A Retrospective Multicentric Study of 34 Patients with Niemann-Pick Type C Disease and Early Liver Involvement in France.

OBJECTIVE: To describe the clinical features and course of liver involvement in a cohort of patients with Niemann-Pick type C disease (NP-C), a severe lysosomal storage disorder. STUDY DESIGN: Patients with genetically confirmed NP-C (NPC1, n = 31; NPC2, n = 3) and liver involvement before age 6 months were retrospectively included. Clinical, laboratory test, and imaging data were collected until the last follow-up or death; available liver biopsy specimens were studied using anti-CD68 immunostaining. RESULTS: At initial evaluation (median age, 17 days of life), all patients had hepatomegaly, 33 had splenomegaly, and 30 had neonatal cholestasis. Portal hypertension and liver failure developed in 9 and 4 patients, respectively. Liver biopsy studies, performed in 16 patients, revealed significant fibrosis in all 16 and CD68+ storage cells in 15. Serum alpha-fetoprotein concentration measured in 21 patients was elevated in 17. Plasma oxysterol concentrations were increased in the 16 patients tested. Four patients died within 6 months of life, including 3 from liver involvement. In patients who survived beyond age 6 months (median follow-up, 6.1 years), cholestasis regressed in all, and portal hypertension regressed in all but 1; 25 patients developed neurologic involvement, which was fatal in 16 patients. CONCLUSIONS: Liver involvement in NP-C consisted of transient neonatal cholestasis with hepatosplenomegaly, was associated with liver fibrosis, and was responsible for death in 9% of patients. The combination of liver anti-CD68 immunostaining, serum alpha-fetoprotein measurement, and studies of plasma biomarkers should facilitate early identification of NP-C.

Application of a glycinated bile acid biomarker for diagnosis and assessment of response to treatment in Niemann-pick disease type C1.

Niemann-Pick disease type C (NPC) is a neurodegenerative disease in which mutation of NPC1 or NPC2 gene leads to lysosomal accumulation of unesterified cholesterol and sphingolipids. Diagnosis of NPC disease is challenging due to non-specific early symptoms. Biomarker and genetic tests are used as first-line diagnostic tests for NPC. In this study, we developed a plasma test based on N-(3ß,5α,6ß-trihydroxy-cholan-24-oyl)glycine (TCG) that was markedly increased in the plasma of human NPC1 subjects. The test showed sensitivity of 0.9945 and specificity of 0.9982 to differentiate individuals with NPC1 from NPC1 carriers and controls. Compared to other commonly used biomarkers, cholestane-3ß,5α,6ß-triol (C-triol) and N-palmitoyl-O-phosphocholine (PPCS, also referred to as lysoSM-509), TCG was equally sensitive for identifying NPC1 but more specific. Unlike C-triol and PPCS, TCG showed excellent stability and no spurious generation of marker in the sample preparation or aging of samples. TCG was also elevated in lysosomal acid lipase deficiency (LALD) and acid sphingomyelinase deficiency (ASMD). Plasma TCG was significantly reduced after intravenous (IV) 2-hydroxypropyl-ß-cyclodextrin (HPßCD) treatment. These results demonstrate that plasma TCG was superior to C-triol and PPCS as NPC1 diagnostic biomarker and was able to evaluate the peripheral treatment efficacy of IV HPßCD treatment.

A combination of 7-ketocholesterol, lysosphingomyelin and bile acid-408 to diagnose Niemann-Pick disease type C using LC-MS/MS.

BACKGROUND: Niemann-Pick disease type C (NPC) is an autosomal recessive disorder caused by mutations of NPC1 or NPC2, which encode the proteins that are responsible for intracellular cholesterol trafficking. Loss of this function results in the accumulation of cholesterol-related products, such as oxysterols, sphingolipids, and NPC-related bile acids, which were recently used as biochemical biomarkers for the diagnosis of NPC. Bile acid-408 is a new significant compound we found in Japanese NPC patients, and it likely belongs to the category of bile acids. However, the diagnosis of NPC using a single biomarker is not satisfactory for clinical application because of the high instance of false negatives or positives. Therefore, we proposed an application of NPC diagnosis using a combination of 7-ketocholesterol (7-KC), lysosphingomyelin (lysoSM), bile acid-408 and/or glucosylsphingosine (lysoGL-1). METHODS AND FINDINGS: 7-KC, lysoSM and lysoGL-1 in sera and bile acid-408 in dried blood spots (DBS) were quantified within 17 minutes using tandem mass spectrometry and high-resolution mass spectrometry, respectively. We measured these biomarkers in NPC patients (n = 19), X-linked adrenoleukodystrophy (X-ALD) patients (n = 5), patients with other lysosomal diseases (n = 300), newborns (n = 124) and healthy people (n = 74). Our results showed a promising accuracy (97%) for NPC diagnosis using the combination of 7-KC, lysoSM and bile acid-408. However, contrary to our expectations, lysoGL-1 levels did not present at a significantly greater amount in NPC patients than other patients and negative controls. CONCLUSIONS: The combination of 7-KC, lysoSM and bile acid-408 improves the accuracy of NPC diagnosis and is feasible for mass screening due to its simple sample preparation and measurement. Future research should investigate the chemical structure of bile acid-408 to further facilitate its advantage in diagnosis.

Cholestane-3ß, 5α, 6ß-triol: Further insights into the performance of this oxysterol in diagnosis of Niemann-Pick disease type C.

In recent years the oxysterol species cholestane-3ß, 5α, 6ß-triol (C-triol) has found application as a diagnostic biomarker for Niemann-Pick disease type C. Other studies have described increased C-triol in patients with Niemann-Pick disease type A/B and milder increases in lysosomal acid lipase deficiency (LALD), whereas they note normal C-triol levels in Smith-Lemli-Opitz syndrome (SLOS) and familial hypercholesterolaemia (FH) patients. Herein, we review data collected in our laboratory during method evaluation along with 5 years of routine analysis and present findings which differ from those reported by other groups with respect to LALD, SLOS and FH in particular, whilst providing further evidence regarding the clinical sensitivity and specificity of this biomarker, which are difficult to accurately assess. All of our Wolman disease (severe LALD) patients have demonstrated gross elevations of C-triol at diagnosis, with reduction to normal levels after induction of enzyme replacement therapy. In diagnostic specimens from SLOS patients we observed very low or undetectable C-triol levels whereas in post-therapeutic SLOS patients demonstrated normalised levels; we also describe a homozygous FH patient in which C-triol is significantly elevated. Upon investigation, we found that C-triol was formed artefactually from cholesterol during our sample preparation, i.e. this is a false positive of analytical origin; at present it is unclear whether similar effects occur during sample preparation in other laboratories. Our data demonstrates clinical sensitivity of 100% during routine application to diagnostic specimens; this is in keeping with other estimates, yet in a small proportion of patients diagnosed prior to C-triol measurement, either by Filipin staining of fibroblasts or molecular genetics, we have observed normal C-triol concentrations. Clinical specificity of C-triol alone is 93.4% and 95.3% when performed in conjunction with lysosomal enzymology. These performance statistics are very similar to those achieved with Filipin staining of cultured fibroblasts in the 5 years preceding introduction of C-triol to routine use in our laboratory. It is increasingly apparent to us that although this analyte is a very useful addition to the diagnostic tools available for NPC, with considerable advantages over more invasive and time-consuming methods, the interpretation of results is complex and should be undertaken only in light of clinical details and results of other analyses including enzymology for lysosomal acid lipase and acid sphingomyelinase.

Diagnosis of niemann-pick C1 by measurement of bile acid biomarkers in archived newborn dried blood spots.

BACKGROUND: Niemann-Pick disease type C1 (NPC1) is a rare, neurodegenerative cholesterol storage disorder. Diagnostic delay of >5 years is common due to the rarity of the disease and non-specific early symptoms. To improve diagnosis and facilitate early intervention, we previously developed a newborn screening assay based on newly identified plasma bile acid biomarkers. Because the newborn screen had been validated using dried blood spots (DBS) from already diagnosed NPC1 patients, an unanswered question was whether the screen would be able to detect individuals with NPC1 at birth. METHODS: To address this critical question, we obtained the newborn DBS for already diagnosed NPC1 subjects (n = 15) and carriers (n = 3) residing in California, New York, and Michigan states that archive residual DBS in biorepositories. For each of the DBS, we obtained two neighbor controls - DBS from patients born on the same day and in the same hospital as the NPC1 patients and carriers. 3ß,5α,6ß-trihydroxycholanic acid (bile acid A) and trihydroxycholanic acid glycine conjugate (bile acid B) were measured in the DBS using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. RESULTS: Bile acid B, the more specific biomarker for which the fully validated DBS assay was developed, was detected in 8/15 NPC1 patients, and elevated above the cut-off in 2/15 patients (the two samples with the shortest storage time). Bile acid B was detected in 2/2, 6/10, and 0/7 NPC1 samples that have been stored for <10.5 years, 13-20 years, and > 20 years, respectively, indicating that the glycine conjugate is detectable in DBS but may have reduced long-term stability compared with bile acid A, the precursor trihydroxycholanic acid, which was elevated in 15/15 NPC1 subjects, but not in carriers and controls. CONCLUSIONS: These results demonstrate that newborn screening for NPC1 disease is feasible using bile acid biomarkers.

The impact of biomarkers analysis in the diagnosis of Niemann-Pick C disease and acid sphingomyelinase deficiency.

BACKGROUND: Although representing two distinct disease entities, Niemann-Pick disease type C (NP-C) disease and acid sphingomyelinase deficiency (ASMD) share several phenotypic features. The lack of biomarkers was responsible in the past of diagnostic delay. Recently, plasma oxysterols, cholestan-3ß,5α,6ß-triol (Triol) and 7-ketocholesterol (7-KC) and lysosphingolipids, Lyso-sphingomyelin (Lyso-SM) and Lysosphingomyelin-509 (Lyso-SM-509), have been proposed as diagnostic biomarkers. We aimed to assess the diagnostic power of the two biomarkers categories and to evaluate possible correlations with patients' age and clinical phenotypes. PATIENTS AND METHODS: We analyzed plasma oxysterols and lysosphingolipids in patients affected by NP-C and ASMD, and compared with healthy controls. RESULTS: Oxysterols were always increased in both NP-C and ASMD. In NP-C, Lyso-SM and Lyso-SM-509 were increased in 70%, and 100% of patients, respectively. Biomarkers negatively correlated with patients' age, with highest levels in early-infantile, intermediate in the late-infantile and lowest in the juvenile phenotype. In ASMD, lysosphingolipids were both increased, with a greater order of magnitude than in NP-C, with highest levels in chronic-neurovisceral vs visceral phenotype. CONCLUSIONS: Lysosphingolipids are useful biomarkers for a rapid and precise diagnosis, allowing clear distinction between NP-C and ASMD. They are more reliable biomarkers than oxysterols and correlate with patients' age and clinical phenotype.

Niemann-Pick Disease, Type C1 Gene Expression in PBMCs is Associated with Interleukin 10 Serum Concentration: a Case-Control Study.

BACKGROUND: Recent studies showed that atherosclerosis is a lysosomal storage disease (LSD) and Niemann-Pick disease type C1 (NPC1) is the most important protein of the lysosomal membrane that is involved in the removal of FC from lysosomes. Whereas several in vitro and in vivo studies have described the crosstalk between lysosomal cholesterol accumulation and increased inflammation, there is no study addressing the correlation between NPC1 gene expression and an anti-inflammatory cytokine, interleukin 10 (IL-10) serum concentration in atherosclerotic patients. METHODS: IL-10 and 25-hydroxyvitamin D serum concentrations were quantified by enzyme-linked immunosorbent assay (ELISA) in atherosclerotic patients (n = 40) and a control group (n = 40). NPC1 gene expression analysis was performed by quantitative real-time PCR, and correlation between the two parameters was assessed. RESULTS: Mean IL-10 serum concentration and peripheral blood mononuclear cells' (PBMCs) gene expression of NPC1, adjusted for drug consumption, age, and BMI, was not significantly different between the patient and control groups (p = 0.6 and 0.67 respectively). However, NPC1 gene expression showed positive significant correlation with IL-10 serum concentration (p = 0.04, r = 0.29). We also observed lower serum concentration of IL-10 in the subjects with lower 25-hydroxyvitamin D serum concentration (p = 0.034). CONCLUSIONS: Our findings supported the previous observations showing the contribution of lysosomal lipid homeostasis of PBMCs to inflammation and pathogenesis of atherosclerosis.

Unravelling new pathways of sterol metabolism: lessons learned from in-born errors and cancer.

PURPOSE OF REVIEW: To update researchers of recently discovered metabolites of cholesterol and of its precursors and to suggest relevant metabolic pathways. RECENT FINDINGS: Patients suffering from inborn errors of sterol biosynthesis, transport and metabolism display unusual metabolic pathways, which may be major routes in the diseased state but minor in the healthy individual. Although quantitatively minor, these pathways may still be important in healthy individuals. Four inborn errors of metabolism, Smith-Lemli-Opitz syndrome, cerebrotendinous xanthomatosis and Niemann Pick disease types B (NPB) and C (NPC) result from mutations in different genes but can generate elevated levels of the same sterol metabolite, 7-oxocholesterol, in plasma. How this molecule is metabolized further is of great interest as its metabolites may have an important role in embryonic development. A second metabolite, abundant in NPC and NPB diseases, cholestane-3ß,5α,6ß-triol (3ß,5α,6ß-triol), has recently been shown to be metabolized to the corresponding bile acid, 3ß,5α,6ß-trihydroxycholanoic acid, providing a diagnostic marker in plasma. The origin of cholestane-3ß,5α,6ß-triol is likely to be 3ß-hydroxycholestan-5,6-epoxide, which can alternatively be metabolized to the tumour suppressor dendrogenin A (DDA). In breast tumours, DDA levels are found to be decreased compared with normal tissues linking sterol metabolism to cancer. SUMMARY: Unusual sterol metabolites and pathways may not only provide markers of disease, but also clues towards cause and treatment.

Intrathecal 2-hydroxypropyl-ß-cyclodextrin decreases neurological disease progression in Niemann-Pick disease, type C1: a non-randomised, open-label, phase 1-2 trial.

BACKGROUND: Niemann-Pick disease, type C1 (NPC1) is a lysosomal storage disorder characterised by progressive neurodegeneration. In preclinical testing, 2-hydroxypropyl-ß-cyclodextrins (HPßCD) significantly delayed cerebellar Purkinje cell loss, slowed progression of neurological manifestations, and increased lifespan in mouse and cat models of NPC1. The aim of this study was to assess the safety and efficacy of lumbar intrathecal HPßCD. METHODS: In this open-label, dose-escalation phase 1-2a study, we gave monthly intrathecal HPßCD to participants with NPC1 with neurological manifestation at the National Institutes of Health (NIH), Bethesda, MD, USA. To explore the potential effect of 2-week dosing, three additional participants were enrolled in a parallel study at Rush University Medical Center (RUMC), Chicago, IL, USA. Participants from the NIH were non-randomly, sequentially assigned in cohorts of three to receive monthly initial intrathecal HPßCD at doses of 50, 200, 300, or 400 mg per month. A fifth cohort of two participants received initial doses of 900 mg. Participants from RUMC initially received 200 or 400 mg every 2 weeks. The dose was escalated based on tolerance or safety data from higher dose cohorts. Serum and CSF 24(S)-hydroxycholesterol (24[S]-HC), which serves as a biomarker of target engagement, and CSF protein biomarkers were evaluated. NPC Neurological Severity Scores (NNSS) were used to compare disease progression in HPßCD-treated participants relative to a historical comparison cohort of 21 NPC1 participants of similar age range. FINDINGS: Between Sept 21, 2013, and Jan 19, 2015, 32 participants with NPC1 were assessed for eligibility at the National Institutes of Health. 18 patients were excluded due to inclusion criteria not met (six patients), declined to participate (three patients), pursued independent expanded access and obtained the drug outside of the study (three patients), enrolled in the RUMC cohort (one patient), or too late for the trial enrolment (five patients). 14 patients were enrolled and sequentially assigned to receive intrathecal HPßCD at a starting dose of 50 mg per month (three patients), 200 mg per month (three patients), 300 mg per month (three patients), 400 mg per month (three patients), or 900 mg per month (two patients). During the first year, two patients had treatment interrupted for one dose, based on grade 1 ototoxicity. All 14 patients were assessed at 12 months. Between 12 and 18 months, one participant had treatment interrupted at 17 months due to hepatocellular carcinoma, one patient had dose interruption for 2 doses based on caregiver hardship and one patient had treatment interrupted for 1 dose for mastoiditis. 11 patients were assessed at 18 months. Between Dec 11, 2013, and June 25, 2014, three participants were assessed for eligibility and enrolled at RUMC, and were assigned to receive intrathecal HPßCD at a starting dose of 200 mg every 2 weeks (two patients), or 400 mg every two weeks (one patient). There were no dropouts in this group and all 3 patients were assessed at 18 months. Biomarker studies were consistent with improved neuronal cholesterol homoeostasis and decreased neuronal pathology. Post-drug plasma 24(S)-HC area under the curve (AUC8-72) values, an indicator of neuronal cholesterol homoeostasis, were significantly higher than post-saline plasma 24(S)-HC AUC8-72 after doses of 900 mg (p=0·0063) and 1200 mg (p=0·0037). CSF 24(S)-HC concentrations in three participants given either 600 or 900 mg of HPßCD were increased about two fold (p=0·0032) after drug administration. No drug-related serious adverse events were observed. Mid-frequency to high-frequency hearing loss, an expected adverse event, was documented in all participants. When managed with hearing aids, this did not have an appreciable effect on daily communication. The NNSS for the 14 participants treated monthly increased at a rate of 1·22, SEM 0·34 points per year compared with 2·92, SEM 0·27 points per year (p=0·0002) for the 21 patient comparison group. Decreased progression was observed for NNSS domains of ambulation (p=0·0622), cognition (p=0·0040) and speech (p=0·0423). INTERPRETATION: Patients with NPC1 treated with intrathecal HPßCD had slowed disease progression with an acceptable safety profile. These data support the initiation of a multinational, randomised, controlled trial of intrathecal HPßCD. FUNDING: National Institutes of Health, Dana's Angels Research Trust, Ara Parseghian Medical Research Foundation, Hope for Haley, Samantha's Search for the Cure Foundation, National Niemann-Pick Disease Foundation, Support of Accelerated Research for NPC Disease, Vtesse, Janssen Research and Development, a Johnson & Johnson company, and Johnson & Johnson.

LC-MS/MS multiplex analysis of lysosphingolipids in plasma and amniotic fluid: A novel tool for the screening of sphingolipidoses and Niemann-Pick type C disease.

BACKGROUND: The biological diagnosis of sphingolipidoses currently relies on the measurement of specific enzymatic activities and/or genetic studies. Lysosphingolipids have recently emerged as potential biomarkers of sphingolipidoses and Niemann-Pick type C in plasma. METHODOLOGY: We developed a sensitive and specific method enabling the simultaneous quantification of lysosphingolipids by LC-MS/MS: lysoglobotriaosylceramide for Fabry disease, lysohexosylceramide (i.e. lysoglucosylceramide and/or lysogalactosylceramide) for Gaucher and Krabbe diseases, lysosphingomyelin and its carboxylated analogue lysosphingomyelin-509 for Niemann-Pick type A or B, and C diseases, lysoGM1 ganglioside for GM1gangliosidosis and lysoGM2 ganglioside for GM2 gangliosidosis. FINDINGS: The diagnostic performances were validated in plasma samples analysing a large series of patients affected with sphingolipidoses and Niemann-Pick type C disease (n = 98), other inborn errors of metabolism (n = 23), and controls (n = 228). The multiplex measurement of lysosphingolipids allowed the screening of Fabry (including female patients and late-onset variants), Gaucher and infantile Krabbe, Niemann-Pick type A/B and C diseases with high sensitivity and specificity. LysoGM1 and LysoGM2 were elevated in most of the patients affected with GM1 and GM2 gangliosidosis respectively. In amniotic fluid supernatant from pregnancies presenting non-immune hydrops fetalis (n = 77, including previously diagnosed Gaucher (n = 5), GM1 gangliosidosis (n = 4) and galactosialidosis (n = 4) fetuses) and from normal pregnancies (n = 15), a specific and dramatic increase of lysohexosylceramide was observed only in the Gaucher amniotic fluid samples. INTERPRETATION: This multiplex assay which allows the simultaneous measurement of lysosphingolipids in plasma modifies the diagnostic strategy of sphingolipidoses and Niemann-Pick type C. Furthermore, in pregnancies presenting non-immune hydrops fetalis, lysohexosylceramide measurement in amniotic fluid offers a rapid screening of fetal Gaucher disease without waiting for glucocerebrosidase activity measurement in cultured amniocytes.

Quantitation of plasmatic lysosphingomyelin and lysosphingomyelin-509 for differential screening of Niemann-Pick A/B and C diseases.

Acid sphingomyelinase deficiency (ASMd, Niemann-Pick disease A/B) and Niemann-Pick type C disease (NPC) share core clinical symptoms. Initial diagnostic discrimination of these two rare lysosomal storage diseases is thus difficult. As sphingomyelin accumulates in ASMd as well as NPC, lysosphingomyelin (sphingosylphosphorylcholine) and its m/z 509 analog were suggested as biomarkers for both diseases. Herein we present results of simultaneous LC-ESI-MS/MS measurements of lysosphingomyelin and lysosphingomyelin 509 in plasma and dried blood spots (DBS) collected from ASMd and NPC patients and suggest that the plasma but not DBS levels of the two analytes allow differential biochemical screening of ASMd and NPC.

Assessment of plasma chitotriosidase activity, CCL18/PARC concentration and NP-C suspicion index in the diagnosis of Niemann-Pick disease type C: a prospective observational study.

BACKGROUND: Niemann-Pick disease type C (NP-C) is a rare, autosomal recessive neurodegenerative disease caused by mutations in either the NPC1 or NPC2 genes. The diagnosis of NP-C remains challenging due to the non-specific, heterogeneous nature of signs/symptoms. This study assessed the utility of plasma chitotriosidase (ChT) and Chemokine (C-C motif) ligand 18 (CCL18)/pulmonary and activation-regulated chemokine (PARC) in conjunction with the NP-C suspicion index (NP-C SI) for guiding confirmatory laboratory testing in patients with suspected NP-C. METHODS: In a prospective observational cohort study, incorporating a retrospective determination of NP-C SI scores, two different diagnostic approaches were applied in two separate groups of unrelated patients from 51 Spanish medical centers (n = 118 in both groups). From Jan 2010 to Apr 2012 (Period 1), patients with ≥2 clinical signs/symptoms of NP-C were considered 'suspected NP-C' cases, and NPC1/NPC2 sequencing, plasma chitotriosidase (ChT), CCL18/PARC and sphingomyelinase levels were assessed. Based on findings in Period 1, plasma ChT and CCL18/PARC, and NP-C SI prediction scores were determined in a second group of patients between May 2012 and Apr 2014 (Period 2), and NPC1 and NPC2 were sequenced only in those with elevated ChT and/or elevated CCL18/PARC and/or NP-C SI ≥70. Filipin staining and 7-ketocholesterol (7-KC) measurements were performed in all patients with NP-C gene mutations, where possible. RESULTS: In total across Periods 1 and 2, 10/236 (4%) patients had a confirmed diagnosis o NP-C based on gene sequencing (5/118 [4.2%] in each Period): all of these patients had two causal NPC1 mutations. Single mutant NPC1 alleles were detected in 8/236 (3%) patients, overall. Positive filipin staining results comprised three classical and five variant biochemical phenotypes. No NPC2 mutations were detected. All patients with NPC1 mutations had high ChT activity, high CCL18/PARC concentrations and/or NP-C SI scores ≥70. Plasma 7-KC was higher than control cut-off values in all patients with two NPC1 mutations, and in the majority of patients with single mutations. Family studies identified three further NP-C patients. CONCLUSION: This approach may be very useful for laboratories that do not have mass spectrometry facilities and therefore, they cannot use other NP-C biomarkers for diagnosis.

Determination of serum cholestane-3ß,5α,6ß-triol by gas chromatography-mass spectrometry for identification of Niemann-Pick type C (NPC) disease.

Niemann-Pick type C (NPC) is a neurological disease caused by an intracellular cholesterol accumulation. Cholesterol oxidation product cholestane-3ß,5α,6ß-triol (C-triol) serves as diagnostic biomarker for NPC, but its measurement in the routine laboratory remains difficult. We developed an isotope dilution gas chromatography-mass spectrometry (GC-MS) method permitting screening for NPC in plasma. 1440 plasma samples obtained from clinically suspicious patients were subjected to alkaline saponification. C-triol was extracted with carbon tetrachloride, transformed into the trimethylsilylethers, separated on a fused silica capillary column with a nonpolar silicone stationary phase, and analyzed by GC-MS. NPC diagnosis was confirmed by DNA sequencing. The method was linear over a concentration range of 0.03-200ng/mL with a mean recovery rate of 98.6%. The intra- and inter-day variation coefficients assessed at two concentrations were below 15%. Limits of quantification (LOQ) and detection (LOD) were 0.03ng/mL and 0.01ng/mL, respectively. Receiver operating characteristic (ROC) analysis estimated that the area under curve was 0.997 implying a significant discriminatory power to identify subjects with NPC. Nevertheless, 13 NPC patients and 29 control subjects confirmed by sequencing showed false negative or positive results, respectively. Two patients with cerebrotendinous xanthomatosis showed a 5-10-fold increase in C-triol levels. We developed a quick and sensitive GC-MS method for determination of C-triol, which may serve as a simple and inexpensive diagnostic tool aiding NPC diagnosis in a routine hospital laboratory. As C-triol elevation is not limited to NPC, the NPC diagnosis has to be confirmed by DNA sequencing.

Diagnostic tests for Niemann-Pick disease type C (NP-C): A critical review.

Niemann-Pick disease type C (NP-C) is a neurovisceral lysosomal cholesterol trafficking and lipid storage disorder caused by mutations in one of the two genes, NPC1 or NPC2. Diagnosis has often been a difficult task, due to the wide range in age of onset of NP-C and clinical presentation of the disease, combined with the complexity of the cell biology (filipin) laboratory testing, even in combination with genetic testing. This has led to substantial delays in diagnosis, largely depending on the access to specialist centres and the level of knowledge about NP-C of the physician in the area. In recent years, advances in mass spectrometry has allowed identification of several sensitive plasma biomarkers elevated in NP-C (e.g. cholestane-3ß,5α,6ß-triol, lysosphingomyelin isoforms and bile acid metabolites), which, together with the concomitant progress in molecular genetic technology, have greatly impacted the strategy of laboratory testing. Specificity of the biomarkers is currently under investigation and other pathologies are being found to also result in elevations. Molecular genetic testing also has its limitations, notably with unidentified mutations and the classification of new variants. This review is intended to increase awareness on the currently available approaches to laboratory diagnosis of NP-C, to provide an up to date, comprehensive and critical evaluation of the various techniques (cell biology, biochemical biomarkers and molecular genetics), and to briefly discuss ongoing/future developments. The use of current tests in proper combination enables a rapid and correct diagnosis in a large majority of cases. However, even with recent progress, definitive diagnosis remains challenging in some patients, for whom combined genetic/biochemical/cytochemical markers do not provide a clear answer. Expertise and reference laboratories thus remain essential, and further work is still required to fulfill unmet needs.

[Blood 7-ketocholesterol level, clinical features and gene mutation analysis of 18 children with Niemann-Pick disease type C].

OBJECTIVE: To investigate 7-ketocholesterol (7-KC) level in the blood, clinical features and gene mutation of Niemann-Pick disease type C (NPC). METHOD: Eighteen patients diagnosed as NPC in Shanghai Xinhua Hospital seen from February 2013 to October 2014 were enrolled in this study. They included 13 males and 5 females and aged from 5 months to 21 years. The plasma 7-KC concentrations, clinical features and gene mutations of NPC patients were reviewed retrospectively. RESULT: Fourteen NPC patients had neurological symptoms with the age of neurological onset from 1 year to 16 years. In seven cases the disease was early-infantile subtype, in 1 late-infantile subtype, in five juvenile subtype and in one adult subtype. The 7-KC value in the plasma of NPC patients was higher than the normal range, (348.5±168.7) µg/L in the early-infantile subtype, 150.6 µg/L in the late-infantile subtype, (145.0±46.3) µg/L in the juvenile subtype, and 32.0 µg/L in the adult subtype, respectively, additionally, four NPC patients had no observable neuropsychiatric disability when confirmed to be NPC by genetic testing, with the plasma 7-KC value (345.6±134.2) µg/L; 16 of 18 patients had splenomegaly or hepatosplenomegaly. Among 18 patients, 34 different mutations in the NPC1 gene were identified including 27 reported mutations, 1 novel small deletion 3609_3610delAC, five novel exonic point mutations, c. 3683T>C(M1228T), c. 3679A>T(R1227W), c. 1070C>T(S357L), c. 1456A>C(N486H) and c. 1142G>A(W381X) and 1 novel intronic mutation c. 881+ 3A>G. CONCLUSION: The 7-KC levels in the blood of patient was remarkably increased, and there was a tendency that 7-KC levels inversely correlated with the age of neurological onset. Most NPC patient had splenomegaly or hepatosplenomegaly. Among 18 patients, 34 different mutations in the NPC1 gene were identified including seven novel mutations, which enriched the gene mutation spectrum.

Identification of novel bile acids as biomarkers for the early diagnosis of Niemann-Pick C disease.

This article describes a rapid UPLC-MS/MS method to quantitate novel bile acids in biological fluids and the evaluation of their diagnostic potential in Niemann-Pick C (NPC). Two new compounds, NPCBA1 (3ß-hydroxy,7ß-N-acetylglucosaminyl-5-cholenoic acid) and NPCBA2 (probably 3ß,5α,6ß-trihydroxycholanoyl-glycine), were observed to accumulate preferentially in NPC patients: median plasma concentrations of NPCBA1 and NPCBA2 were 40- and 10-fold higher in patients than in controls. However, NPCBA1 concentrations were normal in some patients because they carried a common mutation inactivating the GlcNAc transferase required for the synthesis of this bile acid. NPCBA2, not containing a GlcNAc moiety, is thus a better NPC biomarker.

Development of a bile acid-based newborn screen for Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, it is imperative to improve diagnostics and facilitate early intervention. We used metabolomic profiling to identify potential markers and discovered three unknown bile acids that were increased in plasma from NPC but not control subjects. The bile acids most elevated in the NPC subjects were identified as 3ß,5α,6ß-trihydroxycholanic acid and its glycine conjugate, which were shown to be metabolites of cholestane-3ß,5α,6ß-triol, an oxysterol elevated in NPC. A high-throughput mass spectrometry-based method was developed and validated to measure the glycine-conjugated bile acid in dried blood spots. Analysis of dried blood spots from 4992 controls, 134 NPC carriers, and 44 NPC subjects provided 100% sensitivity and specificity in the study samples. Quantification of the bile acid in dried blood spots, therefore, provides the basis for a newborn screen for NPC that is ready for piloting in newborn screening programs.

Rapid Diagnosis of 83 Patients with Niemann Pick Type C Disease and Related Cholesterol Transport Disorders by Cholestantriol Screening.

Niemann Pick type C (NP-C) is a rare neurodegenerative disorder caused by an impairment of intracellular lipid transport. Due to the heterogeneous clinical phenotype and the lack of a reliable blood test, diagnosis and therapy are often delayed for years. In the cell, accumulating cholesterol leads to increased formation of oxysterols that can be used as a powerful screening parameter for NP-C. In a large scale study, we evaluated the oxysterol cholestane-3ß,5α,6ß-triol (c-triol) as potential biomarker for a rapid diagnosis of NP-C. Using GC/MS, c-triol has been analyzed in 1902 plasma samples of patients with the suspicion for NP-C. Diagnosis in patients with elevated oxysterols was confirmed by genetic analysis. 71 new NP-C patients (69 NP-C1 and two NP-C2) and 12 Niemann Pick type A/B patients were identified. 24 new mutations in NPC1, one new mutation in NPC2 and three new mutations in the SMPD1 gene were found. Cholestane-3ß,5α,6ß-triol was elevated in Niemann Pick type C1, type C2, type A/B and in CESD disease. No other study has ever identified so many NP-C patients, proving that c-triol is a rapid and reliable biomarker to detect patients with NP-C disease and related cholesterol transport disorders. It should replace the filipin test as the first-line diagnostic assay.

Chronic administration of an HDAC inhibitor treats both neurological and systemic Niemann-Pick type C disease in a mouse model.

Histone deacetylase inhibitors (HDACi) are approved for treating rare cancers and are of interest as potential therapies for neurodegenerative disorders. We evaluated a triple combination formulation (TCF) comprising the pan-HDACi vorinostat, the caging agent 2-hydroxypropyl-ß-cyclodextrin (HPBCD), and polyethylene glycol (PEG) for treating a mouse model (the Npc1(nmf164) mouse) of Niemann-Pick type C (NPC) disease, a difficult-to-treat cerebellar disorder. Vorinostat alone showed activity in cultured primary cells derived from Npc1(nmf164) mice but did not improve animal survival. However, low-dose, once-weekly intraperitoneal injections of the TCF containing vorinostat increased histone acetylation in the mouse brain, preserved neurites and Purkinje cells, delayed symptoms of neurodegeneration, and extended mouse life span from 4 to almost 9 months. We demonstrate that the TCF boosted the ability of HDACi to cross the blood-brain barrier and was not toxic even when used long term. Further, the TCF enabled dose reduction, which has been a major challenge in HDACi therapy. TCF simultaneously treats neurodegenerative and systemic symptoms of Niemann-Pick type C disease in a mouse model.