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Confirming a diagnosis of Niemann-Pick type C disease

Diagnosing Niemann-Pick type C (NP-C) has traditionally been a complex process; however, recent advances in biomarker and molecular genetic (NGS) testing have made rapid, sensitive and specific analyses possible.1 A combination of positive biomarker and genetic testing results is now deemed sufficient to diagnose NP-C in most cases.1 Figure 1 provides an algorithm for the laboratory diagnosis of NP-C in clinical practice.

Figure 1:The algorithm for laboratory diagnosis of NP-C (Marquardt et al, 2016).1 It should be noted that local factors (e.g. availability of, and access to, services) may determine the choice and order of diagnostic tests).

aAt-risk patient populations are defined in Table 1; bSingle-gene sequencing (exons or known mutations) or other; c2 different alleles; dCovers deep intronic sequencing and if possible gene transcription regulatory regions; eBiomarker(s) profiling (if not initially conducted), or extended biomarkers profiling (in addition to those already conducted). Note: In spite of comprehensive investigations it may not be possible to definitively confirm a diagnosis of NP-C in a few patients. In such cases, a thorough reappraisal of longitudinal clinical data, more in‑depth genomic analyses (e.g., whole exome and whole genome sequencing) and cell biological assessments could be considered.
cDNA, complementary DNA; MLPA, multiplex ligation-dependent probe amplification; NP‑C, Niemann-Pick disease Type C; VUS, variant of unknown significance.



Biomarker testing

Currently available biomarker tests

Oxysterol testing

Oxidative stress at the cellular level causes the non-enzymatic production of cholesterol autoxidation products (oxysterols).2 These metabolites, namely cholestane-3β,5α,6β-triol (C-triol) and 7-ketocholesterol (7‑KC), can be measured in blood plasma, and have been demonstrated to be elevated in patients with NP-C compared with controls.2–4 Plasma oxysterol testing is now an established and readily available routine test in many laboratories worldwide.

However, oxysterol testing results should be interpreted with caution, as oxysterols have also been shown to be elevated in patients with other diseases, such as acid lipase deficiency, NP-A and NP-B.2,5,6

Diagnosticians should also be aware that prolonged storage of plasma samples at room temperature can lead to autoxidation of cholesterol, which may then lead to false-positive results.

Testing procedure

·       Analysis of oxysterol levels can be performed using a variety of methods, including gas chromatography-mass spectrometry (GC/MS), or liquid chromatography-tandem mass spectrometry (LC-MS/MS).2

·       Oxysterol testing can be performed using blood samples collected during routine clinical examination.

·       Samples can be sent at room temperature if their delivery from patient to testing laboratory can be guaranteed within 48 hours (to prevent cholesterol autoxidation and the production of false-positive results), although sending samples on dry ice is advised to avoid complication.

For further guidance regarding how testing should be conducted, please contact your local laboratory, or local Actelion Pharmaceuticals Ltd representative.


Lysosphingolipids have been found to be elevated in the plasma of patients with NP-C. In particular, lysosphingomyelin (lyso-SM) was shown to be significantly elevated by 2.8-fold in patients NP-C compared with control subjects,2,8 but is also elevated in many other diseases, and alone it is not sufficiently sensitive to be a biomarker for NP-C.2 A less-well characterized lyso-SM derivative, lysosphingomyelin-509 (lyso-SM-509), has been shown to be elevated in patients with NP-C compared with control subjects.2,9 However, lyso‑SM‑509 is also increased, albeit to a greater extent, in patients with NP-A and NP-B, with some overlap between patients with NP-C and patients with NP-A/B.2,9 Therefore, co-measurement of lyso-SM and lyso‑SM‑509 as part of a multi-analyte panel, may allow distinction between patients with NP-C and those with NP-A/B.

Lysosphingolipids are also available as a test to aid the diagnosis of patients with suspicion of NP-C. For further guidance regarding how testing should be conducted, please contact your local laboratory, or local Actelion Pharmaceuticals Ltd representative.

Testing centers worldwide

For further information on the latest available testing centers, please see the NP-C Professional Network.

Future biomarker tests

Bile acids

Certain bile acids, namely 3β,5α,6β-trihydroxy-cholanoyl-glycine, a metabolite of C-triol (an oxysterol also elevated in NP-C), have been found to be elevated in patients with NP-C compared with control subjects.10 A high-throughput mass spectrometry-based method has been developed and validated to analyze bile acids in dried blood spots, and could also be applicable to plasma and urine.1,10

Preliminary investigations have shown greater specificity for patients with NP-C compared with oxysterols,10 and pre-analytical autoxidation is not a concern with bile acids. Bile acids therefore hold future promise as a valuable additional biomarker for NP-C screening and diagnosis.

Genetic testing

It is of utmost importance to genetically confirm a diagnosis in patients with high clinical suspicion and/or a biomarker profile consistent with NP-C.2 In newly diagnosed patients, it can also be useful to identify affected siblings, detect carriers in blood relatives and identify patients with NP-C2 who may benefit from hematopoietic stem cell transplantation. Parental DNA testing can also confirm allele segregation and homozygous status.11

Traditional methods

Traditionally, Sanger sequencing of genomic DNA or complementary DNA was performed in parallel with filipin staining (the historical ‘gold standard’ diagnostic test) to confirm NP-C diagnosis, and to determine the presence of identifiable pathological NP-C gene mutations.1

Next-generation sequencing methods and gene panels

Next-generation sequencing technologies are now widely available, providing faster, more cost-effective and more accurate genetic analysis.2 Targeted gene and multi-gene panels, including NPC1 and NPC2, can be used to screen clinical patient groups with an increased risk of NP-C.1 However, this method does not allow the identification of deep intronic mutations or large structural variants, and results from a primary genetic test may also be negative or heterozygous. In such circumstances, additional molecular studies should be conducted to detect complete or partial gene deletions/duplications and deep intronic mutations.

Additional genetic tests

Array comparative genomic hybridization (array CGH) can be used to detect large DNA dosage alterations. Quantitative real-time polymerase chain reaction or multiplex ligation-dependent probe amplification should be used to specifically quantify NPC1 and NPC2 copy numbers.1 To detect deep intronic mutations, full-gene sequencing (cDNA analysis followed by the sequencing of specific intronic regions within the gDNA) should be conducted.1

Online databases should be used to record mutations (e.g. NPC-db2 database in Tübingen).

Biochemical testing

The filipin staining test assesses the functional significance of new NPC1 or NPC2 genetic variants and was traditionally the ‘gold standard’ assay for NP-C diagnosis; however, it is no longer favored as a first-line diagnostic test. Current recommendations indicate the filipin staining test when genetic testing has not allowed identification of two pathogenic alleles in a patient, and to assist in the determination of the pathogenicity of novel mutations. For more information and guidance on how to perform the filipin staining test, please refer to Vanier and Latour 2015.12

Control subject
No marking

NP-C subject
Fluorescent vacuoles in lysosomes marked
with the accumulation of cholesterol

Supportive assessments

Bone marrow biopsy

Aspiration and examination of bone marrow may show foam cells and provide a measure of disease burden in late disease. It can provide a rapid screening test in cases where bone marrow is readily available.1

Electron microscopy

Electron microscopy of skin or liver biopsy can show polymorphous cytoplasmic bodies.


Magnetic resonance imaging (MRI) and positron emission tomography (PET) may be useful, supportive assessments for NP-C diagnosis, but are not specific for NP-C.1 It should be noted that the absence of abnormal MRI findings does not exclude a diagnosis of NP-C, as changes in MRI results are highly variable, nonspecific, and of uncertain sensitivity.1

For more details on MRI findings observed in patients with NP-C, please see the Neuropathology section of this website.


  1. Marquardt T, Clayton P, Gissen P, et al, on behalf of the NP-C  Diagnostics Working Group. New consensus recommendations for the detection and diagnosis of Niemann-Pick disease type C. J Inherit Metab Dis 2016;39:35.
  2. Vanier MT, Gissen P, Bauer P, et al. Diagnostic tests for Niemann-Pick disease Type C (NP-C): a critical review. Mol Genet Metab 2016;118:224–54.
  3. Boenzi S, Deodato F, Taurisano R, et al. A new simple and rapid LC-ESI-MS/MS method for quantification of plasma oxysterols as dimethylaminobutyrate esters. Its successful use for the diagnosis of Niemann-Pick Type C disease. Clin Chim Acta 2014;437:93–100.
  4. Reunert J, Fobker M, Kannenberg F, et al. Rapid diagnosis of 83 patients with Niemann Pick Type C disease and related cholesterol transport disorders by cholestantriol screening. EBioMedcine 2016;4:170–5
  5. Boenzi S, Deodato F, Taurisano R, Goffredo BM, Rizzo C. Evaluation of plasma cholestane-3β,5α,6β-triol and 7-ketocholesterol in inherited disorders related to cholesterol metabolism. J Lipid Res 2016;57:361–7.
  6. Polo G, Burlina A, Furlan F, et al. High level of oxysterols in neonatal cholestasis: a pitfall in analysis of biochemical markers for Niemann-Pick type C disease. Clin Chem Lab Med 2015;54:1221–9.
  7. Jiang X, Sidhu R, Porter FD, et al. A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma. J Lipid Res 2011;52:1435–45.
  8. Welford RW, Garzotti M, Lourenco C, Mengel E, Marquardt T, Reunert J. Plasma lysosphingomyelin demonstrates great potential as a diagnostic biomarker for Niemann-Pick disease type C in a retrospective study. PLoS One 2014;9:e114669.
  9. Giese AK, Mascher H, Grittner U, et al. A novel, highly sensitive and specific biomarker for Niemann-Pick Type C1 disease. Orphanet J Rare Dis 2015;10:78.
  10. Jiang X, Sidhu R, Mydock-McGrane L, et al. Development of a bile acid-based newborn screen for Niemann-Pick disease type C. Sci Transl Med 2016;8:337ra3636.
  11. Patterson MC, Hendriksz CJ, Walterfang M, et al., on behalf of the NP-C Guidelines Working Group. Recommendations for the diagnosis and management of Niemann–Pick disease type C: An update. Mol Genet Metab 2012;106:330–44.
  12. Vanier MT, Latour P. Laboratory diagnosis of Niemann-Pick disease type C: the filipin staining test. Methods Cell Biol 2015;126:357–75.