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Meta-Analysis
. 2012 Feb;9(2):e1001177.
doi: 10.1371/journal.pmed.1001177. Epub 2012 Feb 21.

Homocysteine and coronary heart disease: meta-analysis of MTHFR case-control studies, avoiding publication bias

Robert Clarke  1 , Derrick A Bennett, Sarah Parish, Petra Verhoef, Mariska Dötsch-Klerk, Mark Lathrop, Peng Xu, Børge G Nordestgaard, Hilma Holm, Jemma C Hopewell, Danish Saleheen, Toshihiro Tanaka, Sonia S Anand, John C Chambers, Marcus E Kleber, Willem H Ouwehand, Yoshiji Yamada, Clara Elbers, Bas Peters, Alexandre F R Stewart, Muredach M Reilly, Barbara Thorand, Salim Yusuf, James C Engert, Themistocles L Assimes, Jaspal Kooner, John Danesh, Hugh Watkins, Nilesh J Samani, Rory Collins, Richard Peto; MTHFR Studies Collaborative Group
Collaborators, Affiliations
Meta-Analysis

Homocysteine and coronary heart disease: meta-analysis of MTHFR case-control studies, avoiding publication bias

Robert Clarke et al. PLoS Med. 2012 Feb.

Abstract

Background: Moderately elevated blood levels of homocysteine are weakly correlated with coronary heart disease (CHD) risk, but causality remains uncertain. When folate levels are low, the TT genotype of the common C677T polymorphism (rs1801133) of the methylene tetrahydrofolate reductase gene (MTHFR) appreciably increases homocysteine levels, so "Mendelian randomization" studies using this variant as an instrumental variable could help test causality.

Methods and findings: Nineteen unpublished datasets were obtained (total 48,175 CHD cases and 67,961 controls) in which multiple genetic variants had been measured, including MTHFR C677T. These datasets did not include measurements of blood homocysteine, but homocysteine levels would be expected to be about 20% higher with TT than with CC genotype in the populations studied. In meta-analyses of these unpublished datasets, the case-control CHD odds ratio (OR) and 95% CI comparing TT versus CC homozygotes was 1.02 (0.98-1.07; p = 0.28) overall, and 1.01 (0.95-1.07) in unsupplemented low-folate populations. By contrast, in a slightly updated meta-analysis of the 86 published studies (28,617 CHD cases and 41,857 controls), the OR was 1.15 (1.09-1.21), significantly discrepant (p = 0.001) with the OR in the unpublished datasets. Within the meta-analysis of published studies, the OR was 1.12 (1.04-1.21) in the 14 larger studies (those with variance of log OR<0.05; total 13,119 cases) and 1.18 (1.09-1.28) in the 72 smaller ones (total 15,498 cases).

Conclusions: The CI for the overall result from large unpublished datasets shows lifelong moderate homocysteine elevation has little or no effect on CHD. The discrepant overall result from previously published studies reflects publication bias or methodological problems.

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Conflict of interest statement

The Clinical Trial Service Unit has a policy of not accepting honoraria or other payments from the pharmaceutical industry, except for reimbursement of costs to participate in scientific meetings (RCl, DAB, SP, JCH, RCo, RP). PV and MDK are employees of Unilever R&D Vlaardingen, The Netherlands. Unilever makes no claims regarding B-vitamins, homocysteine, and CVD on their food products, and PV and MDK have worked on the paper due to their expertise and data from previous academic life. PV and MDK therefore do not consider this to be a competing interest but declare it for reasons of transparency. HH is an employee of deCode, a biotechnology company that produces genetic testing services. JD and RCo are on the Editorial Board of PLoS Medicine. All other authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Mean serum folate concentrations in 81 population surveys, by calendar year and region.
White squares, no folate supplementation; black squares, after folate supplementation; broken vertical line, 1995–1996, when folate supplementation began in the United States, Canada, Australia, New Zealand (US & ANZ), and some but not all European countries. No Asian surveys were in supplemented populations.
Figure 2
Figure 2. Homozygote CHD OR (TT versus CC MTHFR C677T genotype) in 19 unpublished datasets, yielding 24 parts that are classified by genotyping panel size.
For these datasets, being unpublished introduces a negligible bias (less than 0.3% for each OR and about 0.1% for the overall OR: eAppendix 1). Black squares indicate OR (with areas inversely proportional to the variance of log OR), and horizontal lines indicate 99% CIs. The subtotals and their 99% CIs are indicated by black diamonds. The overall OR and its 95% CI is indicated by a white diamond. The weight (defined as the inverse of the variance of the maximum likelihood estimate of the log OR) and the product of the weight times OR indicates how much each study has contributed to the subtotals and totals. Because the weights and products are approximately additive, they can be used to estimate the effects of ignoring particular studies, or of grouping studies in different ways.
Figure 3
Figure 3. Homozygote CHD OR (TT versus CC MTHFR C677T genotype) in each probable folate status category, from meta-analyses of 19 unpublished datasets (all large).
Average homocysteine difference (in the non-CHD general population) for all areas and periods is weighted in proportion to the numbers of TT CHD cases in all 19 unpublished datasets. Nonpublication involves negligible bias: Appendix S2 in Text S1.
Figure 4
Figure 4. Homozygote CHD OR (TT versus CC MTHFR C677T genotype) in each probable folate status category, from meta-analyses of 86 published studies, 14 large (i.e., variance of log OR less than 0.05) and 72 smaller studies.
Black squares indicate OR (with areas inversely proportional to the variance of log OR in each subdivision), and horizontal lines indicate 99% CIs. The overall OR and its 95% CI are indicated by a black diamond. Average homocysteine difference (in the non-CHD general population) for all areas and periods is weighted in proportion to the numbers of TT CHD cases in all 86 studies.
Figure 5
Figure 5. Effects of folic acid on major coronary events (nonfatal myocardial infarction or coronary death) in a meta-analysis of the published results of all large randomized trials of homocysteine reduction.
Data for the VITATOPS trial are for myocardial infarction only. Data for FAVORIT are for all cardiovascular disease outcomes. Symbols and conventions as in Figure 2.
See this image and copyright information in PMC

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