Laboratory Diagnosis of Lysosomal Diseases: Newborn Screening to Treatment

Abstract

The goal of screening programs for inborn errors of metabolism (IEM) is early detection and timely intervention to significantly reduce morbidity, mortality and associated disabilities. Phenylketonuria exemplifies their success as neonates are identified at birth and then promptly treated allowing normal neurological development. Lysosomal diseases comprise about 50 IEM arising from a deficiency in a protein required for proper lysosomal function. Typically, these defects are in lysosomal enzymes with the concomitant accumulation of the enzyme's substrate as the cardinal feature. None of the lysosomal diseases are screened at birth in Australia and in the absence of a family history, traditional laboratory diagnosis of the majority, involves demonstrating a deficiency of the requisite enzyme. Diagnostic confusion can arise from interpretation of the degree of residual enzyme activity causative of disease and is impractical when the disorder is not due to an enzyme deficiency per se. Advances in mass spectrometry technologies has enabled simultaneous measurement of the enzymes' substrates and their metabolites which facilitates the efficiency of diagnosis. Employing urine chemistry as a reflection of multisystemic disease, individual lysosomal diseases can be identified by a characteristic substrate pattern complicit with the enzyme deficiency. Determination of lipids in plasma allows the diagnosis of a further class of lysosomal disorders, the sphingolipids. The ideal goal would be to measure biomarkers for each specific lysosomal disorder in the one mass spectrometry-based platform to achieve a diagnosis. Confirmation of the diagnosis is usually by identifying pathogenic variants in the underlying gene, and although molecular genetic technologies can provide the initial diagnosis, the biochemistry will remain important for interpreting molecular variants of uncertain significance.

Figure 1

Schematic of the biochemistry of lysosomal diseases. The X on the DNA strand marks the molecular defect resulting in a defective gene product of either an enzyme, ancillary protein (often a transporter) or a cofactor (often required for enzyme activity) which is required for proper metabolism. Consequently, the substrate that cannot be metabolised or transported accumulates resulting in disease.

Figure 2

Flowchart outlining the typical steps involved in the laboratory diagnosis of lysosomal disorders. Of note, if one of the family of sulphatase enzymes is deficient it is necessary to test for another sulphatase to show that it is normal to rule out a diagnosis of multiple sulphatase deficiency.

Figure 3

Mass spectrometry to identify each subtype of mucopolysaccharidosis (MPS) in urine. GAG, glycosaminoglycans; LC-ESI-MS/MS, liquid chromatography-electrospray ionisation-tandem mass spectrometry; IS, internal standard; PMP, 1-phenyl-3-methyl-5-pyrazolone.

Figure 4

Thin layer chromatography (TLC) of oligosaccharide patterns in urine samples. Lane 1, α-mannosidosis patient; Lane 2 and 3, unaffected; Lane 4, sialidosis patient.

Figure 5

Multiplex mass spectrometry platform for Fabry disease, Gaucher disease, metachromatic leukodystrophy (MLD) and Niemann-Pick type C. LC-ESI-MS/MS, liquid chromatography-electrospray ionisation-tandem mass spectrometry; IS, internal standard; lyso-CTH for Fabry disease; lysoSM509 for Niemann-Pick type C; 18:0 sulphatide for MLD; GluSph for Gaucher disease.

Figure 6

Amplification-refractory mutation system (ARMS) for the identification of the N370S molecular variant in a patient with Gaucher disease. N denotes the PCR with a primer to the wild type (WT) sequence and M for the N370S sequence. WT DNA yields a PCR product with only the N primer, DNA homozygous for N370S yields a PCR product with only the M primer (hom) and heterozygous DNA yields a product with both primers (het). Note the patient is tested in duplicate and analysed in two different lanes on the agarose gel.