INTRODUCTION

The cobalamin C (cblC) complementation group is the most common inborn error of vitamin B₁₂ metabolism, affecting both methylcobalamin and adenosylcobalamin biosynthesis.¹ The resulting functional deficiency of methylmalonyl-CoA mutase and methionine synthase activities results in combined methylmalonic aciduria/hyperhomocysteinemia (MMA/HCys) (Figure 1). This disorder is often associated with both neurologic and systemic abnormalities, but recent work suggests early-onset (first year of life) and late-onset subgroupings with somewhat different clinical outcomes.² In the early-onset group, the clinical course is characterized by progressive global neurologic disability (hypotonia, seizures, developmental arrest) along with ophthalmologic and hematologic abnormalities. Childhood onset cblC defect is associated with milder hematologic abnormalities (often affecting only erythropoiesis), isolated extrapyramidal neurologic abnormalities and improved survival with only mild-to-moderate disability. Therapeutic intervention with hydroxycobalamin may improve biochemical abnormalities but may not completely prevent permanent neurologic sequelae.

Figure 1.

Homocysteine and methylmalonyl-CoA metabolism and the cblC defect in cobalamin metabolism. Cobalamin is essential for the metabolism of homocysteine and methylmalonyl-CoA. Methionine synthase catalyzes the conversion of homocysteine to methionine and requires the cofactor methylcobalamin (MeCbl) as a methyl group donor. The metabolism of methylmalonyl-CoA to succinyl-CoA is accomplished by the enzyme methylmalonyl-CoA mutase and requires the presence of adenosylcobalamin (AdoCbl). The cblC defect in cobalamin metabolism prevents the synthesis of MeCbl and AdoCbl from dietary vitamin B₁₂, causes functional deficiencies of methionine synthase and methylmalonyl-CoA mutase, and results in combined MMA/Hcys.

Full figure and legend (22K)

In the current report, we present two infants whose clinical presentation raised significant suspicion for acute onset neonatal bacterial or viral sepsis. An inborn error of metabolism was not initially regarded with high likelihood in the differential diagnosis, most likely because of an absence of any acute metabolic derangement detectable by routine chemistry studies. In addition, during biochemical genetic evaluation, we detected a massive increase in HCys in cerebrospinal fluid (CSF) from one case. This finding may provide insight into the underlying cause of neurologic deterioration in these and other cblC patients.

MATERIALS AND METHODS

All procedures followed standard laboratory practice, employing reagents of the highest available purity. Plasma amino acids and urine organic acids were quantified using ion-exchange chromatography and gas chromatography–mass spectrometry, respectively.^3,4 HCys in plasma or CSF was quantified by reversed-phase HPLC with electrochemical detection.⁵ Determination of [⁵⁷Co]-cyanocobalamin uptake and distribution, ¹⁴C-propionate and ¹⁴C-methyltetrahydrofolate uptake (with and without hydroxycobalamin), and cobalamin complementation studies in cultured fibroblasts were performed as described.² Routine fibroblast culture followed established methods.⁶

CASE REPORTS

Patient 1, a 3-week-old male, presented to the emergency department with lethargy, decreased oral intake and dehydration. Parents were first cousins of Saudi Arabian descent. Initial physical examination was remarkable for lethargy, diffuse hypotonia, and poor perfusion. Routine laboratory testing was significant only for leukopenia (total WBC=1500/mm³) and thrombocytopenia (platelet count=29,000/mm³). Hgb, Hct, and MCV were all normal (12.9 g/dl, 37.5%, and 97.8 fl, respectively) for age. Laboratory evaluation for bacterial sepsis was normal. The baby received empiric intravenous antibiotic therapy. Although perfusion improved, the lethargy did not respond to standard supportive care. At 1 week after hospital admission and prior to diagnosis of combined MMA/HCys, several episodes of generalized motor seizures in association with apnea occurred. EEG was mildly abnormal with a discontinuous pattern and occasional intermittent sharp transients seen bilaterally. Cranial CT scan was normal without evidence of hydrocephalus or hemorrhage. Seizures responded to treatment with phenobarbital and phenytoin. Diagnostic evaluation for inborn errors of metabolism revealed the presence of homocystine in plasma (67 mol/l) by amino-acid analyzer and of MMA in the urine organic acid analysis.

Patient 2, a Hispanic female, presented at day 19 of life with lethargy, diffuse hypotonia, dehydration, cough, and decreased oral intake. She had been seen twice in the emergency department and clinic, and had undergone a laboratory evaluation for bacterial sepsis with empiric parenteral antibiotic therapy prior to her NICU admission. Physical examination revealed mild hepatomegaly without splenomegaly, severe lethargy, diffuse hypotonia, but mildly hyperactive symmetric deep tendon reflexes. She was afebrile and normotensive. Laboratory evaluation demonstrated thrombocytopenia (platelet count=55,000/mm³) and mild neutropenia (Total WBC=5600/mm³, absolute neutrophil count=1000). Hgb and Hct were normal (13.1 g/dl and 37% respectively), while MCV=99 fl. Shortly after admission, recurrent generalized motor seizures developed in association with severe obtundation and extensor posturing. Both cranial CT and MRI scans were normal. Therapy with multiple anticonvulsants was required to prevent seizures during the first week of hospitalization. Plasma amino-acid analysis revealed the presence of homocystine (54 mol/l), and MMA was detected in urine by organic acid analysis.

Once the diagnosis of combined MMA/HCys was suspected, treatment with intramuscular hydroxycobalamin (1 mg/day) was initiated in both infants. Oral L-carnitine (100 mg/kg/day) and betaine (1.5 g/kg/day) therapies were added later to assist with the disposal of MMA and HCys, respectively. On this regimen, seizures were well-controlled, abnormal motor movements and muscle tone abnormalities improved, and full consciousness returned over a 2-week convalescence. Currently, both children are clinically stable, and have not suffered recurrent episodes of acute neurologic deterioration. Both children remain mildly diffusely hypotonic, and both motor and cognitive developmental parameters are moderately delayed.

RESULTS AND SIGNIFICANCE

Pertinent metabolite and enzyme findings are displayed in Table 1. The data verify decreased uptake of [⁵⁷Co]cyanocobalamin in cultured fibroblasts derived from both patients, and concomitant inability to convert cyanocobalamin to functional species, including adenosylcobalamin (the cofactor for methylmalonyl-CoA mutase activity) or methylcobalamin (the cofactor for methionine synthase). Inability of fibroblasts to incorporate ¹⁴C-propionate and ¹⁴C-methyltetrahydrofolate into acid-precipitable proteins is further evidence for these enzyme deficiencies. These data, in conjunction with complementation studies employing polyethylene glycol fusion of fibroblasts (not shown), indicate both patients belong to the cblC subgroup.

Table 1 - Metabolic and Intact Fibroblast Analyses in Two Patients with cblC Methylmalonic Aciduria/Hyperhomocysteinemia.

Full table

During routine metabolic workup of patient 2, CSF amino acids were measured in a search for the underlying cause of her seizure activity. To our knowledge, this is the first examination of CSF HCys levels in cblC deficiency. HCys concentration measured 35 M (normal <0.03 to 0.08 for age) in CSF, an approximate 400-fold increase over normal, and a 10-fold increase over that we have seen in an infant with nutritional cobalamin deficiency (3.4 M, data not published).

Highly increased CSF HCys is likely to be associated with neurotoxicity in cblC patients. Increased HCys will complex with free adenosine, thereby decreasing adenosine pools and impacting purine biosynthesis.⁷ Rates of transmethylation reactions (affecting DNA, RNA, and protein biosynthesis) are controlled by adenosylmethionine (AdoMet; methyl donor) and adenosylhomocysteine (AdoHCys) concentrations. Moreover, AdoHCys (the product of methyltransferase reactions) is a potent inhibitor of methyltransferase reactions, with K_i values of 0.1 to 10 M (considerably lower than K_m values for AdoMet). Thus, it is highly probable that increased CSF HCys will have a major impact on cerebral metabolism.⁸

Our results also suggest the prudence of pursuing a full laboratory evaluation for inborn errors of metabolism in neonates who present with the combination of deteriorating neurologic status and systemic findings such as thrombocytopenia or pancytopenia, even in the absence of more stereotypical laboratory signs of metabolic disease (metabolic acidosis, ketosis, hypoglycemia, or hyperammonemia). Inborn errors of metabolism should be included in the differential diagnosis of any critically ill infant but especially in those instances in which the laboratory evaluation for bacterial sepsis is negative.

References

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