A novel, highly sensitive and specific biomarker for Niemann-Pick type C1 disease
- Anne-Katrin Giese†1,
- Hermann Mascher†2,
- Ulrike Grittner3,
- Sabrina Eichler4,
- Guido Kramp4,
- Jan Lukas1,
- Danielle te Vruchte5,
- Nada Al Eisa5,
- Mario Cortina-Borja6,
- Forbes D Porter7,
- Frances M Platt5 and
- Arndt Rolfs1, 4Email author
© Giese et al. 2015
Received: 9 January 2015
Accepted: 24 April 2015
Published: 17 June 2015
Lysosomal storage disorders (LSDs), are a heterogeneous group of rare disorders caused by defects in genes encoding for proteins involved in the lysosomal degradation of macromolecules. They occur at a frequency of about 1 in 5,000 live births, though recent neonatal screening suggests a higher incidence. New treatment options for LSDs demand a rapid, early diagnosis of LSDs if maximal clinical benefit is to be achieved.
Here, we describe a novel, highly specific and sensitive biomarker for Niemann-Pick Type C disease type 1 (NPC1), lyso-sphingomyelin-509. We cross-validate this biomarker with cholestane-3β,5α,6β-triol and relative lysosomal volume. The primary cohort for establishment of the biomarker contained 135 NPC1 patients, 66 NPC1 carriers, 241 patients with other LSDs and 46 healthy controls.
With a sensitivity of 100.0% and specificity of 91.0% a cut-off of 1.4 ng/ml was established. Comparison with cholestane-3β,5α,6β-triol and relative acidic compartment volume measurements were carried out with a subset of 125 subjects. Both cholestane-3β,5α,6β-triol and lyso-Sphingomyelin-509 were sufficient in establishing the diagnosis of NPC1 and correlated with disease severity.
In summary, we have established a new biomarker for the diagnosis of NPC1, and further studies will be conducted to assess correlation to disease progress and monitoring treatment.
Lysosomal storage disorders (LSDs) are a group of over 70 different inborn errors of metabolism with a combined incidence around 1 in 5,000 live births . Symptoms depend on tissue distribution and function of the accumulating substrates . Niemann-Pick Type C disease (NPC) is an autosomal-recessive LSD, that is reported to affect 1:120,000 live births , though the relative frequency in at-risk cohorts such as patients with neurologic and psychiatric symptoms, is reported to be as high as 1.2% . In 95% of cases NPC is caused by mutations in the NPC1 gene (OMIM: *607623), only in 5% of cases have mutations in the NPC2 gene been reported (OMIM: *601015). Currently, Miglustat is the only EMEA-approved drug for the treatment of NPC [5,6], though novel approaches such as cyclodextrin  and histone-deacetylase inhibitors  are being investigated. Despite clinical evaluation of patients new clinical studies will also require assessment of independent, objective surrogate parameters to evaluate disease progression and response to treatment. In addition to the classical Filipin staining test, which is not always conclusive in patients with NPC , two biomarkers for NPC have been reported to date, oxysterols  and measurement of intracellular acidic compartment volume (mean equivalent of fluorescence - MEFL/LysoTracker) . While oxysterols facilitate the primary diagnosis of NPC, MEFL is mainly useful in assessing the follow-up and treatment response of patients.
Here, we report the establishment of a novel biomarker for NPC1 and cross-validate our findings with published cholestane-3β,5α,6β-triol and MEFL data analyzing the same plasma data . As a first step we screened ten NPC1 patients and 10 age- and gender-matched healthy controls utilizing HPLC and tandem mass spectrometry and carefully assessed differences in mass spectra. This resulted in the detection of lyso-sphingomyelin-509 (lyso-SM-509) as a potential biomarker for NPC. In the following step, we screened 135 NPC1 patients, 66 NPC1 carriers, 241 patients with other LSDs and 46 healthy controls for lyso-SM-509. The structure is very similar to lyso-sphingomyelin (lyso-SM), the chemical formula being (C24H49N2O7P), though so far we were not able to elucidate the exact structure of lyso-SM-509. In keeping with this finding, lyso-SM-509 is also highly elevated in patients with NP-A/B, though the disease is easier to diagnose due to availability of enzyme activity assays for sphingomyelinase.
In a second line assessment we compared lyso-SM-509 measurements with those published for cholestane-3β,5α,6β-triol and MEFL. Here, we report on the result of the establishment of lyso-SM-509 as a biomarker and the cross-validation with the previously published biomarkers.
Patients and blood samples
Blood samples were obtained from patients enrolled in the “Biomarker for Niemann-Pick Type C Disease (BioNPC)” trial (ClinicalTrials.gov identifier: NCT01306604) and underwent biochemical analysis or genetic testing for verification of a suspected metabolic disease by the Albrecht-Kossel-Institute for Neuroregeneration (AKos). Additionally, blood plasma samples collected in the scope of the investigation of relative acidic compartment volume in NPC have been analysed for retrospectively for lyso-sphingomyelin-509 levels .
Genetic analysis, Oxysterol and LysoTracker measurements
Identification of lyso-sphingomyelin-509 as a biomarker for NPC
Screening for potential biomarkers was carried out using HPLC-MS/MS of blood plasma samples of 10 healthy subjects and 10 NPC1 patients with accurate mass MS-systems (Orbitrap XL) and tandem MS systems. Differences between NPC1 patients and healthy controls were carefully assessed with regards to substances strongly increased only in patients. Molecular weight and structure of such substances were determined carefully. Peaks were identified by accurate mass MS-system and measured by tandem MS/MS systems using already known internal standards as reference. After validating the robustness of lyso-SM-509, measurements in healthy controls, NPC1 carriers, NPC1 patients and in patients with other LSDs ensued (For details see Additional file 1: Figure S1 and S2). Chemical formula determination was done on an LTQ Orbitrap XL, parent ion as well as fragment ions were determined with accurate mass and high resolution leading to the chemical formula described. Purification steps were performed in order to receive larger and purer compound (SPE and liquid-liquid extraction steps-for purification only) to gain higher concentrated compound for HPLC determination and infusion experiments. Sodium aduct ions as well as negatively charged ions of the substance had also been determined and studied more in depth on a Q/TOF system. Stress tests of the substance in plasma had been performed, it appears that the substance is stable in plasma over several hours at room temperature, furthermore strongly acidic or alkaline conditions did not show significant degradation. Also several freeze/thaw cycles in human plasma showed stability of the compound.
Method for determination of free lyso-sphingomyelin and lyso-sphingomyelin-509 in plasma
50 μL of the sample were mixed with 100 μL of Internal Standard (lyso-Gb2 (Matreya LLC, USA)) working solution (in EtOH) and were then mixed subsequently using a DVX-2500 Multi-tube vortex device at 2500 rpm for about 30 seconds. After centrifugation at 4000 rpm for 2 minutes the clear supernatant was transferred into auto-sampler vials and injected into the HPLC-MS/MS system. Mobile phase used for gradient elution was 50 mM formic acid in water and 50 mM formic acid in acetonitrile/acetone (1/1, v/v). HPLC flow was set at 0.9 mL/min on an ACE 3 C8 column (50 × 2.1 mm) at 60°C, the injection volume used was 5 μL. Retention time for the analytes were approximately 3.2 minutes for lyso-SM and 3.6 minutes for lyso-SM-509, and for the internal standard (lyso-Gb2 (Matreya LLC, USA)) approximately 3.2 minutes. Lyso-Gb2 was checked in regards to native concentrations in plasma and was found to be at very low levels only, a sufficient amount was added therefore during sample preparation. The MS/MS system used was an API 4000 using electrospray ionization in MRM mode in positive mode at 500°C for determination of free Lyso-Sphingomyelin in plasma. Quadrupole resolution was set at unit /unit, MRM transitions used were 465 → 184 m/z for the analyte, 509 → 184 m/z for 509, and 624 → 282 for the internal standard. Calibration was done from 2 ng/mL to 200 ng/mL in aqueous/methanolic solution; QC samples were spiked in plasma at levels of 15 and 150 ng/mL.
Statistics for lyso-sphingomyelin-509
Data were aggregated by taking the first measured value according to genotype (NPC1 patients, NPC1 carriers, NP- A/B patients, P-A/B carriers and healthy controls). Data is given in median and interquartile range where indicated. In order to analyze the diagnostic value of lyso-sphingomyelin-509 a receiver operating characteristic (ROC) curve was employed and the area under the curve (AUC) with 95%CI was calculated. Descriptive statistics and ROC curve analysis were done using SPSS Release Version 22 (© SPSS, Inc., 2013, Chicago, IL, www.spss.com).
For comparison with MEFL and cholestane-3β,5α,6β-triol data
Logistic regression models were fitted to compare proportions, Wilcoxon-Mann-Whitney and Kruskal-Wallis tests were used to compare medians, and ANOVA was used to compare means where appropriate. Linear regression models were fitted with maximum likelihood. A locally adaptive super smoother was used to extract the data structure without a regression model . These calculations were performed in the R language and environment for statistical computing (version 2.14.2; http://www.R-project.org). Graphs with error bars represent mean ± SD. A p-value smaller than 0.05 was considered significant.
The protocol of the BioNPC trial has been approved by the Research Ethics Committee of the University Rostock (ClinicalTrials.gov identifier: NCT01306604). Patients undergoing therapy were treated according to standard protocols. Written informed consent was obtained from all participants or their legal guardians.
Results and discussion
Establishment of lyso-sphingomyelin-509 as a biomarker for NPC and characterization of the study cohort
The recently emerging novel treatment approaches for treating NPC including cyclodextrin , HSP70 and HDAC inhibitors  strengthen the need for early diagnosis and monitoring of disease progression. Biomarkers for LSDs have been investigated for some time to facilitate diagnosis and monitoring response to therapy. In, 1989 Rosengren and colleagues investigated the glycosphingolipid pattern in late infantile metachromatic leukodystrophy (MLD) . Lysosulfatide had been identified in MLD and normal brain tissue subsequently, the authors arguing for a de novo synthesis of lysoglycosphingolipids from sphingosine and arrive at the conclusion that they do not play a role in disease pathology .
Since then the perception of lysoglycosphingolipids has changed dramatically. For example several lysosphingolipid biomarkers having been established recently, e.g. lyso-Globotriaosylceramide (lyso-Gb3) in Fabry disease , Glucosylsphingosine (lyso-GL-1) in Gaucher disease , Galactosylsphingosine (Psychosine) in Krabbe disease [17,18] and lyso-sphingomyelin in Niemann-Pick B patients .
Characteristics of investigated populations
Individuals ( n )
Measurements ( n )
Age (first value)
Males ( n )
Females ( n )
(median, IQR, number of cases)
Age (median, IQR)
Age (median, IQR)
(n = 67)
(n = 20)
(n = 14)
7 (min-max: 2-35)
(n = 3)
(n = 12)
51 (min-max: 4-71))
Medians and interquartile ranges for lyso-SM-509 in ng/ml for all investigated sub-cohorts
Lyso-SM-509 in ng/ml
NP- A/B patients
NP- A/B carrier
Sensitivity, specificity and accuracy of lyso-SM-509 in NPC1 patients
NPC1 patients ( n )
Cut point/sensitivity range (ng/ml)
AUC and 95%CI in ROC Analysis
Relationship to MEFL and cholestane3β,5α,6β‐triol
For NPC1, two other biomarkers have already been established – non-enzymatically generated Oxysterols and the LysoTracker (by assessing MEFL as a measure of relative lysosomal volume) [10,11]. In the second part of our paper, the newly established lyso-SM-509 has been compared to both in the same plasma samples. Even though, two different biomarkers have been established and can be utilized for different aspects of the disease (Oxysterols for primary diagnosis by measurement of either Cholestane-3β,5α,6β-triol or 7-ketocholesterol, which allows for discrimination between controls and NPC1 subjects  vs. MEFL for monitoring of disease progression and treatment response, which has been assessed in a 5-year prospective study ), an ideal biomarker for the primary diagnosis, monitoring of disease progression and treatment response has not yet been established. Lyso-sphingomyelin-509 as a biomarker for NPC adds discriminatory power to identify and will further facilitate early disease diagnosis. In fact, many diseases – even common diseases such as myocardial infarction – involve several biomarkers being used for primary diagnosis and follow-up. Therefore, our findings significantly contribute to the current literature on diagnosing NPC by adding a new biomarker.
Correlation of biomarkers with severity scores
In summary, we have established a novel, sensitive and specific biomarker for the primary diagnosis of NPC1. The major advantage of lyso-SM-509 is that it is quick and easy to measure and standardize. As a consequence, lyso-SM-509 is definitely a useful biomarker for simple diagnostic in blood plasma, future investigations will analyze the sensitivity and specificity of lyso-SM-509 on dried blood spot samples, which would further simplify the diagnostic process.
The authors would like to thank all patients and clinical partners who have contributed to the recruitment of the patients. Special thanks are extended to Susanne Zielke, Sabine Rösner, Frances König, Vivian Kersten and Lea Maciolek for important support in the organization of the clinical study. F.M.P. is a Royal Society Wolfson Research Merit Award holder. The University College London Institute of Child Health receives a portion of its funding from the UK Department of Health´s NIH Research Biomedical Research Funding Centres funding scheme. DtV is supported by Action Medical Research and an unrestricted grant from Actelion. FDP is supported by the intramural research program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH.
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