Plasma lysosphingomyelin demonstrates great potential as a diagnostic biomarker for Niemann-Pick disease type C in a retrospective study

Richard W D Welford, Marco Garzotti, Charles Marques Lourenço, Eugen Mengel, Thorsten Marquardt, Janine Reunert, Yasmina Amraoui, Stefan A Kolb, Olivier Morand, Peter Groenen, Richard W D Welford, Marco Garzotti, Charles Marques Lourenço, Eugen Mengel, Thorsten Marquardt, Janine Reunert, Yasmina Amraoui, Stefan A Kolb, Olivier Morand, Peter Groenen

Abstract

Niemann-Pick disease type C (NP-C) is a devastating, neurovisceral lysosomal storage disorder which is characterised by variable manifestation of visceral signs, progressive neuropsychiatric deterioration and premature death, caused by mutations in the NPC1 and NPC2 genes. Due to the complexity of diagnosis and the availability of an approved therapy in the EU, improved detection of NP-C may have a huge impact on future disease management. At the cellular level dysfunction or deficiency of either the NPC1 or NPC2 protein leads to a complex intracellular endosomal/lysosomal trafficking defect, and organ specific patterns of sphingolipid accumulation. Lysosphingolipids have been shown to be excellent biomarkers of sphingolipidosis in several enzyme deficient lysosomal storage disorders. Additionally, in a recent study the lysosphingolipids, lysosphingomyelin (SPC) and glucosylsphingosine (GlcSph), appeared to be elevated in the plasma of three adult NP-C patients. In order to investigate the clinical utility of SPC and GlcSph as diagnostic markers, an in-depth fit for purpose biomarker assay validation for measurement of these biomarkers in plasma by liquid chromatography-tandem mass spectrometry was performed. Plasma SPC and GlcSph are stable and can be measured accurately, precisely and reproducibly. In a retrospective analysis of 57 NP-C patients and 70 control subjects, median plasma SPC and GlcSph were significantly elevated in NP-C by 2.8-fold and 1.4-fold respectively. For miglustat-naïve NP-C patients, aged 2-50 years, the area under the ROC curve was 0.999 for SPC and 0.776 for GlcSph. Plasma GlcSph did not correlate with SPC levels in NP-C patients. The data indicate excellent potential for the use of lysosphingomyelin in NP-C diagnosis, where it could be used to identify NP-C patients for confirmatory genetic testing.

Conflict of interest statement

Competing Interests: RW, MG, SK, OM and PG are employees of Actelion Pharmaceuticals Ltd; CML, EM, YA, JR and TM have received travel reimbursements, consulting fees or honoraria from Actelion Pharmaceuticals Ltd, Allschwil, Switzerland. The competing interests do not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Chemical structures of sphingolipids.
Figure 1. Chemical structures of sphingolipids.
The lysosphingolipids SPC and GlcSph measured in plasma for this study, along with their N-acetylated counterparts, sphingomyelin and the monohexosylceramide glucosylceramide which are known to accumulate in the organs of NP-C patients.
Figure 2. Stability of GlcSph and SPC…
Figure 2. Stability of GlcSph and SPC in plasma.
A and B show the stability of the plasma QC samples under different conditions for SPC and GlcSph respectively. Percentage measured compared to the average concentration of the samples determined in the three validation batches is shown. Data are reported +/− standard deviation for 3 replicate measurements. Note the observation of all data being a little under 100% represents a systematic bias from this particular batch; Stability of SPC and GlcSph in EDTA-plasma from five donors incubated at room temperature (C and D) and 4°C (E and F) for up to 96 hours. Each trace represents an individual donor.
Figure 3. Plasma SPC and GlcSph in…
Figure 3. Plasma SPC and GlcSph in NP-C patients and controls.
One sample per patient is shown, where multiple samples from one patient were available the first was used. The dotted horizontal line represents the 95% percentile of the entire control group for SPC (10.3 nM) and GlcSph (2.4 nM). The bar is the median. A and B, SPC and GlcSph in the entire cohort; C and D, SPC and GlcSph separated by age (years); E and F, SPC and GlcSph separated by miglustat status (0–50 years). The miglustat treated patients had been on treatment for 2.8±1.4 years (average ± standard deviation), 0.7–6 years (Min-Max).
Figure 4. ROC analysis of plasma SPC…
Figure 4. ROC analysis of plasma SPC and GlcSph.
SPC (green) and GlcSph (blue) for the age range 0–50 years with miglustat-naïve NP-C patients compared to the control group. The area under the curve (95% CI) was 0.9994 (0.9972 to 1.002) and 0.7764 (0.6479 to 0.9048) for SPC and GlcSph respectively.
Figure 5. Biomarker correlation plots A: SPC…
Figure 5. Biomarker correlation plots A: SPC and GlcSph.
The control subjects are black circles, the NP-C patients are colored by miglustat treatment status as for Figure 3E. A Spearman correlation analysis gave (r, p value) for control (0.48, B: SPC and cholestan-3β,5α,6β-triol. For controls (black circles) and NP-C (red squares). The 2 markers did not correlate for the NP-C patients (Spearman r = 0.265, p = 0.273). The horizontal dotted line represents the 95 percentile of normal for cholestan-3β,5α,6β-triol (0.04 ng/µL). The vertical cut-off is 95 percentile of normal for SPC. Values below the assay LOQ were set at the LOQ for SPC (5 nM) and cholestan-3β,5α,6β-triol (0.001 ng/uL).
Figure 6. Glucosylsphingosine not galactosylsphingosine is increased…
Figure 6. Glucosylsphingosine not galactosylsphingosine is increased in the plasma of NP-C patients.
LC-MS/MS chromatograms showing separation of glucosyl- and galactosyl-sphingosine with HILIC chromatography. (A) QC2; (B) QC2 spiked with 5 nM glucosylsphingosine; (C) QC2 spiked with 5 nM galactosylsphingosine; (D) control sample 1; (E) control sample 2; (F) control sample 3; (G) NP-C sample 1; (H) NP-C sample 2; (I) NP-C sample 3.

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