BACKGROUND: Prediagnostic steps in suspected metachromatic leukodystrophy (MLD) rely on clinical chemical methods other than enzyme assays. We report a new diagnostic method which evaluates changes in the spectrum of molecular types of sulfatides (3-O-sulfogalactosyl ceramides) in MLD urine. METHODS: The procedure allows isolation of urinary sulfatides by solid-phase extraction on DEAE-cellulose membranes, transportation of a dry membrane followed by elution and tandem mass spectrometry (MS/MS) analysis in the clinical laboratory. Major sulfatide isoforms are normalized to the least variable component of the spectrum, which is the indigenous C18:0 isoform. This procedure does not require the use of specific internal standards and minimizes errors caused by sample preparation and measurement. RESULTS: Urinary sulfatides were analyzed in a set of 21 samples from patients affected by sulfatidosis. The combined abundance of the five most elevated isoforms, C22:0, C22:0-OH, C24:0, C24:1-OH, and C24:0-OH sulfatides, was found to give the greatest distinction between MLD-affected patients and a control group. CONCLUSIONS: The method avoids transportation of liquid urine samples and generates stable membrane-bound sulfatide samples that can be stored at ambient temperature. MS/MS sulfatide profiling targeted on the most MLD-representative isoforms is simple with robust results and is suitable for screening.

• Keywords
• ASA, CV, DEAE, DEAE-cellulose membrane, DUS, Diethylaminoethyl, Dry urinary samples, IPN, Isoforms, MLD, MS/MS, PTFE, Psap-d, S/N, SRM, Screening for metachromatic leukodystrophy, Tandem mass spectrometry, Urinary sulfatide, arylsulfatase A, coefficient of variation, dry urinary sample, isoform profile number (ratio of the sum of the major five isoforms and the C18:0 sulfatide), metachromatic leukodystrophy, polytetrafluoroethylene, prosaposin deficiency, selected reaction monitoring, signal to noise ratio, tandem mass spectrometry,
• MeSH
• DEAE-Cellulose MeSH
• Child MeSH
• Solid Phase Extraction MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Membranes, Artificial MeSH
• Leukodystrophy, Metachromatic diagnosis   urine   MeSH
• Adolescent MeSH
• Specimen Handling standards   MeSH
• Child, Preschool MeSH
• Reference Standards MeSH
• Case-Control Studies MeSH
• Sulfoglycosphingolipids urine   MeSH
• Tandem Mass Spectrometry MeSH
• Desiccation MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DEAE-Cellulose MeSH
• Membranes, Artificial MeSH
• Sulfoglycosphingolipids MeSH

Prosaposin deficiency (pSap-d) and saposin B deficiency (SapB-d) are both lipid storage disorders caused by mutations in the PSAP gene that codes for the 65-70 kDa prosaposin protein, which is the precursor for four sphingolipid activator proteins, saposins A-D. We report on two new patients with PSAP gene defects; one, with pSap-d, who had a severe neurovisceral dystrophy and died as a neonate, and the other with SapB-d, who presented with a metachromatic leukodystrophy-like disorder but had normal arylsulfatase activity. Screening for urinary sphingolipids was crucial to the diagnosis of both patients, with electrospray ionization tandem mass spectrometry also providing quantification. The pSap-d patient is the first case with this condition where urinary sphingolipids have been investigated. Multiple sphingolipids were elevated, with globotriaosylceramide showing the greatest increase. Both patients had novel mutations in the PSAP gene. The pSap-d patient was homozygous for a splice-acceptor site mutation two bases upstream of exon 10. This mutation led to a premature stop codon and yielded low levels of transcript. The SapB-d patient was a compound heterozygote with a splice-acceptor site variant exclusively affecting the SapB domain on one allele, and a 2 bp deletion leading to a null, that is, pSap-d mutation, on the other allele. Phenotypically, pSap-d is a relatively uniform disease of the neonate, whereas SapB-d is heterogeneous with a spectrum similar to that in metachromatic leukodystrophy. The possible existence of genotypes and phenotypes intermediate between those of pSap-d and the single saposin deficiencies is speculated.

• MeSH
• Child MeSH
• Heterozygote MeSH
• Homozygote MeSH
• Infant MeSH
• Skin pathology   MeSH
• Humans MeSH
• Magnetic Resonance Imaging MeSH
• Leukodystrophy, Metachromatic genetics   metabolism   pathology   MeSH
• RNA Splice Sites genetics   MeSH
• Brain abnormalities   pathology   MeSH
• Mutation * MeSH
• DNA Mutational Analysis MeSH
• Codon, Nonsense MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Saposins deficiency   genetics   MeSH
• Sequence Deletion MeSH
• Sphingolipids urine   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA Splice Sites MeSH
• Codon, Nonsense MeSH
• PSAP protein, human MeSH Browser
• Saposins MeSH
• Sphingolipids MeSH