Neuropsychiatric Disturbances in Presumed Late-Onset Cobalamin C Disease FREE

TISSUE VITAMIN B₁₂ deficiency can be due to inadequate intake (as seen in vegans), acquired malabsorption (as seen in pernicious anemia), or various inborn errors of cobalamin (Cbl) metabolism. Depending on the level of the metabolic block, the inborn errors may include congenital malabsorption due to intrinsic factor deficiency or defective transport by enterocytes (Imerslund-Grasbeck disease), defective cell delivery due to transcobalamin II (TCII) deficiency, and failure of cellular adenosylcobalamin (AdoCbl) and/or methylcobalamin (MetCbl) synthesis, which are the 2 active coenzyme forms of Cbl.^{1 - 2} These congenital diseases are typically characterized by a megaloblastic anemia and variable accumulation of methylmalonic acid (MMA) and/or homocysteine in the blood and urine (Figure 1).³ Tissue vitamin B₁₂ deficiency is associated with a variable level of serum vitamin B₁₂. It is always low in vegans or in those who have malabsorption and is usually normal in those with TCII deficiency or intracellular defects of AdoCbl or MetCbl synthesis.

Patients with congenital malabsorption develop megaloblastic anemia and failure to thrive in the first years of life and may later develop a myelopathy.^{2 - 3} In the first months of life, most patients with TCII deficiency present with severe megaloblastic anemia, failure to thrive, and diarrhea, but neurological involvement is not present at diagnosis.^{2 - 3} On the basis of complementation studies performed in cultured skin fibroblasts, failure in the synthesis of cellular AdoCbl and/or MetCbl has been divided into disease groups, Cbl A to Cbl H.^{1 ,4} Isolated increased levels of MMA in the blood and urine characterizes Cbl A, Cbl B, and Cbl H diseases. Most of these patients present in infancy with recurrent episodes of ketoacidosis without megaloblastic anemia. Hyperhomocysteinemia and hypomethioninemia without methylmalonic aciduria characterize Cbl E and Cbl G diseases. Most patients who have these 2 diseases present in the first months of life with megaloblastic anemia, poor feeding, and, if their disease is not promptly diagnosed, with various neurological deficits such as tonus abnormalities or seizures. Cobalamin C, Cbl D, and Cbl F diseases are due to defective synthesis of both MetCbl (resulting in hyperhomocysteinemia and hypomethioninemia) and AdoCbl (resulting in methylmalonic aciduria). Most of these patients present in the neonatal period with feeding difficulties, failure to thrive, neurological deterioration, and megaloblastic anemia. In addition, they may have renal and liver failure, cardiomyopathy, pneumonia, and retinopathy.^{3 ,5}

Late-onset forms are rare and difficult to diagnose because they are usually restricted to the nervous system, thus lacking the indicative hematological signs.^{5 - 6} Given the few reported cases and their possible genetic heterogeneity, the clinical spectrum of late-onset Cbl C disease needs to be clarified. We report 2 cases in a sibship with presumed Cbl C disease. The suspected pathophysiology of neurological and psychiatric disturbances is also discussed.

A 24-year-old woman was admitted because of a progressive gait disorder. Her medical history was unremarkable. She was born to nonconsanguineous parents and was 1 of 4 siblings. She reported frequent falls, inability to run, and difficulty in ascending and descending stairs. Findings on neurological examination were bilateral proximal weakness, hyperreflexia, spasticity, and impaired proprioception of the lower limbs, with a positive bilateral Babinski sign. Serologic test results for human immunodeficiency virus and human T-cell lymphotrophic virus 1 were negative as were those from tests for syphilis. Magnetic resonance imaging of the brain and the spinal cord showed no abnormalities, and fundoscopy findings and visual evoked responses were normal. Routine laboratory investigation findings were normal including hematological indices (hemoglobin, 11.9 g/dL; white blood cell count, 5.7 ×10⁹/L; platelet count, 281 ×10⁹/L; and mean corpuscular volume, 89.5 µm³). Serum vitamin B₁₂ levels (490 pg/mL; reference range, >200 pg/mL) and cerebrospinal fluid examination results were normal. Nine months after onset, corticosteroids were administered without benefit. During the following 18 months, her condition remained stable. When her sister (case 2) was diagnosed as having Cbl C disease, we performed an analysis of urinary organic acid levels and measured plasma homocysteine levels. The findings—a high urinary MMA level (3330 µmol/mmol of creatinine; reference range, <50 µmol/mmol of creatinine) and a raised homocysteine level level (reference range, 125 µmol/L; <15 µmol/L)—suggested that she had the same disease as her sister. Defective synthesis of AdoCbl and MetCbl was confirmed in cultured Epstein-Barr virus (EBV)–infected lymphocytes (Table 1). A combined daily treatment with intramuscular hydroxocobalamin (1 mg), oral betaine (9 g), L-carnitine (3 g), and folinic acid (10 mg) was started. After 3 months there was a marked decrease in urinary MMA and plasma homocysteine levels (Table 2), but the clinical examination findings were unchanged. However, the patient described a mild improvement in walking and a decreased frequency of falls.

Two years after the onset of symptoms in the patient in case 1, her 16-year-old sister was initially seen with a 3-month history of dissociative symptoms and delusions of persecution with visual and auditory hallucinations. One month prior to hospital admission, she had also developed an unsteady gait and urinary incontinence. She was previously healthy and had been an average student. Physical examination on admission showed an areflexic paraparesis with an extensor plantar response on the right side and impaired vibration and position sense in the lower limbs. Fundoscopy findings were normal. Neurophysiological studies showed intermediate conduction velocity with reduction of amplitude and denervation. A sural nerve biopsy specimen displayed demyelination and wallerian degeneration (Figure 2). Routine laboratory examination findings were normal including hematological indices (hemoglobin, 12.0 g/dL; white blood cell count, 6.0 ×10⁹/L; platelet count, 375 ×10⁹/L; and mean corpuscular volume, 81 µm³). Tests for common causes of peripheral neuropathy, including values for serum vitamin B₁₂ (417 pmol/L; reference range, >200 pmol/L) and urine porphyrin, and cerebrospinal fluid examination findings were normal. Electroencephalography showed diffuse slow waves. Magnetic resonance imaging brain study showed mild cortical atrophy and bilateral hyperintensity in the periventricular white matter on T2-weighted and flair images (Figure 3). Over the following 6 months, the motor deficit progressed to respiratory failure requiring mechanical ventilatory support. She also developed a deep venous thrombosis in the left femoral and pelvic veins, which was treated with antivitamin K. At this stage, she had persistent psychotic features, lethargy, and a complete tetraplegia characterized by spasticity of the upper limbs and flaccidity of the lower limbs. She was fully dependent on mechanical ventilatory support. A metabolic workup displayed a cellular vitamin B₁₂ deficiency with a high degree of methylmalonic aciduria (MMA level, 3890 µmol/mmol of creatinine; reference range, <50 µmol/mmol of creatinine), an elevated plasma total homocysteine level (205 µmol/L; reference range, <15 µmol/L), and hypomethioninemia (methionine levels, 7 µmol/L; reference range, >20 µmol/L]) with normal serum TCII levels. Measurement of AdoCbl and MetCbl synthesis in cultured skin fibroblasts and in cultured EBV-infected lymphocytes confirmed the defective synthesis of MetCbl and AdoCbl (Table 1). A combined daily treatment with intravenous hydroxocobalamin (2 mg), oral betaine (9 g), L-carnitine (3 g), and folinic acid (10 mg) was started. After 6 weeks, clinical improvement was dramatic with the recovery of arm function and the disappearance of psychotic features and lethargy. She also regained enough axial strength to allow her to sit but remained wheelchair bound. She was weaned off mechanical ventilatory support on day 45. Concurrently, biochemical abnormalities markedly improved (Table 2).

Screening of her 2 healthy brothers showed normal results for the presence of homocystinemia and methylmalonic aciduria and they were considered free of the disease.

Synthesis and measurement of AdoCbl and MetCbl were done on fibroblasts, EBV-infected lymphocytes, or both, as previously described.^{12 - 13} In case 1, fibroblast culture failed despite the addition of both methionine (15 mg/L) and 30% fetal calf serum, an occurrence that we have previously seen in some other cases of Cbl C disease. Briefly, cells undergoing exponential growth were transferred to RPMI medium deprived of vitamin B₁₂, supplemented with glutamine and 10% AB serum labeled with cobalt 57–labeled cyanocobalamin, and then incubated for 72 hours in a carbon dioxide incubator. Subsequently, cells were washed and centrifuged. The radioactivity was evaluated on a part of the pellet corresponding to the total intracellular uptake; the remainder was purified and fractionated by thin-layer chromatography. Distribution of the different forms of Cbl was then measured.

Our patients presented no evidence of having defective vitamin B₁₂ supplies, malabsorption, or transport since they had normal vitamin B₁₂ and TCII levels in their serum. Despite the absence of megaloblastic anemia and the presence of normal vitamin B₁₂ serum levels, they were both clearly affected with a neurological form of vitamin B₁₂ deficiency only diagnosed by the demonstration of homocystinuria and methylmalonic aciduria. Patient 1 exhibited a subacute combined degeneration of the spinal cord, which is highly suggestive of a vitamin B₁₂ deficiency. Nevertheless, the diagnosis of functional vitamin B₁₂ deficiency had been long delayed because of normal serum vitamin B₁₂ levels although a metabolic investigation of the cellular vitamin B₁₂ status had not been performed.

The metabolic disorder of these sisters can probably be included in the Cbl C group, although no complementation studies were done with fibroblasts from patients identified with Cbl D or Cbl F. First, Cbl C is the most common of these diseases; to our knowledge, only 2 cases in a sibship have been reported as Cbl D disease and fewer than 10 cases as Cbl F disease.⁶ Second, in contrast to fibroblasts from patients with Cbl F, which accumulate excess unmetabolized cyanocobalamin, fibroblasts from our patient had a low incorporation of cyanocobalamin (in accordance with our findings in other patients with Cbl C disease) compared with healthy control subjects. Similar therapeutic approaches are proposed for these 3 groups of Cbl disease and aim to normalize all metabolite values including methionine levels.

Most patients who have Cbl C disease have an early clinical onset, in the neonatal period or in early infancy. The neonates usually display feeding difficulties and rapidly progressive neurological deterioration that proceeds to coma. They often are initially seen with megaloblastic anemia and thrombocytopenia. They may also develop hemolytic uremic syndrome due to thrombotic microangiopathy. Most of them follow a downhill course with liver, cardiac, and pulmonary involvement. Survivors have mental retardation and a peculiar retinopathy with nystagmus.^{5 - 6} Late-onset Cbl C disease occurs more rarely as only 6 cases of patients with symptoms beginning after the age of 10 years have been reported in the literature.^{7 - 11} Table 3 summarizes the main manifestations of the disease in this age group. Late-onset Cbl C disease can manifest with various neurological or psychiatric disturbances, but all 8 patients (including the 3 described herein) had normal vitamin B₁₂ levels; none had megaloblastic anemia. Although a rapidly progressive disease is usual, a relapsing-remitting course may occur.⁸ As exemplified by the findings in our patients, disease expression is variable, even in a sibship. At present, neither this variability nor the existence of later-onset forms can be explained.

The subacute myelopathy with demyelination and peripheral neuropathy observed in these patients are similar to those observed in acquired forms of vitamin B₁₂ deficiency, such as pernicious anemia. The clinical neuromyelopathy related to vitamin B₁₂ deficiency has been described for more than a century, but the link between these neurological signs and the vitamin deficiency was not discovered until later.¹⁴ This neuromyelopathy is due to demyelination, as evidenced in our patient 2 in findings on both sural nerve biopsy and magnetic resonance imaging examination of the brain. A reduced supply of the methyl group has previously been implicated as a cause of central nervous system demyelination.^{15 - 16} In fact, methylation of homocysteine to form methionine is impaired. This leads to a deficiency of S-adenosylmethionine (SAM), which is a key intermediary in methylation reactions.¹⁷ In inborn errors of the methyltransfer pathway, demyelination has been shown to be associated with low SAM levels in the cerebrospinal fluid whereas normalization of SAM levels has been associated with evidence of remyelination.¹⁸ Myelin basic protein, which is a major component of the myelin sheath, is methylated by a specific methyltransferase requiring SAM as the methyl donor.¹⁹ It has, therefore, been hypothesized that demyelination may be due to decreased methylation of myelin basic protein, causing conformational changes in the protein and subsequent splitting of the myelin sheath.¹⁶

Among 8 late-onset cases of patients with Cbl C disease (Table 3), 4 exhibited signs of psychosis or dementia, isolated or in association with myeloneuropathy, but without any hematological abnormalities. In adults, vitamin B₁₂ deficiency has been described in neuropsychiatric disorders in the absence of anemia and/or macrocytosis, but in association with increased levels of homocysteine and urinary MMA.^{20 - 22} Metabolic evidence for vitamin B₁₂ deficiency has also been found more frequently in elderly patients with Alzheimer-type dementia than in controls.²³ Whatever its cause (congenital or acquired), hyperhomocystinemia and methylmalonic aciduria are the metabolic hallmarks of vitamin B₁₂ deficiency and measurements of their degree of severity seem (by measuring homocysteine and methionine levels) to be the most sensitive and accurate markers of intracellular vitamin B₁₂ status.^{24 - 25} Dementia and psychosis in patients with Cbl C disease are similar to those observed in adults intially seen with acquired vitamin B₁₂ deficiency. These psychiatric signs are most likely related to defective methylation. This hypothesis is sustained by the description of similar signs of myeloneuropathy variously associated with schizophrenia in patients affected with either a Cbl G disease or a methylenetetrahydrofolate reductase deficiency.^{26 - 28} Indeed, methylenetetrahydrofolate reductase is the enzyme responsible for the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, which carries the methyl group essential for methionine synthesis and, thus, for SAM synthesis (Figure 1).¹⁸ Methylenetetrahydrofolate reductase deficiency and Cbl G disease thus share the remethylation defect with Cbl C disease.

In addition, to neuropsychiatric disturbances, 1 of the 2 sisters described herein also developed a deep venous thrombosis of the femoral and pelvic veins, as previously described in 2 other cases of late-onset Cbl C disease (Table 3). Although the peripheral neuropathy (in 2 of these 3 patients noted in Table 3)—and the subsequent loss of mobility—could have favored the thrombotic complication, it is more likely to be related to the extreme hyperhomocysteinemia present in these patients. Patients initially seen with inborn errors of metabolism resulting in hyperhomocysteinemia and homocystinuria are known to have increased susceptibility to premature occlusive vascular disease and, in some cases, autopsy has revealed multiple thrombi involving both arteries and veins.^{29 - 30}

Most patients with Cbl C disease respond biochemically and clinically when treated with high-dose systemic hydroxocobalamin.^{7 - 11} As with pernicious anemia, myelopathy and peripheral neuropathy improved more slowly and less completely than cortical signs (Table 3). Because this treatable condition can lead to death or irreversible neurological damage, it requires prompt diagnosis and treatment. This article emphasizes the need, at any age, for extensive investigation of the vitamin B₁₂ status in patients with neurological symptoms whose clinical picture is consistent with vitamin B₁₂ deficiency and in whom the serum vitamin B₁₂ level is normal.

Corresponding author: Francis Bolgert, MD, Service de Neurologie 1, Groupe Hospitalier Pitié-Salpêtrière, Unité de Réanimation Neurologique, 47-83 Boulevard de l'hôpital, 75651 Paris CEDEX 13, France (e-mail: sophie.demeret@psl.ap-hop-paris.fr).

Accepted for publication December 30, 2002.

Author contributions: Study concept and design (Drs Roze, Gervais, Demeret, Ogier de Baulny, Zittoun, and Bolgert); acquisition of data (Drs Roze, Zittoun, Benoist, Said, and Bolgert); analysis and interpretation of data (Drs Roze, Demeret, Ogier de Baulny, Zittoun, Pierrot Deseilligny, and Bolgert); drafting of the manuscript (Drs Roze and Zittoun); critical revision of the manuscript for important intellectual content (Drs Roze, Gervais, Demeret, Ogier de Baulny, Zittoun, Benoist, Said, Pierrot Deseilligny, and Bolgert); obtained funding (Dr Roze); administrative, technical, and material support (Drs Benoist, Pierrot Deseilligny, and Bolgert); study supervision (Drs Gervais, Demeret, Ogier de Baulny, Zittoun, Pierrot Deseilligny, and Bolgert).

We thank Nicole Baumann and Katia Youssov for help with the clinical care of the patients. We also thank Thierry Maisonobe who performed the neurophysiological studies and Kristin Roze for support.