Laboratory diagnosis of Niemann-Pick disease type C: the filipin staining test

Abstract

Niemann-Pick disease type C (NPC) is an atypical neurovisceral lysosomal storage disorder resulting from mutations in either the NPC1 or the NPC2 gene, currently conceived as a lipid trafficking disorder. Impaired egress of cholesterol from the late endosomal/lysosomal (LE/L) compartment is a key element of the pathogenesis. The resulting accumulation of unesterified cholesterol in the LE/L compartment can be visualized by fluorescence microscopy after staining with filipin. The "filipin test," performed on cultured fibroblasts, is the historical gold standard method to establish the diagnosis in patients. The authors provide methodological details of the protocol developed and used in their laboratory since 1988, in which two sources of low-density lipoproteins (LDL) (total serum and pure LDL) are used in parallel to facilitate the final interpretation. Methodological caveats and variability of patterns encountered in patients with proven Niemann-Pick C disease (typical "classic" or "intermediate," atypical "variant") are described. An overview of the past 5 years referrals (533 subjects tested, 57 NPC cases, but also 74 mildly/weakly positive tests not due to NPC) is discussed, leading to a proposed algorithm for interpretation of results in the filipin test. This tool takes into account the limits of the method. In up to 15% of all referrals, the filipin test was inconclusive in absence of molecular analysis. Patients diagnosed in the adult age preferentially showed an "intermediate" or "variant" pattern. Well conducted, the filipin test remains an efficient approach for diagnosing NPC, and it is a good functional test to study the pathogenicity of novel mutations.

Keywords: Cholesterol homeostasis; Filipin; Intracellular cholesterol; Intracellular trafficking; Laboratory diagnosis; Late endosome; Lysosome; NPC1; NPC2; Niemann–Pick C disease.