.0. This Homocysteine and MTHFR finding indicates that the significance of the pooled Based on the published estimate from a large meta- OR was not excessively influenced by any single analysis on homocysteine and MTHFR studies,25 we study. There was no significant heterogeneity among assumed that the average homocysteine concentration

Molecular Psychiatry Homocysteine metabolism and schizophrenia: a meta-analysis JW Muntjewerff et al 147 is 2.7 mmol/l higher in individuals with the TT tion of folate enrichment of foods in North-America genotype than in subjects with the CC genotype. In since 1997. In our meta-analysis, no risk estimates by line with this, the pooled OR of 1.36 (95% CI: 1.07– continent were calculated, because of the low number 1.72) for TT versus CC genotype is equivalent to an of included North-American studies. OR of 1.77 (95% CI: 1.13–2.73) for the standard Studies of genetic variants that affect homocysteine 5 mmol/l increase in homocysteine concentration levels would reflect long-term exposure to elevated (raising 1.36 to the power of 5/2.7). This risk estimate homocysteine, and be independent of confounding is similar to the observed OR of 1.70 in our meta- and concerns about reverse causality.44 Apart from the analysis. effect modification by dietary intake of folate and riboflavin, it is unlikely that the effects of these Discussion genotypes on schizophrenia will be influenced by lifestyle or other confounders. Genetic confounding The overall result of the present meta-analysis by linkage disequilibrium is possible, whereby a gene demonstrates that for each 5 mmol/l increase in linked to the MTHFR gene controls an unknown risk homocysteine concentration the relative risk of factor for schizophrenia and also increases the schizophrenia is roughly doubled (OR ¼ 1.70; 95% homocysteine level. Nevertheless, no such linkage CI: 1.27–2.29). This finding must be interpreted with has been found thus far. The studies included in this some caution because of the observed significant meta-analysis involved less than a few hundred cases heterogeneity among the homocysteine studies in this for each study, and so the CI around the individual meta-analysis. However, the association of homozygo- ORs were wide. The present study provides more sity of the 677C4T polymorphism in the MTHFR reliable evidence as to the importance of the TT gene and schizophrenia with an OR of 1.36 (95% genotype for the risk of schizophrenia. The concor- CI: 1.07–1.72) is suggestive for a causal relation bet- dance of the risk estimates from homocysteine studies ween aberrant homocysteine metabolism and schizo- and MTHFR 677C4T polymorphism studies con- phrenia. cerning schizophrenia supports the hypothesis that A limitation of using a meta-analytic approach for the association of elevated homocysteine levels with population-based observational studies is the fact that this disease is in part causal. these studies may yield estimates of associations that Hyperhomocysteinemia is not specific for schizo- are influenced by confounding due to age, sex or phrenia, and has been reported as a risk factor for ethnic admixture (population stratification) either various diseases such as cardiovascular diseases,25 between studies or between cases and controls within neural tube defects,45 and neuropsychiatric disorders each study.42 In this meta-analysis, only the unad- including Alzheimer’s disease,46 Parkinson’s dis- justed ORs could be calculated for each study because ease47 and depression,48–50 stressing the pivotal role the adjusted ORs were not routinely provided. There of the single-carbon mechanism. The pathogenic might also be confounding because of the clinical mechanisms underlying these moderate hyperhomo- heterogeneity of the patients included. Most studies cysteinemic diseases are not yet fully understood. were restricted to schizophrenia patients, but some Interestingly, accumulating evidence from in vitro studies also included cases with schizophreniform or and animal studies suggests that the nervous system schizoaffective disorders. It is unlikely that this has (neuronal homeostasis) is, in particular, sensitive to been an important moderating variable, because of the folate deprivation and raised homocysteine levels.51,52 low number of included patients with these diag- Extra-cellular homocysteine has been found to be noses. toxic to cultured neurons and neuron cells via Plasma homocysteine concentrations reflect genetic stimulation of NMDA receptors, enhancing oxidative and environmental factors such as diet, B-vitamin stress and inducing DNA damage eventually resulting intake and life-style factors like coffee consumption in apoptosis and adverse effects on synaptic and glial and smoking.43 None of the studies on homocysteine function.51,53,54 Recently, experiments with mice have levels and risk of schizophrenia explicitly reported on shown that these mechanisms are not only important all critical factors in the assessment of homocysteine. in cultured embryonic premitotic neurons, but in the The distribution of these confounders may have adult postmitotic neurons as well.55 The deleterious differed among studies and by introducing systematic effect of MTHFR dysfunction on brain development error it might explain the observed heterogeneity was observed in individuals with a severe deficiency among studies. In order to gain further insight in the of this enzyme, resulting in delayed psychomotor association between homocysteine and schizophre- development, mental retardation and psychiatric nia, it will be of utmost importance to standardize the symptoms.56 measurement of homocysteine. The most obvious The 70% higher risk of schizophrenia for a 5 mmol/l factor that contributes to heterogeneity of homocys- increase in homocysteine observed in our meta- teine levels is the folate and riboflavin status of the analysis suggests a dose–response relationship. This study population.43 In a meta-analysis on MTHFR and finding provides a rational of using homocysteine venous a clear difference was seen levels as a biological parameter for intervention between European studies and North-American stu- studies. A meta-analysis of short-term trials compar- dies,26 which could be explained by the implementa- ing the effects on homocysteine levels of different

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