An update on vitamin B12-related gene polymorphisms and B12 status
An update on vitamin B12-related gene polymorphisms and B12 status
S. Surendran 0
A. Adaikalakoteswari 1 2
P. Saravanan 1 2
I. A. Shatwaan 0
J. A. Lovegrove 0
K. S. Vimaleswaran 0
0 Hugh Sinclair Unit of Human Nutrition, Department of Food and Nutritional Sciences and Institute for Cardiovascular and Metabolic Research (ICMR), University of Reading , PO Box 226, Whiteknights, Reading RG6 6AP , UK
1 UK Academic Department of Diabetes and Metabolism, George Eliot Hospital , Nuneaton , UK
2 Warwick Medical School - Population Evidence and Technologies, University of Warwick , Coventry CV4 7AL , UK
Background: Vitamin B12 is an essential micronutrient in humans needed for health maintenance. Deficiency of vitamin B12 has been linked to dietary, environmental and genetic factors. Evidence for the genetic basis of vitamin B12 status is poorly understood. However, advancements in genomic techniques have increased the knowledge-base of the genetics of vitamin B12 status. Based on the candidate gene and genome-wide association (GWA) studies, associations between genetic loci in several genes involved in vitamin B12 metabolism have been identified. Objective: The objective of this literature review was to identify and discuss reports of associations between single-nucleotide polymorphisms (SNPs) in vitamin B12 pathway genes and their influence on the circulating levels of vitamin B12. Methods: Relevant articles were obtained through a literature search on PubMed through to May 2017. An article was included if it examined an association of a SNP with serum or plasma vitamin B12 concentration. Beta coefficients and odds ratios were used to describe the strength of an association, and a P < 0.05 was considered as statistically significant. Two reviewers independently evaluated the eligibility for the inclusion criteria and extracted the data. Results: From 23 studies which fulfilled the selection criteria, 16 studies identified SNPs that showed statistically significant associations with vitamin B12 concentrations. Fifty-nine vitamin B12-related gene polymorphisms associated with vitamin B12 status were identified in total, from the following populations: African American, Brazilian, Canadian, Chinese, Danish, English, European ancestry, Icelandic, Indian, Italian, Latino, Northern Irish, Portuguese and residents of the USA. Conclusion: Overall, the data analyzed suggests that ethnic-specific associations are involved in the genetic determination of vitamin B12 concentrations. However, despite recent success in genetic studies, the majority of identified genes that could explain variation in vitamin B12 concentrations were from Caucasian populations. Further research utilizing larger sample sizes of non-Caucasian populations is necessary in order to better understand these ethnic-specific associations.
Vitamin B12; Vitamin B12 levels; Cobalamin; Genetic epidemiology; Polymorphisms; Genetics of vitamin B12
Vitamin B12, also known as cobalamin (Cbl), is an essential
water-soluble micronutrient required to be ingested by
humans to maintain health. The nutritional deficiency of
vitamin B12 has been linked to many complications
including an increased risk of macrocytic anaemia,
neuropsychiatric symptoms [
], cardiovascular diseases [
the onset of different forms of cancer [
]. To maintain
adequate vitamin B12 status, individuals must ingest
sufficient dietary vitamin B12 and retain the ability to absorb
vitamin B12. The absorption, transport and cellular uptake
of vitamin B12 is dependent upon the co-ordinated action
of the binding proteins: haptocorrin (HC), intrinsic factor
(IF), transcobalamin II (TC) and other specific cell
receptors. After vitamin B12 binds to HC in the stomach and IF
in the duodenum, it binds to TC within the enterocyte and
is then released into the blood stream. The vitamin
B12TC complex then binds to the transcobalamin receptor
(TC-R) and is taken up by cells via endocytosis .
Genetic variants may alter vitamin B12 tissue status by
affecting the proteins involved in vitamin B12
absorption, cellular uptake and intracellular metabolism [
a study using monozygotic and dizygotic twins, the
heritability of B12 levels was estimated to be 59%, indicating
that the magnitude of genetic influence on vitamin B12
levels are considerable [
]. At present, genetic studies of
vitamin B12 status suggest that it is a multifactorial trait,
where several single-nucleotide polymorphisms (SNPs)
in multiple genes interact with the environment to cause
the altered B12 status [
]. Most of the SNPs related to
vitamin B12 status have been examined using a
candidate gene approach [
]. However, it is now possible to
use an unbiased genome-wide association (GWA) study
to associate DNA sequence variations across the human
genome with the risk factors of developing a disease [
Despite a number of informative genome-wide
association studies and candidate gene analyses, the complex
relationship between an individual’s genotype and their
vitamin B12 status remains poorly understood. This
article is the first literature review to discuss the results
of genetic studies associated with vitamin B12 status in
healthy individuals. Understanding the possible
underlying genetic factors of vitamin B12 metabolism will lead
to an increased understanding of the biological
mechanisms underlying vitamin B12 status.
Materials and methods
In order to identify published articles, literature searches
were completed using the PubMed database (https://
www.ncbi.nlm.nih.gov/pubmed/), from the earliest date
of indexing until May 2017. The following keywords
were used to identify articles from PubMed: ‘vitamin
B12 and genetics’ (n = 2792), ‘vitamin B12 and gene
polymorphisms’ (n = 447), ‘genetic variants of vitamin
B12’ (n = 115), ‘genetic variants of cobalamin’ (n = 95),
‘genetics of cobalamin’ (n = 2574), ‘genetics of vitamin
B12’(n = 2721) ‘vitamin B12 and genes’ (n = 932) and
‘cobalamin and genes’ (n = 858). In addition, reference
lists of identified publications were hand searched to
identify other studies potentially eligible for inclusion.
No limits on geographical location were placed in the
literature search, and only articles written in English
were selected. After inclusion and exclusion criteria were
applied, a comprehensive list of relevant articles was
included in this review.
The abstracts of all articles with relevant titles were
reviewed first and were further assessed if they reported
original data on testing for an association of a SNP with
plasma or serum vitamin B12 concentrations. Articles
were excluded if (1) they included non-human subjects,
(2) they were limited to a subset of the population (e.g.
pregnant women/carrying a disease) and (3) the sample
size of the population was less than 10.
Based on the search criteria and keywords used, 10,534
articles were identified from the PubMed database.
Following this, 10,482 articles were excluded according to
the established exclusion criteria, and 52 articles were
then considered as potentially relevant for the review. The
full text of the 52 articles was read, which resulted in the
exclusion of a further 29 articles. As a result, only 23
articles were selected for analysis (Fig. 1). A P < 0.05 was
considered as statistically significant.
The studies were identified by a single investigator (SS),
and the following data were double-extracted
independently by two reviewers (VKS and IAS): first author,
publication year, location or ethnicity of participants, sample size,
mean age, study design, SNP position, name and rs ID,
genotype and allele distribution by vitamin B12 status. For
the outcome data, the beta coefficients of vitamin B12
concentrations per risk allele, odds ratios (ORs) with their
corresponding 95% confidence intervals (95% CIs) were
extracted. Any discrepancies over extracted data were settled
through discussion between the two independent reviewers
(VKS and IAS). Finally, corresponding authors were
contacted to provide any additional information where needed.
Results of database search: genes associated with vitamin
The following section reviews studies of genetic variants
which have been associated with vitamin B12 status.
These variants have been grouped as (a) co-factors or
regulators essential for the transport of vitamin B12, (b)
membrane transporters actively facilitating membrane
crossing (c) involved in the catalysis of enzymatic
reactions in the one carbon cycle (d) involved in cell
cycle regulation, (e) mitochondrial proteins and (f ) other
genes (Figs. 2 and 3). A summary of GWA and
candidate gene association studies that have been reported to
be associated with circulating plasma or serum B12
concentrations are presented in Table 1 and Table 2.
The location and function of the most frequently studied
genes associated with vitamin B12 concentrations are
summarized in Table 3.
Co-factors or regulators of co-factors essential for the transport of vitamin B12
Methylmalonic aciduria and homocystinuria, cblC type
The methylmalonic aciduria and homocystinuria, cblC type
(MMACHC) gene is located in the chromosome region
]. The MMACHC gene encodes a chaperone
protein MMACHC (cblC protein) which binds to vitamin
B12 in the cytoplasm and appears to catalyze the reductive
decyanation of cyanocobalamin into cob(II)alamin [
Among the common variations, SNP rs12272669 has
been associated with vitamin B12 status, where ‘A’ allele
carriers had higher vitamin B12 concentrations
compared with ‘G’ allele carriers (P = 3.00 × 10−9, β =
0.51 pmol/l) in 37,283 Icelandic individuals [
Furthermore, SNP rs10789465 was associated with vitamin
B12 concentrations (P = 1.00 × 10−3) in a candidate gene
association study comprising 262 Caucasian women of
North European descent [
]. Currently, it is unknown
how these variants
Transcobalamin 1 (TCN1)
The transcobalamin 1 (TCN1) gene is located on
chromosome 11 and codes for the vitamin B12 binding
protein, transcobalamin I (TCI; also called haptocorrin
(HC) or R binder) [
]. TCI is involved in facilitating
the entry of vitamin B12 into the cells, via
receptormediated endocytosis [
]. Six studies have reported
associations between variants within the TCN1 gene and
circulating vitamin B12 concentrations [
Nongmaithem et al. [
] investigated the association
between several nucleotide variations within the TCN1
gene and vitamin B12 levels in a GWA study comprising
534 healthy children from Mysore, India. Carriers of the
‘G’ allele of the rs526934 variant were found to have lower
circulating vitamin B12 concentrations (β = − 0.16 pmol/l,
P = 0.02) compared to ‘A’ allele carriers [
]. This finding
was in accordance with the studies conducted in Chinese,
Icelandic, Italian and individuals residing in the US
(predominantly non-Hispanic white) [
Furthermore, additional variants of the TCN1 gene (rs34528912
and rs34324219) were observed to be associated with
vitamin B12 status (P < 0.05) in individuals of Icelandic,
Indian and Danish backgrounds [
Although no functional data are available to confirm
the functional effect of these SNPs on vitamin B12
concentrations, the results from these studies suggest
that the SNPs may have important physiological
consequences for the role of the TCN1 protein in
relation to vitamin B12 levels.
Fucosyltransferase 2 (FUT2)
The fucosyltransferase 2 (FUT2 gene), also known as the
Se gene (secretor) is located on chromosome 19. The
FUT2 gene codes for a secretor enzyme α(1,2)
fucosyltransferase which fucosylates oligosaccharides producing
H type 1 and 2 antigens. H antigens are precursors of
ABO and Lewis b histo-blood group antigens that are
expressed on mucosal surfaces [
]. Recent studies have
shown suggestive associations between variants of FUT2
with diabetes and body mass index [
For the FUT2 gene, seven SNPs including rs281379,
rs492602, rs516316, rs601338, rs602662, rs838133 and
rs1047781 were previously reported to be associated
with vitamin B12 levels [
12, 18–22, 27–29
]. To identify
loci associated with plasma vitamin B12, a meta-analysis
of three genome-wide association scans (n = 4763) was
carried out in a Caucasian population residing in the
]. The SNP rs601338, also known as 428 G/A
nonsecretor variant allele (W143X variant), was
significantly associated with plasma vitamin B12 levels (P =
6.92 × 10−15), with the allele ‘A’ being positively
associated with plasma vitamin B12 levels (β = 0.06 pg/ml)
Grarup et al.
Grarup et al.
SE = 10.35
Effect: A allele
Other: G allele
β = 12.83 pg/ml
SE = 13.24
Effect: A allele
Other: G allele
β = 27.62 pg/ml
SE = 8.15
Effect allele: A
β = − 0.14 pmol/l
Effect allele: A
β = − 0.65 pmol/l
Effect: C allele
Other: A allele
β = 0.21 pmol/l
Effect: C allele
Other: A allele
β = 0.40 pmol/l
Effect: C allele
Other: A allele
β = 0.30 pmol/l
Actin like 9 (ACTL9)
β = 0.17 pmol/l
59 ± 6
n = 3495
SE = 5.53
Effect: T allele
β = 0.13 pmol/l
Effect: T allele
β = 0.22 pmol/l
Effect: T allele
β = 0.23 pmol/l
]. This finding was further confirmed in another
study looking at 37,283 Icelandic adults (P = 2.40 × 10−95,
β = 0.162 pmol/l) [
], as well as in two Indian
populations of children (β = 0.18–0.25 pmol/l) [
the minor allele frequency (MAF) of rs601338 varies
widely between ethnicities, contributing to genetic
heteroegeneity in FUT2-B12 associations. In previous
reports by Grarup et al. [
] and Hazra et al. [
frequency of the minor allele ‘G’ for the associated SNP
(rs601338) was between 38.4 and 49.0%, for Icelandic
and Caucasian populations from the USA, respectively.
In contrast, the allele ‘A’ was found to be the minor allele
in the Indian population (MAF = 23.0%) [
presence of the ‘A’ allele is associated with higher vitamin
B12 concentrations, compared to ‘G’ allele carriers. This
indicates that in the Indian population, a greater number
of individuals carry the ‘G’ allele and hence could partly
explain why Indians are expected to have a lower
vitamin B12 status [
]. The FUT2 rs601338 variant is
less common in East Asians than Europeans [MAF =
3.5%; HapMap HCB (Han Chinese in Beijing, China)
and MAF = 1.2%; HapMap JPT (Japanese in Tokyo,
Japan)] and may explain why the locus has not been
identified in Chinese individuals in previous studies [
Another common non-synomynous SNP rs1047781
(A385T) has been shown to be a potential functional
variant associated with vitamin B12 status and a major
FUT2 secretor defining SNP in East Asians, and has also
been reported to reduce the expression of
]. Lin et al. found that the ‘T’ allele of the
SNP rs1047781 was significantly associated with higher
vitamin B12 concentrations in 3495 Chinese men (P =
3.62 × 10−36, β = 70.21 pg/ml) . This genetic marker is
present only in East-Asians; hence, it could not be
replicated in a study conducted in Icelandic individuals [
To date, three studies have shown an association between
the SNP rs492602 and vitamin B12 concentrations [
]. The SNP rs492602 is in complete linkage
disequilibrium (LD) with FUT2 W143X (rs601338) (r2 = 1), as shown
in the Nurses Health Study [
]. Hazra et al. [
] found that
the ‘A’ allele of the SNP rs492602 variant was associated
with lower vitamin B12 concentrations (β = − 0.06 pg/ml, P
= 1.30 × 10−14) among 4763 Caucasians from the USA, this
finding was similarly observed in a GWA study (2696
women) by the same authors (β = − 0.09 pg/ml, P = 5.36 ×
]. In a subsequent study in 3114 Canadian adults,
the ‘G’ allele was shown to be associated with a lower risk
(P = 2.0 × 10−4, odds ratio 0.60, 95% CI 0.54–0.70) of
vitamin B12 deficiency (< 148 pmol/l) [
Finally, the most commonly studied variant of the FUT2
gene is the SNP rs602662. This SNP was also reported to be
in LD with the SNPs rs601338 (r2 = 0.76) and rs516316 (r2
= 0.83) in Caucasian populations from the USA and Iceland
]. Zinck et al.  reported that ‘A’ allele carriers of
the rs602662 variant were at a lower risk of vitamin B12
deficiency (< 148 pmol/l) (OR 0.61, 95% CI 0.47–0.80, P =
3.0 × 10−4) in a population of 3114 Canadian adults [
Similarly, a higher vitamin B12 status was observed in
carriers of the ‘A’ allele in four different studies looking at
Caucasians (β = 0.04–43.27 pmol/l) [
12, 20, 21, 29
] and Indians
and homocystinuria type C
Replication factor C
subunit 1 (RFC1)
A = 0.430
cblA type (MMAA)
cblA type (MMAA)
cblA type (MMAA)
cblA type (MMAA)
48 ± 13
LMBR1 domain containing rs1457498
LMBR1 domain containing rs3778241
LMBR1 domain containing rs3799105
LMBR1 domain containing rs6455338
LMBR1 domain containing rs9294851
Transcobalamin 2 (TCN2)
n = 797
n = 198
n = 797
n = 2411
Transcobalamin 2 (TCN2)
Reductase (MTHFR) + rs180131
G = 0.120 Not available > 0.05
All studies have a cross-sectional design
SNP single-nucleotide polymorphism
*NORwegian Colorectal CAncer Prevention (NORCCAP) cohort
†Data refers to HapMap European population, with data collected from Utah Residents (CEPH) with Northern and Western European Ancestry
‡The specific data available is not published elsewhere and was obtained by contacting the corresponding authors
(β = 0.10–0.25 pmol/l) [
]. Furthermore, additional
variants of the FUT2 gene were observed to be associated with
vitamin B12 levels (P < 0.05) in the following SNPs:
rs1047781, rs516316, rs838133 and rs281379 [
12, 19, 22
It has been proposed that host genetic variation in the
FUT2 gene may alter the composition of the gut
microbiome. Individuals, who are nonsecretors (homozygous for
the non-functional FUT2 phenotype), lack terminal fucose
residues on mucin glycans [
]. As a result, the gut
microbial community of individuals with FUT2 deficiency
may reduce in composition and diversity, as microbes
cannot adhere or utilize host-derived glycans [
Variations in the FUT2 gene can potentially alter the
susceptibility to Helicobacter pylori (H. pylori) infection and its
related gastric-induced vitamin B12 malabsorption [
Gastric pathogens, such as H. pylori, attach to
α1,2-fucosylated glycan’s on epithelial cells, or structures masked by
fucosylation with the help of these H antigens in individuals
with the secretor status [
]. Infections with H. pylori
in the human intestine have been reported to interfere with
the release of intrinsic factor needed for vitamin B12
absorption . Interestingly, a study in Northern Portugal
found that the SNP rs602662 ‘A’ allele has been linked to a
non-secretor status (null H antigens), and this may decrease
the risk of bacterial infection from pathogens, such as H.
pylori, and explains why subjects who carry ‘A’ allele have a
high vitamin B12 status [
]. Alternatively, independent of
H. pylori-mediated gastritis, individuals who carried FUT2
secretor variants who were also heterozygous for a GIF (a
fucosylated glycoprotein needed for vitamin B12
absorption) mutation, had lower vitamin B12 concentrations [
Fucosyltransferase 6 (FUT6)
The fucosyltransferase 6 (FUT6) gene is located on
chromosome 19 and encodes a Golgi stack membrane
protein, involved in the formation of Sialyl-Lewis X, an
E-selectin ligand [
]. These Lewis associated antigens
are associated with H. pylori adherence to the gastric
and duodenal mucosa [
]. Overgrowth of H. pylori
has been linked to vitamin B12 deficiency, as gastric
bacteria reduces the secretion of IF which is needed to
form the vitaminB12-IF complex [
In light of the potential physiological link between the
FUT6 gene and vitamin B12 deficiency, three studies
investigated the relationship between variants in the
FUT6 gene and vitamin B12 status. Lin et al. first
] that the ‘A’ allele of the rs3760776 variant was
associated with higher vitamin B12 levels (β = 49.78 pg/
ml, P = 3.68 × 10−13) in a sample of 3495 men of Chinese
Han and Chinese descent [
]. Similarly, homozygous ‘A’
allele carriers of Icelandic (β = 0.068 pmol/l, P = 4.4 × 10
] and Indian (β = 0.18–0.30 pmol/l) [
populations had high serum vitamin B12 concentrations.
Interestingly, this gene variant may have the potential to serve
as a genetic marker for type 2 diabetes [
Furthermore, additional variants of the FUT6 gene
], rs78060698 , rs3760775 [
]) were observed to be associated with a
higher vitamin B12 status in individuals of the Indian,
Icelandic and Danish populations (P < 0.05).
Bioinformatic analysis has shown that the FUT3, FUT5 and
FUT6 genes form a cluster on chromosome 19p13.3
]. Interestingly, the SNPs rs3760775, rs10409772,
rs12019136, rs78060698, rs17855739, rs79744308,
rs7250982 and rs8111600 from this cluster were in LD
with the FUT6 SNP rs3760775 (r2 = 0.57–0.84) in South
Asian populations. Available data has shown differences
in the LD structure between South Asian populations
and individuals of East Asian and European origin [
The variation of LD patterns across ethnicities could
account for the heterogeneity of vitamin B12
Nongmaithem et al. [
] noted that alternative allelic
states of the SNP rs78060698 variant may influence the
binding affinity of HNF4α (a key regulator of FUT6
expression) to the FUT6 protein. FUT6 is responsible for
The MMACHC gene encodes a chaperone protein MMAACHC (cblC
protein) which binds to vitamin B12 in the cytoplasm and appears
to catalyze the reductive decyanation of cyanocobalamin into
It encodes a glycoprotein called Transcobalamin 1, also known as
haptocorrin (HC), which binds to vitamin B12. It shields dietary
vitamin B12 from the acidic environment of the stomach
19q13.33 It encodes the enzyme fucosyltransferase 2 (FUT2), which is
involved in the synthesis of antigens of the Lewis blood group [
These antigens mediate the attachment of gastric pathogens to
the gastric mucosa, which can affect the absorption of vitamin B12
It encodes the enzyme fucosyltransferase 6 (FUT6), which is
involved in forming Lewis associated antigens. These antigens
attach gastric pathogens to the gastric mucosa. It has been shown
that these gastric pathogens can reduce the absorption of vitamin
B12 in the gut [
It encodes a transport protein called transcobalamin 2 (TC), which
binds to vitamin B12 within the enterocyte. The TC-B12 complex
enters the portal circulation [
] and makes vitamin B12 available
for cellular uptake in target tissues [
It encodes the intestinal receptor Cubilin, which is expressed in the
renal proximal tubule and intestinal mucosa [
recognizes the vitaminB12-intrinsic factor complex, and binds to an
other protein called Amnionless to facilitate the entry of vitamin
B12 into the intestinal cells [
ABCD4 codes for an ABC transporter. It has been postulated that
ABCD4 is involved in intracellular cobalamin processing [
], and is
involved in transporting vitamin B12 from lysosomes to the cytosol.
In the cytosol, vitamin B12 can be converted into methylcobalamin
(MeCbl) and adenosylcobalamin (AdoCbl) [
It encodes the membrane receptor transcobalamin receptor (TCblR),
which binds to the transcobalamin-vitamin B12 complex, and medi
ates the uptake of vitamin B12 into cells [
MTHFR codes for a critical enzyme involved in homocysteine
remethylation. MTHFR catalyzes the reduction of
5,10methylenetetrahydrofolate to 5-methyltetrahydrofolate in an irre
versible reaction [
This gene is responsible for maintaining adequate levels of activated
vitamin B12 (methylcob(III)alamin), which maintains the enzyme
methionine synthase in its active state [
MS4A3 encodes a protein involved as a hematopoietic cell cycle
]. MS4A3 gene may have a role in the cell-cycle regula
tion in the GI tract, thus affecting the renewal of intestinal and gas
tric epithelial cells, and absorption of vitamin B12 [
MMAA encodes a protein that may be involved in the translocation
of vitamin B12 into the mitochondria [
]. In addition, MMAA could
play an important role in the protection and reactivation of
Methylmalonyl-coA mutase (MCM) in vitro [
It encodes a Mitochondrial enzyme methylmalonyl-CoA mutase
(MUT), which catalyzes the isomerization of methylmalonyl-CoA to
succinyl-CoA. This isomerization requires vitamin B12 as a cofactor
in the form of 5-prime-deoxyadenosylcobalamin (AdoCbl) [
It encodes a human mitochondrial enzyme, which is co-expressed
with other co-enzymes in the mitochondrial B12 pathway [
synthesizing α(1,3) fucosylated glycans, which act as a
biological interface for the host-microbial interaction
]. It is plausible that the SNP rs78060698 maintains
the structure of glycans, which in turn control intestinal
host-microbial interactions leading to altered
concentrations of vitamin B12 [
]. Another hypothesis is that
genetic variants may disrupt the formation of
fucosyltransferases which mediate the glycosylation of B12
binding proteins and their receptors, thus influencing
vitamin B12 concentrations .
Transcobalamin 2 (TCN2)
The TCN2 gene also known as transcobalamin 2 is located
on chromosome 22. This gene has the function of making
a vitamin B12 binding protein called transcobalamin II
(TC) found in human serum [
]. Data suggests that
TCN2 genetic variants are associated with Alzheimer’s
disease and clinical manifestations of autoimmune
gastritis in individuals with low vitamin B12 status [
is involved with absorption and transporting vitamin B12
into the cell. Only 10–20% of vitamin B12 is attached to
TC; the remainder is attached to holo-haptocorrin
(transcobalamin 1) [
18, 52, 53
]. Five studies have reported
associations between variants within the TCN2 gene and
vitamin B12 levels [
12, 18, 22, 52, 54
The most commonly reported TCN2 polymorphism in
Caucasian populations is the SNP rs1801198, where the C
to G substitution at nucleotide 776 (TCN2 776C>G)
results in an amino acid exchange of proline to arginine at
codon 259 (P259R). In a candidate gene association study
of 613 Irish men, a significant association was observed
between the SNP rs1801198 and serum vitamin B12 levels
(P = 0.01). Individuals with the homozygous wildtype ‘CC’
genotype had lower vitamin B12 levels (mean 243.5 pmol/
l) compared to those with ‘GG’ genotype (mean
279.7 pmol/l) [
]. In contrast, it was observed that
holotranscobalamin (Holo-TC) concentrations were
significantly associated with the SNP rs1801198, in a population
of 122 individuals from Portugal, where the G allele
carriers (median 54.2 pmol/l) had lower Holo-TC levels
compared to the C variant (P < 0.05; median 66.3 pmol/l)
]. Four other studies reported no significant
associations between the SNP rs1801198 and vitamin B12
concentrations in Caucasian populations (P > 0.05)
]. It was found that the minor allele frequency (G
allele) of the SNP rs1801198 ranged between 35 and 48%
in Brazillian (36%) , Latino (35%) [
], Nordic (44%)
], Northern Irish (45%)  and Portuguese (48%)
] individuals. Additional variants of the TCN2 gene
(rs757874, rs4820888, rs1131603 and rs5753231) were
associated with vitamin B12 status (P < 0.05) in individuals
of Indian, Canadian, US, African American and
Scandinavian background [
12, 18, 22, 55, 59
It has been suggested that the 776GG homozygous
variant encodes a protein with a lower binding affinity to
vitamin B12 in comparison to the wildtype ‘C’ allele [
Additionally, other studies have indicated that variations
in the TC protein reduce the binding of vitamin B12 to
TC or prevent the TC-R from recognising the vitamin
B12-TC complex [
Genes that code for membrane transporters that actively facilitate membrane crossing
Cubulin (CUBN) also known as the intestinal intrinsic
factor receptor or intrinsic factor-cobalamin (IF-B12) receptor
is located on chromosome 10. CUBN is expressed on the
intestinal and kidney epithelial cells and is involved with
the uptake of the intrinsic factor-vitamin B12
(vitaminB12IF) complex [
20, 60, 61
]. CUBN polymorphisms have been
associated with maternal neural tube defects risk,
megaloblastic anaemia, coronary heart disease and gastric cancer
in individuals with low vitamin B12 status [
Studies of the association between vitamin B12 status
and the variants within CUBN have yielded conficting
results. Hazra et al. [
] was the first to report an association
between the ‘G’ allele of the rs1801222 (Ser253Phe) variant
and higher vitamin B12 status (β = 0.05 pg/ml, P = 2.87 × 10
−9) in 4763 individuals from the US population [
association was confirmed in another study looking at
45,571 Icelandic and Danish individuals (β = 0.10–
0.17 pmol/l; P = 3.3 × 10−75) [
]. In contrast, a study in
3114 Canadian individuals (85% Caucasian and 15%
nonCaucasian) showed that the ‘G’ allele of the rs1801222
variant was associated with a higher risk of vitamin B12
deficiency (OR 1.61 pmol/l, 95% CI 1.24–2.09, P = 3.0 × 10
]. Genotypic frequency of the risk conferring minor
allele ‘A’ was compared between three different studies
(Canadian, Nordic and individuals of European ancestry
living in the USA). It was found that Canadian individuals
carried the lowest frequency of the risk allele ‘A,’ at 10%
]. On the other hand, Hazra et al. [
] and Grarup et al.
] observed that the minor allele frequency ‘A’ was 28.0
and 40.7% in Caucasian individuals residing in the USA
and Nordic populations, respectively. Interestingly, several
other genetic variants within CUBN (rs4748353,
rs11254363 and rs12243895) were found to be either
positively or negatively associated with vitamin B12 levels in
residents from China, [
] Canada [
], USA and Italy [
To date several hypotheses have attempted to explain
how CUBN variants are involved with lower vitamin B12
concentrations. One hypothesis is that CUBN is
coexpressed with the protein amnionless (AMN,
chromosome 14) forming the cubam complex [
]. Cubilin has
additionally been suggested to function together with
megalin (LRP2, chromosome 2) [
], thus any
polymorphisms in either AMN or LRP2 genes can affect B12
absorption leading to B12 malabsorption and deficiency.
Another hypothesis is that polymorphisms affecting
CUBN decrease the transport and the absorption of
vitamin B12 in the ileum [
]. Functional studies on
rs11254363, rs1801222, rs12243895 and rs4748353 are
required to explain how these variants affect the
regulation of the CUBN gene.
ATP-binding cassette subfamily D member 4 (ABCD4)
The ATP-binding cassette subfamily D member 4
(ABCD4) gene is located on chromosome 14. This gene
codes for the ABCD4 protein, which is a membrane
transporter involved in transporting vitamin B12 out of
]. It has been shown that polymorphisms of
the ABCD4 gene affect the functioning of the ABCD4
protein and the intracellular processing of vitamin B12 [
There has been only one study to date investigating
the association between vitamin B12 status and ABCD4
variants. Grarup et al. [
] examined 45,571 Nordic
adults and 25,960 Icelandic adults in a GWA study [
where the ‘T’ allele of the rs3742801 and ‘C’ allele of the
rs4619337 variants were associated with higher vitamin
B12 levels (β = 0.045–0.093 pmol/l, P = 5.3 × 10−8; β =
0.05, P = 3.4 × 10−8, respectively), suggesting an impact
of this gene on vitamin B12 status.
Previous research has shown that the protein LMBD1
(which is responsible for the lysosomal export of vitamin
B12) interacts with the ABCD4 protein. The
mechanisms of interaction between LMBD1 and ABCD4
remain unclear, but it is believed that polymorphisms in
human LMBRD1 gene and ABCD4 can prevent
translocation of the vitamin B12 from the lysosome to the
CD320 molecule (CD320)
The CD320 gene also known as the ‘CD320 molecule’
gene is located on chromosome 19. This gene codes for
the transcobalamin receptor (TCblR), which binds and
engulfs Holo-TC by endocytosis [
]. At present, two
SNPs, rs2336573 and rs8109720, have shown association
with vitamin B12 levels [
12, 18, 59
The most commonly studied variant of the CD320
gene is the rs2336573 variant, a missense polymorphism,
that results in an amino acid change from glycine to
arginine, at the codon position 220. Zinck et al. found that
the ‘C’ allele of the rs2336573 variant was associated
with a lower risk (OR 0.62, 95% CI 0.45–0.86, P = 0.003)
of vitamin B12 below adequate (< 220 pmol/l) among
3114 Canadian adults [
]. In contrast, an earlier study
looking at a population of 45,571 adults from Iceland
and Denmark found that the ‘T’ allele was associated
with higher B12 levels (β = 0.22–0.32 pmol/l; P = 8.4 × 10
]. A previous study has shown that this
polymorphism is associated with the maternal risk of
developing neural tube defects [
]. Cell culture models
have shown that SNPs in the CD320 receptor can lead
to a reduction in vitamin B12 uptake [
Involved in the catalysis of enzymatic reactions in the one carbon cycle
Methylenetetrahydrofolate reductase (MTHFR)
The methylenetetrahydrofolate reductase (MTHFR) gene
is located on chromosome 1 [
] and codes for a critical
enzyme involved in homocysteine remethylation.
MTHFR catalyzes the reduction of
5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate in an irreversible
]. The two most well-known MTHFR gene
polymorphisms are the C677T (rs1801133) and A1298C
(rs1801131) variants. Both variants have been associated
with reduced enzyme activity and an altered distribution
of intracellular folate [
The majority of candidate gene association studies
have shown no association (P > 0.05) with MTHFR gene
polymorphisms (rs1801131 and rs1801133) and vitamin
B12 concentrations in Brazillian [
European , French [
], Norweigian [
] and Spanish
] populations. However, Thuesen, et al. reported that
‘T’ allele carriers of the C677T genotype variant were
associated with an increased prevalence of low-serum
vitamin B12 (OR 1·78; 95% CI 1·25, 2·54; P = 0·003) in a
population of 6784 Danish adults [
]. There are no
explanations to date, which have linked the biological
mechanism of TT homozygosity and B12 deficiency. It
could be postulated that the C677T polymorphism is
associated with a decrease in intestinal absorption of
vitamin B12 [
Methioninesynthase reductase (MTRR)
The MTRR gene, also known as the ‘methionine
synthase reductase’ gene is located on chromosome 5. This
gene is responsible for maintaining adequate levels of
activated vitamin B12 (methylcob(III)alamin), which
maintains the enzyme methionine synthase in its active
]. Currently, four SNPs, rs162036, rs162048,
rs1532268 and rs3776455, have shown associations with
vitamin B12 levels in healthy individuals [
The first SNP MTRR rs162036 (Lys350Arg) is a
missense polymorphism [
], which was found to be
associated with vitamin B12 levels (P = 4.00 × 10−2) in 262
women of North European descent (no effect size
]. The same authors, also identified a significant
association (P < 0.05) between the SNPs rs162048,
rs1532268 and rs3776455 with vitamin B12 levels. This
study provides the first evidence that MTRR
polymorphisms (rs162036, rs162048, rs1532268 and rs3776455)
significantly influence the circulating vitamin B12
Involved in cell cycle regulation
Membrane-spanning 4-domains A3 (MS4A3)
The membrane-spanning 4-domains A3 (MS4A3) gene is
located on chromosome 11, and codes for the MS4A3
protein (also called HTm4). It has been suggested from
limited studies that the MS4A3 protein may play a role in
cell cycle regulation of hematopoietic cell development by
inhibiting the G(1)-S cell cycle transition [
]. The only
studied variant within this gene in relation to vitamin B12
concentrations is rs2298585, which was investigated in
3495 men, all of Chinese origin. In this study [
], the ‘T’
allele of the rs2298585 variant was associated with higher
serum vitamin B12 concentrations (β = 71.80 pg/ml, P =
2.64 × 10−15) [
]. Another study investigated this SNP in
37,283 Icelandic individuals but found no statistical
significance (β = 0.214 pmol/l, P = 0.075) [
It has been suggested that polymorphisms of the
MS4A3 gene may affect the cell-cycle regulation in the GI
tract, thus affecting the renewal of intestinal and gastric
epithelial cells leading to vitamin B12 malabsorption [
However, data from animal studies have demonstrated
that MS4A3 is restricted to differentiating cells in the
central nervous system and hematopoietic cells [
Methylmalonic aciduria (cobalamin deficiency) cb1A type
The MMAA gene also known as the ‘methylmalonic
aciduria (cobalamin deficiency) cb1A type’, is located on
chromosome 4q31.1-2 [
]. MMAA encodes a protein
(MMAA) that may be involved in the translocation of
vitamin B12 into the mitochondria [
]. In addition,
MMAA could play an important role in the protection
and reactivation of methylmalonyl-coA mutase (MCM)
in vitro [
]. Three studies have reported associations
between variants within the MMAA gene and vitamin
B12 concentrations [
12, 13, 22
Andrew et al. was first to report that the SNP rs4835012
was significantly associated with vitamin B12
concentrations (P = 3.00 × 10−2) in 262 Caucasian women of North
European descent (no effect size available) [
recently in a GWA study looking at 534 Indian children, the
‘C’ allele of the SNP rs2270655 was significantly associated
with lower vitamin B12 concentrations (β = − 0.20 pmol/l,
P = 2.00 × 10−2) [
]. This association was confirmed in
another study looking at 45,576 Danish and Icelandic
adults (β = − 0.07 to − 0.30, P = 2.20 × 10−13) [
these SNPs might be involved with determination of
vitamin B12 concentrations, their precise biochemical role
provides instructions for the formation of
methylmalonylCoA mutase (MUT), which is a mitochondrial enzyme.
MUT acts as a catalyst which isomerizes
methylmalonylCoA to succinyl-CoA [
]. MUT requires
5-primedeoxyadenosylcobalamin (AdoCbl), which is a form of B12
that works with MUT to form succinyl-CoA.
SuccinylCoA participates in the TCA cycle (tricarboxylic cycle) to
yield energy [
]. The MUT gene is involved in
homocysteine metabolism, and it is dependent on vitamin B12 for its
]. Four studies have reported associations
between variants within the MUT gene (chr6:49,508,102,
rs1141321, rs9473555, rs6458690 and rs9381784) and
vitamin B12 status [
12, 13, 19, 20
In a meta-analysis of data from 4763 Caucasian
individuals from the USA, participants homozygous for the
rs9473558 (now merged into rs1141321) ‘T’ allele (β = −
0.04 pg/ml, P = 4.05 × 10−8) and MUT rs9473555 ‘C’ allele
(β = − 0.04 pg/ml, P = 4.91 × 10−8) were inversely
associated with plasma vitamin B12 levels [
]. These findings
were confirmed in other studies involving Icelandic (β = −
0.061 pmol/l; β = − 0.062 pmol/l, repectively) [
Chinese populations (β = − 30.34 pg/ml; β = − 31.0 pg/ml,
Citrate lyase beta like (CLYBL)
The citrate lyase beta like (CLYBL) gene is located at
chromosome 13 and codes for a human mitochondrial
protein. The functions of CLYBL include metal ion
binding, carbon-carbon lyase activity and citrate (pro-3s)-lyase
]. Approximately, 5% of humans have a stop
codon polymorphism in CLYBL which is associated with
low levels of plasma vitamin B12, but the mechanistic link
of this to vitamin B12 is currently unknown [
The association between the CLYBL variant rs41281112
and vitamin B12 levels has been studied in two different
populations. Lin et al. [
] found that the ‘T’ allele was
associated with lower serum vitamin B12 levels among 3495
men of Chinese Han and Chinese descent (β = − 83.60 pg/
ml, P = 9.23 × 10−10) [
]. Similarly, Grarup et al. [
found that the ‘T’ allele of the SNP rs41281112 variant
was associated with lower serum vitamin B12 levels (β =
− 0.29 to − 0.17 pmol/l, P = 8.9 × 10−35) in 45,571 adults,
all of Icelandic and Danish origin [
At present, molecular functioning studies have
elucidated that the polymorphism rs41281112 (G<A) changes
the amino acid from Arginine to a stop codon resulting
in a loss of protein expression [
]. As a result, Lin et al.
] proposed that the rs41281112 variant interferes with
the binding of CLYBL protein to metal ions, potentially
leading to a lower uptake of vitamin B12 [
Methylmalonyl-CoA mutase (MUT)
The MUT gene also known as the methylmalonyl-CoA
mutase is located on chromosome 6. The MUT gene
Our review also identified that SNPs in actin like 9
(ACTL9, rs2340550) [
], serum paraoxonase/arylesterase
1 (PON1, rs391757) [
], cystathionine beta synthase
(CBS, rs2124459) [
], carbamoyl-phosphate synthase 1
(CPS1, rs1047891) [
] and DNA methyltransferase gene/
tRNA aspartic acid methyltransferase 1 (DNMT2/
TRDMT1, rs56077122 [
] and rs2295809 [
were associated with vitamin B12 status in Canadian,
Chinese, Danish and Icelandic populations. The SNPs in
the intergenic regions [rs583228, rs10515552, rs12377462
], rs117456053, rs62515066 and Chr6:88,792,234 [
were found to be associated with vitamin B12 status,
however, plausible underlying biological mechanism as to why
these SNPs were associated with vitamin B12
concentrations have not been identified.
Ethnic-specific genetic differences in B12 deficiency
In the past, vitamin B12 deficiency within populations in
the Indian subcontinent, Mexico, Central and South
America and certain regions of Africa was solely attributed to
dietary habits/low consumption of meat [
]. We now
know that genetic factors also influence vitamin status in
]. Indian populations have a high prevalence
of vitamin B12 deficiency, typically attributed to the high
number of vegetarians present in the population. However,
non-vegetarians in India have been observed to have lower
vitamin B12 concentrations compared to Caucasian
]. In addition, a recent systematic review
showed that B12 deficiency is common during pregnancy
in other populations where vegetarianism is rare . Poor
dietary intake, low bioavailable B12 in meat products (i.e.
food processing and reheating of food) and a possible
underlying genetic predisposition to vitamin B12 status
could be the reasons for such observation in
nonvegetarian populations [
Although several studies have explored the association
of SNPs with vitamin B12 status, only a limited number of
genetic loci have been reported to support the presence of
ethnic differences in vitamin B12 status in non-European
]. We can assume four genetic
mechanisms which possibly account for these differences: (1)
difference in effect allele frequencies, (2) genetic
heterogeneity across different ethnic groups, (3) variance in LD
structure and (4) gene-gene and gene-environment
interactions . A key example of ethnic specificity has been
demonstrated in the FUT2 gene, whereby different
mutations leading to nonsecretor status have been identified
(the secretor status of FUT2 gene is associated with a low
vitamin B12 status) [
]. The 428G→A polymorphism
(rs601338) is the characteristic for the nonsecretor allele
in Europeans and appears in about 20% of the Caucasian
]. In South-East and East-Asians
populations, the SNP rs601338 is rare and the more common
FUT2 missense mutation rs1047781 is associated with
nonsecretor status [
Genetic variants associated with circulating vitamin B12
have been studied in the following populations: African
American (n = 1) [
], Brazilian (n = 4) [
58, 77, 78, 105
Canadian (n = 1) , Caucasian (n = 4) [
20, 28, 29, 59
Chinese (n = 1) , Danish (n = 2) [
ancestry (n = 1) , French (n = 1) [
], Icelandic (n = 1)
], Indian (n = 2) [
], Italian ancestry and residents
of the USA (n = 1) , Latino (n = 2) [
Irish (n = 1) , Norwegian (n = 2) [
Portuguese (n = 1) . To date, the majority of genetic
association studies of vitamin B12 status have been performed in
Caucasian populations, and a few have reported
associations in high-risk populations such as Mexico and India
]. More studies exploring a wider range of
ethnicities with large sample sizes may help to identify novel SNPs
that may be associated with vitamin B12 status. Studying
the genetic structure of chromosomal regions that are
associated with variability in vitamin B12 levels in different
populations can help us understand the evolutionary
aspects of B12 associations and their relationship with
environmental exposures. It is important that before any
dietrelated recommendations based on genotypes are given at
the population level, associations between the SNPs and
various health outcomes need to be confirmed .
In summary, our review has identified significant
associations of vitamin B12 status with 59 B12-related SNPs
from 19 genes. Among these genes, five were co-factors
or regulators for the transport of vitamin B12 (FUT2,
FUT6, MMACHC, TCN1 and TCN2); three were
membrane transporters actively facilitating the membrane
crossing of vitamin B12 (ABCD4, CUBN and CD320);
three were involved in the catalysis of enzymatic
reactions in the one-carbon cycle (CBS, MTHFR and MTRR);
one was involved in cell cycle regulation (MS4A3); three
were mitochondrial proteins (CLYBL, MMAA and
MUT) and lastly four genes had an unknown function
(ACTL9, CPS1, DNMT2/TRDMT1 and PON1). Our
review highlights the complex nature of the B12 genetics
where several genes/SNPs from various parts of B12
metabolic pathway contribute to the susceptibility to
vitamin B12 deficiency. Identification of gene variants
involved in this metabolic pathway using large-scale
genetic association studies in diverse ethnic populations
would contribute to our understanding of the
pathophysiology of B12 deficiency and help in discovering
biomarkers of vitamin B12-related chronic diseases.
Dr. Karani S Vimaleswaran acknowledges support from the British Nutrition
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