X-Linked Cobalamin Disorder (HCFC1) Mimicking Nonketotic Hyperglycinemia With Increased Both Cerebrospinal Fluid Glycine and Methylmalonic Acid.

BACKGROUND: Autosomal recessive or X-linked inborn errors of intracellular cobalamin metabolism can lead to methylmalonic aciduria and homocystinuria. In neonates, both increased cerebrospinal fluid glycine and cerebrospinal fluid/plasma glycine ratio are biochemical features of nonketotic hyperglycinemia. METHODS: We describe a boy presenting in the neonatal period with hypotonia, tonic, clonic, and later myoclonic seizures, subsequently evolving into refractory epilepsy and severe neurocognitive impairment. RESULTS: Increased cerebrospinal fluid glycine and cerebrospinal fluid to plasma glycine ratio were indicative of nonketotic hyperglycinemia. Early magnetic resonance imaging showed restricted diffusion and decreased apparent diffusion coefficient values in posterior limb of internal capsules and later in entire internal capsules and posterior white matter. Sequencing did not show a mutation in AMT, GLDC, or GCSH. Biochemical analysis identified persistently increased cerebrospinal fluid levels of glycine and methylmalonic acid and increased urinary methylmalonic acid and plasma homocysteine levels, which improved on higher parenteral hydroxocobalamin dose. Exome sequencing identified a known pathogenic sequence variant in X-linked cobalamin (HCFC1), c.344C>T, p. Ala115Val. In addition, a hemizygous mutation was found in the ATRX (c. 2728A>G, p. Lys910Glu). Retrospective review of two other patients with X-linked cobalamin deficiency also identified increased cerebrospinal fluid glycine levels. CONCLUSIONS: This boy had X-linked cobalamin deficiency (HCFC1) with increased cerebrospinal fluid glycine and methylmalonic acid and increased cerebrospinal fluid to plasma glycine ratio suggesting a brain hyperglycinemia. Putative binding sites for HCFC1 and its binding partner THAP11 were identified near genes of the glycine cleavage enzyme, providing a potential mechanistic link between HCFC1 mutations and increased glycine.

Cobalamin deficiency-induced changes in magnetic resonance imaging of cerebrospinal fluid volume in the cervical tract in the rat.

We wanted to verify the magnetic resonance imaging (MRI) abnormalities that occur in the central nervous system (CNS) of cobalamin-deficient (Cbl-D) rats. The rats were made Cbl-D by means of total gastrectomy or feeding a Cbl-D diet. MR images of the cervical tract of the vertebral canal were recorded using a vertical spectrometer, and the volume of cerebrospinal fluid (CSF) in this part of the vertebral canal was calculated. The findings of the present study demonstrate that: (i) there was a significant decrease in cervical tract CSF volume regardless of the way in which the vitamin deficiency was induced; (ii) this volume normalized in the totally gastrectomized rats after chronic Cbl treatment; (iii) no blood-brain or blood-CSF barrier lesions were found in Cbl-D rats, using either MRI with a paramagnetic contrast agent or calculating the albumin CSF/serum concentration quotient. Cbl deficiency decreases CSF volume in the cervical tract of the vertebral canal of the rat, without apparently impairing the blood-brain barrier.

Increased levels of the CD40:CD40 ligand dyad in the cerebrospinal fluid of rats with vitamin B12(cobalamin)-deficient central neuropathy.

The levels of the soluble (s) CD40:sCD40 ligand (L) dyad, which belongs to the tumor necrosis factor (TNF)-alpha:TNF-alpha-receptor superfamily, are significantly increased in the cerebrospinal fluid (CSF), but not the serum of cobalamin (Cbl)-deficient (Cbl-D) rats. They were normalized or significantly reduced after treatment with Cbl, transforming growth factor-beta1 or S-adenosyl-L-methionine, and the normal myelin ultrastructure of the spinal cord was concomitantly restored. The concomitance of the two beneficial effects of these treatments strongly suggests that the increases in CSF sCD40:sCD40L levels may participate in the pathogenesis of purely myelinolytic Cbl-D central neuropathy in the rat. In keeping with this, an anti-CD40 treatment prevented myelin lesions.

New insights into the pathophysiology of cobalamin deficiency.

Cobalamin-deficient (Cbl-D) central neuropathy in the rat is associated with a locally increased expression of neurotoxic tumour necrosis factor-alpha (TNF-alpha) and a locally decreased expression of neurotrophic epidermal growth factor (EGF). These recent findings suggest that cobalamin oppositely regulates the expression of TNF-alpha and EGF, and raise the possibility that these effects might be independent of its coenzyme function. Furthermore, adult Cbl-D patients have high levels of TNF-alpha and low levels of EGF in the serum and cerebrospinal fluid. Serum levels of TNF-alpha and EGF of cobalamin-treated patients normalize concomitantly with haematological disease remission. These observations suggest that cobalamin deficiency induces an imbalance in TNF-alpha and EGF levels in biological fluids that might have a role in the pathogenesis of the damage caused by pernicious anaemia.

Increased spinal cord NGF levels in rats with cobalamin (vitamin B12) deficiency.

We have recently demonstrated that the neuropathological morphological alterations caused by cobalamin (Cbl) deficiency in the rat central nervous system are related to the vitamin's inability to modulate the synthesis of some neurotoxic and neurotrophic agents in opposite directions. In the present study, we measured nerve growth factor (NGF) levels in the spinal cord (SC) and cerebrospinal fluid (CSF) of rats made Cbl-deficient (Cbl-D) by means of total gastrectomy (TG) or a Cbl-D diet. In both cases, Cbl deficiency increased SC and CSF NGF levels after the appearance of myelinolytic lesions in the SC white matter (SCWM) (i.e. after the second post-TG month), and these changes were normalised by Cbl treatment in the 4-month-totally-gastrectomised (TGX) rats. Intracerebroventricular (i.c.v.) anti-NGF-antibody treatment prevented the onset of the myelinolytic SCWM lesions in the 2-month-TGX rats (i.e. when SC and CSF NGF levels are still normal) and normalised the ultrastructure of the SCWM in the 4-month-TGX rats, which was however worsened by the i.c.v. administration of NGF. These findings demonstrate that: (i) Cbl deficiency increases SC and CSF NGF levels; and (ii) endogenous NGF seems to play a noxious role in the progression of rat Cbl-D central neuropathy.

High tumor necrosis factor-alpha [corrected] levels in cerebrospinal fluid of cobalamin-deficient patients.

We studied 14 patients with neurological manifestations of subacute combined degeneration (SCD) and 40 control patients not cobalamin (Cbl)-deficient. The cerebrospinal fluid (CSF) markers of Cbl deficiency (Cbl and total homocysteine [tHCYS] levels) and the CSF levels of tumor necrosis factor (TNF)-alpha and epidermal growth factor (EGF) were measured. Significantly higher levels of tHCYS and TNF-alpha, and significantly lower levels of Cbl and EGF were found in the SCD patients. In human CSF, as in human serum and the rat central nervous system, decreased Cbl concentrations are concomitant with an increase in TNF-alpha and a decrease in EGF-levels. Ann Neurol 2004;56:886-890.

Cobalamin (vitamin B12)-deficiency-induced changes in the proteome of rat cerebrospinal fluid.

We studied the changes in the proteome of CSF (cerebrospinal fluid) in two animal models of Cbl (cobalamin) deficiency: TGX (totally gastrectomized) rats and rats fed a Cbl-D (Cbl-deficient) diet. Two-dimensional PAGE was used to detect qualitative and quantitative variations in proteins in the CSF samples. The peak increase in total CSF protein concentration was observed 4 months after TG (total gastrectomy) and after 6 months of eating a Cbl-D diet. There is a specific increase 4 months after TG in the spots corresponding to alpha1-antitrypsin and the de novo presence of thiostatin and haptoglobin beta. Cbl-replacement treatment in 4-month-TGX rats corrected these alterations in the CSF proteome. However, most of the CSF proteome alterations attenuated in Cbl-untreated 8-month-TGX rats and in rats fed a Cbl-D diet for 16 months. Transthyretin concentration varied slightly in the CSF of both types of Cbl-D rat, whereas the relative abundance of prostaglandin D synthase rose sharply in the CSF of the rats fed a Cbl-D diet for 16 months. We have demonstrated previously that the histological and ultrastructural CNS (central nervous system) damage in both types of Cbl-D rat appears within 2-3 months of Cbl deficiency, and thus appears to precede the alterations in the CSF proteome. The CSF proteome patterns of rats in which phlogosis was induced in or outside the CNS are quite different from those of the CSF of Cbl-D rats. All these findings demonstrate that the alterations in the CSF proteome of Cbl-D rats are specifically linked to Cbl deficiency.

Nature and nurture in vitamin B12 deficiency.

We report on a child in whom severe nutritional vitamin B12 deficiency was exacerbated by a genetic impairment of the folate cycle, causing reduced CSF concentrations of the methyl group donor 5-methyltetrahydrofolate. Some patients with vitamin B12 deficiency may benefit from high dose folic acid supplementation, even if plasma concentrations are high.

Cobalamin (vitamin B(12)) positively regulates interleukin-6 levels in rat cerebrospinal fluid.

We have previously demonstrated that the repeated intracerebroventricular (i.c.v.) microinjection of interleukin-6 (IL-6) prevented the myelinolytic lesions of cobalamin-deficient (Cbl-D) central neuropathy [or subacute combined degeneration (SCD)] in totally gastrectomized (TGX) rats. We therefore hypothesized that cobalamin (Cbl) may actually regulate IL-6 levels in rat cerebrospinal fluid (CSF). We measured IL-6 levels in the CSF of rats made Cbl-D by means of total gastrectomy (TG) or chronic feeding with a Cbl-D diet and killed at different times from the beginning of the experiment, and found that IL-6 levels significantly and progressively decreased over time. Chronic 2-month Cbl administration started 1 week after surgery prevented the decrease in IL-6 levels and, when it was started 2 months after surgery, it significantly increased IL-6 levels, but not to presurgical values. We also investigated whether IL-6 decrease might be ultimately due to the Cbl-deficiency-linked decrease in epidermal growth factor (EGF) synthesis. Repeated i.c.v. administrations of EGF to TGX rats did not modify CSF IL-6 levels. These results, together with those of a previous study showing the preventive effect of IL-6 treatment on SCD lesions, demonstrate that: (i) Cbl selectively regulates CSF IL-6 levels; and (ii) decreased IL-6 availability plays a role in the pathogenesis of the experimental SCD, in which no evidence of inflammatory and/or immunological reaction has been observed.

Elevation of 2-methylcitric acid I and II levels in serum, urine, and cerebrospinal fluid of patients with cobalamin deficiency.

Citrate synthase catalyzes the condensation of acetyl-coenzyme A (CoA) and oxaloacetic acid to form citric acid. The enzyme also catalyzes the condensation of propionyl-CoA and oxaloacetic acid with a maximal reaction velocity (Vmax) approximately 10(-4) times that of acetyl-CoA to form 2-methylcitric acid, which contains two asymmetric carbon atoms and exists as two pairs of related enantiomers designated as 2-methylcitric acid I and II. Cobalamin (Cbl) deficiency can lead to increases in intracellular levels of propionyl-CoA. To assess the magnitude of increased synthesis of 2-methylcitric acid in Cbl deficiency, we developed a new capillary gas chromatographic-mass spectrometric assay and measured 2-methylcitric acid levels in serum and cerebrospinal fluid (CSF) of normal subjects and patients with clinically confirmed Cbl deficiency. The normal range for 2-methylcitric acid level was 60 to 228 nmol/L for serum in 50 normal blood donors and 323 to 1,070 nmol/L for CSF in 19 normal subjects. In 50 patients with clinically confirmed Cbl deficiency, values for 2-methylcitric acid in serum ranged from 93 to 13,500 nmol/L; 44 (88%) had values above the normal range. In five patients with clinically confirmed Cbl deficiency, levels of the sum of 2-methylcitric acid I and II ranged from 1,370 to 16,300 nmol/L in CSF, and all five (100%) patients had levels above the normal range. We conclude that levels of 2-methylcitric acid are elevated in serum and CSF of most patients with Cbl deficiency.

Decreased vitamin B12 and folate levels in cerebrospinal fluid and serum of multiple sclerosis patients after high-dose intravenous methylprednisolone.

Twenty-one patients (15 women, 6 men) with definite multiple sclerosis (MS) were treated with 1000 mg intravenous methylprednisolone-succinate (MP) daily for 10 days. Before MP treatment there was a negative correlation (r = 0.59, P = 0.0084) between serum vitamin B12 and progression rate, defined as the ratio of the score on Kurtzke's Expanded Disability Status Scale and disease duration. A significant decrease was demonstrated in the cerebrospinal fluid (CSF) and serum levels of folate and in the CSF level of vitamin B12 after MP treatment. The decrease in serum B12 was not statistically significant. After MP treatment all median levels of vitamin B12 and folate were below the reference medians. We hypothesize that low or reduced vitamin B12/folate levels found in MS patients may be related to previous corticosteroid treatments. Otherwise a more causal relationship between low vitamin B12/folate and MS cannot be excluded. Further studies may be required to clarify the vitamin B12 and folate metabolism in patients with MS.

Vitamin B12 in CSF: reduced CSF/serum B12 ratio in demented men.

Vitamin B12 concentrations were determined in serum and cerebrospinal fluid (CSF) of 32 controls and 102 patients with dementia. The dementias were classified as Alzheimer's disease (AD), senile dementia of Alzheimer type (SDAT) and multi-infarct dementia (MID). A substantial number of patients (n = 42) could not be assigned to any of these diagnostic groups, as their dementias were of non-AD/SDAT and non-MID types. They were instead assigned to a group called non ultra descriptum (NUD). CSF B12 correlated significantly with serum B12. There were no statistically significant differences in serum B12 levels between the groups. Although with considerable overlap, CSF B12 concentrations and CSF/serum B12 ratios were significantly lower in the NUD group than in the control group. The NUD group had significantly lower CSF/serum B12 ratios than the group of patients with AD/SDAT. There was significant male predominance in the group of demented patients that had low CSF/serum B12 ratios outside the bivariate reference region. CSF and serum B12 levels appear insufficient as measures of the true brain vitamin B12 status. It may be a more dynamic approach to use the CSF/serum B12 ratio as an indication of transport function across the blood brain barrier, and possibly also across the CSF brain cell barrier.

Cerebrospinal fluid methylmalonic acid levels in normal subjects and patients with cobalamin deficiency.

We measured methylmalonic acid, which accumulates in the blood and tissues of patients with cobalamin deficiency, in the CSF of 65 patients using capillary-gas chromatography and mass spectrometry. In 58 control patients, methylmalonic acid concentrations were always higher in CSF than in serum (mean CSF: serum ratio, 2.65; range, 1.17 to 7.78). In contrast, in six patients with elevated serum methylmalonic acid levels due to renal failure, CSF concentrations were normal in five and the CSF: serum ratio was less than one in four. In three patients with neuropsychiatric syndromes due to cobalamin deficiency and one patient with a normal serum cobalamin level who was an abuser of nitrous oxide, CSF concentrations were markedly increased (mean level, 600 times that of controls), out of proportion to those in the serum (mean CSF: serum ratio, 8.38; range, 3.5 to 13.5). The potential usefulness of CSF metabolite levels in the diagnosis of cobalamin deficiency is undetermined.

1H NMR characterization of normal human cerebrospinal fluid and the detection of methylmalonic acid in a vitamin B12 deficient patient.

1H NMR was used to analyze human cerebrospinal fluid (CSF) from a group of neurological disease controls and from a vitamin B12 deficient patient. The spectra were acquired at either 7.06 or 9.40 T at ambient temperature with CSF freeze dried and reconstituted in 2H2O. 3-Trimethylsilyl propionate was used as an internal chemical shift and concentration reference. All of the CSF samples showed peaks for lactate, L-alanine, acetate, glutamine, citrate, creatine/creatinine and sugar resonances. There was good agreement between the metabolite concentrations as determined by NMR with those obtained using conventional chemical methods. 1D and 2D 1H NMR techniques along with J-coupling and T1 analysis were used to confirm the peak assignments. Methylmalonic acid could be detected and quantitated (ca 150 microM) in the CSF from the vitamin B12 deficient patient.

Effect of folic acid and vitamin B12 deficiencies on 5-hydroxyindoleacetic acid in human cerebrospinal fluid.

Indoles were measured in cerebrospinal fluid (CSF) from control patients, from patients suffering from folate deficiency, and from patients with vitamin B12 deficiency. The folate-deficient patients were classified according to whether they exhibited a neuropsychiatric syndrome, consisting of organic mental changes, polyneuropathy, and depression, which responded to folate administration. CSF 5-hydroxyindoleacetic acid was low in the vitamin B12-deficient patients and in those folate-deficient patients whose symptoms were not related to folate deficiency. CSF 5-hydroxyindoleacetic acid returned to normal with folate treatment in the patients exhibiting folate-responsive neuropsychiatric signs. The data indicate a close association between folate-responsive neuropsychiatric symptoms and changes in 5-hydroxytryptamine metabolism in the central nervous system.

[Demyelinating diseases and the cerebrospinal fluid cytoproteinogram]. / Afecções desmielinizantes e citoproteinograma do líquido cefalorraqueano.

Neuromyelitis of cyanocobalamin deficiency (group 1) post vaccination myelopathy (group 2), multiple sclerosis and neuromyelitis optica (group 3) are studied comparatively as to the respective cerebrospinal fluid changes on cytology, proteins and gamma globulins. Changes were found to be more intense and frequent in the last two groups. Local immunecompetent phenomena are considered in the interpretation of findings and related to modifications that may be induced in myelin macromolecules by factors of diverse order which may be involved in demyelination in each one of the three groups.