Overview of genotype-phenotype mapping and disease model panel.

Disease modelling as genotype-phenotype mapping in humans and a model organism. Arrows show progression from symptom identification (a phenotype outside the healthy range) to genotyping (placing a patient in genotype space by identifying a genetic variant), disease model creation (making a corresponding mutation in a model organism), and model organism phenotyping. Phenotypic drug screens identify compounds that move a disease model towards the wild-type phenotype (green arrow). These candidates can then be tested in humans (dashed green arrow). Thin lines show other symptom-gene-ortholog-phenotype connections. (B) Venn diagram showing number of conserved genes (Ortholist 2), those involved in neuron or muscle function (Wormbase), and those associated with human genetic disorders according to the Online Mendelian Inheritance in Man (OMIM) database. (C) Sequence similarity between human and C. elegans genes and the total number of orthology programs predicting that the gene is an ortholog.

Summary of the associated human ortholog(s), predicted functional class and key associated human disease phenotypes (Human Phenotype Ontology database) for each C. elegans disease model mutant.

Diverse multidimensional behavioural phenotypes are obtained across the diverse panel of disease model mutants. (A)

Representative field of view for a single camera channel with individual skeletonised worm images of bbs-1(syb1588) (top) and N2 (bottom) imaged on the same plate viewed using Tierpsy Tracker. (B) Representative behavioural phenotypes extracted by Tierpsy, representing changes in morphology, posture and locomotion of different disease mutant strains. P-values are for comparisons to wild-type N2 worms, block permutation t-test, corrected for multiple comparisons. (C) Hierarchical clustering of behavioural fingerprints. Features are Z-normalised. The top barcode shows the period of image acquisition where the behavioural feature was extracted by Tierpsy: pre-stimulation (pink), blue light (blue) and post-stimulation (green). (D) Principal component analysis of the disease model mutants and N2 reference (blue). Strains move in phenospace between pre-stimulus (circular points), blue light (crosses) and post-stimulation (squares) recordings. Strains with an aberrant blue light response are shown in red. Error bars represent standard error of the mean. (E) The total number of statistically significant behavioural features for each strain compared to N2 (from the total set of 8289 features extracted by Tierpsy). P-values for each feature were calculated using block permutation t-tests, using n = 100,000 permutations and P < 0.05 considered statistically significant after correcting for multiple comparisons using the Benjamini-Yekutieli method.

Ciliopathies disease model phenologs. (A-D)

Key behavioural features altered in loss-of-function mutant strains associated with ciliopathies: bbs-1(syb1588), bbs-2(syb1547), tub-1(syb1562) and tmem-231(syb1575), under baseline (pre-stimulus) imaging conditions. Individual points marked on the box plots are well averaged values (three worms per well) for each feature across the independent days of tracking. P-values are for comparisons to wild-type N2 worms using block permutation t-tests (n = 100,000 permutations, correcting for multiple comparisons using the Benjamini-Yekutieli method). (E-H) Changes in selected features in response to stimulation with a single 10-second blue light pulse (blue shaded region). Feature values were calculated using 10 second windows centred around 5 seconds before, 10 seconds after, and 20 seconds after the beginning of each blue light pulse. (I) Heatmap of the entire set of 8289 behavioural features extracted by Tierpsy for the disease model strains associated with ciliopathies and N2. The ‘stim type’ barcode denotes when during image acquisition the feature was extracted: pre-stimulation (pink), blue light stimulation (blue) and post-stimulation (green). Asterisks show the location of selected features present in A-E.

NALCN disease model phenologs. (A-D)

Key behavioural and postural features altered in loss-of-function mutant strains associated with NALCN mutants: nca-2(syb1612), unc-77(syb1688) and unc-80(syb1531), under baseline (pre-stimulus) imaging conditions. Individual points marked on the box plots are well averaged values (three worms per well) for each feature across the independent days of tracking. P-values are for comparisons to wild-type N2 worms using block permutation t-tests (n = 100,000 permutations correcting for multiple comparisons using the Benjamini-Yekutieli method). (E-H) Changes in selected features in response to stimulation with a single 10-second blue light pulse (blue shaded region). Feature values were calculated using 10 second windows centred around 5 seconds before, 10 seconds after, and 20 seconds after the beginning of each blue light pulse. (E) A representative ‘fainting phenotype’ for unc-80(syb1531) and nca-2(syb1612), characterised by an increase in pausing following the cessation of stimulation with blue light. (I) Heatmap of the entire set of 8289 behavioural features extracted by Tierpsy for the disease model strains associated with NALCN disease and N2. The ‘stim type’ barcode denotes when during image acquisition the feature was extracted: pre-stimulation (pink), blue light stimulation (blue) and post-stimulation (green). Asterisks show the location of selected features present in A-D.

Drug repurposing screening (A)

Phenotypes of unc-80(syb1531) mutant (red star) and N2 (blue star) worms treated with 1% DMSO, and unc-80(syb1531) mutants treated with a library of 743 FDA approved drugs at a concentration of 100 μM for 4 hours (circles). Each point represents an average of 3 well replicates, across 3 independent days of tracking (n = 9 total). Blue points are the top 30 compounds that significantly improved all three of the core behavioural features, pushing the unc-80 mutant strain towards the control in phenospace. (B) Confirmation screen of the top 30 compounds identified in the initial library screen. Again unc-80(syb1531) and N2 DMSO treated controls are represented by red and blue stars, respectively, and each circular point represents unc-80(syb1531) treated with 100 μM compound for 4 hours. The 13 compounds coloured in yellow lead to the worsening of >1000 behavioural features (see below). Liranaftate and atorvastatin both lead to a rescue of the core mutant phenotype with a low number of side effects. (C) Total number of behavioural side effects following treatment of unc-80(syb1531) with the 30 compounds in the confirmation screen. Side effects are defined as features that are not significant between unc-80 mutants and wild-type N2 worms treated with 1% DMSO but where there is a significant difference between unc-80 mutants treated with a drug compared to N2. Red dashed line separates drug treatments that lead to a worsening of >1000 behavioural features that correspond to the points coloured in yellow in the 3D scatterplot.