Variants in the GBA1 gene represent the most frequent genetic etiology of Parkinson disease (PD). The GBA1 gene encodes the lysosomal enzyme beta-glucocerebrosidase (GCase), and GCase activity represents an important, modifiable therapeutic target and potential biomarker. Ysslestein et al. recently reviewed the advantages and limitations of multiple strategies for measuring GCase activity, including blood analyses.1 While they conclude that total lysate from blood includes lysosomal and non-lysosomal GCase protein, and therefore may not optimally capture intracellular lysosomal GCase activity, blood GCase offers promise as a biomarker for use in future clinical trials. We add our experience measuring GCase activity in specimens from PD harboring either a GBA1 variation (GBA-PD) or a LRRK2-G2019S variation (LRRK2-PD), and from idiopathic-PD (IPD) with no variation in GBA1 and LRRK2-G2019S, using four different approaches: enzymology combined with liquid-chromatography tandem mass spectrometry (methodologies 1-3); methodology 1) DBS1: in whole blood dried blood spot (DBS) specimens with CDC reagents2; 2) DBS2: using Perkin-Elmer reagents3; 3) in frozen plasma, thawed and spotted to filter paper (plasma); and 4) in leukocytes using a 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) based fluorometric assay (previously reported)4. Sample quality in DBS was deemed poor by visual inspection or concurrent sub-optimal activities of control enzymes, and samples were excluded in 27.7% of DBS1 and 6.7% of DBS2.
As anticipated, GCase activity measured in both DBS studies was lower in GBA-PD compared with both IPD and LRRK2-PD4-6, and GCase activity measured in plasma was lower in GBA-PD vs. IPD (Table 1). The stability of GCase activity over time was estimated in a subset of participants with repeated measurements using the interclass correlation coefficient (ICC), and was moderate (DBS1, n=104 observations, ICC=0.66; 95% CI: 0.27, 0.91). However, there was a small increase in GCase activity over time (B=0.004 umol/L/h/day, 95% CI: 0.001, 0.008), which may be explained by a shorter freezer storage time in samples collected closer to the assay date.7 Methods for statistical analyses are described in Table 1.
Table 1:
Summary of cross-sectional measurements of GCase activity using different measurement approaches.
| Study | Groups | ||||
|---|---|---|---|---|---|
| GCase | Idiopathic PD | Gaucher Disease-PD |
GBA-PD | LRRK2-PD | p-value |
| Dried blood spot 1 (DBS1) * | |||||
| N | 45 | 1 | 29 | 5 | IPD vs GBA PD: <0.01 |
| Median (IQR) | 5.33 (3.11, 7.07) | 1.20 | 3.21 (1.27, 4.68) | 5.92 (5.33. 7.35) | IPD vs LRRK2 PD: 0.40 |
| Range | 0.24, 23.96 | - | 0.39, 9.47 | 4.26, 8.22 | GBA PD vs LRRK2 PD: 0.01 |
| Dried blood spot 2 (DBS2) * | |||||
| N | 44 | 1 | 18 | 21 | IPD vs GBA PD: 0.04 |
| Median (IQR) | 5.28 (3.79, 7.39) | 0.15 | 3.55 (0.90, 5.35) | 6.69 (4.62, 7.75) | IPD vs LRRK2 PD: 0.13 |
| Range | 0.21, 13.05 | - | 0.11, 10.16 | 0.23, 11.11 | GBA PD vs LRRK2 PD: 0.007 |
| Plasma * | |||||
| N | 143 | 1 | 84 | 17 | IPD vs GBA PD: 0.04 |
| Median (IQR) | 0.70 (0.42, 1.24) | 0.13 | 0.56 (0.30, 1.36) | 0.97 (0.45, 2.21) | IPD vs LRRK2 PD: 0.26 |
| Range | 0.11, 22.95 | - | 0.11, 99.44 | 0.11, 4.63 | GBA PD vs LRRK2 PD: 0.06 |
| 4-MUG ** | |||||
| N | 8 | 3 | 15 | NA | IPD vs GBA PD: 0.01 |
| Median (IQR) | 28.5 (22.5, 37.5) | 6.40 (5.10, 11.0) | 16.0 (15.0, 22.0) | ||
| Range | 16.0, 56.0 | - | 11.0, 40.0 | ||
DBS study 1, DBS study 2, and plasma unit of measure: umol/L/h
4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) unit of measure: nmol/mg protein/hour.
1: Methods: p-values are from non-parametric, unadjusted comparisons (Wilcoxon Rank Sum). Interclass correlation coefficient (ICC) was estimated using mixed effects model adjusting for days since baseline collection, genetic status, and participant (random effect) (Stata 16).
GBA-PD: PD harboring a GBA1 variation; LRRK2-PD: PD harboring a LRRK2-G2019S variation; idiopathic-PD: PD with no variation in GBA1 and LRRK2-G2019S
In agreement with Ysselstein et al., although differences in GCase activity between methods were observed, the ranges of GCase activities overlapped considerably between groups (Table 1). Furthermore, while we agree that the use of DBS to measure GCase activity can be advantageous due to ease of collection, our data revealed specimen integrity issues using both DBS approaches. We speculate that this might be attributable to poor specimen collection technique, introduction of moisture prior to specimen storage, and storage duration. In DBS1, the specimens that were flagged for poor quality had a higher median storage time compared with those without integrity issues (median (IQR): 183.5 days (131, 266) vs. 132 (62, 241)), which would suggest that increased duration of storage may be a contributing factor to reduced GCase activity.7
In conclusion, we recommend the standardization of methods used to measure GCase activity in DBS, including the establishment of standard collection protocols and site reliability. In an effort to decrease variability, we advocate repeat measurement of GCase activity and consideration of the effect of leukocytosis and storage time on GCase activity.
Acknowledgements
The authors are grateful to the study participants who graciously donated their time and energy for this study and to Mariel Pullman, MD, Amanda Glickman, MD, and Sarah Simon for their work contributing to this study. This study was supported by National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (NINDS) (grant Nos. U01 NS107016 and U01 NS094148), and the Bigglesworth Family Foundation, Bachman-Strauss Chair.
Financial Disclosures:
RSP received funding for this project from the National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (NINDS) (U01NS107016-01A1 and U01NS094148-01), and the Bigglesworth Family Foundation. The following authors have no disclosures relevant to this letter: RAO, RWAP, OB, DR, SBB.
Footnotes
Conflict of interest statement: None of the authors reports any conflict of interest.
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