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. 2021 Mar 18;11(1):6332.
doi: 10.1038/s41598-021-85471-4.

Global network analysis in Schizosaccharomyces pombe reveals three distinct consequences of the common 1-kb deletion causing juvenile CLN3 disease

Affiliations

Global network analysis in Schizosaccharomyces pombe reveals three distinct consequences of the common 1-kb deletion causing juvenile CLN3 disease

Christopher J Minnis et al. Sci Rep. .

Erratum in

Abstract

Juvenile CLN3 disease is a recessively inherited paediatric neurodegenerative disorder, with most patients homozygous for a 1-kb intragenic deletion in CLN3. The btn1 gene is the Schizosaccharomyces pombe orthologue of CLN3. Here, we have extended the use of synthetic genetic array (SGA) analyses to delineate functional signatures for two different disease-causing mutations in addition to complete deletion of btn1. We show that genetic-interaction signatures can differ for mutations in the same gene, which helps to dissect their distinct functional effects. The mutation equivalent to the minor transcript arising from the 1-kb deletion (btn1102-208del) shows a distinct interaction pattern. Taken together, our results imply that the minor 1-kb deletion transcript has three consequences for CLN3: to both lose and retain some inherent functions and to acquire abnormal characteristics. This has particular implications for the therapeutic development of juvenile CLN3 disease. In addition, this proof of concept could be applied to conserved genes for other mendelian disorders or any gene of interest, aiding in the dissection of their functional domains, unpacking the global consequences of disease pathogenesis, and clarifying genotype-phenotype correlations. In doing so, this detail will enhance the goals of personalised medicine to improve treatment outcomes and reduce adverse events.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
General overview of SGA analysis of btn1 mutants versus ade6 control. (A) Representative images of the SGA plates for control (ade6) and query mutants (btn1∆, btn1D363G, btn1102–208del), with empty control quadrants shown for ade6 (yellow boxes). (B) Exclusion of small colonies for ade6 control across batches as they represent high variability therefore reducing noise. (C) Principle component biplot of the variance within the SGA data for ade6 control (yellow) and query-mutants btn1∆ (blue), btn1D363G (orange), btn1102–208del (red), with experimental batch indicated. (D) Cluster analysis for each strain and all the genes with their normalised colony size difference against ade6 control with batch effects removed. Interactions are coloured in blue for negative interactions (< − 0.5) and yellow for positive interactions (> 0.5). (E) Gene linkage of normalised fitness score for ade6 control and query mutants btn1∆, btn1D363G, btn1102–208del from one experiment. Vertical dashed line represents ade6 or btn1 gene location, red points represent interaction scores excluded from data since less than 500 kb/500,000 bps from query gene location.
Figure 2
Figure 2
Venn diagrams and comparative volcano plots for strains btn1∆, btn1102–208del (1-kb) and btn1D363G (D363G) against control ade6. (A) Numbers represent robust hits across three independent SGAs for each query mutant. These include the subsets of genes that are shared between two or all mutants as well as their unique interactions. (BD) Colony size difference plotted for each gene and each strain against the ade6 control respectively (B) btn1∆, (C) btn1102–208del (D) btn1D363G. Every gene had quad intra-repeats and the experiment was done in triplicate. All genes are plotted on the adjusted p-value on a log scale. The strongest interacting genes are highlighted as negative (red) or positive (green) interactors.
Figure 3
Figure 3
Comparative 2D-volcano plots for SGA queries of strains btn1∆ and btn1D363G. (A) Biplot of colony size difference between btn1∆ vs ade6∆ on the x-axis and btn1D363G vs ade6∆ on the y-axis for each gene. Clustering of genes along the diagonal line highlights the similarities between the strains. Upper right quadrant represents shared positive interactions, lower left quadrant represents shared negative interactions. (B) Biplot of colony size difference between btn1∆ vs ade6∆ on the x-axis and btn1D363G vs btn1∆ on the y-axis. Clustering of most genes around the centre is due to the similarities between the two strains. Upper left and lower right quadrants represent residual functionalities. Genes away from the centre/at the extremes represent biological differences between the two strains. Gene points are represented by their max adjusted p-value score, with a logarithmic colour distribution.
Figure 4
Figure 4
Comparative 2D-volcano plots for SGA queries of strains btn1102–208del against btn1∆ and btn1D363G. (A) Biplot of colony size difference between btn1∆ vs ade6∆ on the x-axis and btn1102–208del vs ade6∆ on the y-axis. Upper right quadrant represents shared positive interactions, lower left quadrant represents shared negative interactions. (B) Biplot of colony size difference between btn1∆ vs ade6∆ on the x-axis and btn1102–208del vs btn1∆ on the y-axis. Upper left and lower right quadrants represent residual functionalities in btn1102–208del relative to btn1∆. Upper right and lower left quadrants represent gain of functionalities in btn1102–208del relative to btn1∆. (C) Biplot of colony size difference between btn1D363G vs ade6∆ on the x-axis and btn1102–208del vs ade6∆ on the y-axis. Upper right quadrant represents shared positive interactions, lower left quadrant represents shared negative interactions. (D) Biplot of colony size difference between btn1D363G vs ade6∆ on the x-axis and btn1102–208del vs btn1D363G on the y-axis. Upper left and lower right quadrants represent residual functionalities in btn1102–208del relative to btn1D363G. Upper right and lower left quadrants represent gain of function relative to btn1D363G. Gene points are represented by their max adjusted p-value score and a logarithmic colour distribution with significant genes annotated. For (A) and (C) genes away from the diagonal represent biological differences between the two strains. For (B) and (D) genes away from the centre/at the extremes represent biological differences between the two strains.
Figure 5
Figure 5
Gain of function interactions unique to btn1102–208del. This 2D volcano plot of colony size difference between btn1∆ and ade6∆ (x-axis) against colony size difference between btn1∆ vs btn1102–208del (y-axis) highlights the gain of function interactions unique to btn1102–208del. Points highlighted around the vertical axis represent genes that had no colony difference between btn1∆ vs ade6∆ control, however were significant for btn1102–208del. For simplicity any interaction that did not meet the criteria for unique gain of function was changed to 1 adjusted p-value (yellow). Gene points are represented by their adjusted p-value score for interaction btn1102–208del vs ade6∆, colour represented by a logarithmic colour distribution with significant genes (adjusted p-value < 1e−3) annotated. Full list of genetic interactions in S10: supplementary tables.
Figure 6
Figure 6
Genetic network of unique btn1102–208del interactions. (A) Genetic networks of unique btn1102–208del interactions represented in terms of positive (green) and negative (red) interacting gene and their corresponding biological processes GO term. Grey GO terms represent terms with both interactions, with a scale of red or green dependent on the number of interactions of either side associated with that specific GO term. (B) Genetic networks of unique btn1102–208del interactions represented in terms of positive (green) and negative (red) interacting gene and their corresponding KEGG GO term. Kappa score = 0.4. Generate using ClueGO.

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