Genetic disorders of vitamin B 12 [601296]

Genetic disorders of vitamin B 12
metabolism: eight complementation
groups –eight genes
D. Sean Froese1and Roy A. Gravel2,*
Vitamin B 12(cobalamin, Cbl) is an essential nutrient in human metabolism.
Genetic diseases of vitamin B 12utilisation constitute an important fraction of
inherited newborn disease. Functionally, B 12is the cofactor for methionine
synthase and methylmalonyl CoA mutase. To function as a cofactor, B 12must
be metabolised through a complex pathway that modifies its structure andtakes it through subcellular compartments of the cell. Through the study ofinherited disorders of vitamin B
12utilisation, the genes for eight
complementation groups have been identified, leading to the determination of
the general structure of vitamin B 12processing and providing methods for
carrier testing, prenatal diagnosis and approaches to treatment.
Vitamin B 12, also known as cobalamin (Cbl), is a
micronutrient that is synthesised only bymicroorganisms, yet is essential to human
health. Cobalamin was first isolated by Smith
(Ref. 1) and Rickes (Ref. 2), after Minot andMurphy (Ref. 3) showed that pernicious
anaemia could be treated with oral liver extract.
Later, vitamin B
12deficiency as a result of
genetic disease was described despite adequate
vitamin intake (Ref. 4). Some patients responded
successfully to very high doses of vitamin B 12,
suggesting blocks in vitamin processing. These
patients had homocystinuria and /or methylmalonic
aciduria, implicating dysfunctional methioninesynthase (MS) and /or methylmalonyl-CoA mutase
(MUT or MCM). We now know that blocks inthe intracellular processing of cobalamin into
cofactor forms, methylcobalamin (MeCbl) forMS and adenosylcobalamin (AdoCbl) for MCM,
or in the functional activity of MS or MCM
result in inborn errors. These genetic blocks maybe devastating in newborns or in early
childhood. Understanding the genes, gene
products and subcellular transport of vitaminB
12is important for minimising the disease
burden from these disorders. This review
outlines the present knowledge of cobalaminmetabolism, with a focus on steps related to the
intracellular human pathway and the initial
cataloguing of cobalamin-utilisation disordersinto complementation groups and biochemically
distinct classes. Key to these discoveries have
This manuscript is dedicated to the memory of Aarif Edoo.
1Structural Genomics Consortium, University of Oxford, Oxford, UK.
2Department of Biochemistry & Molecular Biology, Faculties of Medicine and Kinesiology, University
of Calgary, Calgary, Alberta, Canada.
*Corresponding author: Roy A. Gravel, Department of Biochemistry & Molecular Biology, Faculties of
Medicine and Kinesiology, University of Calgary, Room 250 Heritage Medical Research Building,
3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail: [anonimizat] reviews
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Genetic disorders of vitamin B 12metabolism:
eight complementation groups –eight genes

been the hundreds of patients who have been the
source of cell cultures and DNA samples that havegiven us our current understanding of vitamin B
12
utilisation in humans.
Vitamin B 12structure
The structure of cobalamin was first solved byHodgkin (Ref. 5) using x-ray crystallography. It
is a large organometallic molecule,
∼1300 –1500 Da in size, and is the most
chemically complex vitamin known. The focal
point of vitamin B
12is the central cobalt atom,
which has up to six ligands bound to it. Four ofthe ligands are the nitrogen atoms of the planar
corrin ring that surround the cobalt atom
(Fig. 1). The α-axial ligand, extending below
the corrin ring, is a nitrogen of the 5,6-
dimethylbenzimidazole (DMB) phosphoribosyl
moiety that also attaches back to the corrin ringthrough one of its propionamide side chains.
The upper or β-axial ligand varies, depending
on the modification state of cobalamin (R-groupin Fig. 1a). Functional β-axial ligands are methyl
(MeCbl) or 5
′-deoxyadenosyl (AdoCbl) groups.
Additionally, a hydroxyl group (OHCbl) or acyano group (CNCbl) can be bound as
physiologically relevant β-axial ligands.
There are three important, inter-related factors
that contribute to cobalamin reactivity and
function: the oxidation state of cobalt; whether
the DMB is coordinated to cobalt in the loweraxial position; and the identity of the R-group
bound in the upper axial position. The cobalt
atom of cobalamin may exist in the +3
[cob(III)alamin], +2 [cob(II)alamin] or +1
[cob(I)alamin] oxidation state. AdoCbl, MeCbl,
CNCbl and OHCbl, all of which arecob(III)alamins, prefer to adopt a configuration
where the DMB nitrogen base is coordinated to
the cobalt in the lower axial position (referred toas ‘base-on ’) (Fig. 1). Some enzymes, however,
are able to shift these cob(III)alamins to the
‘base-off ’configuration. Interestingly, MS and
MCM, which use MeCbl and AdoCbl as
cofactors, respectively, bind the cobalamin so
that the DMB nitrogen is displaced from thecobalt and replaced by a histidine of the enzyme
(Fig. 1b). This type of binding is considered
‘base-off /His-on ’and is important for the
catalytic activity of the enzymes. Cob(II)alamin
generally has no R-group bound, binding only
DMB as the lower axial ligand to make its
preferred five-coordinate state. However, in thepresence of ATP , MMAB, the human
adenosyltransferase (ATR) enzyme, is able tobind cob(II)alamin in a novel four-coordinate
state, where neither axial position is occupied
(Ref. 51). Cob(I)alamin usually has no axialligand. It is a highly reactive nucleophile that
very easily oxidises to cob(II)alamin (Ref. 52).
The challenge to the cell is how to productivelyproduce cob(I)alamin, as in the reaction cycle of
MS, without exposing the local environment to
nucleophillic attack and risk of free-radicaldamage. The nature of the axial ligands also
affects the ease with which the central cobalt is
reduced. Strongly coordi nating ligands stabilise
cobalt against reduction, whereas weakly
coordinating ligands allow cobalt to be reduced
more easily (Ref. 53). Base-on cobalamin fallsinto the former category, protecting the cobalt
from reduction because DMB has greater
electron-donating character than the H
2O
molecule that binds in its absence (Ref. 54).
Vitamin B 12origins
Vitamin B 12is an extremely old molecule in
evolution. It has even been suggested that B 12
was synthesised prebiotically (Ref. 55).
Accordingly, vitamin B 12utilisation is dispersed
throughout evolution, occurring in bothEukaryota and Prokaryota, perhaps having been
passed on from the ‘breakthrough organism ’–
the last organism to use RNA as the solegenetically encoded catalyst (Ref. 56).
Interestingly, although cobalamin utilisation is
distributed widely among phyla, cobalaminsynthesis seems limited to only a select few
Archaea and Eubacteria. Perhaps this is because
the synthesis of cobalamin is so complex –
involving in excess of 25 steps, which can
proceed either aerobically ( cob genes) or
anaerobically ( cbigenes) (Ref. 57). Therefore,
although mammals and other higher organisms
require vitamin B
12for life, they ostensibly
acquire it from their prokaryotic counterparts.Another interesting facet of the evolution of
cobalamin is that vitamin B
12users seem more
scattered than logically spread out throughevolution, with whole phyla sometimes gaining
or losing vitamin B
12dependency (Ref. 58).
Additionally, although mammals and otherhigher eukaryotes are restricted to two
cobalamin-dependent enzymes, MS and MCM,
prokaryotes use a plethora of enzymes requiring
this cofactor. These enzymes include threeexpert reviews
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Genetic disorders of vitamin B 12metabolism:
eight complementation groups –eight genes

classes of AdoCbl-dependent mutases, the
isomerases (e.g. MCM, ribonucleotide reductase,
glutamate mutase), the eliminases (e.g. dioldehydratase) and the aminomutases (e.g.
D-
lysine-5,6-aminomutase), as well as the
MeCbl-dependent methyltransferases (e.g. MS)and the vitamin B
12-dependent reductive
dehalogenases (e.g. 3 –6 chloro-4-hydroxybenzoate
dehalogenase) (Refs 59, 60). Therefore, becausehigher eukaryotes share common vitamin B
12
ancestry with prokaryotes, but have apparentlylimited use of the vitamin and no biosynthesis,prokaryotes in general and a few specific bacteria
andArchaea in particular have proved to be veryuseful models to understand vitamin B
12
metabolism.
Human vitamin B 12ingestion and
absorption
Because vitamin B 12is made by only a few
microorganisms, it is acquired through dietaryuptake in animals. Human dietary sources
include milk, eggs, fish and meat in quantities in
excess of a few micrograms a day (Ref. 61). Inhumans, the absorption, transport and cellular
uptake of cobalamin is complex. Food-bound
cobalamin is released in the stomach with the
help of peptic activity, where it is subsequently
a
b
Structure of vitamin B 12 (cobalamin)
Expert Reviews in Molecular Medicine © Cambridge University Press 2010
Figure 1. Structure of vitamin B 12(cobalamin). (a) The ‘R group ’corresponds to substitutions at the upper or
β-axial ligand (5′-deoxyadenosyl-, methyl-, hydroxo-, cyano-). The dimethylbenzimidazole constituent (DMB)
is shown coordinated to the cobalt in the lower α-axial position ( ‘base-on ’structure). DMB is linked to the corrin
ring through a phosphoribosyl attached to a propionamide side chain. (b) Structure of methylcobalamin(MeCbl) with DMB displaced from the cobalt by a histidine residue in methionine synthase (MS; the ‘base-
off/His-on ’structure). A similar configuration is observed for adenosylcobalamin (AdoCbl) bound to
methylmalonyl-CoA mutase. Structures are from http://www.genome.jp using the ‘SIMCOMP Search ’utility
(query C00576, vitamin B
12; C06410, MeCbl-MS).expert reviews
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Genetic disorders of vitamin B 12metabolism:
eight complementation groups –eight genes

bound by haptocorrin (HC) (Ref. 62). In the small
intestine, cobalamin is released from HC bypancreatic protease digestion and bound by
intrinsic factor (IF) to form an IF –Cbl complex.
IF is very specific for cobalamin (i.e. for formsthat have the lower DMB intact) and
presumably acts as an early screening
mechanism to prevent degraded cobalaminsfrom intracellular access (Ref. 63). The IF –Cbl
complex passes through the small intestine,
where it is bound on the apical surface of ilealepithelial cells by a receptor composed of a
heterodimer of amnionless and cubilin, called
cubam, which aids in the endocytosis of IF –Cbl
(Refs 64, 65). Once inside the cell, IF is degraded
in the lysosomes and cobalamin is released into
the cytosol (Ref. 66), where it is transportedacross the ileal receptor cell and released into
the bloodstream, possibly by the recently
identified (with respect to vitamin B
12transport)
multidrug resistance protein MRP1 (Ref. 67). In
the bloodstream, cobalamin binds to either HC
or transcobalamin (TC) (Ref. 68). Although HCbinds the bulk of plasma cobalamin (75 –90%), it
is not involved in cellular cobalamin uptake
apart from uptake in hepatocytes (Ref. 68).Therefore, individuals who have deficient or
absent HC have serum cobalamin values in the
deficient range, but show no sign of cobalamindeficiency (Ref. 61). Although TC binds only a
minor fraction of circulating cobalamins
(10 –25%), it is the protein responsible for
facilitating the uptake of cobalamin by cells
(Refs 69, 70). Mutations in the gene encoding TC
(TCN1 ) result in severe tissue cobalamin
insufficiency, megaloblastic anaemia, failure to
thrive and often neurological complications,
despite normal plasma cobalamin concentrations(Refs 71, 72). Additionally, TC acts as a final
screening mechanism because, like IF, TC is very
specific for cobalamin forms that have the lowerDMB intact (Refs 73, 74). Treatment of TC
deficiency requires very high serum cobalamin
levels, ranging from 1000 to 10 000 pg /ml,
achieved by oral or intramuscular delivery of
0.5–1.0 mg of CNCbl or HOCbl once or twice
weekly (Refs 60, 75). There is some evidencethat at sufficiently high concentrations, at least
some tissues are capable of taking up unbound
cobalamin (Ref. 61). From the bloodstream,cobalamin is taken up into cells through
receptor-mediated endocytosis as a complex of
Cbl –TC bound to the TC receptor (TCblR)(Refs 76, 77, 78). A mutation in the gene
encoding TCblR ( CD320 ) was recently identified
in asymptomatic newborns whose fibroblasts
showed decreased Cbl uptake, where restoration
of the missing codon by site-directedmutagenesis (c.262 –264) resulted in normal
TCblR function (Ref. 79). In the lysosome, the
Cbl –TC complex is digested to create free
cobalamin, which is subsequently transported
into the cytosol probably as a mixture of
cob(III)alamin and cob(II)alamin. Once in thecytosol, cobalamin is processed by many
proteins, some known, others perhaps still
unknown, to produce the cofactors MeCbl andAdoCbl. Failure to produce the cobalamin
cofactors results in a lack of functional enzymes
and causes the constellation of biochemical,developmental and neurological manifestations
associated with intracellular pathway defects.
Complementation analysis for cataloguing
the intracellular pathway of vitamin B 12
disorders
The considerable range of clinical and biochemical
heterogeneity observed in patients with vitaminB
12pathway disorders led to a need to sort
them into genetically defined groups. The
question of whether severe and mild disease orB
12-responsive and -unresponsive forms could
be explained by mutations in different genes
was addressed early on by complementationanalysis. This is a powerful technique that
permits the identification of specific genes
through their expression in fibroblastheterokaryons, multinucleate cells produced by
the fusion of fibroblast strains from different
patients, which could then be tested forrestoration of function. To examine
complementation, the incorporation of
[
14C]propionate or [14C]methyltetrahydrofolate
[or [14C]formate to methionine and serine
(Ref. 22)] into trichloroacetic acid (TCA)-
precipitable material was monitored byautoradiography of cells in situ or by direct
scintillation counting of the TCA precipitate
(Refs 80, 81). Initially, four distinctcomplementation groups were identified,
cblA–cblC and mut. However, over the years, the
method came to be used diagnostically withhundreds of cell lines being analysed, mainly in
the McGill University laboratory of David
Rosenblatt, which became a dominant
diagnostic centre using these techniques. So far,expert reviews
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Genetic disorders of vitamin B 12metabolism:
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eight complementation groups, cblA–cblG and
mut, have been described that have blocks in the
production or utilisation of MeCbl, AdoCbl or
both cofactors. Although all the genes
corresponding to these disorders have now beendescribed, many of their functions remain
unclear. Figure 2 illustrates the known or
predicted functional location of the proteinproducts of these genes. Three complementationgroups –cblF,cblC and cblD –correspond to
blocks in steps that are common to the synthesisof both cofactors with resulting deficiency of MS
and MCM activities. Patients from these groups
have combined homocystinuria andmethylmalonic aciduria. Three groups, cblD
variant 1, cblE and cblG, have blocks in the
cytosolic pathway leading to MeCbl synthesis orapo-MS and result in deficient MS activity and
Methylmalonic aciduria
Methylmalonyl-CoA mutase– AdoCbl
Methionine synthase– MeCblAdoCbl
MeCblCob(II) Cbl-R TC-Cbl-RLysosomeCytosolMitochondrion
cblA::mut
cblE::cblGcblB
cblD
cblD-1cblCMethylmalonyl-CoA Succinyl-CoA
Methionine
HomocystinuriaCH3-THF+
Homocysteine
Intracellular processing of vitamin B 12 showing sites of defects in
complementation groups
Expert Reviews in Molecular Medicine © Cambridge University Press 2010cblD-2 (cblH )cblF
Figure 2. Intracellular processing of vitamin B 12showing sites of defects in complementation groups.
Complementation groups are in blue and are positioned at sites of metabolic blocks (shown in red).Cobalamin intermediates are in red. Excreted metabolites due to genetic defects are in shaded boxes.Pathway details are described in the text. In the lysosome, cobalamin is released from transcobalamin (TC)through its degradation (arrow pointing to dots). In the cytosol, R groups are released by the cblC protein
with the cob(II)alamin [Cob(II)] product remaining bound (dotted line emanating from the cblC protein
denotes complex with cobalamin forms). The three versions of the cblD protein ( cblD ,cblD-1 ,cblD-2 )
illustrate the role of the protein in directing cobalamin to the mitochondrial or cytosolic pathway. In themitochondrion, the cblB protein adds the 5
′-deoxyadenosyl group, generating the active cofactor
[adenosylcobalamin (AdoCbl)], which is transferred to the mut [methylmalonyl-CoA mutase (MCM)] protein.
The cblA protein is proposed to act as a gatekeeper to ensure that the cofactor form that is accepted andretained by MCM is AdoCbl. In the cytosolic pathway, cob(II)alamin is bound to the cblG [methionine
synthase (MS)] protein. The cblE [methionine synthase reductase (MSR)] protein catalyses generation of the
active cofactor, methylcobalamin (MeCbl), or its regeneration if oxidised to cob(II)alamin during reaction cycles.expert reviews
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Genetic disorders of vitamin B 12metabolism:
eight complementation groups –eight genes

homocystinuria. The final groups, cblD variant 2,
cblA,cblB and mut, affect steps occurring in the
mitochondrion leading to AdoCbl synthesis or
apo-MCM and result in deficient MCM activity
and methylmalonic aciduria.
Complementation groups affecting both
methionine synthase and methylmalonyl-
CoA mutase
cblF
cblF was initially assigned to describe an infant
with developmental delay and mild
methylmalonic aciduria who was vitamin B 12
responsive (Ref. 82). Her fibroblasts were found
to accumulate free cobalamin, failing to attach to
MS or MCM. By electron microscopy andsubcellular fractionation, it was shown that most
of the cobalamin was trapped in the lysosome,
with only a small portion reaching the cytosol ormitochondria (Ref. 83). Complementation
studies confirmed the genetically distinct
disorder (Ref. 84). It was proposed that cblF
represents a defect in the export of cobalamin
out of the lysosome into the cytosol (Ref. 82).
The gene responsible for cblF was very recently
cloned by homozygosity mapping and
microcell-mediated chromosome transfer and
was found to correspond to LMBRD1 , which
encodes the lysosomal membrane protein
LMBD1 (Ref. 27). LMBD1 shares homology with
a family of membrane proteins that internaliselipocalins, which are carriers of small
hydrophobic molecules such as steroids and
lipids, leading to the suggestion that a lipocalin-like molecule may bind vitamin B
12in the
lysosome on its release following TC
degradation (Ref. 27). Transfection of cblF
fibroblasts with intact cDNA corrected
intracellular cobalamin processing and restored
functional MS and MCM. The nature of theprotein, with nine predicted transmembrane
domains, and localisation of the protein to the
lysosomal membrane, is consistent with a role inthe export of cobalamin from the lysosome.
Only 13 patients have been described with cblF
(Refs 27, 28). Six mutations have been identified,and, interestingly, all of them have been chain-
terminating frameshift mutations, with one,
1056delG, accounting for 18 of 26 analysedalleles (Fig. 3). Clinically, it is a highly variable
disorder. Most patients show failure to thrive in
infancy and mild to severe developmental delay,
but respond to vitamin B
12therapy. Althoughmost patients presented with homocystinuria
and methylmalonic aciduria, the index case,mentioned above, showed no evidence of
megaloblastic anaemia or homocystinuria. This
individual was reportedly asymptomatic on B
12
therapy in adulthood, despite being homoallelic
for the 1056delG mutation (Ref. 27).
cblC
The cblC complementation group originally
corresponded to the first set of patients who
failed to produce either AdoCbl or MeCbl
(Ref. 85). Since then, approximately 400 patientshave been described with cblC, making it the
most common disorder of intracellular vitamin
B
12metabolism (Ref. 14). The gene responsible
for the cblC group, called MMACHC ,w a s
identified by homozygosity mapping in 2006
(Ref. 15). More than 50 different disease-causingmutations have been identified and are
summarised in Ref. 14 (Fig. 3). The most
common is the c.271dupA mutation, whichcauses a frameshift truncation, accounting for
42% of pathogenic alleles (Ref. 14). Additionally,
the c.394C >T (R132X) and c.331C >T (R111X)
mutations are found commonly, at 20% and 5%
of alleles, respectively. Although all cblC patients
have combined homocystinuria andmethylmalonic aciduria and often have
haemotological, neurological and ophthalmic
abnormalities to some degree, they tend to fallinto either of two distinct phenotypes
correlating with age of onset (Ref. 86). Early-
onset patients present in the first year of lifewith severe disease and rarely respond clinically
to treatment, whereas late-onset patients present
in childhood to adulthood, are more likely tohave less severe symptoms, and usually respond
better to treatment (Ref. 86). A strong
genotype –phenotype correlation can be found
with some mutations: the c.271dupA and
c.331C >T (R111X) mutations usually cause the
much more prevalent early-onset disease,whereas some missense mutations [e.g. c.482G >
A (R161Q)] and, bewilderingly, the c.394C >T
(R132X) nonsense mutation usually result inlate-onset disease (Refs 14, 15). The cblC protein
(MMACHC) was predicted to have a vitamin
B
12binding site and a TonB-like domain; the
latter is a protein associated with bacterial
cobalamin uptake (Ref. 15). Although initial
studies suggested that MMACHC had base-on
CNCbl binding (Ref. 87), it was laterexpert reviews
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Genetic disorders of vitamin B 12metabolism:
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Mutations in the genes underlying the defects of the eight complementation
groups
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Figure 3. Mutations in the genes underlying the defects of the eight complementation groups. (See next
page for legend. )expert reviews
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Genetic disorders of vitamin B 12metabolism:
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demonstrated to bind CNCbl in the base-off
conformation (Ref. 88) and to reductively cleave
the CN group to form MMACHC-bound
cob(II)alamin (Ref. 87). It has also been shownto catalyse glutathione-dependent dealkylation
of cobalamins containing C2 –C6 alkanes, Ado-,
or Me- as the upper axial ligand (Ref. 89). Theseresults suggest that MMACHC might be
involved in intracellular cobalamin transport
and reductive dealkylation or decyanation,perhaps interacting with the cblF protein for
export of cobalamin out of the lysosome and
beginning the initial processing of cobalamin,yielding cob(II)alamin, before passing it along
for distribution to the rest of the pathway
(Refs 14, 62) (Fig. 2).
Late-onset cblC patients are cobalamin
responsive but, as observed initially in
fibroblasts and later in patients, they respondpoorly to CNCbl compared with OHCbl, a
phenomenon that is unique among the vitamin
B
12disorders (Refs 86, 90, 91, 92). Investigations
of cobalamin binding by wild-type and mutant
protein, the latter containing the R161Q
mutation commonly associated with OHCblresponsiveness in cblC patients, revealed
reduced binding of CNCbl but not OHCbl by
the mutant protein compared with the wild type(Ref. 93). Further, thermolability studies showed
that MMACHC protein is strongly stabilised by
cobalamin binding, whereas mutant protein wasmuch less stabilised and only minimally or not
at all with CNCbl (Ref. 87). These results
suggest that OHCbl responsiveness in patientswith the R161Q mutation is due to a
combination of better affinity for OHCbl than
CNCbl and a much better stabilisation of themutant protein by OHCbl. It may well be that
the high-dose OHCbl treatment generally used
with cblC patients protects the protein from
degradation in vivo. An interesting outcome of
these studies was the finding that the cobalamin
cofactors, AdoCbl and MeCbl, were far moreprotective of the mutant protein than OHCbl.
Although treatment with AdoCbl or MeCbl has
been tried before, it was found that they are not
incorporated directly as cofactors of theircognate enzymes but are dealkylated and
processed anew, results that have been
confirmed in vitro with MMACHC (Refs 89, 94).However, the increased stabilisation of mutant
protein afforded by the cofactors suggests that
their use should be re-examined in some cases.
cblD
The cblD complementation group was first
described in two siblings with combined
homocystinuria and methylmalonic aciduria anddeficiency of MCM and MS activities (Ref. 95),
although the designation ‘cblD ’was not given
until many years later (Ref. 81). For over 25years, they remained the only cblD patients
described, and biochemical analysis revealed
that only this complementation group seemed tobehave in a manner similar to cblC, but with less
severe defects (Ref. 91). However, in 2004,
Suormala et al. (Ref. 96) described three newcases of cblD: two had only MS deficiency
(called cblD variant 1) and one had only MCM
deficiency ( cblD variant 2). These results
suggested that the cblD protein might be
responsible for branching of the cobalamin
metabolism pathways to the cytosolic ormitochondrial compartments (Fig. 2). The same
group cloned the cblD gene 4 years later
(Ref. 20), naming it MMADHC . With an
additional four patients they showed a clear
genotype –phenotype relationship, whereby
truncation mutations in the 5
′region resulted in
only methylmalonic aciduria (MCM deficiency),
truncation mutations in the middle and 3′
regions resulted in combined methylmalonic
aciduria and homocystinuria (MCM and MS
deficiency), and missense mutations in the 3′
region resulted in only homocystinuria (MSdeficiency) (Fig. 3). Transfection experimentsFigure 3. Mutations in the genes underlying the defects of the eight complementation groups. (See
previous page for figure. ) For each complementation group, the gene name is given in brackets. Mutations
are shown as cDNA position with corresponding amino-acid change in brackets. The numbering for each isbased on the cDNA sequence: +1 corresponds to the A of the ATG translation initiation codon. Nonsense
and frameshift (fs) mutations are displayed above the gene whereas missense and possible splice site orcryptic splice site (ss) mutations are displayed below. Mutations are based on cblA (Refs 6, 7, 8, 9, 10),
cblB (Refs 11, 12, 13), cblC (Refs 14, 15, 16, 17, 18, 19), cblD (Refs 20, 21), cblE (Refs 22, 23, 24, 25, 26),
cblF (Refs 27, 28), cblG (Refs 29, 30, 31, 32) and mut (Refs 9, 13, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50).expert reviews
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Genetic disorders of vitamin B 12metabolism:
eight complementation groups –eight genes

demonstrated correction of the defect in mutant
fibroblasts. In particular, a cDNA constructedwith a 5
′-truncation mutation could correct the
synthesis of MeCbl, suggesting that an internal
translation initiation site is probably functional.Additionally, they demonstrated that the cblH
complementation group, which had been
previously described for one patient withunidentified methylmalonic aciduria (Ref. 97),
was actually an example of cblD variant 2
(Fig. 2). MMADHC has a predictedmitochondrial leader sequence and a putative
vitamin B
12binding sequence and shows limited
homology to a bacterial ABC transporter(Ref. 20). Although no functional or biochemical
data are yet available for MMADHC, it is
currently speculated to interact with MMACHCas part of a chaperone role to present cobalamin
to the cytosolic or mitochondrial pathways
(Refs 20, 62).
Complementation groups affecting only
methionine synthase
In 1984, Schuh and colleagues (Ref. 98) described
an infant with homocystinuria, megaloblastic
anaemia and developmental delay who,
although treatable with cobalamin, showed noevidence of methylmalonic aciduria, a novel
outcome at the time. This suggested that the
infant had a unique block in MS or the synthesisof MeCbl. Subsequently, two other patients were
described with the same symptoms (Refs 99,
100). Broken cell extracts from the originalpatient revealed that with added reducing
agents, MS worked perfectly. However, this was
not the case for MS from the next two patients.Their cell extracts were defective regardless of
additions. Ultimately, Watkins and Rosenblatt
(Ref. 101) used complementation analysis toshow that the original patient, designated as
cblE, had a block in a separate genetic locus to
the other two patients, which they designated ascblG (Fig. 2).
cblG
The gene responsible for cblG was cloned by
three separate groups based on the
identification of human sequences homologous
to the E. coli vitamin-B 12-dependent MS,
encoded by the metH gene, and other bacterial
and Caenorhabditis elegans sequences (Refs 29,
102, 103). The human gene is designated
MTR , for methyltransferase, as it wasnamed when initially mapped to human
chromosome 1 (Ref. 91), or more formallyas 5-methyltetrahydrofolate:homocysteine
methyltransferase. Twenty different mutations
have been identified in MTR (Fig. 3). The most
common is c.3518C >T (P1173L), which is
present in 16 of 24 cblG cell lines surveyed
(Ref. 30). The clinical disease is highlyvariable, with onset ranging from neonatal to
adulthood, although most patients present
with homocystinuria, hypomethioninaemia,megaloblastic anaemia and developmental delay
(Refs 101, 104, 105, 106). In addition to its
importance in protein synthesis, MS is a keyenzyme of the methionine cycle, which maintains
the cellular level of the methionine derivative, S-
adenosylmethionine, the methyl donor in a widearray of cellular processes including DNA, RNA
and protein methylation. It is also uniquely
involved in the folate cycle, because it is the onlymammalian enzyme to use 5-
methyltetrahydrofolate as a methyl donor
(Ref. 30). MS catalyses the methylation ofhomocysteine to form methionine in a reaction
that requires the presence of enzyme-bound
MeCbl for activity (Refs 107, 108). The reactionproceeds by methyl transfer from 5-
methyltetrahydrofolate to MS-bound
cob(I)alamin to form MeCbl, followed by transferof the methyl group to homocysteine to form
methionine and regeneration of cob(I)alamin
(Refs 109, 110). Mammalian MS and Escherichia
coli MetH are 55% identical (Ref. 103). The
sequence homology extends to the domain
structure of MetH in which linearly arrayeddomains of the enzyme contain binding sites for
the various substrates and cofactors (Refs 111,
112, 113). These domains seem to be faithfullymaintained in mammalian MS.
cblE
Because the reaction catalysed by MS regeneratescob(I)alamin in every reaction cycle, the cofactorrisks occasional oxidation to cob(II)alamin with
consequent inactivation of the enzyme
(Ref. 114). In E. coli , the restoration of a
functional cofactor is dependent on two
flavoproteins, flavodoxin and flavodoxin
reductase, containing flavin mononucleotide(FMN) and flavin adenine dinucleotide (FAD)
prosthetic groups, respectively (Refs 115, 116).
The corresponding human reducing system
proved to be encoded by a single gene, MTRR ,expert reviews
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Genetic disorders of vitamin B 12metabolism:
eight complementation groups –eight genes

which is mutated in cblE patients (Fig. 3); it
encodes a single protein containing FMN- andFAD/NADPH-binding sites (Ref. 117). This
enzyme, named methionine synthase reductase
(MSR), is a linear array of ‘flavodoxin ’at the N-
terminus, an intervening linker sequence in the
middle and ‘flavodoxin reductase ’at the C-
terminus. It restores MS activity by catalysingthe reductive methylation of cob(II)alamin on
the inactivated MS in which S-
adenosylmethionine is the source of the methylgroup (Refs 118, 119). Additionally, MSR has
been shown to catalyse the reduction of free B
12
[as aquocob(III)alamin] to cob(II)alamin
(Ref. 120), making it an aquocobalamin
reductase and possibly functioning as such in
the cytosolic pathway. MSR has also beensuggested to function as a cob(II)alamin
reductase in the mitochondrial pathway. This
hypothesis was based on a potentialmitochondrial leader sequence found by
alternative splicing in the MTRR gene, as well as
in vitro results demonstrating the production ofcob(I)alamin through physical interaction with
the mitochondrial MMAB protein (Refs 117,
121). However, evidence demonstrating MSRexpression only in the cytosol (Ref. 122) and
separate evidence suggesting that MMAB does
not require a distinct cobalamin reductase(Ref. 123) have strongly countered this
argument. The clinical presentation of cblE
patients is similar to that of cblG patients. It is
usually impossible to separate the two
disorders, except for one patient who
unexpectedly also had methylmalonic aciduria(Ref. 105), which remains unexplained.
Complementation groups affecting only
methylmalonyl-CoA mutase
Patients with methylmalonic aciduria without
elevated homocysteine or abnormalities of
circulating vitamin B 12have defects in the
mitochondrial pathway of AdoCbl synthesis andfunctional MCM. Clinically, patients have the
following common features: failure to thrive,
lethargy, vomiting of protein feeds, dehydration,respiratory distress and hypotonia (Ref. 124).
Early on, three genes were implicated in the
mitochondrial pathway, corresponding tocomplementation groups cblA,cblB and mut
(Refs 80, 85) (Fig. 2). A detailed understanding
of the mitochondrial processing of vitamin B
12
is only now beginning to emerge, basedlargely on studies completed on bacterial model
systems.
cblA
cblA was initially the designation for patient
fibroblasts that failed to accumulate AdoCbl in
intact cells, but showed restored AdoCblsynthesis in a broken-cell assay with OHCbl,
ATP and a reducing system (Ref. 85). This
block was hypothesised to correspond to adefect in mitochondrial cob(II)alamin
reductase because of the ability of an external
reducing system to alleviate the block (Ref. 85).The human gene, named MMAA , was identified
in a search for genes clustered in proximity to
MCM ( mut) in microbial genomes and while
searching for orthologous sequences in the
human genome (Ref. 6). Examination of the
sequence of MMAA , however, revealed that it
did not encode a reductase, but rather a protein
that belonged to the G3E family of P-loop
GTPases, a group of proteins that participate inthe assembly or function of the metal centres in
metalloenzymes (Ref. 6). More than 30 cblA
patient mutations have been described in theMMAA gene, with most of them corresponding
to nonsense or frameshift mutations (Fig. 3).
Although most cblA patients present in infancy
or childhood with methylmalonic aciduria
and potentially life-threatening acidotic crises
(Ref. 104), they often respond to vitamin B
12
therapy despite the severity of mutations
(Refs 104, 124, 125). Reasons for this are
becoming clearer because of our increasedunderstanding of the role of MMAA. Studies
have focused on the bacterial orthologues of
MMAA, including key research by Banerjee andcolleagues working with MeaB, the MMAA
orthologue from Methylobacterium extorquens .
Initial bacterial studies showed that MMAAorthologues form a complex with MCM and
that GTP binding and hydrolysis contribute to
cobalamin processing (Refs 126, 127, 128).Further, it was shown that MeaB protects MCM
from inactivation and that the state of MeaB
(apo, GDP or GTP bound) alters the affinity ofMCM for AdoCbl (Ref. 129). Finally, recent
studies suggest that MeaB acts as a regulator of
MCM cofactor binding and ejection, where thebinding and hydrolysis of GTP by MeaB are
important in the discrimination of MCM
binding to AdoCbl versus cob(II)alamin, and
promotes ejection of the latter, inactive cofactorexpert reviews
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Genetic disorders of vitamin B 12metabolism:
eight complementation groups –eight genes

from MCM (Ref. 130). These studies are
summarised below in the proposed model forcobalamin processing.
cblB
Patients in the cblB group are also deficient in
AdoCbl synthesis and are metabolically similartocblA (Ref. 86). However, cblB patients tend to
present earlier, respond more poorly to vitamin
B
12and, consequently, may produce a more
severe clinical course with more profound
neurological and metabolic complications
(Refs 124, 125). The cblB disorder was separated
biochemically from cblA by the failure to
synthesise AdoCbl in broken cell extracts
containing a reducing system (Ref. 85). On thebasis of this early work, cblB had long been
expected to correspond to a defect in the
ATP:cob(I)alamin ATR. Like MMAA , the gene
was identified in the survey of MCM-containing
gene clusters in microbial genomes (see the
previous section) and was named MMAB
(Ref. 11). It was shown to correspond to an ATR
based on sequence and functional similarity
with a class of bacterial ATRs called PduO(Refs 11, 131). The MMAB protein catalyses the
transfer of the 5
′-deoxyadenosyl group of ATP
to cob(I)alamin to form AdoCbl. To assessfunction, cob(II)alamin is the usual cobalamin
added in vitro, with a reducing system, often
MSR and NADPH, added to facilitate thereaction. Unexpectedly, in the presence of ATP ,
MMAB binds cob(II)alamin in an unusual base-
off, four-coordinate state (Ref. 51). This unusualbinding elevates the redox potential of
cob(II)alamin to the physiological range of
possible reduction by reduced flavin, perhapsin the form of an electron transfer protein, thus
obviating the need for a specific cobalamin
reductase to generate the reactive cob(I)alaminintermediate (Ref. 123). Most of the mutations
identified in cblB disease were found to cluster
in exon 7, which encodes the active site of theenzyme (Refs 12, 132) (Fig. 3). Several
mutations have been mo delled in human and
microbial ATRs, identifying defects in substrateor cofactor binding, active site functions or
protein dynamics (Refs 132, 133, 134, 135, 136).
Interestingly, two of the mutations (R186W,E193K) resulted in absent protein in western
blots of patient cell extracts, suggesting
protein instability as a major contributor to
disease phenotype (Ref. 137). Human MMABh a sb e e nc r y s t a l l i s e di nt h ep r e s e n c eo fA T P
(Ref. 132). It was found to exist as a trimer withthree active sites, only two of which contained
ATP . Crystallisation in the presence of both ATP
and cob(II)alamin has been accomplished for theLactobacillus reuteri ATR ( LrPduO) (Ref. 135). The
two enzymes are highly similar and,
interestingly, AdoCbl was detected in LrPduO
crystals, underscoring the capacity of reduced
flavin, generated in the incubation mix, to drive
the four-coordinate cob(II)alamin intermediate tocob(I)alamin and the formation of AdoCbl.
mut
Themutcomplementation group is representative
of mutations in the MUT (MCM) gene. MCM is
important for the metabolism of branched-chain
amino acids, odd-chain fatty acids and
cholesterol (Ref. 104). It catalyses the reversibleisomerisation of
L-methylmalonyl-CoA to L-
succinyl-CoA (Fig. 2), which is important for the
breakdown of propionate and for replenishingthe tricarboxylic acid cycle. The MUT gene was
the earliest of the human B
12pathway genes to
be cloned, accomplished by antibody screeningof a human liver λgt11 expression library
(Ref. 138). Nearly 200 disease-causing mutations
have been identified in MUT (Ref. 33) (Fig. 3).
Two distinct classes of mutations have been
described: mut
−(‘mut-minus ’), when there is
residual enzyme activity or detectable [14C]
propionate incorporation by mutant fibroblasts;
and mut0(‘mut-zero ’), when protein or enzyme
activity is not detected, as found, for example, inframeshift or chain-terminating mutations and
some amino-acid substitutions (Refs 139, 140).
Unsurprisingly, these two groups separatepatients clinically. mut
0patients have a
higher occurrence of morbidity, mortality and
neurological complications than mut−,a n d mut−
patients are more responsive to B 12therapy
(Ref. 124). MCM from Proprionibacterium shermanii
has been crystallised in the presence of AdoCbland substrate (PDB accession number 4REQ)
( R e f s1 4 1 ,1 4 2 ,1 4 3 ) .T h eh u m a ne n z y m e( P D B
accession number 3BIC) is structurally similar totheP . shermanii enzyme, with which it shares 60%
identity in the α-subunit (Ref. 144). Human MCM
exists as a homodimer in the mitochondrialmatrix with 1 mol of AdoCbl bound per subunit
(Refs 145, 146). Studies of the Methylobacterium
extorquens enzyme, as described above, suggest
that human MCM does not exist alone butexpert reviews
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Genetic disorders of vitamin B 12metabolism:
eight complementation groups –eight genes

functions as a complex with other proteins, notably
MMAA and possibly MMAB.
Model for the intracellular processing of
vitamin B 12
The genes and proteins corresponding to all eight
complementation groups defined in patients haveprovided most of the elements required to describe
the intracellular processing of vitamin B
12(Fig. 2).
Cobalamin, generallyas OHCbl or CNCbl, is takeninto the lysosome as a Cbl –TC complex, where
digestion of the transcobalamin releases free
cobalamin. The cobalamin is transported intothe cytosol through the LMBD1 protein,
possibly drawn through the lysosomal
transporter by interaction of the cytosol face ofLMBD1 with the MMACHC protein. It appears
that cobalamins are bound to MMACHC in the
base-off state, poised for cleavage of the upperaxial ligand if one is present. MMACHC may
act as an intracellular cobalamin carrier for
delivery of the cofactor to the MMADHCprotein for targeting to the cytosolic (MS) or
mitochondrial (MCM) pathways. Evidence of
interaction of MMACHC with either LMBD1 orMMADHC has yet to be demonstrated. In the
cytosolic compartment, cob(II)alamin is expected
to be bound to MS, where it is reductivelymethylated by MSR, using NADPH as an
electron donor, to generate the MeCbl active
cofactor of MS. MMADHC also participates inthe targeting of cobalamin to the mitochondrial
pathway, although the specific transporter has
yet to be identified. Cob(II)alamin, on entry intothe mitochondrial matrix, is bound by MMAB
for the generation of AdoCbl, with the reducing
equivalents probably coming from an electrontransfer protein rather than a cobalamin
reductase, as had been previously anticipated.
The subsequent transfer of AdoCbl to MCM ispredicted to be an exquisitely complicated
process involving a complex of MMAA and
MCM and possibly MMAB.
First, the complex of MCM:MMAA –GTP
prevents the binding of cob(II)alamin, which
would otherwise inactivate MCM. Second,AdoCbl is transferred directly from MMAB to
the MCM –MeaB –GTP complex in a process
requiring ATP binding to MMAB and GTPhydrolysis by MMAA. Third, in reaction cycles
in which the radical 5
′deoxyadenosyl
intermediate is lost, leaving MCM with an
inactive cob(II)alamin cofactor, the GTP-boundMMAA causes displacement of the cofactor,
making the enzyme available for renewedAdoCbl binding. The proposed role for MMAA
derives principally from studies of MeaB by
Padovani and Banerjee (Refs 127, 129, 130). Thecrystal structure of MeaB has been determined
and carries the expected nucleotide-binding
domains and domains predicted to be involvedin MCM binding at the N-terminus and a
dimerisation domain at the C-terminus (PDB
accession number 4REQ) (Ref. 147). The recentlycrystallised human MMAA (PDB accession
number 2WWW), although it has a similar
overall structure, seems to adopt a slightlydifferent mode of assembly, which may have
implications for the three-way interaction with
MMAB and MCM.
Research in progress and outstanding
research questions
Although all eight genes predicted through
complementation analysis have been identified,additional genes are anticipated. Most notably,
the mechanism of the mitochondrial transport of
B
12remains unknown. The involvement of cblD
defects in both the cytosolic and mitochondrial
pathways suggests that the MMADHC protein
is an accessory to the mitochondrial uptake ofvitamin B
12. The lysosomal delivery of vitamin
B12to the cytosol might also involve additional
genes. The similarity of LMBD1 to the lipocalinfamily of membrane receptors predicts the
involvement of a lipocalin-like molecule that
might act as a vitamin B
12carrier after digestion
of the Cbl –TC complex in the lysosome.
Although a mitochondrial cobalamin reductase
cannot be fully ruled out, studies on PduO-typeATRs argue against such a protein in human
cells. Finally, if confirmed in human cells, the
MMAA –MCM or MMAA –MCM –MMAB
complex predicted by studies on MeaB proteins
might account for the gene set required for
mitochondrial cobalamin processing andutilisation. All these suggestions recognise the
absence of additional disease states among
human vitamin B
12processing disorders. The
general view is that unidentified genes would
probably not tolerate mutation (and therefore be
lethal embryonically), might be associated withshared functions (and therefore might not reveal
a vitamin B
12disease phenotype) or might be
redundant with genes encoding proteins of
similar function (allowing a bypass of a geneticexpert reviews
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Genetic disorders of vitamin B 12metabolism:
eight complementation groups –eight genes

defect). Therefore, completion of the human
pathway requires either finding new genes ordemonstrating that the pathway is fully
functional without them. One approach might be
to investigate the pathway in a model eukaryoticorganism carrying orthologues of most (or all)
the human genes, as suggested in studies of
the methylmalonate pathway in C. elegans ,a n
organism with knockout mutants of several
vitamin B
12pathway genes (Refs 148, 149, 150).
Although we have gained much insight into the
pathway of vitamin B 12metabolism, the goal of the
medical geneticist has been to gain insight into
managing the vitamin B 12disorders, providing
access to carrier testing and prenatal diagnosis,
and, in the best of outcomes, preventing or
successfully treating symptomatic disease. Theremarkable feature of vitamin B
12utilisation
disorders has been their potential for treatment.
The discovery that high-dose vitamin B 12can
overcome pathway deficits in some patients has
given new life to individuals with an otherwise
potentially severe or fatal disease. The earlydiscovery that OHCbl is effective in the
treatment of cblC disorder while CNCbl is not is
a powerful illustration of the complexity ofvitamin B
12biochemistry. The more recent
finding that AdoCbl or MeCbl may have a
significant stabilising effect on MMACHCprotein, despite ultimately being hydrolysed to
cob(II)alamin, reminds us that there is still much
to be learned on behalf of the patient. Strikingly ,the most recent success with gene therapy to treat
mice with knockout of the Mut gene (Ref. 151)
has opened up a new avenue for treatmentthat might ultimately benefit patients with
metabolically ‘unresponsive ’disorders. The
application of widespread newborn screening forhomocysteine and methylmalonate underscores
the opportunity to identify and treat these patients
before the onset of potentially irreversible disease.
Acknowledgements and funding
We are grateful to the peer reviewers for their
helpful comments and corrections. Contributing
research and the preparation of this review weresupported by a Canadian Institutes for Health
Research (CIHR) grant, MOP-44353, to R.A.G.
Scholarship support to D.S.F. was provided bythe CIHR Training Program in Genetics, Child
Development and Health at the University of
Calgary and by the Structural Genomics
Consortium, Oxford University.References
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Further reading, resources and contacts
Reviews
There are many excellent reviews that address different aspects of vitamin B 12metabolism, the proteins
involved and clinical correlates:
Banerjee, R., Gherasim, C. and Padovani, D. (2009) The tinker, tailor, soldier in intracellular B12 trafficking.
Current Opinion in Chemical Biology 13, 484-491
Li, F ., Watkins, D. and Rosenblatt, D.S. (2009) Vitamin B(12) and birth defects. Molecular Genetics and
Metabolism 98, 166-172
Fowler, B., Leonard, J.V . and Baumgartner, M.R. (2008) Causes of and diagnostic approach to methylmalonic
acidurias. Journal of Inherited Metabolic Disease 31, 350-360
(continued on next page)expert reviews
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Genetic disorders of vitamin B 12metabolism:
eight complementation groups –eight genes

Further reading, resources and contacts (continued)
Dali-Youcef, N. and Andres, E. (2009) An update on cobalamin deficiency in adults. Quarterly Journal of
Medicine 102, 17-28
Websites
The Organic Acidemia Association. A volunteer organisation for the provision of support and information on
organic acidurias, including the methylmalonic acidurias:

Home

National Institutes of Health: Office of Rare Diseases Research. A highly informative site covering basic
information, patient advocacy, research and clinical trials, and research resources for the public andscientific community:
http://rarediseases.info.nih.gov/
National Center for Biotechnology Information: Online Mendelian Inheritance in Man. Compilation of gene and
phenotype information on all known Mendelian genetic disorders searchable by disease or gene name:
http://www.ncbi.nlm.nih.gov/omim
Features associated with this article
Figures
Figure 1. Structure of vitamin B 12(cobalamin).
Figure 2. Intracellular processing of vitamin B 12showing sites of defects in complementation groups.
Figure 3. Mutations in the genes underlying the defects of the eight complementation groups.
Citation details for this article
D. Sean Froese and Roy A. Gravel (2010) Genetic disorders of vitamin B 12metabolism: eight complementation
groups –eight genes. Expert Rev. Mol. Med. Vol. 12 e37, November 2010, doi:10.1017/S1462399410001651expert reviews
http://www.expertreviews.org/ in molecular medicine
20
Accession information: doi:10.1017/S1462399410001651; Vol. 12; e37; November 2010
© Cambridge University Press 2010. Re-use permitted under a Creative Commons Licence –by-nc-sa.
Genetic disorders of vitamin B 12metabolism:
eight complementation groups –eight genes