Increased Clinical Sensitivity and Specificity of Plasma Protein N-Glycan Profiling for Diagnosing Congenital Disorders of Glycosylation by Use of Flow Injection-Electrospray Ionization-Quadrupole Time-of-Flight Mass Spectrometry.

BACKGROUND: Congenital disorders of glycosylation (CDG) represent 1 of the largest groups of metabolic disorders with >130 subtypes identified to date. The majority of CDG subtypes are disorders of N-linked glycosylation, in which carbohydrate residues, namely, N-glycans, are posttranslationally linked to asparagine molecules in peptides. To improve the diagnostic capability for CDG, we developed and validated a plasma N-glycan assay using flow injection-electrospray ionization-quadrupole time-of-flight mass spectrometry. METHODS: After PNGase F digestion of plasma glycoproteins, N-glycans were linked to a quinolone using a transient amine group at the reducing end, isolated by a hydrophilic interaction chromatography column, and then identified by accurate mass and quantified using a stable isotope-labeled glycopeptide as the internal standard. RESULTS: This assay differed from other N-glycan profiling methods because it was free of any contamination from circulating free glycans and was semiquantitative. The low end of the detection range tested was at 63 nmol/L for disialo-biantennary N-glycan. The majority of N-glycans in normal plasma had <1% abundance. Abnormal N-glycan profiles from 19 patients with known diagnoses of 11 different CDG subtypes were generated, some of which had previously been reported to have normal N-linked protein glycosylation by carbohydrate-deficient transferrin analysis. CONCLUSIONS: The clinical specificity and sensitivity of N-glycan analysis was much improved with this method. Additional CDGs can be diagnosed that would be missed by carbohydrate-deficient transferrin analysis. The assay provides novel biomarkers with diagnostic and potentially therapeutic significance.

PTC124 improves readthrough and increases enzymatic activity of the CPT1A R160X nonsense mutation.

Deficiency of carnitine palmitoyltransferase 1A (CPT1A) results in impaired hepatic long-chain fatty acid oxidation and ketogenesis. We have previously described a patient with a severe CPT1A phenotype who is homozygous for the nonsense mutation 478 C > T (R160X). It has been known for some time that gentamicin can promote readthrough of nonsense codons. Recently, a new compound (PTC124) with less clinical toxicity than gentamicin has been indicated as a therapy for patients with nonsense mutations for multiple genetic diseases. The study is designed to investigate whether PTC124 can promote readthrough of the R160X CPT1A mutation and increase normal sized CPT1 protein expression and activity in the patient's skin fibroblasts. Our study demonstrated that after both PTC 124 and gentamicin treatment, there was an increase in CPT1 activity in patient fibroblasts to levels that are similar to that of the mild Inuit P479L variant. Our results provide additional evidence for proof of principle that PTC124 is a potential therapeutic agent for treating patients with any genetic condition that results from a nonsense mutation.