An update on vitamin B12-related gene polymorphisms and B12 status

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.


Background
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 [1], cardiovascular diseases [2] and the onset of different forms of cancer [3,4]. 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 B12-TC complex then binds to the transcobalamin receptor (TC-R) and is taken up by cells via endocytosis [5].
Genetic variants may alter vitamin B12 tissue status by affecting the proteins involved in vitamin B12 absorption, cellular uptake and intracellular metabolism [6]. In 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 [7]. 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 [8]. Most of the SNPs related to vitamin B12 status have been examined using a candidate gene approach [8]. 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 [9]. 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.
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.

Study selection
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.

Data extraction
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 B12 status
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 (MMACHC) The methylmalonic aciduria and homocystinuria, cblC type (MMACHC) gene is located in the chromosome region 1p34.1 [10]. 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 [11].
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 [12]. 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 [13]. Currently, it is unknown how these variants affect the regulation of the MMACHC gene.
Nongmaithem et al. [22] 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 [22]. This finding was in accordance with the studies conducted in Chinese, Icelandic, Italian and individuals residing in the US (predominantly non-Hispanic white) [12,[19][20][21]. 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 [12,22].
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 [5]. Recent studies have shown suggestive associations between variants of FUT2 with diabetes and body mass index [23][24][25][26].
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 Fucosyltransferases [30,31]. 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) [19]. This genetic marker is present only in East-Asians; hence, it could not be replicated in a study conducted in Icelandic individuals [12].
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 (r 2 = 0.76) and rs516316 (r 2 = 0.83) in Caucasian populations from the USA and Iceland [12,29]. Zinck et al. [18] 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 [18]. 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        (β = 0.10-0.25 pmol/l) [22,27]. 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 [32,33]. 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 [33,34]. Variations in the FUT2 gene can potentially alter the susceptibility to Helicobacter pylori (H. pylori) infection and its related gastric-induced vitamin B12 malabsorption [35][36][37][38][39][40]. 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 [35][36][37][38][39][40]. Infections with H. pylori in the human intestine have been reported to interfere with the release of intrinsic factor needed for vitamin B12 absorption [40]. 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 [41]. 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 [42].

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 [19]. These Lewis associated antigens are associated with H. pylori adherence to the gastric and duodenal mucosa [43,44]. 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 [19,40].
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 observed [19] 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 [19]. Similarly, homozygous ' A' allele carriers of Icelandic (β = 0.068 pmol/l, P = 4.4 × 10 −6 ) [12] and Indian (β = 0.18-0.30 pmol/l) [22] 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 [26].
Furthermore, additional variants of the FUT6 gene (rs708686 [12,22], rs78060698 [22], rs3760775 [22] and rs7788053 [12]) 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 [45]. Interestingly, the SNPs rs3760775, rs10409772, rs12019136, rs78060698, rs17855739, rs79744308, rs7250982 and rs8111600 from this cluster were in LD with the FUT6 SNP rs3760775 (r 2 = 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 [22]. The variation of LD patterns across ethnicities could account for the heterogeneity of vitamin B12 concentrations [46].
Nongmaithem et al. [22] 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 cob(II)alamin [11].
Fucosyltransferase 2 (FUT2) 19q13.33 It encodes the enzyme fucosyltransferase 2 (FUT2), which is involved in the synthesis of antigens of the Lewis blood group [5]. These antigens mediate the attachment of gastric pathogens to the gastric mucosa, which can affect the absorption of vitamin B12 [109].
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 [43,44].
Membrane transporters that actively facilitates membrane crossing It encodes the intestinal receptor Cubilin, which is expressed in the renal proximal tubule and intestinal mucosa [20,60,61]. Cubilin 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 [67].
ATP binding cassette subfamily D member 4 (ABCD4) 14q24.3 ABCD4 codes for an ABC transporter. It has been postulated that ABCD4 is involved in intracellular cobalamin processing [69], 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) [70].
Proteins involved in the catalysis of enzymatic reactions in the one carbon cycle Methylenetetrahydrofolate reductase (MTHFR) 1p36 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 [74].
Methionine synthase reductase (MTRR) 5p15.31 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 [83].

Proteins involved in cell cycle regulation
Membrane-spanning 4-domains A3 (MS4A3) 11q12.1 MS4A3 encodes a protein involved as a hematopoietic cell cycle regulator [85]. 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 [86].
Mitochondrial protein Methylmalonic aciduria (cobalamin deficiency) cb1A type (MMAA) 4q31 MMAA encodes a protein that may be involved in the translocation of vitamin B12 into the mitochondria [88]. In addition, MMAA could play an important role in the protection and reactivation of Methylmalonyl-coA mutase (MCM) in vitro [90].
Citrate lyase beta like (CLYBL) 13q32.3 It encodes a human mitochondrial enzyme, which is co-expressed with other co-enzymes in the mitochondrial B12 pathway [111].
synthesizing α(1,3) fucosylated glycans, which act as a biological interface for the host-microbial interaction [47]. 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 [22,48]. 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 [22].
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 [56]. 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 [5].
Genes that code for membrane transporters that actively facilitate membrane crossing
Studies of the association between vitamin B12 status and the variants within CUBN have yielded conficting results. Hazra et al. [20] 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 [20]. This 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 ) [12]. In contrast, a study in 3114 Canadian individuals (85% Caucasian and 15% non-Caucasian) 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 −4 ) [18]. 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% [18]. On the other hand, Hazra et al. [20] and Grarup et al. [12] 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, [19] Canada [18], USA and Italy [21].
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 [67]. Cubilin has additionally been suggested to function together with megalin (LRP2, chromosome 2) [68], 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 [20]. 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 lysosomes [69]. It has been shown that polymorphisms of the ABCD4 gene affect the functioning of the ABCD4 protein and the intracellular processing of vitamin B12 [70].
There has been only one study to date investigating the association between vitamin B12 status and ABCD4 variants. Grarup et al. [12] examined 45,571 Nordic adults and 25,960 Icelandic adults in a GWA study [12], 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 cytoplasm [70,71].
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 [18]. 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 −59 ) [12]. A previous study has shown that this polymorphism is associated with the maternal risk of developing neural tube defects [62]. Cell culture models have shown that SNPs in the CD320 receptor can lead to a reduction in vitamin B12 uptake [72].
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 [73] and codes for a critical enzyme involved in homocysteine remethylation. MTHFR catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate in an irreversible reaction [74]. 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 [75,76].
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 [77,78], North European [28], French [79], Norweigian [80] and Spanish [81] 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 [57]. 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 [82].

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 state [83]. Currently, four SNPs, rs162036, rs162048, rs1532268 and rs3776455, have shown associations with vitamin B12 levels in healthy individuals [13].
The first SNP MTRR rs162036 (Lys350Arg) is a missense polymorphism [84], 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 available) [13]. 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 concentrations.

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 [85]. 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 [19], 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 ) [19]. Another study investigated this SNP in 37,283 Icelandic individuals but found no statistical significance (β = 0.214 pmol/l, P = 0.075) [12].
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 [86]. However, data from animal studies have demonstrated that MS4A3 is restricted to differentiating cells in the central nervous system and hematopoietic cells [87].
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) [13]. More 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 ) [22]. 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 ) [12]. While these SNPs might be involved with determination of vitamin B12 concentrations, their precise biochemical role is unknown.

Methylmalonyl-CoA mutase (MUT)
The MUT gene also known as the methylmalonyl-CoA mutase is located on chromosome 6. The MUT gene provides instructions for the formation of methylmalonyl-CoA mutase (MUT), which is a mitochondrial enzyme. MUT acts as a catalyst which isomerizes methylmalonyl-CoA to succinyl-CoA [91]. MUT requires 5-primedeoxyadenosylcobalamin (AdoCbl), which is a form of B12 that works with MUT to form succinyl-CoA. Succinyl-CoA participates in the TCA cycle (tricarboxylic cycle) to yield energy [92]. The MUT gene is involved in homocysteine metabolism, and it is dependent on vitamin B12 for its function [93]. 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].

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 activity [19]. 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 [94].
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 [94]. As a result, Lin et al. [19] proposed that the rs41281112 variant interferes with the binding of CLYBL protein to metal ions, potentially leading to a lower uptake of vitamin B12 [19].

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 [95]. We now know that genetic factors also influence vitamin status in individuals [96]. 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 populations [27,97]. In addition, a recent systematic review showed that B12 deficiency is common during pregnancy in other populations where vegetarianism is rare [98]. 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 [99,100].
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 populations [19,22]. 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 [101]. 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) [102]. The 428G→A polymorphism (rs601338) is the characteristic for the nonsecretor allele in Europeans and appears in about 20% of the Caucasian population [103]. 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 [104].

Conclusion
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.