Reliable Assay of Acid Sphingomyelinase Deficiency with the Mutation Q292K by Tandem Mass Spectrometry.

Niemann-Pick-A/B disease is a lysosomal storage disease caused by deficiency of acid sphingomyelinase (1 ). Efforts are underway to develop novel therapies for this disease, and some newborn screening centers have started to test for acid sphingomyelinase enzymatic activity in dried blood spots on newborn screening cards. Available assays for newborn screening include tandem mass spectrometry (MS/MS) (2 ) and fluorometry (3 ). Diagnostic laboratories have typically used a radiometric assay with radiolabeled sphingomyelin or the fluorometric assay (4 ). However, in a detailed study of 24 patients confirmed as having Niemann-Pick-A/B disease, 4 had the Q292K missense allele. Fibroblasts from these 4 patients showed a normal to increased acid sphingomyelinase activity when assayed with the fluorometric substrate but displayed activity in the radiometric assay with natural sphingomyelin substrate in the low end of the range of the affected samples (4 ). The age of onset of symptoms in these 4 patients was 1, 1, 2, and 3 years, suggesting that the disease can be quite severe in patients harboring this allele (4 ). This led to the conclusion of a “diagnostic pitfall” in the use of the fluorometric substrate for analysis of Niemann-Pick-A/B disease. This issue was also reported in the original paper describing the fluorometric reagent (3 ), and it was shown that this mutant displayed a decreased affinity for sphingomyelin, which presumably explained the low activity on the natural substrate (3 ). Fig. 1 shows the natural substrate and the substrates used in the MS/MS and fluorometric assays. The MS/MS substrate is a close structural analog of the natural substrate sphingomyelin, the only difference being the length of the fatty acyl chain linked to the sphingosine amino group. The fluorometric substrate has some structural resemblance to sphingomyelin but is substantially different owing to the need to incorporate a fluorogenic group in the substrate. Apparently the Q292K mutation causes a deformation in the active site of acid sphingomyelinase that reduces the affinity of the natural substrate but not the unnatural substrate. To explore the assay behavior of wild-type human acid sphingomyelinase and the Q292K mutant, we transfected HEK293 cells with a plasmid expressing either wild-type or mutant protein. Twenty-four hours posttransfection, the culture medium © 2015 American Association for Clinical Chemistry 1 Nonstandard abbreviations: MS/MS, tandem mass spectrometry; HMU-PC, 6-hexadecanoylamino-4methylumbelliferylphosphorylcholine. Fig. 1. The left panel shows thefluorimetric assay results for acid sphingomyelinase using HMU-PC substrate (upper right) and the cell culture medium indicated below each bar. The right panel showsMS/MS results using C6-sphingomyelin substrate (upper right along with natural sphingomyelin) together with C4-sphingomyelin internal standard (IS) (not shown). Error bars are the SDs from triplicate assays. P/IS is the mass spectrometry ion response of the product divided by that of the internal standard. Nontransf., nontransfected. Clinical Chemistry 61:5 771–778 (2015) Letters to the Editor