++
Cobalamin (vitamin B12) is a complex organometallic
molecule that is synthesized by many bacteria and is obtained in
the human diet from meat, fish, and dairy products. It is not present
in plant foods, so strict vegetarians are at risk for dietary deficiency.
Derivatives of cobalamin are required for the activity of two enzymes: (1) methylcobalamin
(MeCbl) is generated during the catalytic cycle of methionine synthase,
a cytoplasmic enzyme that catalyzes methylation of homocysteine
to methionine (see Chapter 138),
and (2) 5′-deoxyadenosylcobalamin (AdoCbl)
is required for the mitochondrial enzyme methylmalonyl-CoA mutase
to catalyze the conversion of methylmalonyl-CoA, formed during catabolism
of branched-chain amino acids and odd-chain fatty acids, to succinyl-CoA
(see Chapter 137). Therefore, inborn errors of cobalamin metabolism
result in isolated methylmalonic aciduria, isolated homocystinuria,
or combined methylmalonic aciduria and homocystinuria, depending
on which step in cobalamin metabolism is affected.1-4 In
these disorders, hypomethioninemia usually occurs together with
hyperhomocysteinemia. Elevated homocysteine levels are associated
with an increased risk of thrombosis; decreased methionine is associated
with abnormalities of the white matter of the nervous system. Elevated
levels of methylmalonic acid are usually associated with metabolic
acidosis.
+++
Metabolism of Cobalamin
++
Cobalamin consists of a planar corrin ring with a central cobalt
atom, a 5,6-dimethylbenzimidazole base, and an upper axial ligand
attached to the cobalt atom that varies in different forms of cobalamin.5 Physiologically important
cobalamins include hydroxycobalamin (OHCbl), methylcobalamin (MeCbl), and
adenosylcobalamin (AdoCbl). The most common commercially available
form of vitamin B12 contains a cyano group in the upper axial
position (CNCbl). The central cobalt can exist in the oxidized Co3+ state (cob[III]alamin),
the Co2+ state (cob[II]alamin),
or the fully reduced Co1+ state (cob[I]alamin).
Converting exogenous cobalamin, typically in the form of cob(III)alamin,
to its biologically active coenzyme forms involves reducing the
central cobalt to the cob(I)alamin form, then adding the appropriate
upper axial ligand (eFig. 147.1).
++
++
Absorption of dietary cobalamin is a complex process that is
dependent on a cobalamin-binding protein, intrinsic factor (IF),
secreted by the parietal cells of the stomach.6 Cobalamin
in food exists bound to proteins. After proteins are digested, cobalamin
becomes bound in the stomach to haptocorrin, a cobalamin-binding
protein secreted in the saliva. Haptocorrin is broken down in the
intestine and cobalamin binds the IF. The IF-cobalamin complex is
taken up by enterocytes in the distal ileum in a process mediated
by a receptor, cubam, composed of the proteins cubilin and amnionless.
After uptake by enterocytes, cobalamin is released into the circulation,
bound to the transport protein transcobalamin (TC).
++
TC-bound cobalamin is available for uptake by most cell types.
Following endocytosis, mediated by an as-yet uncharacterized cell-surface receptor,
the cobalamin-TC complex dissociates in lysosomes, and free cobalamin
is transferred to the cytoplasm. Subsequently, cobalamin may become
associated with methionine synthase in the cytoplasm, or it may
be transported to the mitochondria, where it is converted to adenosylcobalamin
and becomes associated with methylmalonyl-CoA mutase.1 The
early steps of cellular cobalamin metabolism common to synthesis
of both coenzyme derivatives remain poorly understood. Two proteins
that play a role in these early steps, the products of the MMACHC
and MMADHC genes, have been identified on the basis of patients
with inborn errors, but their functions are not known.
++
The catalytic cycle of methionine synthase involves transfer
of a methyl group from 5-methyl-THF to an enzyme-bound cob(I)alamin
molecule, forming methylcobalamin (MeCbl); the methyl group is then
transferred from MeCbl to homocysteine, forming methionine and regenerating
cob(I)alamin. Occasionally the cob(I)alamin is oxidized to cob(II)alamin;
when this occurs, regeneration of MeCbl requires the activity of
a second protein, methionine synthase reductase, which uses adenosylmethionine
as its methyl group donor. Remethylation of homocysteine to form
methionine thus requires the activity of both methionine synthase
and methionine synthase reductase.7,8
++
The system by which cobalamin enters the mitochondria is unknown,
as is the form of cobalamin that crosses the mitochondrial membrane.
Mitochondrial reductases that can support reduction of cob(II)alamin
to cob(I)alamin have been identified in vitro, but the enzyme that
catalyzes this reaction in vivo has not been identified. Cob(I)alamin
reacts with adenosine triphosphate (ATP) to form adenosylcobalamin
(AdoCbl) in a reaction catalyzed by cob(I)alamin adenosyltransferase,
encoded by the MMAB gene.9 This is a member of
the PduO family of cobalamin adenosyltransferases, and it has been
argued that, in addition to catalyzing adenosylation of cob(I)alamin,
it also binds cobalamin in an activated base-off conformation (in
which the dimethylbenzimidazole base no longer coordinates with
the central cobalt atom) and directly transfers the activated AdoCbl
product to methylmalonyl-CoA mutase.10 Another
protein, the product of the MMAA gene, appears to play a role in
supporting the formation of holo-methylmalonyl-CoA mutase and in maintaining
the enzyme-bound cobalamin in its active form.11,12 Presence
of methylmalonyl-CoA mutase, cobalamin adenosyltransferase, and
the MMAA protein, possibly in association with a cob(II)alamin reductase,
is required for conversion of methylmalonyl-CoA to succinyl-CoA
in vivo.
+++
Isolated Methylmalonic Aciduria
+++
Metabolic Derangement
++
Isolated methylmalonic aciduria is seen in patients with mutations
affecting methylmalonyl-CoA mutase itself (the mut disorder [OMIM 251000] due
to mutations at the MUT locus on chromosome 6p21; see Chapter 137) or with mutations resulting in decreased
synthesis of adenosylcobalamin (the cblA disorder [OMIM 251100],
caused by mutations at the MMAA gene on chromosome 4q31.1-31.2,
and the cblB disorder [OMIM 251110], caused by
mutations in the MMAB gene on chromosome 12q24). The mut disorder
has been subdivided into two classes: (1) in mut0 there
is no stimulation mutase activity in cultured cells on incubation
with hydroxycobalamin (OHCbl), and there is often no detectable
mutase protein; (2) in mut–, mutase activity
is stimulated by addition of OHCbl.1
+++
Clinical Presentation
++
Patients with isolated methylmalonic aciduria are prone to episodes
of life-threatening acidosis, often in response to infection or
to increased protein intake. Initial presentation is most frequently
during infancy, with vomiting, hypotonia, irritability, and lethargy,
then progressing to coma and death if not treated. In the most severely
affected individuals, there may be intractable acidosis during the
neonatal period that leads to death, but in many patients the disorder
is characterized by recurrent acidotic crises characterized by severe ketoacidosis,
hyperammonemia and hyperglycemia, or hyperglycemia with anemia,
neutropenia, thrombocytopenia or pancytopenia, failure to thrive,
or metabolic stroke. Prolonged elevation of plasma methylmalonic
acid may lead to end-stage renal failure necessitating transplantation.
Studies of series of patients have shown that presentation is typically
most severe in individuals with mut0, while cblB
is less severe and cblA and mut– patients have
the least severe presentation and the best prognosis.13,14
++
Organic acid analysis in these patients reveals elevated levels
of methylmalonic acid and of metabolites such as methylcitric acid, 3-hydroxypropionic
acid, and tiglylglycine. Tandem mass spectroscopy of acylcarnitines shows
elevated propionyl carnitine. Elevated glycine may be present in
blood and urine. Elevation of methylmalonic acid levels may also
be seen in individuals with methylmalonyl-CoA epimerase (racemase)
deficiency and in patients with mutations affecting either subunit
of succinyl-CoA ligase; however, in these orders, the levels are
lower than in mut, cblA, and cblB.
++
Differentiation between the different forms of methylmalonic
aciduria has depended on studies of cultured fibroblasts. In all
three groups, mutase function (assessed by measuring the incorporation
of label from [14C]propionate
into cellular macromolecules) is decreased in the presence of normal
methionine synthase function (assessed by measuring the incorporation
of label from [14C]methyl-THF
or [14C]formate into cellular
macromolecules). There is deficient conversion of exogenous [57Co]CNCbl
into adenosylcobalamin (AdoCbl) in cblA and cblB fibroblasts in
the presence of normal methylcobalamin synthesis; AdoCbl synthesis
may be normal or reduced in mut fibroblasts. The cblA and cblB disorders
can be differentiated on the ability of cell extracts to support
conversion of [57Co]CNCbl to
AdoCbl, but this test is rarely performed. Differentiation between
the three classes is typically carried out by complementation analysis.1
++
All three disorders are inherited as autosomal recessive traits. Over
180 mutations in the MUT gene have been identified in patients with
mut, the most common of the disorders. The mutations in most families
are private, but several mutations are relatively common within
a specific ethnic group. These include a c.2150G→T
(p.G717V) mutation in African patients, c.349GÆT
(p.E117X) in Japanese patients, and c.323CÆT (p.R108C)
in American Hispanic patients.15 Thirty MMAA mutations
have been identified in cblA patients. The most common of these,
c.433CÆT (p.R145X), represents 43% of mutant alleles in
Western patients but is less frequent in Japanese patients, where
a c.503delC predominates.16,17Twenty-three MMAB
mutations have been identified in cblB patients. A c.556CÆT
(p.R186W) mutation was the most common. In X-ray crystallographic
studies, virtually all identified MMAB mutations clustered in an
area of the gene product that is the active site of the enzyme.18
+++
Isolated Homocystinuria
+++
Metabolic Derangement
++
Homocystinuria in the absence of methylmalonic aciduria is seen
in patients with the cblE (OMIM 250940) and cblG (OMIM 236270) disorders.
In both disorders, methionine synthase function is impaired; in
the cblG disorder, synthase-specific activity in cell extracts is
decreased under all assay conditions, while in the cblE disorder,
specific activity is decreased only in the presence of limiting
concentrations of exogenous reducing agent.19 The
cblG disorder is caused by mutations affecting the MTR gene on chromosome
1q43, which encodes methionine synthase.20,21 The
cblE disorder is caused by mutations of the MTRR gene on chromosome
5p15.3-15.2, which encodes methionine synthase reductase.22
+++
Clinical Presentation
++
Patients with these disorders are characterized by megaloblastic
anemia and neurological problems, including developmental delay,
cerebral atrophy, hypotonia, microcephaly, and seizures. Homocystinuria
is less severe in patients with these disorders than in patients
with homocystinuria due to cystathionine β-synthase
deficiency. Presentation is typically within the first 2 years of
life but can present in adulthood as well.23
++
Decreased methionine synthase activity results in increased serum
total homocysteine and in homocystine in the urine, combined with
reduced levels of methionine and elevated cystathionine. Serum cobalamin
levels are normal. These disorders can be differentiated from homocystinuria
due to cystathionine β-synthase deficiency, which
presents with higher levels of homocysteine, elevated methionine,
and decreased cystathionine levels (see Chapter 138, Table 138-1). Fibroblasts are characterized
by decreased methyl-THF incorporation with normal propionate incorporation
and by decreased methylcobalamin synthesis with normal adenosylcobalamin
synthesis. The disorders can be differentiated by the response of
methionine synthase–specific activity to the titration
of a reducing agent but more typically are differentiated by complementation
analysis.
++
Both disorders are inherited as autosomal recessive traits. Eighteen
MTRR mutations have been identified in cblE patients. The most common
of these is an intronic c.903+469TÆC mutation
that results in a 140 bp insertion of intronic sequence in mRNA;
a c.1361CÆT (p.S454L) mutation is common among patients
of Iberian origin and appears to be associated with a mild form
of the disease.24,25 Twenty MTR mutations
have been identified in cblG patients. The most common of these,
c.3158CÆT (p.P1173L), has never been observed in homozygous
form.26
+++
Combined Methylmalonic Aciduria
and Homocystinuria
+++
Metabolic Derangement
++
Three inborn errors of metabolism that affect early steps in
cobalamin metabolism result in combined methylmalonic aciduria and
homocystinuria. The cblC disorder (OMIM 277400) is caused by mutations
at the MMACHC locus on chromosome 1p34.1,27 and the
cblD disorder (OMIM 277410) is caused by mutations at the MMADHC
locus on chromosome 2q23.2.28 The function of neither
of these gene products is understood. Although the original patients
with the cblD disorder had combined methylmalonic acid and homocystinuria,
additional patients with isolated homocystinuria (cblD variant 1)
and isolated methylmalonic aciduria (cblD variant 2) have subsequently
been recognized.29 The cblF disorder results in
inability to transfer endocytosed cobalamin from the lysosome to
the cytoplasm.30 The gene underlying this disorder
has not yet been identified.
+++
Clinical Presentation
++
The large majority of patients with combined methylmalonic aciduria
and homocystinuria belong to the cblC class, with over 300 patients identified.
Individuals with this disorder typically present during the first
year of life with megaloblastic anemia, failure to thrive, developmental delay,
hypotonia, seizures, macrocephaly, and cerebral atrophy. Despite
the presence of methylmalonic aciduria, metabolic decompensation is
infrequent. Other patients present later in life or in adulthood
with ataxia, dementia, or psychosis. A distinctive salt-and-pepper
retinopathy has been described in some cblC patients. Presentation
in classic cblD disease is similar to that of cblC. Presentation
of the cblF disease is variable. Frequent findings have included
feeding difficulties, failure to thrive, growth retardation, and persistent
stomatitis.
++
Patients with these disorders have the biochemical characteristics
of both methylmalonic aciduria and homocystinuria, although methylmalonic
acid levels are generally lower than in patients with isolated methylmalonic
aciduria. Fibroblasts show decreased function of both methylmalonyl-CoA
mutase and methionine synthase, and decreased synthesis of both
adenosylcobalamin and methylcobalamin (Table 147-1).
Fibroblasts from patients with the cblC or cblD disorder are unable
to accumulate cobalamin in cells, reflecting the inability of cells
to retain cobalamin that is not bound to one of the cobalamin-dependent
enzymes. In cblF disorder, fibroblasts accumulate large amounts
of cobalamin, but most of this is unmetabolized cobalamin that is
trapped within the lysosomes.
++
++
Both disorders are inherited as recessive autosomal traits. Over
40 mutations in the MMACHC gene have been identified. The most common of
these is a c.271dupA mutation that represents 40% of identified
mutant alleles, primarily in patients of European or western Asian origin.
It is associated with early onset severe disease when homozygous.
Several other mutations show evidence of clustering in specific ethnic
groups, and genotype-phenotype correlations have been observed.27,31 A
small number of mutations in the MMADHC gene have been identified
in patients with the much rarer cblD disorder. The classical cblD
disorder is associated with truncating mutations, while mutations
at the C-terminal domain are associated with isolated homocystinuria
(variant 1), and mutations in the N-terminal domain are associated
with isolated methylmalonic aciduria (variant 2).28 The
gene underlying the cblF disorder remains unknown.
++
Patients with inborn errors of cobalamin are treated with large
doses (up to 1 mg per day) of cobalamin administered intramuscularly.
In the cblC disorder, hydroxycobalamin is more effective than CNCbl,
the standard form of pharmacological vitamin B12)32;
this may be true for other disorders as well. In patients with methylmalonic
aciduria, protein restriction represents an important aspect of
treatment. Supplementation with carnitine and with antibiotics (to
reduce propionate production by gut bacteria) has also been used.
Supplementation with betaine, which supports conversion of homocysteine
to methionine by the liver enzyme betaine-homocysteine methyltransferase,
has proved effective in patients with homocystinuria.
+++
Inborn Errors
of Cobalamin Uptake and Transport
++
Combined methylmalonic aciduria and homocystinuria is also seen
in patients with defects affecting intestinal uptake of cobalamin and
its transport in the serum. Decreased uptake occurs in individuals
with intrinsic factor deficiency (OMIM 261000), caused by mutations
at the GIF gene on chromosome 11q13,33,34 and in those
with Imerslund-Gräsbeck syndrome (IGS; OMIM 261100). The
latter disorder is the result of dysfunction of the cubam receptor in
the distal ileum, which can be caused by mutations at the CUBN gene
on chromosome 10p12.1 or the AMN gene on chromosome 14q32, which
encode the cubilin and amnionless components of cubam; additional
IGS families are not linked to either chromosome and represent further
genetic heterogeneity in this disorder.35,36 Patients
usually present between 1 and 5 years of age with decreased serum
cobalamin levels, megaloblastic anemia, and neurological impairment.
IGS patients often have proteinuria as well.37 Fewer
than 30 patients with intrinsic factor deficiency have been identified,
and approximately 300 patients with IGS have been identified. Clusters of
IGS have been identified in Finland (CUBN mutations), Norway (AMN
mutations), and in the eastern Mediterranean (mutations in both genes). In
the absence of readily available clinical tests to assess cobalamin
uptake, specific diagnosis in patients with disorders of cobalamin
uptake depends on sequencing of the GIF, CUBN, and AMN genes.
++
Patients with transcobalamin deficiency (OMIM 275350) usually
present in the first months of life with megaloblastic anemia; failure
to thrive; weakness; and, frequently, immunologic dysfunction; without
treatment, neurological impairment develops.1 Because
a majority of cobalamin circulates bound to haptocorrin (which is
not available for uptake by most types of cells) rather than to
transcobalamin, circulating cobalamin levels may be normal in this
disorder. Mutations in the TCN2 gene on chromosome 22q11.2 have
been identified in a small number of patients.36 Treatment
involves maintaining very high serum levels of cobalamin by either
oral or intramuscular route.