Download A Patient With an Inborn Error of Vitamin B12

CASE REPORT
A Patient With an Inborn Error of Vitamin B12
Metabolism (cblF) Detected by Newborn Screening
AUTHORS: Christine M. Armour, MD,a Alison Brebner, BSc,b
David Watkins, PhD,b Michael T. Geraghty, MD,c Alicia Chan,
MD,d and David S. Rosenblatt, MDb
aDepartment
of Medical Genetics, Children’s Hospital of Eastern
Ontario, Ottawa, Canada; bDepartment of Human Genetics, McGill
University, Montreal, Canada; cDepartment of Pediatrics,
Metabolics Unit, Children’s Hospital of Eastern Ontario, Ottawa,
Canada; and dDepartment of Medical Genetics, University of
Alberta Hospital, Edmonton, Canada
KEY WORDS
cobalamin, cblF, methylmalonic acidemia, hyperhomocysteinemia,
lysosome, newborn screening, LMBRD1
ABBREVIATIONS
AdoCbl—adenosylcobalamin
CNCbl—cyanocobalamin
MeCbl—methylcobalamin
MMA—methylmalonic acid
NBS—newborn screening
OHCbl—hydroxocobalamin
Dr Armour cared for the patient, collected data, drafted the
initial manuscript, and approved the final manuscript as
submitted; Ms Brebner compiled the data, drafted the initial
manuscript, and approved the final manuscript as submitted;
Dr Watkins collected the data, analyzed the data, wrote the
manuscript, and approved the final manuscript as submitted;
Dr Geraghty contributed to the diagnosis and care of the patient
and approved the final manuscript; Dr Chan contributed to the
ongoing care of the patient, contributed clinical data, and
approved the final manuscript; and Dr Rosenblatt designed the
study, reviewed and revised the manuscript, and approved the
final manuscript as submitted.
abstract
A neonate, who was found to have an elevated C3/C2 ratio and minimally elevated propionylcarnitine on newborn screening, was subsequently identified as having the rare cblF inborn error of vitamin
B12 (cobalamin) metabolism. This disorder is characterized by the
retention of unmetabolized cobalamin in lysosomes such that it is
not readily available for cellular metabolism. Although cultured fibroblasts from the patient did not show the expected functional abnormalities of the cobalamin-dependent enzymes, methylmalonyl-CoA
mutase and methionine synthase, they did show reduced synthesis
of the active cobalamin cofactors adenosylcobalamin and methylcobalamin. Mutation analysis of LMBRD1 established that the patient
had the cblF disorder. Treatment was initiated promptly, and the
patient showed a robust response to regular injections of cyanocobalamin, and she was later switched to hydroxocobalamin. Currently,
at 3 years of age, the child is clinically well, with appropriate development. Adjusted newborn screening cutoffs in Ontario allowed
detection of a deficiency that might not have otherwise been identified, allowing early treatment and perhaps preventing the adverse
sequelae seen in some untreated patients. Pediatrics 2013;132:e257–
e261
www.pediatrics.org/cgi/doi/10.1542/peds.2013-0105
doi:10.1542/peds.2013-0105
Accepted for publication Mar 13, 2013
Address correspondence to David Watkins, PhD, Department of
Medical Genetics, McGill University Health Centre/Montreal
General Hospital, 1650 Cedar Ave L3.319, Montreal, Quebec,
Canada, H3G 1A4. E-mail: [email protected]
PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).
Copyright © 2013 by the American Academy of Pediatrics
FINANCIAL DISCLOSURE: The authors have indicated they have
no financial relationships relevant to this article to disclose.
FUNDING: Dr Rosenblatt is supported by an operating grant
(MOP-15078) from the Canadian Institutes of Health Research.
PEDIATRICS Volume 132, Number 1, July 2013
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e257
Derivatives of vitamin B12 (cobalamin) are
required for the function of 2 essential cellular enzymes. Adenosylcobalamin
(AdoCbl) is required by the mitochondrial enzyme methylmalonyl-CoA mutase,
which converts methylmalonyl-CoA to
succinyl-CoA. Methylcobalamin (MeCbl) is
required by the cytoplasmic enzyme methionine synthase in the conversion of
homocysteine to methionine.1 Mutations
in the genes encoding either of these
enzymes, or encoding proteins required
for the synthesis of either cobalamin cofactor, result in inborn errors of cobalamin metabolism. There are currently
9 known disorders caused by mutations
in genes coding for proteins involved in
the pathway: cblA-cblG, cblJ, and mut.1,2
Affected patients typically have elevated
serum and urine levels of the precursor
metabolites for one or both of these
reactions, including methylmalonic acid
(MMA) and/or homocysteine, depending
on where in the pathway the defect
occurs.
The cblF inborn error (Online Mendelian Inheritance in Man database
number 277380) is caused by mutations
in the LMBRD1 gene on chromosome
6q13, which encodes the lysosomal
membrane protein LMBD1.3 Endocytosis
of transcobalamin-bound circulating
cobalamin is mediated by the transcobalamin receptor, with dissociation
of the transcobalamin-cobalamin complex and proteolysis of transcobalamin
occurring in the lysosome. LMBD1 is
thought to play a role in transporting
cobalamin from the lysosome into
the cytoplasm after its release from
transcobalamin. Fibroblasts from
patients with cblF accumulate free cobalamin, unbound to protein, within
lysosomes, with decreased synthesis
of both cobalamin cofactors and decreased function of both cobalamindependent enzymes. This results in increased levels of MMA and homocysteine in the blood and urine. To date
there are 15 reported cblF patients.3–5
e258
Several have had decreased serum vitamin B12 levels, apparently reflecting
a role for the lysosome in intestinal
uptake of ingested cobalamin; the
Schilling test for cobalamin absorption
has been reported to be abnormal in
cblF patients.6 A second disorder with
an identical biochemical and cellular
phenotype, cblJ, caused by mutations
in the gene which codes for a different
lysosomal membrane protein, ABCD4,
has recently been described in 3
patients.2,7
There is a great deal of variability in
clinical presentation among the small
number of known cblF patients. Common clinical findings include failure
to thrive, developmental delay, congenital heart defects, macrocytic anemia, neutropenia, thrombocytopenia,
pancytopenia, and minor facial abnormalities such as pegged teeth.3 Patients
have generally responded well to therapy with hydroxocobalamin (OHCbl) by
intramuscular injection. When cultured
patient fibroblasts are incubated in the
presence of transcobalamin-bound labeled cyanocobalamin (CNCbl), they
accumulate high levels of cobalamin,
almost all of which is unmetabolized
CNCbl localized to the lysosome. There
is relatively little synthesis of AdoCbl or
MeCbl and decreased function of both
methylmalonyl-CoA mutase and methionine synthase.8,9
CLINICAL REPORT
The proband, the product of an unremarkable pregnancy, was a full-term
female infant with a birth weight of
3175 g. There was no family history of
metabolic disease or consanguinity.
Her parents were of mixed white European descent, and she has a healthy
older sister. She was exclusively
breastfed and clinically well at the time
of ascertainment. She was identified
after a positive newborn screen showing
a borderline elevation of C3 acylcarnitine and increased ratio of C3/C2 acylcarnitines (Table 1). Follow-up testing
12 days after birth demonstrated a
continued elevation of C3 acylcarnitine,
with increased excretion of MMA without
methylcitric acid. Serum amino acids
revealed an elevated homocysteine
TABLE 1 Diagnostic Laboratory Data
Age
Test
Patient Value
Reference
24 h (NBS)
C3 acylcarnitine
C3/C2 ratio
Plasma C3 acylcarnitine
Serum total homocysteine
Hemoglobin
Serum cobalamin
Urine MMA
Serum total homocysteine
Serum cobalamin
Serum total homocysteine
Urine MMA
Plasma C3 acylcarnitine
Serum cobalamin
Serum total homocysteine
Plasma C3 acylcarnitine
Urine MMA
Serum cobalamin
Mean corpuscular vol
Urine MMA
Plasma C3 acylcarnitine
5.73 mM
0.27
2.55 mM
39 mM
117
62 pM
54 mmol/mol Cr
34 mM
306 pM
17 mM
25 mmol/mol Cr
1.11 mM
243 pM
13 mM
0.97 mM
14 mmol/mol Cr
158 pM
83.2 fL
8.4 mmol/mol Cr
0.69 mM
,5.5 mM
,0.2
,0.65 mM
4–15 mM
135–206
165–740 pM
4.3 6 2.3 mmol/mol Cr
4–15 mM
165–740 pM
4–15 mM
4.3 6 2.3 mmol/mol Cr
,0.65 mM
165–740 pM
4–15 mM
,0.65 mM
4.3 6 2.3 mmol/mol Cr
165–740 pM
85–123 fL
4.3 6 2.3 mmol/mol Cr
,0.65 mM
12 d
26 d
34 da
51 db
83 d
C3, propionyl carnitine; Cr, creatinine.
a Four days after 1 mg CNCbl intramuscular injection.
b Three weeks after 1 mg CNCbl intramuscular injection.
ARMOUR et al
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CASE REPORT
concentration. Hemoglobin and mean
corpuscular volume values were within
the reference range, but the serum vitamin B12 concentration was low. Simultaneous maternal investigations
were performed, which included complete blood count and serum vitamin
B12 and homocysteine concentrations,
all of which were within the reference
ranges. Given the infant’s low serum
vitamin B12, she was given an intramuscular injection of 1 mg CNCbl,
the only form available at the time.
This served both as treatment and as
an aid in determining the cause of the
abnormal metabolite excretion.
Urine MMA was elevated before
CNCbl injection. Four days after injection, serum vitamin B12 was within
the reference range, and serum MMA
and C3 acylcarnitine had improved
but had not completely normalized.
Three weeks later, the serum vitamin
B12 and homocysteine levels were still
within the reference range, but plasma
acylcarnitines and urine MMA, considered one of the most sensitive indicators of vitamin B12 status, remained
elevated. Two months after the first
injection, the serum vitamin B12 concentration had fallen slightly below the
reference range, and other values had
normalized.
Although the biochemical phenotype
was responsive to CNCbl, the infant
was switched to 1 mg OHCbl by intramuscular injection every 2 weeks,
once this preferred therapeutic form
became available. All biochemical
fibroblasts was measured by incubating them for 96 h with [ 57Co]
CNCbl bound to transcobalamin, then
performing a hot ethanol extraction
and high performance liquid chromatography as previously described.11
There was decreased synthesis of
AdoCbl and MeCbl and accumulation of
CNCbl. Restriction endonuclease analysis and sequencing of the LMBRD1
gene in patient genomic DNA identified
the common c.1056delG (p.L352fsX18)
mutation and a novel c.1339-1G.T
splice site mutation, confirming the
diagnosis of the cblF disorder.
parameters subsequently normalized,
and she was maintained on this schedule.
At 3 months of age, her length was
54.6 cm (3rd–10th percentile), weight
5.55 kg (50th percentile) and head
circumference 39.2 cm (25th–50th
percentile). Apart from mild diffuse,
patchy eczema, she had a completely
normal physical examination. She was
reported to be colicky but slept 6 to
7 hours overnight. Echocardiogram
was normal, with only a patent foramen ovale. There was no evidence
of any neurologic abnormalities. At
3 years of age, her development is
appropriate in all spheres, and there
is no evidence of any developmental
delay, or any ongoing health issues.
DISCUSSION
Newborn screening by tandem mass
spectrometry identified a small increase in blood C3 acylcarnitine concentration as well as an increase in the
ratio of C3 to C2 acylcarnitine. The latter
measure has been shown to offer
a higher specificity and sensitivity than
measurement of C3 acylcarnitine concentration alone12–14 and had only
recently been incorporated into the
newborn screening algorithm used
for determining the cutoff between
positive and negative values in Ontario.
This combination of abnormal C3 acylcarnitine and C3/C2 ratio allowed the
patient to be initially detected and referred for additional testing regarding
her abnormal metabolic profile. The
presence of elevated MMA and homocysteine levels suggested an inborn
error of cobalamin metabolism. The
mother was not cobalamin-deficient
LABORATORY STUDIES AND
FINDINGS
Incorporation of the radiolabeled
substrates [14C]propionate and [14C]
methyltetrahydrofolate into cellular
macromolecules were measured both
with and without the addition of
3.75 mM OHCbl to the media as previously reported.10 Both [14C]propionate incorporation, a measure of
methylmalonyl-CoA mutase function,
and from [14C]methyltetrahydrofolate
incorporation, a measure of methionine synthase function, were within the
reference range (Table 2). Incorporation of labeled propionate was stimulated by incubation with OHCbl,
consistent with a defect in cobalamin
metabolism. The distribution of the
cobalamin cofactors in the patient’s
TABLE 2 Studies of Patient Fibroblasts
Assay
Patient
cblF patients (n = 11)
Reference range
[14C]-propionate Incorporation
(nmol/mg protein/18 h)
[14C]-methylTHF Incorporation
(nmol/mg protein/18 h)
Cobalamin Distribution
(% of total Cbl)
–OHCbl
+OHCbl
–OHCbl
+OHCbl
CNCbl
AdoCbl
MeCbl
6.9; 7.6
1.9 6 1.8
10.8 6 3.7
11.1; 12.2
7.2 6 3.2
10.9 6 3.5
75; 69
43 6 11
225 6 165
670; 637
297 6 173
305 6 125
73.2; 78.6
80.7 6 7.2
11.27 6 6.9
2.5; 2.7
2.4 6 0.6
15.29 6 4.2
4.3; 4.7
1.3 6 1.1
57.95 6 6.7
Fibroblast cultures were incubated for 18 h in a medium containing labeled methyl tetrahydrofolate and propionate with and without OHCbl. Cellular macromolecules were precipitated in 5%
trichloroacetic acid and radioactivity determined by liquid scintillation counting. The distribution of the cobalamin cofactors in the patient’s fibroblasts was measured by incubating them with
[57Co]CNCbl bound to transcobalamin, then performing a hot ethanol extraction and high performance liquid chromatography.
PEDIATRICS Volume 132, Number 1, July 2013
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e259
nor was the decreased serum cobalamin level in the baby the result of
dietary deficiency.
Studies of cultured fibroblasts provided information about the metabolic
block in this patient but did not allow
definitive assignment to one of the
known inborn errors of cobalamin
metabolism. Analysis of uptake of
labeled CNCbl and its conversion to
the active cobalamin cofactor forms
showed a pattern similar to that of
other cblF patients, with accumulation
of large amounts of unmetabolized
CNCbl and decreased synthesis of
AdoCbl and MeCbl (Table 2). However,
indirect measures of methylmalonylCoA mutase and methionine synthase
function gave values within the reference ranges, precluding assignment of
the patient’s disorder by complementation analysis. Diagnosis of the cblF
disorder was made by analysis of the
LMBRD1 gene, which demonstrated
the presence of 2 disease-causing
mutations: the common c.1056delG
(p.L352fsX18) mutation and a novel
splice site mutation, c.1339-1G.T,
which resulted in production of a
transcript missing exon 14.5 Analysis of cDNA from patient fibroblasts
demonstrated the presence of a small
amount of LMBRD1 mRNA of the normal
size in addition to the misspliced product.5 It is possible that the presence of
a “leaky” splice site mutation in combination with the common LMBRD1
mutation in this patient was responsible for her milder phenotype.
However, another cblF patient heterozygous for the common mutation, and
a different splice site mutation had
a typical biochemical and clinical
phenotype.5 The results of fibroblast
studies indicate that measurement of
cobalamin cofactor synthesis is more
sensitive than indirect measures of
cobalamin-dependent enzyme function
in detecting inborn errors of metabolism. Similar findings have been reported in a patient with the cblJ disorder,
who had abnormal cobalamin cofactor
synthesis in the presence of normal
methylmalonyl-CoA mutase and methionine synthase function.7
cblF patients and in patients with the
phenotypically similar cblJ disorder,
although diagnosis has occurred as
late as 11 years of age in one cblF patient.15 Similarly, a patient with a biochemically milder form of the cblJ
disorder came to medical attention at 8
years of age.7
At the time that this patient was
ascertained, cblF was the only known
inborn error to cause accumulation
of unmetabolized cobalamin in the
lysosomes. Since this time, the cblJ
disorder, which has an identical
clinical and biochemical phenotype,
has been identified,2 and if a patient
similar to ours came to medical attention today, molecular analysis of
both the LMBRD1 and ABCD4 genes
would need to be undertaken.
Early treatment of the patient may have
prevented the onset of adverse clinical symptoms typically seen in cblF
patients because the patient is currently doing well at 3 years of age.
Onset of symptoms has typically occurred in the first months of life in
ACKNOWLEDGMENTS
We thank Gail Dunbar for growth and
storage of fibroblasts and Jocelyne
Lavallée for the somatic cell clinical
investigations. Thanks to Isabelle R.
Miousse and Maria Galvez for the
mutation analysis of the patient. Dr
Rosenblatt is a member of the Research Institute of the McGill University Health Centre.
in a Turkish patient. J Inherit Metab Dis.
2010;33(1):17–24
5. Miousse IR, Watkins D, Rosenblatt DS. Novel
splice site mutations and a large deletion
in three patients with the cblF inborn error
of vitamin B12 metabolism. Mol Genet
Metab. 2011;102(4):505–507
6. Laframboise R, Cooper BA, Rosenblatt DS.
Malabsorption of vitamin B12 from the intestine in a child with cblF disease: evidence
for lysosomal-mediated absorption. Blood.
1992;80(1):291–292
7. Kim JC, Lee NC, Hwu PWL, et al. Late onset of
symptoms in an atypical patient with the
cblJ inborn error of vitamin B12 metabolism:
diagnosis and novel mutation revealed by
exome sequencing. Mol Genet Metab. 2012;
107(4):664–668
8. Rosenblatt DS, Hosack A, Matiaszuk NV,
Cooper BA, Laframboise R. Defect in vitamin
B12 release from lysosomes: newly described inborn error of vitamin B12 metabolism. Science. 1985;228(4705):1319–1321
9. Vassiliadis A, Rosenblatt DS, Cooper BA,
Bergeron JJM. Lysosomal cobalamin accumulation in fibroblasts from a patient with
an inborn error of cobalamin metabolism
(cblF complementation group): visualization
by electron microscope radioautography.
Exp Cell Res. 1991;195(2):295–302
10. Watkins D. Cobalamin metabolism in
methionine-dependent human tumour and
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CASE REPORT
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e261
A Patient With an Inborn Error of Vitamin B12 Metabolism (cblF) Detected by
Newborn Screening
Christine M. Armour, Alison Brebner, David Watkins, Michael T. Geraghty, Alicia
Chan and David S. Rosenblatt
Pediatrics; originally published online June 17, 2013;
DOI: 10.1542/peds.2013-0105
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PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly
publication, it has been published continuously since 1948. PEDIATRICS is owned, published,
and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk
Grove Village, Illinois, 60007. Copyright © 2013 by the American Academy of Pediatrics. All
rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275.
Downloaded from by guest on June 17, 2017
A Patient With an Inborn Error of Vitamin B12 Metabolism (cblF) Detected by
Newborn Screening
Christine M. Armour, Alison Brebner, David Watkins, Michael T. Geraghty, Alicia
Chan and David S. Rosenblatt
Pediatrics; originally published online June 17, 2013;
DOI: 10.1542/peds.2013-0105
The online version of this article, along with updated information and services, is
located on the World Wide Web at:
/content/early/2013/06/12/peds.2013-0105
PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly
publication, it has been published continuously since 1948. PEDIATRICS is owned,
published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point
Boulevard, Elk Grove Village, Illinois, 60007. Copyright © 2013 by the American Academy
of Pediatrics. All rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275.
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