We found an observational association between decreased serum vitamin B12 levels and elevated BMI in two Danish cohorts. This led to the hypothesis that low serum vitamin B12 levels are causally involved in body mass regulation, and we investigated this by applying a Mendelian randomization design, including a formal instrumental variable analysis using a B12 GRS to model an unbiased genetically induced decrease of serum vitamin B12. However, our results provided no clear evidence for a causal role of low serum vitamin B12 levels in obesity.
The Mendelian randomization design applied in this study may be likened to a randomized clinical trial, where individuals are randomized by nature to carry genetic variants associating with low or high circulating levels of vitamin B12. The purpose of the randomization process in a clinical trial is to obtain an even distribution of potential confounders across the treatment arms, and likewise the Mendelian randomization design should result in an equal distribution of known and unknown confounders across genotypes. Additionally, reverse causality (here that higher BMI causes lower serum vitamin B12 levels) is circumvented since genotypes are non-modifiable. Hence, in the setting of a Mendelian randomization study, genotypes or a GRS are used as an unconfounded instrument for the risk factor under investigation and allows a largely unbiased evaluation of the causal role of the risk factor. To obtain a proper instrument for serum vitamin B12 levels, we constructed a GRS consisting of eight vitamin B12 decreasing SNPs in genes encoding proteins from the pathway responsible for the absorption, processing, and coenzymatic activity of vitamin B12 [
16]. This B12 GRS associated strongly with serum vitamin B12 and accordingly explained ~6% of the phenotypic variance in serum vitamin B12 levels. For comparison, 97 BMI associated SNPs from the GIANT consortium cumulatively explain only 2.7% of the phenotypic variance in BMI [
2], which among many reasons may be due to the unspecific representation of numerous different pathways where the variants are not directly linked functionally. The fact that the B12 GRS associated strongly and specifically with serum vitamin B12 levels and the fact that the eight SNPs included in the B12 GRS all have well-characterized roles in vitamin B12 functioning, are coding and thus potentially the true functional SNP, qualifies the B12 GRS as a useful instrument for evaluating causality. If serum vitamin B12 levels are causally linked to BMI, the B12 GRS should be related to BMI to the extent predicted by its influence on serum vitamin B12 levels. Although we did not have sufficient statistical power to completely rule out causality, our instrumental variable analyses provided no evidence that low serum vitamin B12 levels
cause increased BMI.
A crucial assumption underlying the Mendelian randomization design is that the genotype is associated with the outcome only via the risk factor under investigation [
17]. However, genetic variants may have pleiotropic effects and thus influence the outcome through other pathways than the one under investigation. To circumvent issues of pleiotropy, we therefore additionally applied the MR-Egger approach. Inclusion of
FUT2 rs602662 and
FUT6 rs778805 as instruments for genetically induced decreased serum vitamin B12 levels did not support introduction of directional pleiotropy, but the statistical power seemed to decrease markedly when applying the MR-Egger method. Nevetheless,
FUT2 rs602662 which showed the strongest individual SNP effect on serum vitamin B12, is
known to have pleiotropic effects. Thus, our finding that the G-allele of
FUT2 rs602662 was associated with decreased levels of serum vitamin B12 as well as increased BMI, does not necessarily support a causal role of decreased vitamin B12 levels in obesity. This finding may be explained by the strong linkage disequilibrium (r
2 = 0.81, D′ = 1.0) between
FUT2 rs602662 and
FUT2 rs601338 which encodes a nonsense mutation (W143X) causing a lack of presentation of blood group antigens A, B and H on mucosal surfaces and in secretions. This SNP could potentially modulate the composition and function of the gut microbiota, a hypothesis that is supported by various studies. Homozygous carriers of the stop codon (
FUT2 rs601338 AA carriers corresponding to
FUT2 rs602662 AA carriers) are nonsecretors and they have been shown to be resistant to infections with
Helicobacter pylori [
27,
28] and Norovirus, the major cause of viral acute gastroenteritis worldwide [
29‐
32]. Infection with
H. pylori may cause chronic gastritis [
33] leading to decreased secretion of intrinsic factor and therefore impaired absorption of vitamin B12. Intriguingly, the resistance to gastrointestinal infections suggests that lack of blood group antigens on the intestinal mucosa not only inhibits the infectious potential of pathogenic gastrointestinal microbes, but also may inhibit the habitual interaction with commensal microbes in the gastrointestinal tract. This is supported by studies showing that secretor status influences the composition of the gut microbiota [
34‐
36]. Also, several studies have reported a link between gut microbes and human obesity [
37‐
39] and it has been demonstrated that germ-free mice are leaner than conventionally raised mice and that conventionalization of germ-free mice with microbiota from conventionally raised mice results in an increased body fat content [
37]. Therefore, the slightly
lower BMI observed among AA carriers of
FUT2 rs602662 (accompanied by
higher serum vitamin B12 levels) in our study may be explained by the lack of expression of blood group antigens in the gastrointestinal tract and therefore impaired cross-talk with commensal microbes. In support of this interpretation, results from GIANT showed that the
FUT2 rs601338 A-allele (coding for the stop codon) associated nominally with decreased BMI (
P = 0.007). However, mechanistic studies in rodents and statistically powered studies of humans involving characterization of host genotype, microbiota composition, and BMI, must be conducted to fully address this hypothesis.
In conclusion, we did not find support for a causal role of decreased serum vitamin B12 levels in obesity. However, our study gives rise to the hypothesis that the FUT2 locus explains a part of the observational association between serum vitamin B12 and BMI through its regulation of the cross-talk between gut microbes and the human host. Furthermore, the B12 GRS constructed in this study may be used a valid instrument in future Mendelian randomization studies evaluating of the causal role of serum vitamin B12 levels in other disorders.