Cultured diploid fibroblasts from a patient with a previously undescribed inborn error of cobalamin metabolism accumulate unmetabolized, nonprotein-bound vitamin B12 in lysosomes. These cells are able to endocytose the transcobalamin II-B12 complex and to release B12 from transcobalamin II. The freed vitamin B12 is not released from lysosomes into the cytoplasm of the cell. This suggests that there is a specific lysosomal transport mechanism for vitamin B12 in the human.

This paper reports clinical and metabolic studies of two Italian siblings with a novel form of persistent isolated hypermethioninaemia, i.e. abnormally elevated plasma methionine that lasted beyond the first months of life and is not due to cystathionine beta-synthase deficiency, tyrosinaemia I or liver disease. Abnormal elevations of their plasma S-adenosylmethionine (AdoMet) concentrations proved they do not have deficient activity of methionine adenosyltransferase I/III. A variety of studies provided evidence that the elevations of methionine and AdoMet are not caused by defects in the methionine transamination pathway, deficient activity of methionine adenosyltransferase II, a mutation in methylenetetrahydrofolate reductase rendering this activity resistant to inhibition by AdoMet, or deficient activity of guanidinoacetate methyltransferase. Plasma sarcosine (N-methylglycine) is elevated, together with elevated plasma AdoMet in normal subjects following oral methionine loads and in association with increased plasma levels of both methionine and AdoMet in cystathionine beta-synthase-deficient individuals. However, plasma sarcosine is not elevated in these siblings. The latter result provides evidence they are deficient in activity of glycine N-methyltransferase (GNMT). The only clinical abnormalities in these siblings are mild hepatomegaly and chronic elevation of serum transaminases not attributable to conventional causes of liver disease. A possible causative connection between GNMT deficiency and these hepatitis-like manifestations is discussed. Further studies are required to evaluate whether dietary methionine restriction will be useful in this situation.

A s bstract. Cultured fibroblasts from a recently described patient with homocystinuria and megaloblastic anemia of infancy without methylmalonic aciduria were previously shown to have normal cobalamin uptake and a specific decrease in the proportion of intracellular methylcobalamin. As in control cells but unlike in those from patients with combined homocystinuria and methylmalonic aciduria (cobalamin C and cobalamin D), accumulated 57Co-labeled cobalamin was bound in appropriate amounts and proportion to intracellular binders which are known to be the two vitamin B12-dependent enzymes, methionine synthetase and methylmalonylCoA mutase. Despite the association of a normal quantity of intracellular cobalamin with methionine synthetase, the proportion of intracellular cobalamin which was methyl-B12 was below normal and in the range observed in cobalamin C and D cells. This methyl-B12 was decreased by exposure of fibroblasts in culture to nitrous oxide as was observed with control cells. Exposure of control fibroblasts during culture, but not of fibroblasts from this patient, to nitrous oxide significantly reduced

MethylmalonylCoA mutase (MCM) is a mitochondrial homodimer responsible for the isomerization of methylmalonylCoA to succinylCoA. Apomutase defects are traditionally divided into muto and mut- classes on the basis of residual mutase activity. Clinical findings were reviewed in 20 patients with methylmalonic aciduria secondary to MCM deficiency. All 11 muto patients had an early neonatal presentation; 6 of these patients died in infancy and 3 of 5 survivors had a poor neurological outcome as evidenced by severe delay or spastic quadriparesis with dystonia. The 2 other survivors include a 27-month-old child with a mild delay in verbal and fine motor skills and an adolescent with low normal intelligence. Of the 9 mut- patients, 7 became symptomatic in late infancy or childhood and 2 were picked up on screening. Two of the 9 patients have never had an episode of metabolic decompensation yet both are neurologically compromised; one severely retarded and autistic, the other mildly delayed. Four mut- patients have had episodic acidosis and are neurologically moderately affected, while 3 have had episodic acidosis and are neurologically intact. These results confirm phenotypic pleomorphism without a consistent pattern of neurological injury and suggest some broad correlation between mutase class and phenotype. Survival with good outcome is possible among muto patients as is significant morbidity among mut- patients. Acidosis and metabolic imbalance are not necessary preconditions for significant morbidity.

The mut' mutation resulting in methylmalonyl CoA mutase (MCM) apoenzyme deficiency and methylmalonic aciduria is characterized by undetectable enzyme activity in cell extracts and low incorporation of propionate into cultured cells which is not stimulated by hydroxycobalamin. A muto fibroblast cell line (WG1681) from an African-American male infant complemented another mut0 cell line (WG 1130). Cloning and sequencing of cDNA from WG 1681 demonstrated compound heterozygosity for two novel changes at highly conserved sites: G623R and G703R. In addition, two previously described homozygous polymorphisms, H532R and V6711, were found. Hybridization of allele-specific oligonucleotides to PCR amplified MCM exons from the proband and family members identified a clinically normal mother, half-sister, and half-brother as carriers of the G703R change in cis with both polymorphisms.Transfection of each change into a mut' cell line with very low

Conclusions-These results strongly suggest that the cblA variant represents a novel complementation class, which we have designated cblH and which represents a mutation at a distinct gene. They also suggest that the cblA gene encodes a protein that functions as a multimer, allowing for extensive interallelic complementation. (J Med Genet 2000;37:510-513)

Methylmalonic aciduria (MMA) is an autosomal recessive inborn error of metabolism that results from functional defects in methylmalonyl CoA mutase (MCM), a nuclear-encoded, mitochondrial enzyme that uses the vitamin B12 derivative, adenosylcobalamin (AdoCbl) as a cofactor. To date, 23 mutations have been identified at the MUT locus on the short arm of chromosome 6, causing the mut forms of MMA (mut complementation group; mut MMA, McKusick #251000). We now report seven novel mutations. Three were found inmut0 patients: R228Q (c759G-->A) was found as a heterozygous change; G312V (c1011G-->T) and 346delL (c1112delCTT) were both found as homozygous changes. Four mutations were found in mut patients: A191E (c648C-->A) and V633G (c1974T-->G) were found in the same patient; 684insL (c2128insCTC) and L685R (c2130T-->G) were both found as homozygous changes. The recent modelling of the human methylmalonyl CoA mutase allowed for an interpretation of the identified mutations.

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