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J. Inher. Metab. Dis. 22 (1999) 599È607 ( SSIEM and Kluwer Academic Publishers. Printed in the Netherlands

Progressive neurological deterioration and MRI changes in cblC methylmalonic acidaemia treated with hydroxocobalamin G. M. ENNS1*, A. J. BARKOVICH2, D. S. ROSENBLATT4, D. R. FREDRICK3, K. WEISIGER1, C. OHNSTAD1 and S. PACKMAN1¤ 1 Division of Medical Genetics, Department of Pediatrics, 2 Department of Radiology, 3 Department of Ophthalmology, University of California, San Francisco, California, USA ; 4 Departments of Human Genetics, Medicine and Pediatrics, McGill University, Montreal, Quebec, Canada. * Current address : Stanford University, Department of Pediatrics, Division of Medical Genetics, Stanford, CA, USA ¤ Correspondence : University of California San Francisco, Division of Medical Genetics, Department of Pediatrics, 533 Parnassus Avenue, San Francisco, CA 94143-0748, USA MS received 16.06.98

Accepted 11.01.99

Summary : Cobalamin C (cblC) defects result in decreased activity of both methylmalonyl-CoA mutase and N5-methyltetrahydrofolate :homocysteine methyltransferase (methionine synthase), with subsequent methylmalonic aciduria and homocystinuria. Patients typically show failure to thrive, developmental delay and megaloblastic anaemia. Vitamin B therapy has been 12 beneÐcial in some cases. We report a now 4-year-old Hispanic girl with cblC disease documented by complementation analysis, with progressive neurological deterioration and worsening head MRI changes while on intramuscular hydroxocobalamin begun at age 3 weeks. Oral carnitine and folic acid were added at age 1 year. Blood levels of methylmalonic acid were reduced to treatment ranges. In the absence of acute metabolic crises, she developed microcephaly, progressive hypotonia and decreased interactiveness. Funduscopic examination was normal at age 13 months. At age 19 months, she developed nystagmus, and darkly pigmented fundi and sclerotic retinal vessels were observed on examination. Her neonatal head MRI was normal. By age 1 year, the MRI showed di†use white-matter loss, with secondary third and lateral ventricle enlargement, a thin corpus callosum, and normal basal ganglia. At age 15 months, progression of the white-matter loss, as well as hyperintense globi pallidi, were present. Interval progression of both grey- and white-matter loss was seen at age 27 months. We therefore caution that progressive neurological deterioration and head MRI abnormalities may still occur in cblC disease, despite early initiation of hydroxocobalamin therapy and improvement in toxic metabolite concentrations in physiological Ñuids. 599

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Methylmalonic acidaemia may result either from defects in methylmalonyl-CoA mutase (EC 5.4.99.2) or from processing of vitamin B (cobalamin, cbl). Cobalamin 12 C (cblC) defects (McKusick 277400) result in impaired synthesis of methylcobalamin and adenosylcobalamin and secondary decreased activity of both methylmalonylCoA mutase and N5-methyltetrahydrofolate :homocysteine methyltransferase (methionine synthase) (EC 2.1.1.13). Patients present with combined methylmalonic aciduria and homocystinuria. Two distinct phenotypes have been delineated (Rosenblatt et al 1997). Most patients present in the Ðrst year of life and have a severe course characterized by neurodegeneration, failure to thrive, retinopathy, and haematological abnormalities including neutropenia and thrombocytopenia. A minority of patients present in childhood and have a milder clinical course characterized by extrapyramidal signs and psychosis, and less severe haematological abnormalities. Whereas survival with mild to moderate neurological impairment is typical in later-onset cases, mortality or severe neurological dysfunction are usual in cases with onset in infancy (Rosenblatt et al 1997). Decreased activities of cobalamin reductase and cyanocobalamin b-ligand transferase have been documented in Ðbroblasts from patients with cblC disease, but the speciÐc molecular mechanism underlying the disease remains unknown (Mellman et al 1979 ; Pezacka 1993 ; Watanabe et al 1996). Treatment with intramuscular hydroxocobalamin results in improvement, but not complete correction, of the biochemical parameters associated with cblC disease. However, the e†ect of therapy on disease progression has been equivocal (Bartholomew et al 1988 ; Carmel et al 1980 ; Cogan et al 1980 ; Robb et al 1984 ; Rosenblatt et al 1997), with some cases showing improvement (Andersson and Shapira 1998 ; Bartholomew et al 1988 ; Mitchell et al 1986). Betaine, carnitine and folinic acid have also been used in treatment, but without clear additional clinical beneÐt (Bartholomew et al 1988 ; Mitchell et al 1986). In mut0 and mut~ forms of methylmalonic acidaemia, basal ganglia lesions have been documented (de Sousa et al 1989 ; Heidenreich et al 1988 ; Roodhooft et al 1990 ; Yamaguchi et al 1995). Globus pallidus lesions have also been reported in patients with cblA disease (McKusick 251100), cblB disease (McKusick 251110), or cobalamin-responsive disease of unknown type (Andreula et al 1991, Brismar and Ozand 1994 ; Heidenreich et al 1986 ; Korf et al 1986 ; de Sousa et al 1989). Other reports have emphasized delayed myelination, di†use leukoencephalopathy and cerebral atrophy in mut0 and mut~ methylmalonic acidaemia patients in the absence of globus pallidus lesions (Brismar and Ozand 1994 ; Gebarski et al 1983). In cblC disease, CNS imaging has been reported for single time points in only a few cases. These cblC patients have shown cortical atrophy, hydrocephaly and prominent lateral ventricles and interhemispheric Ðssures, but no basal ganglia involvement (Brandstetter et al 1990 ; Carmel et al 1980 ; Cogan et al 1980 ; Rosenblatt et al 1997 ; Traboulsi et al 1992). We herein report a study of the actual progression of the pathological anatomy of CNS and ophthalmological changes in a patient with cblC disease, in the face of ongoing hydroxocobalamin supplementation begun at age 3 weeks.

J. Inher. Metab. Dis. 22 (1999)

MRI changes in cblC disease

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METHODS Plasma amino acid analyses and determinations of free homocystine levels were performed using an automated Beckman System 6300 Amino Acid Analyzer. Plasma methylmalonic acid (MMA) levels and urine organic acid levels were determined by GC-MS analysis (Hewlett-Packard). Plasma total homocysteine levels were determined using an HPLC system with Ñuorescence detection (Mayo Medical Lab, Rochester, MN, USA). Fibroblast [14C]propionate, [14C]methyltetrahydrofolate, and [57Co]cyanocobalamin uptake determinations and complementation analyses were performed as described previously (Rosenblatt and Cooper 1987 ; Rosenblatt et al 1984). Head MRI imaging consisted of 3 mm saggital spin echo T1-weighted images (repetition time (TR) \ 550 ms, echo time (TE) \ 16 ms), 4È5 mm spin echo dual echo T2-weighted axial images (TR \ 2500 ms, TE \ 30, 80 ms), and 4È5 mm axial spin echo T1-weighted images (TR \ 500 ms, TE \ 16 ms).

CLINICAL HISTORY The index patient is a 4-year-old Hispanic girl with cblC methylmalonic acidaemia documented by complementation analysis (Howard et al 1997). She was born at term to a G P SAb mother via spontaneous vaginal delivery. Her birthweight was 2 1 1 2900 g (25%) and Apgar scores were 7 and 9 at 1 and 5 minutes, respectively. From birth, she was hypotonic, had transient hypoglycaemia and fed poorly. Soon after admission to the Intensive Care Nursery at age 9 days, she developed an erythematous, desquamative rash, with hair loss (Howard et al 1997), and experienced one episode of apnoea. She was neutropenic (neutrophils 0.16 ] 109/L) and thrombocytopenic (platelets 74 ] 109/L). Serum electrolytes, liver enzymes and blood ammonia were normal. Urine MMA level at age 19 days was 368 mmol/mol creatinine (Cr) (normal 0È13) and urine methylcitric and 3-hydroxypropionic acids were also elevated. Plasma free homocystine level was 40 kmol/L (normal 0). Plasma branchedchain amino acid concentrations were normal, as were thyroid function studies, serum zinc level, serum biotinidase activity and chromosome analysis. A diagnosis of cblC or cblF (McKusick 277380) methylmalonic acidaemia was suspected, and branched-chain amino acid restriction and intramuscular hydroxocobalamin (500 kg 3 times weekly) were started. Resolution of the rash and haematological abnormalities, and initial improvement in neurological status, were observed shortly thereafter. ConÐrmation of the diagnosis of cblC disease was made by complementation analysis and measurement of adenosylcobalamin and methylcobalamin in cultured skin Ðbroblasts. Fibroblast uptake of [14C]propionate (1.3 nmol/mg protein per 18 h versus control 15), [14C]methyltetrahydrofolate (16 pmol/mg protein per 18 h versus control 101) and [57Co]cyanocobalamin (334 cpm/106 cells versus control 1964) were low. Fibroblast uptake of [14C]propionate and [14C]methyltetrahydrofolate improved approximately 3-fold following addition of hydroxycobalamin to the cell culture medium. When patient Ðbroblasts were fused with mut0 or cblD (McKusick 277410) cell lines, increased uptake of [14C]propionate was observed. J. Inher. Metab. Dis. 22 (1999)

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However, no increase in [14C]propionate uptake was seen when the patient Ðbroblasts were fused with a known cblC cell line, indicating the presence of cblC disease (data not shown). In spite of treatment, her neurological examination worsened progressively, with choreoathetosis and brisk reÑexes appearing by age 13 months. At that time, carnitine and folic acid were added to the treatment regimen. At age 15 months, betaine (250 mg/kg per day) was started. At the time of initiating betaine therapy, the plasma total homocysteine level was 87 kmol/L (normal 4È17). Eight days after starting betaine, she developed a seizure disorder and betaine was discontinued, because the new-onset seizures were considered to present a possible idiosyncratic reaction to betaine. In the absence of acute metabolic crises, she subsequently developed microcephaly, progressive hypotonia, and decreased interactiveness. At age 17 months, hydroxocobalamin therapy was increased to daily dosing. At age 4 years, she is profoundly hypotonic and noninteractive. She did not, however, fail to thrive, with her most recent length being at the 75th centile and weight [ 95th centile. Her neonatal head MRI was normal. By age 1 year, the MRI showed di†use white-matter loss, with secondary third and lateral ventricle enlargement, a thin corpus callosum, and normal basal ganglia (Figure 1a). At age 15 months, progression of the white-matter loss as well as hyperintense globi pallidi were present (Figure 1b). Further interval progression of the grey- and white-matter disease was seen at age 27 months (Figure 1c). Funduscopic examinations as a neonate and at age 13 months were normal. At age 19 months, nystagmus, darkly pigmented fundi and sclerotic retinal arterioles were present bilaterally. Subsequent ophthalmological evaluations showed the same degree of pigmentary retinopathy, but there was also progressive bilateral retinal arteriole sclerosis and decreased visual attentiveness. DISCUSSION Cobalamin defects have been classiÐed into subgroups based on complementation analyses. Patients in cblA and cblB complementation groups fail to synthesize

Figure 1 (a) Axial T2-weighted image at age 12 months shows slight dilatation of the lateral ventricles with delayed myelination and some abnormal high signal intensity adjacent to the tips of the frontal horns. The globus pallidus is slightly hyperintense bilaterally (open arrows). The volume of white matter is slightly reduced compared to normal. (b) Axial T2-weighted image at age 15 months shows a notable reduction in the volume of white matter around the trigones and, to a lesser extent, the frontal horns of the lateral ventricles. The a†ected white matter is of abnormally high signal intensity. Some abnormal high signal intensity is present in the medial globus pallidus bilaterally, adjacent to the posterior limbs of the internal capsule (open arrows). (c) Axial T2-weighted image at age 27 months shows signiÐcant progression of the loss of volume of the white matter in the cerebral hemispheres, which also shows an increase in the abnormal hyperintensity. In addition, the cerebral cortex looks thinner than on the previous images. The abnormal hyperintensity of the medial globus pallidus remains unchanged. The internal capsule appears normal (closed arrows)


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deoxyadenosylcobalamin, a cofactor in the mutase reaction. CblC and cblD defects result in defective synthesis of both deoxyadenosylcobalamin and methylcobalamin. As methylcobalamin is a cofactor for methionine synthase, homocystinuria is also present in cblC and cblD disorders. CblC patients fall into two distinct phenotypic categories : an early-onset group with severe neurological deterioration, and lateronset type with a milder course (Rosenblatt et al 1997). Our patient presented as a neonate with a severe clinical course, typical in many ways of previously reported early-onset cblC cases. However, several unique features are present in her case. An erosive, exfoliative dermatitis was present as a neonate (Howard et al 1997). Such a rash had been reported previously in cblF disease or in uncharacterized methylmalonic acidaemia patients (Howard et al 1977). Our patient also showed progressive head MRI changes a†ecting the white matter and basal ganglia, as well as extrapyramidal signs (choreoathetosis). These have been more frequently associated with later-onset cblC cases (Rosenblatt et al 1997). Poor linear growth and weight gain have been seen in patients with cblC disease, but were not present in our patient. To our knowledge, this is the Ðrst report of neurological deterioration in a patient with cblC disease associated with progressive head MRI changes involving the basal ganglia. The basal ganglia have previously been shown to be involved in patients with mut0 or mut~ forms of methylmalonic acidaemia (de Sousa et al 1989 ; Gebarski et al 1983 ; Heidenreich et al 1988 ; Roodhooft et al 1990 ; Yamaguchi et al 1995), and in patients with cblA or cblB disease (Brismar and Ozand 1994 ; de Sousa et al 1989 ; Heidenreich et al 1988 ; Korf et al 1986). The exact cause of the basal ganglia lesions in methylmalonic acidaemia is unknown. Basal ganglia lesions have not been associated with defects in homocysteine remethylation per se. To explain such lesions in methylmalonic acidaemia, investigators have invoked the notion of toxicity of intermediary organic compounds that accumulate in areas of high metabolic activity (Heidenreich et al 1988 ; Wajner and Coelho 1997). MMA competitively inhibits succinate dehydrogenase (SDH) complex activity in brain and liver, blocking aerobic glucose oxidation (Wajner et al 1992). The basal ganglia are particularly susceptible to a decrease in SDH activity (Wajner and Coelho 1997), and such susceptibility may be increased in times of metabolic stress or catabolism. MMA may also inhibit gluconeogenesis, by decreasing entry of gluconeogenic amino acids into the Krebs cycle, or by causing a general decrease in the total energy available to drive gluconeogenesis (Dutra et al 1993 ; Wajner and Coelho 1997). During the Ðrst 18 months of life, our patientÏs mean urine MMA was 213 mmol/ mol Cr (normal 0È13), with 2/3 of the measurements \200 mmol/mol Cr. Thereafter, over a 26-month period, the average plasma MMA level was 2.22 kmol/L (SD ^ 0.91, normal \0.4), well below that in at least four previously reported patients (Bartholomew et al 1988 ; Mitchell et al 1986). Although hydroxocobalamin therapy improved our patientÏs plasma and urine MMA levels, the residual elevations may have had a role in the pathogenesis of the basal ganglia lesions. As well as having basal ganglia lesions, our patient was noted to have di†use demyelination (Figure 1), a Ðnding reported previously in patients with mut0 and J. Inher. Metab. Dis. 22 (1999)


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mut~ methylmalonic acidaemias (Brismar and Ozand 1994 ; Gebarski et al 1983). Demyelination has also been demonstrated in patients with functional methionine synthase deÐciency (cblG disease), 5,10-methylenetetrahydrofolate reductase deÐciency, and methionine adenosyltransferase deÐciency (Surtees 1998). To explain these Ðndings, a deÐciency of S-adenosylmethionine or elevated levels of Sadenosylhomocysteine have been postulated to interfere with essential methylation of, for example, myelin basic protein or of lipids responsible for maintaining the integrity of the myelin sheath (Scott et al 1994 ; Surtees 1998). Because cblC disease results in functional methionine synthase deÐciency and elevated plasma homocysteine levels, defective CNS methylation reactions may have played a signiÐcant role in the pathogenesis of the demyelination and neurological disease seen in our patient. In our patient, plasma total homocysteine levels averaged 105 kmol/L (SD ^ 39, normal 4È17). We could not compare our patientÏs total homocysteine levels to other cases in the literature, as previous studies have not reported the levels of total homocysteine. Betaine therapy was attempted for only a short time because of concern for a possible idiosyncratic reaction. In sum, the degree of demyelination caused by direct MMA toxicity versus the e†ects of cobalamin deÐciency on homocysteine remethylation is unknown. We also do not know whether more prolonged treatment with betaine or methionine supplementation would have resulted in a better clinical outcome, and suggest that such modalities be tested in future patients. Our patient developed nystagmus, progressive pigmentary retinopathy, retinal vessel narrowing and cortical blindness. Similar ocular Ðndings have been reported in other patients with cblC disease (Bartholomew et al 1988 ; Brandstetter et al 1990 ; Carmel et al 1980 ; Mitchell et al 1986 ; Robb et al 1984 ; Traboulsi et al 1992). Optic disc pallor (Bartholomew et al 1988 ; Robb et al 1984 ; Traboulsi et al 1992), atrophic maculopathy (Bartholomew et al 1988 ; Traboulsi et al 1992), retinal granularity (Andersson and Shapira 1998), and abnormal electroretinograms (Bartholomew et al 1988 ; Mitchell et al 1986 ; Robb et al 1984 ; Traboulsi et al 1992) have also been described. Such ophthalmological changes seem to be limited to the cblC (and, possibly, cblD) forms of methylmalonic acidaemia, and may be related to elevated homocystine levels, as retinal changes may be present in homocystinuria (Cross and Jensen 1973). In summary, ophthalmological abnormalities and progressive MRI changes involving the basal ganglia and white matter may occur in cblC disease, despite early initiation of hydroxocobalamin therapy, the prevention of acute encephalopathic episodes, and improvement in measurable clinical biochemical parameters. Such progression in the face of biochemical improvement suggests the possibility of a pathogenic mechanism based on cell autonomous e†ects of the failed cblC function. However, as a number of the speciÐc Ðndings are not restricted to cblC disease, a component of pathogenesis may well be related to toxicity of residual metabolites, including methylmalonic acid and homocysteine. These hypotheses concerning disease mechanisms should be incorporated both into genetic and prognostic counselling of patient families, and in the design of therapeutic approaches in this disorder.


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ACKNOWLEDGEMENT This work was supported by NIH Grant MO1RR01271 to the Pediatric Clinical Research Center, UCSF, and, in part, by a gift from the Genzyme Corporation. G.E. was supported by NIH Training Grant GM 07085.

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