J V Leonard

Urinary bile acids from a 3-mo-old boy with cholestatic jaundice were analyzed by ion exchange chromatography and gas chromatography-mass spectrometry (GC-MS). This suggested the presence of labile sulfated cholenoic acids with an allylic hydroxyl group, a conclusion supported by analysis using fast atom bombardment mass spectrometry (FAB-MS). The compounds detected by FAB-MS were separated by thin layer chromatography and high performance liquid chromatography. The sulfated bile acids could be solvolyzed in acidified tetrahydrofuran, and glycine conjugates were partially hydrolyzed by cholylglycine hydrolase. Following solvolysis, deconjugation, and methylation with diazomethane, the bile acids were identified by GC-MS of trimethylsilyl derivatives. The major bile acids in the urine were 3 beta,7 alpha-dihydroxy-5-cholenoic acid 3-sulfate, 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid monosulfate, and their glycine conjugates. Chenodeoxycholic acid and cholic acid were undetectable in urine and plasma. The family pedigree suggested that abnormal bile acid synthesis was an autosomal recessive condition leading to cirrhosis in early childhood.

Glycogen storage disease type HI (GSD-III), an autosomal recessive disease, is caused by deficient glycogen debranching enzyme (GDE) activity. Most GSD-III patients are GDE deficient in both liver and muscle (type IIIa), and some GSD-III patients have GDE absent in liver but retained in muscle (type IIIb). The molecular basis for this enzymatic variability is largely unknown. In the present study, the analysis of the GDE gene in three GSD-IIIb patients by single-strand conformation polymorphism (SSCP), DNA sequencing, restriction analysis, and family studies, revealed each of them as being a compound heterozygote for two different mutations. The first mutant alleles in all three patients involved mutations in exon 3 at amino acid codon 6 of the GDE protein. Two had an AG deletion at nucleotides 17 and 18 of the GDE cDNA (17delAG) which resulted in change of subsequent amino acid sequence and a truncated protein (25X); the other had a C to T transition at nucleotide 16 of the cDNA which changed a Glutamine codon to a stop codon (Q6X). The 17delAG mutation was also found in 8 of the 10 additional GSD-IIIb patients. The Q6X mutation was found in one of the remaining two GSD-IIIb patients. These two mutations were not found in any of the 31 GSD-IIIa patients, 2 GSD-IIId patients, nor 28 unrelated normal controls. The second mutant alleles in each of the three GSD-IIIb patients were R864X, R1228X, and W68OX. The R864X and R1228X were not unique for GSD-IIIb as they were also found in GSD-IIIa patients (frequency of 10.3% and 5.2% in Caucasian patients, respectively). Our data demonstrated that both IIIa and IIIb had mutations in the same GDE gene and established for the first time the molecular basis of GSD-III that differentially expressed in liver and muscle. The striking and specific association of exon 3 mutations with GSD-IIIb may provide insight into mechanisms controlling tissue-specific expression of the GDE gene. The identification of exon 3 mutations has clinical significance as well because it distinguished GSD-IIIb from IIIa hence permitting diagnosis from a blood sample rather than a more invasive muscle biopsy.

We previously described demyelination in the brain and subacute combined degeneration of the spinal cord in a patient with 5,10-methylenetetrahydrofolate reductase deficiency. To assess the role of methionine, S-adenosylmethionine, folate, and neurotransmitter amine metabolism in the demyelination process, we measured these metabolites in CSF from this patient; the findings are compared with those obtained from three patients in whom neurologic deterioration had been halted by the administration of betaine. Folate concentrations were low, and amine and biopterin metabolism were abnormal in all patients. Methionine and S-adenosylmethionine concentrations were undetectable in the first patient. In those receiving betaine, methionine concentrations were proportional to the dose administered and S-adenosylmethionine concentrations were near normal. The results provide the first evidence for an association between defective S-adenosylmethionine metabolism and demyelination in humans.