Lymphocyte Galactocerebrosidase Activity by LC-MS/MS for Post–Newborn Screening Evaluation of Krabbe Disease

Abstract

BACKGROUND Deficiency of the lysosomal enzyme galactosylcerebrosidase (GALC) causes Krabbe disease. Newborn screening for Krabbe disease is ongoing, but improved methods for follow-up analysis of screen-positive babies are needed to better advise families and to optimize treatment. We report a new assay for the enzymatic activity of GALC in lymphocytes. METHODS T lymphocytes were isolated from venous blood by magnetic bead technology. The assay used a close structural analog of the natural substrate and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the amount of product with the aid of a chemically identical internal standard. RESULTS The analytical range of the assay (ratio of assay response for the quality control high standard to that from all non-enzymatic-dependent processes) was 20-fold greater than that for the conventional radiometric GALC assay. The LC-MS/MS could distinguish cells that were null in GALC from those that contained traces of active enzyme (down to 0.3 % of normal). There was a good correlation between the level of residual GALC activity in lymphocytes and the severity of Krabbe disease. CONCLUSIONS The new assay can measure small amounts of residual GALC activity in leukocytes with high accuracy compared to previous assays and can contribute, along with genotyping, biomarker analysis, and neurological imaging, a better plan for post newborn screening follow-up for Krabbe disease.

Contribution of tandem mass spectrometry to the diagnosis of lysosomal storage disorders

“…This has also been reported for leukocytes when confirming PD (Lin et al 2017). MS/MS also gives more accurate results than the radiometric method for measuring β-D-galactocerebrosidase activity in lymphocytes (Liao et al 2017b). However, digital microfluidic platforms using fluorimetry are also competitive for NBS of LSDs (Sista et al 2013, Millington and Bali 2017.…”

Psychosine, a marker of Krabbe phenotype and treatment effect

“…Blood psychosine concentration was measured by extraction of DBS 3-mm punches with methanol followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a modification of the published method that has been described separately [9, 13]. A Waters TQS-MS/MS instrument was used, which provided a limit of quantification of approximately 0.1 nM, below that reported in a previous study [13].…”

“…Although a new high-resolution mass spectrometry assay to measure GALC activity in leukocytes has been shown in a research lab to better discriminate between KD phenotypes, the limited prognostic value of current enzyme activity assays and molecular genetic tests complicates clinical decision-making [9]. In providing clinical care to infants with positive screening and confirmatory testing results, the most pressing question that must be addressed by clinicians is whether the patient is likely to develop infantile-onset KD, which requires urgent evaluation for HSCT.…”

Achieving Congruence among Reference Laboratories for Absolute Abundance Measurement of Analytes for Rare Diseases: Psychosine for Diagnosis and Prognosis of Krabbe Disease

“…The glycosylsphingolipid psychosine has emerged as a biomarker for diagnosis and prognosis of Krabbe disease, a rare lysosomal storage disorder caused by deficiency of galactocerebrosidase (GALC) [1][2][3]. GALC removes galactose from galactosylceramide as part of the lysosomal re-cycling of glycosphingolipids that are rich in myelin (Figure 1).…”

“…Because the majority of cases with low enzymatic activity samples have pseudodeficient GALC activity due to common pseudodeficiency GALC gene variants [5], reliance on low GALC activity alone is not sufficient for newborn screening. Psychosine has been shown to be a valuable biomarker when measured in DBS to detect newborns with infantile Krabbe disease [1][2][3], and measuring psychosine as a second-tier test in newborn screening can clarify the vast majority of these low-GALC activity cases as false positives [6]. A few newborn screening laboratories include psychosine analysis as a second-tier method [6], but most programs refer patients to diagnostic centers either when GALC activity is found to be reduced or when molecular genetic analysis of the GALC gene is used as a second tier test and reveals a genotype with known pathogenic mutations or variants of uncertain significance.…”