We studied 26 skin fibroblast cell lines of NPC1-deficient patients from 23 non-related families. Twenty-one of the patients were described by Jahnova and co-workers, to which we refer the reader for patients' detailed clinical and laboratory characteristics. These cell lines are highlighted in Table S1. Cell lines from 8 healthy controls were also used.
Analysis of promoter sequence and determination of haplotypes
A 1.7 kb NPC1 promoter fragment was amplified from genomic DNA of all NP-C patients in the cohort and Sanger sequenced on automated capillary sequencers (ABI Prism 3100-Avant or 3500xL Genetic Analyzer; Life Technologies). PCR products containing sequence variants were cloned using a TA cloning kit (TOPO-TA, Thermo Fisher Scientific) and individual clones were sequenced. Haplotypes were determined from variants found in individual clones. Sequence of primers used for amplifications and sequencing are available upon request.
Reporter gene assays – promoter activity
Reporter plasmid constructs were prepared as follows. PCR products containing a 1688 fragment of the predicted NPC1 promoter were amplified using primers with overhangs carrying XhoI sites. Primer sequences were derived from the genomic sequence of chromosome 18 (GenBank accession No. NG_012795.1).
Amplified promoter fragments were inserted in both sense and antisense orientations into the XhoI site of pGL3basic (luc2CP/Hygro) vector (Promega), at the polylinker site upstream from the firefly luciferase reporter gene, generating pGL3-NPC1. Four constructs carrying sequence variants forming the four NPC1 haplotypes in the same manner were created, generating pGL3-NPC1-Hap1 to Hap4.
HepG2 (human hepatoblastoma) cells were grown in Opti-MEM (Agilent Technologies) medium supplemented with 10% (v/v) foetal bovine serum (FBS) in 25 cm2 flasks at 37 °C, and 5% CO2. A total of 5 x 104 HepG2 cells were seeded per well into 24-well culture plates 24 hours prior to transfection. HepG2 cells were transfected with 166 ng per well of each construct or the empty pGL3 using the TfxTM – 20 and FuGene HD Transfection Reagent (Promega). pRL-TK vector (Promega) harboring the Renilla luciferase gene was cotransfected as an internal control to normalize transfection efficiency.
Experiments were done in triplicate and each transfection was repeated independently at least three times. After 48 hours, transfected cells were washed with PBS and lysed with 100 µl of Passive lysis buffer (Promega). Luciferase reporter activity was assayed using a Dual-Luciferase Reporter Assay System (Promega). The intensity of chemiluminescence was measured in the supernatant using a luminometer (Berthold Technologies). The results were analyzed using one-tailed t-test.
Total RNA was isolated from skin fibroblast cultures using standard procedures  and reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Life Technologies).
Two TaqMan Gene Expression Assays (Applied Biosystems), Hs00264835_m1 and Hs00975249_m1, were used for relative qPCR measurements of NPC1 transcript abundance. To identify suitable endogenous controls cDNA samples from 7 NPC1-deficient patients and 7 control individuals using Human Endogenous Control Array (Applied Biosystems) were tested. The readouts were analyzed using NormFinder (Aarhus University Hospital, Aarhus, Denmark) and beta-2 microglobulin B2M gene was selected as an endogenous control. NPC1 relative expression was measured using 2-DDCt method. The results were expressed relative to NPC1 expression of a control reference sample which was assayed simultaneously with each batch of patient samples. All analyses were carried out using Applied Biosystems 7900 Real Time PCR system (Applied Biosystems).
Determination of allele expression ratios by deep sequencing
NPC1 transcript fragments containing pathogenic or non-pathogenic variants were amplified from the patient cDNAs by PCR as described previously. The minimum length of PCR products was 300bp. PCR libraries were prepared using Nextera XT DNA Sample Preparation Kit (Illumina) and indexed using Nextera XT Index Kit (Illumina). The libraries were sequenced using MiSeq Reagent Nano Kit v2 (2*250) and Nano Flow Cell 500 cycles on MiSeq Sequencer (Illumina).
The reads were aligned to the NPC1 reference sequence (NM_000271) and analyzed using NextGENe software package (SoftGenetics). Only samples with depth greater than 800 reads were included in the analysis. The ratio of reads containing wild-type and variant sequences was calculated after removal of reads carrying probable sequencing errors at the site of evaluated variant. Heterozygosity for SNP c.2793C>T rs1140458 was used for the determination of allele ratio in samples #24 and #26 (genotype p.[A927V];[A927V]). Both the variant and the mutation localize to NPC1 exon 18.
Fibroblasts from patients and control subjects were cultured according to routine procedures in DMEM / 10% FBS and 5 g/l glucose and penicillin / streptomycin antibiotics in 25 cm2 culture flasks and maintained in the same medium for all experiments except for diagnostic filipin testing and LDL-induced cholesterol esterification assays. For non-microscopic studies, confluent cells were PBS washed and harvested by scraping into PBS and centrifuged. The cell pellet was kept frozen at -20°C until use.
Western blot analyses
The samples were sonicated and the protein content was determined by Direct Detect spectrometer (Merck Millipore). Samples were mixed with 6X SDS non-reducing sample buffer (0.35M Tris, pH
6.8, 10% SDS, 30% glycerol, 0.012% bromophenol blue) and non-boiled samples (8 µg of whole cell lysates) were resolved by 10% SDS-PAGE electrophoresis under non-reducing conditions. Protein samples were transferred onto Immobilon-P PVDF membrane (Merck Millipore) using a semi-dry electroblotter (Biotec-Fischer). Reversible Ponceau S was applied to check equal loading of gels. Immunodetection of NPC1 and beta-actin proteins was performed using a rabbit monoclonal anti-NPC1 antibody (ab134113, Abcam) at a 1:3000 dilution, and a mouse monoclonal anti-beta-actin antibody (mAbcam 8226, Abcam) at a 1:4000 dilution, respectively. Detection was performed by chemiluminiscence using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific). Image capture was carried out using ChemiGeniusQ analysis system and GeneSnap software (Syngene, Cambridge, UK). Images were analysed using GeneTools software package (Syngene).
For confocal microscopic co-localization studies, the cells were seeded onto BD Falcon Cultures Slides (Becton Dickinson). Next day the fibroblasts were washed, fixed with ice-cold methanol, blocked with 5% FBS in PBS and co-labelled rabbit monoclonal anti-NPC1 (1:100, ab134113, Abcam) and mouse monoclonal anti-LAMP2 (1:500, H4B4, Iowa Hybridoma Bank) antibodies at 4°C overnight. Secondary antibodies were donkey anti-IgG anti-mouse alexafluor555 or anti-rabbit alexafluor488 conjugates (Pierce) diluted 1:1000. Leica SP8X laser scanning confocal system equipped with 470nm-670nm 80 MHz pulse continuum White Light Laser 2 and HC PL APO 63x/1.40 OIL CS2 (W.D. 0.14 mm) objective was used to image the samples. Image acquisition conditions were: excitation at 496 nm or 553 nm, one voxel 42.2 x 42.2 x 130 nm, 7 Z-steps (fulfilling Nyquist sampling theorem), Hybrid detectors at 503-553 nm or 566-650 nm. Acquired confocal images were deconvolved using theoretical point spread function in Huygens Professional software (Scientific Volume Imaging - SVI, Hilversum, The Netherlands). Overlay colocalization maps and Object Pearson’s coefficients were computed using Huscript (SVI), the grayscale maps were converted to colour coding look-up table (LUT) in Fiji / ImageJ software (NIH, Bethesda).
Direct quantitative filipin staining
Cells were seeded onto BD Falcon culture slides and cellular cholesterol accumulation was visualized and quantified after direct filipin staining. Briefly, cells were cultured under steady-state conditions as stated above, washed with PBS, fixed using 4% paraformaldehyde and stained with 0.1 mg/ml filipin (Sigma) in PBS, prepared by dilution of a filipin DMSO stock solution prepared the same day. To decrease filipin photobleaching ProLong Gold Antifade Mountant (LifeTech) was used as antifade mountant. Slides were examined using a Nikon Eclipse TI fluorescence microscope equipped with DAPI filter set and all photographed at constant 100 ms exposure time. The exposure time was optimized using the most intensive cell samples to prevent pixel saturation. The fluorescent signal density of individual cells (N=10 per cell line) was manually acquired using ImageJ (NIH, Bethesda). Average corrected total cell fluorescence per one cell line was calculated. The presented values reflect fold change relative to average of controls.
Diagnostic filipin testing
Diagnostic filipin testing was performed at the time of individual diagnostic process as described in Vanier et al. A main difference with the direct steady-state procedure was that dual culture slides (Lab-Tek chambers) with cells from each fibroblast line were first incubated in medium supplemented with 10% lipoprotein-deficient serum (LPDS) for 3 days, and then challenged for 24h with (1) medium supplemented with LPDS and 50 µg/ml purified human LDL and (2) medium supplemented with 10% fresh total human serum, prior to filipin staining. A control and a typical NP-C cell lines were included in each diagnostic experiment. Fluorescence microscopic examination was performed using a selective DAPI filter and expert visual evaluation done as discussed by Vanier and Latour.
LDL-induced early rates of cholesteryl ester formation
The test, including complex cell culture conditions, was performed exactly as described by Vanier et al. Esterification rates in Table 1 show values of cholesteryl-[3H]oleate formed/4.5h/mg protein. Classification of the cell lines into biochemical classical, intermediate or variant NP-C profiles [18, 20] was based on combined evaluation of diagnostic filipin testing and results of this assay (both performed in MTV's laboratory).
Unesterified cholesterol and cholesteryl ester levels were analysed in fibroblast cultures cultured under steady-state conditions. Briefly, cells were washed with PBS and harvested using trypsinization and centrifugation. The harvested cells were homogenized in water by sonication and extracted in chloroform:methanol (2:1, v/v) mixture. The extract was filtered via hydrophilic PTFE filters (Millex LH filters, Millipore), dried under the stream of nitrogen and processed for mass spectrometry analysis by modified method of Liebisch et al. Major changes were in replacement of chloroform by hexane and acetylchloride with propionylchloride in the derivatization mixture and the rest of the procedure remained unchanged. Mass spectrometry analysis was performed on triple quadrupole tandem mass spectrometer AB/MDS SCIEX API4000 with electrospray ionization coupled with Agilent 1290 Infinity UPLC. 200 pmol of d7-cholesterol and 40 pmol of C17:0 cholesterol ester were used as internal standards for UC and CE quantitative analysis.
Data analysis and statistics
Statistical computing were performed in R software v3.5.1 or STATISTICA v12. P-values < 0.05 were considered statistically significant.