Fig 1.
Schematic diagram of the NPC1 gene showing the location of the candidate causal mutation NM_174758.2: g.33099467C>G with chromatograms from Sanger sequencing data for two affected calves and a wildtype control.
(a) Location of the bovine NPC1 gene, Chr24: 33058694–33105394 on the ARS-UCD1.2 bovine genome assembly. (b) Position of the six overlapping RT-PCR products (blue bars) in relation to the NPC1 cDNA containing 25 exons (grey bars) with a transcript length of 4721 bp (ENSBTAT00000020219.5). The start codon (ATG) is indicated by the green circle and the stop codon (UAG) is indicated by the red circle, with the cDNA location of the first nucleotide of the start and stop codon given. (c) Genomic region containing the C>G missense variant with protein translation frames obtained from NCBI Genomic Data Viewer (NCBI, accessed 12th August 2019, <https://www.ncbi.nlm.nih.gov/genome/gdv/browser/?context=genome&acc=GCF_002263795.1>). The reading frame is identified by a black box. (d) Sanger sequencing chromatograms for the two affected calves and a wildtype control.
Table 1.
Primer sequences for amplification of cDNA for NPC1 and their location in the ENSBTAT00000020219.5 transcript.
Table 2.
Primer and probe sequences for the real-time PCR assay and primer sequences for the RFLP PCR assay.
Fig 2.
Haematoxylin and eosin staining of 5 micron thick sections from two affected and two unaffected calves at 40x magnification.
(a) Thalamus of calf 2 showing nuclei with foamy, granular basophilic cytoplasm (black arrow), degenerate neurons with a condensed and basophilic nucleus (orange arrow) with the ensuing phagocytosis (red arrows) and macrophages with brown-pigmented cellular debris accumulating around a blood vessel (green arrow). (b) Cerebellar white matter of calf 3 showcasing widespread foamy vacuolation of the cytoplasm of neurons and glia, with eosinophilic and axonal swellings (white arrows) and macrophages and lymphocytes surrounding a small blood vessel (grey arrow). (c) Thalamus from a healthy calf and (d) cerebellum from a healthy calf.
Fig 3.
Two Regions of Homozygosity (ROH) for affected calves 2 and 3 and their respective dams showing approximate locations for two NPC candidate genes of interest based on the UMD_3.1 bovine genome assembly.
(a) A shared ROH on chromosome 10 did not contain the NPC2 gene NM_173918:g. 86,170,653–86,179,237 and (b) A shared ROH on chromosome 24 contained the NPC1 gene NM_174758:g. 33,438,455–33,485,188.
Fig 4.
Multiple species NPC1 protein alignment using T-Coffee and BOXSHADE was completed using accessions NP_777183.1 (Bos taurus), NP_000262.2 (Homo sapiens), XP_001155285.1 (Pan troglodytes), XP_002800934.1 (Macaca mulatta), NP_032746.2 (Mus musculus), NP_705888.2 (Rattus norvegicus), NP_001003107.1 (Canis lupus familiaris), XP_419162.3 (Gallus gallus), NP_001230804.1 (Danio rerio) and XP_004915269.1 (Xenopus tropicalis).
CowMT refers to the mutant Bos taurus sequence and CowWT refers to the wildtype Bos taurus sequence. The predicted change from the proline to arginine in the affected calves is highlighted by an asterix (*) in the CowMT sequence.
Table 3.
Variants identified in Sanger sequencing of NPC1 in affected calf 2.
Fig 5.
Characterisation of NPC1-mutant bovine fibroblasts for (i) wildtype (WT) and NPC1-affected calves (NPC), consisting of calves 2 and 3 and (ii) individual profiles.
(A) Total acidic compartment volume measurements with Lysotracker Green fluorescence values (mean, standard deviation (SD)). (B) Total sphingosine levels with samples labeled with o-phthaldialdehyde solution (mean, SD). (C) Total cholesterol levels measured with Amplex Red cholesterol assay, followed by Folch extraction (mean, SD).
Fig 6.
Filipin staining for free cholesterol in cells in wildtype Angus fibroblast cells (WT) and affected calves 2 (NPC1-1) and 3 (NPC1-2), scale bar is equal to 30 microns.
(A) Representative images for filipin staining (yellow) for wildtype and affected calves, with vertical tiles under each sample. (B) Fluorescence quantification of filipin staining. NPC1-1 (calf 2) is represented in red and NPC1-2 (calf 3) in black. Mean gray value was quantified for each cell with Fiji (image J) software.
Fig 7.
Cholera toxin B staining for GM1 localisation in cells in wildtype Angus fibroblast cells (WT) and affected calves 2 (NPC1-1) and 3 (NPC1-2).
(A) Representative images for cholera toxin staining (red). Scale bar 30 micron. (B) Fluorescence quantification of cholera toxin staining. NPC1-1 (calf 2) is represented in red and NPC1-2 (calf 3) in black. Mean gray value was quantified for each cell with Fiji software.
Fig 8.
HPLC of 2AA-labelled GSL-derived glycans from bovine cultured fibroblasts.
Fluorescence profiles for affected calf 2 (NPC1-1) are shown in red (NPC) and a wildtype Angus animal in blue (WT).
Fig 9.
Quantification of Glycosphingolipids (GSLs) species in bovine cultured fibroblasts from HPLC analysis for wildtype (control), calf 2 (NPC1-1) and calf 3 (NPC1-2).
Fig 10.
Structural modelling and simulations of bovine NPC1 wildtype and p.P990R mutant.
(a) The homology model of bovine NPC1 wildtype embedded in a POPC membrane, showing the NTD in tan, the CTD in purple and the P990 position in pink. (b) Coordination of the R980-D994 salt bridge surrounding the P990 position in wildtype (left) and R990 mutant (right), showing how R990 competes for the negative charge of D994. This snapshot comes from an equilibrated state of the homology model. (c) Root mean square deviation (RMSD) plot of all protein Cα atoms for wildtype (blue) and R990 mutant (red), over 500 ns of simulation and for each replicate. (d) Superimposed simulation snapshots (every 100ns) over the simulation replicates shows structural distortions and enhanced conformational sampling of the CTD (purple) and NTD (tan) domains in the presence of R990 (pink). (e) Distance plot that measures the distance between the R980 CZ and D994 CG atoms over time for wildtype (blue) and R990 mutant (red). (f) RMSD plots showing the larger dynamic motion of the CTD (left) and NTD (right) domains in the R990 (red) mutant simulations in comparison to wildtype (blue).