What laboratory tests should be ordered to evaluate a patient for mucopolysaccharidosis (MPS)?

Laboratory Evaluation for Mucopolysaccharidosis (MPS)

The diagnostic workup for MPS requires a three-tiered approach: quantitative urinary glycosaminoglycan (GAG) analysis, specific lysosomal enzyme activity assays, and molecular genetic testing for confirmation and phenotype prediction. 1, 2

Initial Screening Tests

Urinary Glycosaminoglycan (GAG) Analysis

• Order quantitative urinary GAG measurement using spectrophotometric methods (e.g., dimethylmethylene blue assay) or preferably LC-MS/MS-based multiplex assays as the first-line screening test 3, 4, 5
• The LC-MS/MS multiplex assay demonstrates 100% sensitivity compared to 90% for traditional dimethylmethylene blue (DMB) assays, as DMB produces false-negatives in approximately 10% of MPS patients 6
• Avoid semi-quantitative spot tests using cationic dyes on filter paper, as these have high false-positive and false-negative rates and are considered obsolete 5
• Important caveat: A negative urinary GAG assay does not rule out MPS, as some patients have GAG excretion levels that overlap with healthy controls 5

GAG Pattern Analysis

• If quantitative GAG is elevated, confirm with electrophoresis, thin-layer chromatography, or tandem mass spectrometry to identify specific GAG species (heparan sulfate, dermatan sulfate, keratan sulfate) 5
• The specific GAG pattern helps narrow the differential diagnosis among MPS subtypes 3, 4

Confirmatory Enzyme Activity Assays

Sample Types and Methods

Order specific lysosomal enzyme activity assays based on the suspected MPS type using one of the following sample types 1, 2:

• Leukocyte lysates: Provide definitive diagnosis with 30-60 minute incubation times for most MPS types 1
• Dried blood spots (DBS): Acceptable for screening but require longer incubation times; considered screening methods rather than definitive diagnosis 1, 3
• Plasma/serum: Can be used for certain enzyme assays 5
• Cultured skin fibroblasts: Remain the gold standard for diagnosis when other samples are equivocal 5

Assay Methodologies

The laboratory will typically use one of these platforms 1:

• Fluorometric assays using 4-methylumbelliferone (4-MU)-based substrates for traditional enzyme measurements
• MS/MS-based assays offering higher sensitivity, dynamic range, and multiplexing capability for MPS types I, II, IIIA, IIIB, IIIC, IIID, IVA, VI, and VII 1
• Spectrophotometric assays for arylsulfatases (MPS VI, metachromatic leukodystrophy) 1

Specific Enzyme Tests by MPS Type

For MPS I (Hurler/Scheie syndrome): 2

• α-L-iduronidase (IDUA) enzyme activity in leukocytes
• Patients with severe MPS I-H typically have undetectable enzyme activity, while MPS I-HS and I-S have residual activity (as little as 0.4% can produce mild phenotype) 2

For MPS II (Hunter syndrome): 2

• Iduronate-2-sulfatase (IDS) enzyme activity in plasma or leukocytes
• Critical: Simultaneously measure arylsulfatase A (ARSA) or arylsulfatase B (ARSB) to exclude multiple sulfatase deficiency (MSD) caused by SUMF1 mutations 2
• If both IDS and ARSA/ARSB are low, sequence SUMF1 gene 2

For MPS III (Sanfilippo syndrome): 1

• Specific enzyme depending on subtype (IIIA, IIIB, IIIC, IIID)

For MPS IV (Morquio syndrome): 1

• Type IVA: N-acetylgalactosamine-6-sulfatase
• Type IVB: β-galactosidase

For MPS VI (Maroteaux-Lamy syndrome): 1

• Arylsulfatase B (ARSB) activity

For MPS VII (Sly syndrome): 1

• β-glucuronidase activity

Molecular Genetic Testing

Gene Sequencing

Order molecular genetic analysis of the specific gene encoding the deficient enzyme after confirming low enzyme activity 2, 4:

• MPS I: IDUA gene sequencing 2
• MPS II: IDS gene sequencing (note: 20% of mutations are gross rearrangements from recombination with IDS pseudogene) 2
• Other MPS types: corresponding gene for the deficient enzyme

Clinical Utility of Genetic Testing

Molecular analysis serves multiple purposes 2:

• Diagnosis confirmation when enzyme activity results are equivocal
• Phenotype prediction based on genotype-phenotype correlations (though limited for certain mutations like IDUA p.P533R, which spans the entire disease spectrum) 2
• Carrier detection for family members
• Genetic counseling for family planning
• Prenatal diagnosis (preferably combined with enzyme assays in chorionic villi or amniotic fluid) 5

Known Genotype-Phenotype Correlations

For MPS I 2:

• Homozygosity or compound heterozygosity for p.Q70X and p.W402X nonsense mutations predict severe MPS I-H phenotype (these account for 60-70% of mutant alleles in Caucasians)
• p.R89Q missense and c.678-7g>a splice site mutations predict mild phenotype
• p.P533R mutation is unpredictable (all three MPS I subtypes reported)

For MPS II 2:

• Extensive deletions and contiguous-gene deletions typically manifest severe phenotype with higher seizure risk
• Most point mutations lack clear genotype-phenotype correlations due to inconsistent phenotypic reporting

Additional Diagnostic Considerations

Newborn Screening Follow-up

If an infant has a positive newborn screen 2:

1. Confirm with leukocyte enzyme activity (not DBS alone)
2. If enzyme activity is low, refer immediately to metabolic center
3. Proceed with gene sequencing
4. Initiate multidisciplinary evaluations

Overlap with Other Lysosomal Storage Diseases

Consider α-mannosidosis in the differential diagnosis, as it presents with phenotypically similar features to MPS I but results from α-mannosidase deficiency leading to accumulation of mannose-rich oligosaccharides rather than GAGs 7

Female Patients with MPS II

For females identified with low IDS activity 2:

• Perform confirmatory testing to distinguish false-positives from MPS II heterozygotes and MSD
• Provide genetic counseling and family pedigree review
• Follow heterozygotes periodically, as some may develop symptoms
• Note that some heterozygotes have enzyme activity in the "normal" range and cannot be identified by screening alone

Common Pitfalls to Avoid

• Do not rely solely on enzyme activity levels for phenotype prediction, as there is significant overlap between severe and attenuated forms 2
• Do not use enzyme assays for carrier detection in MPS I, as there is overlap between heterozygotes and normal individuals 2
• Do not dismiss MPS based on normal urinary GAG levels alone, particularly when using DMB assays 5, 6
• Do not forget to test additional sulfatases when evaluating MPS II to exclude multiple sulfatase deficiency 2