Complementary vertebrate Wac models exhibit phenotypes relevant to DeSanto-Shinawi Syndrome
- Department of Biology, Chungnam National University, Republic of Korea
- Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, United States
- Department of Molecular Pathology, New York University College of Dentistry, United States
- Department of Psychiatry and Behavioral Sciences, University of California Davis, United States
- Department of Neurobiology, Physiology and Behavior, University of California Davis, United States
- Department of Physiology, Michigan State University, United States
- Neuroscience Program, Michigan State University, United States
- Corewell Health, United States
- Division of Genetics and Genomic Medicine, Department of Pediatrics, Washington University School of Medicine, United States
- Director, Preclinical and Translational Imaging Center, School of Medicine, University of California Irvine, United States
Figures
Figure 1 with 1 supplement
Generation and validation of murine Wac and zebrafish waca mutants.
(A) Floxed mouse Wac genetic locus and genotyping primers. Genotyping primers (magenta lines) reside external to the loxP sites (orange triangles) flanking exon 5 and nearby introns. (B) The recombined DNA band was Sanger-sequenced and results show the start codon (blue) and the novel stop codon introduced by the frameshift mutation resulting from Cre-mediated deletion of the locus used to generate the locus. (C) Example images of adult wild-type (WT) and Wac Het mice. (D) DNA gel of genotyping for WT and recombined (Het) Wac alleles; upper band is ~1.5 kilobases and the lower band is ~500 basepairs. (E) waca KO zebrafish were generated by CRISPR/Cas9. KO target sites for sgRNAs are indicated by red arrowheads and predicted protein structures for KO mutations are indicated below the panel. (F) WT and waca KO zebrafish have grossly normal sizes but waca KO exhibited a shortened jaw (black triangle) at 5 dpf. (G) Example waca genotyping results showing a 116 bp for WT (upper band) and a 111 (lower band) bp for KO. Abbreviations: (aa) amino acid; (bp) base pair; (Chr) chromosome; (dpf) days post fertilization; (WT) wild-type. Scale bars (C) = 1 cm and (F) = 200 µm.
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Figure 1—source data 1
All genotyping.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig1-data1-v1.zip
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Figure 1—source data 2
Mouse Wac genotyping.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig1-data2-v1.zip
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Figure 1—source data 3
Zebrafish genotype.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig1-data3-v1.zip
Figure 1—figure supplement 1
waca and wacb mRNA expression and generation of other zebrafish wac models.
(A) Whole-mount in situ hybridization analysis of waca and wacb mRNA in zebrafish at 72 hr post fertilization. wacb KO zebrafish were generated by CRISPR/Cas9; double KOs were generated by intercrossing waca and wacb zebrafish. KO target site for sgRNA is indicated on the gene structure (arrowhead). Predicted protein structures show premature truncation caused by frameshift deletion mutations (–20 bp for wacb KO). (B) Morphological characterization of wacb KO and waca,b dKOs at 5 dpf. wacb KO zebrafish showed no obvious phenotypes at any stage analyzed, while waca,wacb dKO showed heart edema, dark yolk, and swimbladder defect in addition to shortened jaw. waca,wacb dKO zebrafish did not survive over 7 dpf. Scale bar in both (A) and (B) = 200 µm.
Figure 2
Craniofacial changes in mutants.
(A, B) Dorsal views of the P0 calvaria stained with Alizarin red for bone. (A’, B’) The suture and fontanel areas in (A) and (B) are pseudo-colored green (anterior) and orange (posterior). (C, D) Dorsal views of P30 wild-type (WT) and Wac Het skull bones; arrow (D) points to a gap in the interfrontal suture. (F) Frontal bone and (P) Parietal bone; white dashed lines are widths measured in (G, H). (E, F) Quantification of P0 fontanel suture areas pseudo colored in A’ and B’; WT n=4, Het n=5. Quantification of the skull width across the frontal (G) and parietal (H) bones at P30 in WTs and Wac Hets; WT n=4, Het n=4. (I, J) The waca KO zebrafish show a shortened jaw structure, compared to WT. Lines denote lower jaw length. (K) Quantification of lower jaw length; n=9 WT and 4 waca KO. (L, M) Cartilage staining of zebrafish using alcian blue, ventral view. (N) Measurements of the width of Meckel’s cartilage in waca KO zebrafish at 13 dpf (red line); WT n=10 and KO n=8. Data are expressed as the mean ± SEM. *p<0.05, **p<0.01, and ***p<0.001. Scale bars (B’) = 1 mm, (D) = 4 mm, (J, M) = 200 µm.
Figure 3 with 1 supplement
Behavioral changes in mutants.
(A) Six to eight week WT and Wac Het mice were tested in the 3-chamber social/novel object test, wild-type (WT) n=20 and Het n=20, and the Y-maze (B), n=13 for both groups, with Wac Hets showing deficits in the Y-maze. (C, D) Example heat maps during the 17–19 min timeframe of the social cohesion test in zebrafish; waca KO males and females were more dispersed (E, F), see methods for test details. (G, H) Distance moved was greater in male waca KOs in the novel tank assay, n=11, both groups, compared to females, n=12 (WT) and 11 (KOs). Data are expressed as the mean ± SEM; *p<0.05, **p<0.01, and ***p<0.001.
Figure 3—figure supplement 1
Ambulatory, spatial memory and anxiety-relevant tests in Wac Het mice.
Adult wild-type (WT) and Wac Het mice were assessed for ambulatory activity in the open field test (A); no changes were found in time spent in the center (top left graph) or periphery (top right graph), as well as total distance travelled (bottom left graph) between groups. (B) The elevated plus maze was used to determine whether mice preferred a safe enclosed arm or open arms. While not significant, Wac Het mice trended towards preference for the open arms. (C) A spatial memory test was used to test if Wac Hets had deficits in spatial memory but as a group no significant differences were observed. Data are expressed as the mean ± SEM, n=13 for both groups for open field periphery measurements, n=17 (WT) and 15 (Het) for total distance. For the radial arm water maze, n=22 WTs and 18 Hets for the elevated plus maze.
Figure 4 with 2 supplements
Murine Wac depletion leads to elevated seizure susceptibility and loss of GABAergic markers.
(A) Schema depicting the seizure induction by pentylenetetrazol (PTZ); mouse image was made by BioRender software. Briefly, P30 mice were administered PTZ intraperitoneally and then assessed for the highest seizure severity score over the course of 20 min. The maximum seizure severity score over 20 min was quantified (B); n=16 (wild-types, WTs) and 22 (Hets). (C) Table of cortical interneuron cell counts in the somatosensory cortex at P30. (D–F) Immunofluorescent images of parvalbumin (PV) and cell density quantification in mice somatosensory cortex, n=3, both groups. (G–I) Immunofluorescent images of LHX6 and cell density quantification in mice somatosensory cortex, n=3, both groups. Data are expressed as the mean ± SEM, *p<0.05 and ****p<0.0001. Scale bar = 100 µm (H).
Figure 4—figure supplement 1
Expression pattern of parvalbumin genes (pvalb1, pvalb4, pvalb6, and pvalb7) in waca KO zebrafish at 7 dpf.
(A–D) Lateral view in left panels; dorsal view in right panels; anterior is to the left. No significant change was detected between wild-type (WT) and waca KO. n=7 for WT and n=6 for waca KO for pvalb1. n=5 for WT and n=9 for waca KO for pvalb4. n=4 for WT and n=6 for waca KO for pvalb6. n=7 for WT and n=3 for waca KO for pvalb7. Scale bar in (D) = 200 µm.
Figure 4—figure supplement 2
Expression pattern of the gad1b gene in waca KO zebrafish at 7 dpf.
(A–D) Dorsal view of gad1b labeling, with anterior to the left (in left panels) and to the top (in right panels). Boxes highlight forebrain regions. Brackets demarcate area of gad1b expression. (E) Quantification of the gad1b area of expression between wild-type (WT) and waca KOs. Data are expressed as the mean ± SEM, n=8 for WT and n=4 for waca KO, ****p<0.0001. Scale bars in (C) = 200 µm, (D) = 200 µm.
Figure 5 with 2 supplements
Cell counts and biochemical assessment of key markers in P30 Wac wild-type (WT) and Het mice.
(A) WT and Wac Het somatosensory cortices were counted for major cell classes, including markers of neurons and glia, n=3 each group, parentheses indicate standard error of the mean. (B) P30 somatosensory cortex tissue was probed via western blotting for WAC and proteins known to be altered in other models or of brain markers. (C) Quantification of protein bands were normalized to GAPDH or the total protein if a phosphorylated protein. Data are expressed as the mean ± SEM, n=4 all groups. *p<0.05 and **p<0.01. Abbreviations: (WB) Western blot and (kDa) kiloDaltons.
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Figure 5—source data 1
Compiled western blots.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data1-v1.pdf
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Figure 5—source data 2
Wac western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data2-v1.zip
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Figure 5—source data 3
beta-catenin western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data3-v1.zip
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Figure 5—source data 4
ERK western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data4-v1.zip
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Figure 5—source data 5
GAD65/67 western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data5-v1.zip
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Figure 5—source data 6
GAPDH western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data6-v1.zip
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Figure 5—source data 7
Gephyrin western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data7-v1.zip
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Figure 5—source data 8
mono-ubiquitin H2B western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data8-v1.zip
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Figure 5—source data 9
phospho-ERK western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data9-v1.zip
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Figure 5—source data 10
phospho-S6 western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data10-v1.zip
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Figure 5—source data 11
PSD95 western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data11-v1.zip
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Figure 5—source data 12
S6 western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig5-data12-v1.zip
Figure 5—figure supplement 1
Example images of cortical glial and neuron cell types in wild-type (WT) and Wac Het brains.
Scale bar in F’=100 µm.
Figure 5—figure supplement 2
Normal pyramidal neuron lamination, intrinsic membrane properties, and spontaneous excitatory postsynaptic currents in the somatosensory cortex.
Representative images of pyramidal neuron laminar markers in somatosensory cortices of P30 wild-type (WT) and Wac Hets for SATB2 (A, B), CTIP2 (C, D) and TBR1 (E, F); scale bar in F=100 µm. Quantification of the % marker per layer for SATB2 (G) n=3, CTIP2 (H) n=3 and TBR1 (I) n=4. (J) Example voltage responses of wild-type (WT) and Wac Het L2/3 excitatory neurons to injected current. (K) Frequency-intensity (FI) plots showing the first interspike interval (ISI) frequency for WT and Wac Het L2/3 excitatory neurons following positive current steps (0–1000 pA; 100 pA steps; 1000 ms duration) (WT: 16 cells from 3 mice; Wac Het: 16 cells from 3 mice). Bar graphs show that the mean linear slopes (gain) of the FI curves were not significantly different (WT: 0.35±0.03 Hz/pA; Wac Het: 0.38±0.04 Hz/pA; p=0.54, two-sample t-test). (L) Three representative traces of spontaneous excitatory postsynaptic currents (sEPSCs: 5 s per trace) recorded in voltage-clamp (–94 mV) from a WT and Wac Het L2/3 excitatory neuron. (M) Cumulative probability plots of sEPSC amplitudes (left) and interevent intervals (right) from WT and Wac Het L2/3 excitatory neurons (WT: 540 events, 12 cells from 3 mice; Wac Het: 450 events, 10 cells from 3 mice). Bar graphs show no significant differences in the mean sEPSC amplitude (left) or frequency when averaged from individual L2/3 excitatory neurons (WT amplitude: 22.7±1.3 pA; Wac Het amplitude: 22.8±1.2 pA, P=0.94, two-sample t-test) (WT frequency: 36.4±3.1 Hz; Wac Het: 41.3±2.1 Hz, p=0.23, two-sample t-test). Values are represented as mean ± SEM, *p<0.05.
Figure 6 with 2 supplements
Magnetic resonance imaging (MRI) reveals increased brain volume in mouse Wac Het males.
(A) There was a significant increase in whole brain volumes in HET compared to wild-type (WT) male mice. Female HET mice did not exhibit significant changes in brain volume relative to WT. Data are expressed as the mean ± SEM; males and female WTs n=7 and female Hets n=6, *p<0.05. (B) %change between cortical regions were larger in most HET mice areas, with distinct regions different between sexes. (Bolded bars indicate significant differences p<0.05, t-test).
Figure 6—figure supplement 1
Volumetric limbic regional changes.
(A) Percent change in limbic regions were modestly larger in HET mice than wild-type (WT). In males, the ventral CA3 and dorsal CA1 were larger but not in females. Female HET mice had smaller amygdala than WT female mice. (Bolded bars indicate significant differences P<0.05, t-test). (B) Ventral CA3 was significantly larger in HET male mice but not in female mice. Similarly, the dorsal CA1 also exhibited increased volumes in males HET mice but not WT nor in females. Data are expressed as the mean ± SEM; all groups n=6–7, (*p<0.05; ***p<0.001).
Figure 6—figure supplement 2
Volumetric changes in white matter.
(A) Percent change in white matter regions were larger in male HET mice compared to wild-type (WT), particularly the dorsal fornix. In females, only the lateral olfactory tract was larger in HET mice than WT. (Bolded bars indicate significant differences p<0.05, t-test). (B) There was an increase in dorsal fornix volume in male but not female HET mice compared to WT. There was a robust increase in corpus callosum volume in HET male mice compared to WT but not in female HET mice. Data are expressed as the mean ± SEM; all groups n=6–7, (*p<0.05; **p<0.01).
Figure 7 with 5 supplements
Transcriptomic characterization by bulk RNA-seq identified shared and divergent transcriptomic dysregulation in males and females.
(A) Number of differential expression (DE) genes identified in the surrogate variable analysis (SVA)-corrected DE analysis and sex-stratified DE analysis. Results are separated by effect direction and significance (p-value <0.05 or FDR <0.1). (B) Volcano plot representing DE of the SVA-corrected DE model in (A). (C) Selected GO analysis of significantly (p<0.05) up and down-regulated genes in the SVA-corrected DE analysis. Results are colored by significance, the size indicated the number of DE genes belonging to that term and are ordered by the ratio of enrichment. (D) Examples of significant DE genes identified in the SVA-corrected model Fggy (FDR = 0.014) and Pcdhga11 (FDR = 0.026), stratified by sex. (E) Curated GO terms from stratified GSEA analysis. Colors indicate significance, size the number of genes in the enriched term, and the enrichment score (NES). (F, G) Volcano plots showing the differential expression of autism spectrum disorder (ASD) genes in males and females. (H) Examples of DE genes which show increased effect size and significance in males, Prr12 (females, p=0.99; males, p=0.0006) and Chd2 (females, p=0.39; males, p=0.0008). (I) Western blots showing PCDH-alpha abundance in wild-types (WT)s and Hets. (J) WB quantification comparing PCDH-alpha normalized to alpha-tubulin expression in P30 somatosensory cortices; n=4 for both genotypes and **p<0.01. Data are expressed as the mean ± SEM.
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Figure 7—source data 1
Compiled western blots.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig7-data1-v1.pdf
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Figure 7—source data 2
PCDH alpha western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig7-data2-v1.zip
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Figure 7—source data 3
alpha tubulin western blot.
- https://cdn.elifesciences.org/articles/109104/elife-109104-fig7-data3-v1.zip
Figure 7—figure supplement 1
Sample sequencing depth.
Values represent millions of aligned reads.
Figure 7—figure supplement 2
Principal component analysis (PCA) dimensionality reduction.
(A) PCA sample clustering showing first eight PCs. Samples are colored by genotype, sex, sequencing depth, and Xist gene counts. PCA did not identify sample outliers. (B) Scree plot showing the amount of variance explained by the first five PCs. (C) Correlation heatmaps of PCs 1–10 vs genotype, sex, and sequencing depth. Values in the heatmap represent correlation p-values. The intensity of the heatmap colors represents the Pearson correlation coefficient (r).
Figure 7—figure supplement 3
Validation of Wac heterozygosity in RNA-seq data.
(A) Wac Het samples demonstrate subtle upregulation of Wac transcript, when reads are counted across the entire gene length (log2FC = 0.09, p=0.01, FDR = 0.5) suggesting compensatory upregulation of Wac transcription. (B) Sashimi plots representing Wac read coverage. Dashed rectangle marks floxed exon 5, which coverage is reduced by ~50% in Het samples when compared to surrounding exons.
Figure 7—figure supplement 4
Gene set enrichment and sex concordance in P2 Wac mouse forebrain bulk RNA-seq.
(A) Curated gene ontology (GO) terms from surrogate variable analysis (SVA)-corrected gene set enrichment analysis (GSEA) analysis. Colors indicate significance, size, the number of genes in the enriched term, and the enrichment score (NES). (B) Custom GSEA test looking for enrichment of disease-associated genes in the SVA-corrected DE model. Colors indicate significance, size the number of genes in the enriched term, and the enrichment score (NES). (C) Correlation of the effected sizes (log fold change) of DEGs (p-value <0.05) identified in the SVA-corrected DE analysis between males and females. Line indicated a best-fit linear regression with a 95% confidence interval. (D) Correlation of the effected sizes (log fold change) of DE genes (p-value <0.05) identified in the female DE analysis between males and females. Line indicated a best-fit linear regression with a 95% confidence interval. (E) Correlation of the effected sizes (log fold change) of DE genes (p-value <0.05) identified in the male DE analysis between males and females. Line indicated a best-fit linear regression with a 95% confidence interval.
Figure 7—figure supplement 5
Summary of differential splicing (DS) analysis.
(A) Visualizations of DS events, including splice graphs. Values in the ΔΨ per junction column represent percentage of the expected differential inclusion for matching edges in the splice graph. (B) DS event classification. DS events indicated in gray result from transgenesis.
Tables
Table 1
Electrophysiological properties of L2/3 excitatory cells in the somatosensory cortex of wild-type (WT) and Wac Het mice.
| L2/3 excitatory neurons | P | Test | ||||||
|---|---|---|---|---|---|---|---|---|
| WT | Wac Het | |||||||
| Mean±s.e.m. | Mean±s.e.m. | |||||||
| RMP (mV) | –86.8 | ± | 0.7 | –87.1 | ± | 0.6 | 0.747 | t-test |
| Rm (MΩ) | 66.8 | ± | 3.6 | 59.2 | ± | 3.7 | 0.151 | t-test |
| τm (ms) | 12.9 | ± | 0.5 | 12.3 | ± | 0.4 | 0.209 | t-test |
| Cm (pF) | 197.4 | ± | 8.8 | 218.3 | ± | 13.7 | 0.720 | MW U-test |
| Rheobase (pA) | 170.0 | ± | 11.0 | 167.2 | ± | 12.6 | 0.868 | t-test |
| Spike threshold (mV) | –49.9 | ± | 0.6 | –50.1 | ± | 0.5 | 0.788 | t-test |
| Spike amplitude (mV) | 85.0 | ± | 1.5 | 88.1 | ± | 1.4 | 0.142 | t-test |
| Spike half-width (mV) | 0.63 | ± | 0.02 | 0.61 | ± | 0.02 | 0.323 | t-test |
| AHP amplitude (mV) | –18.3 | ± | 0.6 | –18.5 | ± | 0.5 | 0.794 | t-test |
| Max rate of rise (mV*ms) | 526.0 | ± | 28.8 | 586.3 | ± | 22.4 | 0.109 | t-test |
| Max rate of decay (mV*ms) | –112.8 | ± | 4.2 | –120.0 | ± | 4.5 | 0.243 | t-test |
| n=16 cells, 3 mice | n=16 cells, 3 mice | |||||||
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*See methods for an explanation of how the electrophysiological parameters were defined/measured. All membrane potentials were corrected for a 14 mV liquid junction potential. All statistical tests were two-tailed.
Table 2
General parameters of the bulk-seq brains analyzed.
Columns indicate sample ID (ID), Genotype (WT = wild-type, HET = Wac mutant), Sex (M = Male, F = Female), RNA concentration, RNA integrity number (RIN) from bioanalyzer, A230/A260 value from spectrometer, number of aligned reads, and full sample metadata identifier (ID + Genotype + Sex).
| Antibody (host/dilution) | Company | Category # | Clone | Lot # |
|---|---|---|---|---|
| Alpha-tubulin (mouse, 1:3000) | Abcam | ab7291 | DM1-A | GR310199-6 |
| Beta-catenin (rabbit, 1:4000) | Abcam | ab32572 | E247 | GR184212-83 |
| CTIP2 (rat, 1:500) | Abcam | Ab18465 | n/a | n/a |
| ERK1/2 (rabbit, 1:4000) | Cell Signaling | 4695 | 137F5 | 35 |
| pERK1/2Thr202/Tyr204;Thr185/Tyr187 (rabbit, 1:4000) | Cell Signaling | 9101 | n/a | 31 |
| GAD65/67 (rabbit, 1:4000) | Sigma-Aldrich | G5163 | n/a | n/a |
| GAPDH (rabbit, 1:4000) | Cell Signaling | 2118 | 14C10 | 14 |
| Gephyrin (rabbit, 1:4000) | Cell Signaling | 43928 | D4J4R | 1 |
| IBA1 (goat, 1:500) | Cell Signaling | 17198 | E404W | 1 |
| LHX6 (mouse, 1:200) | Santa Cruz Bio. | sc-271433 | A-9 | B2219 |
| NeuN (mouse, 1:400) | Millipore | MAB377 | A60 | 3061189 |
| OLIG2 (rabbit, 1:400) | Millipore | AB9610 | n/a | 3071572 |
| Parvalbumin (rabbit, 1:400) | Swant | PV27 | n/a | 2014 |
| Protocadherin alpha (rabbit, 1:3000) | SySy | 190 003 | n/a | 1–8 |
| PROX1 (rabbit, 1:500) | Abcam | ab199359 | EPR19273 | GR3216583 |
| PSD95 (rabbit, 1:4000) | Cell Signaling | 3450 | D27E11 | 4 |
| S100beta (rabbit, 1:500) | Proteintech | 15146–1-AP | n/a | 00092556 |
| S6 (rabbit, 1:4000) | Cell Signaling | 2217 | 5G10 | 7 |
| pS6Ser240/244 (rabbit, 1:4000) | Cell Signaling | 5364 | D68F8 | 8 |
| SATB2 (mouse, 1:500) | Abcam | Ab51502 | n/a | n/a |
| Somatostatin (rat, 1:200) | Millipore | MAB354 | YC7 | 2219310 A |
| TBR1 (rabbit, 1:500) | Abcam | Ab31940 | n/a | n/a |
| mUb-H2B (rabbit, 1:4000) | Cell Signaling | 5546 | D11 | 6 |
| WAC (rabbit, 1:4000) | Abcam | ab109486 | n/a | GR175251-1 |
Additional files
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Supplementary file 1
Surrogate variable analysis (SVA)-corrected differential expression analysis of Wac mutants; combined sexes.
Genes are annotated by common name and ENSEMBL gene ID. Effect size indicated by log fold change (logFC) and relative expression by log counts per million (logCPM). Significance is reported by a likelihood ratio test statistic (LR), standard p-value (p-value), and the multiple-testing corrected p-value or false discovery rate (FDR).
- https://cdn.elifesciences.org/articles/109104/elife-109104-supp1-v1.xlsx
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Supplementary file 2
Differential expression analysis of Wac mutants, using only male samples.
Genes are annotated by both common name and ENSEMBL gene ID. Effect size is indicated by log fold change (logFC) and relative expression by log counts per million (logCPM). Significance is reported by a likelihood ratio test statistic (LR), standard p-value (p-value), and the multiple-testing corrected p-value or false discovery rate (FDR).
- https://cdn.elifesciences.org/articles/109104/elife-109104-supp2-v1.xlsx
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Supplementary file 3
Differential expression analysis of Wac mutants, using only female samples.
Genes are annotated by both common name and ENSEMBL gene ID. Effect size is indicated by log fold change (logFC) and relative expression by log counts per million (logCPM). Significance is reported by a likelihood ratio test statistic (LR), standard p-value (PValue), and the multiple-testing corrected p-value or false discovery rate (FDR).
- https://cdn.elifesciences.org/articles/109104/elife-109104-supp3-v1.xlsx
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Supplementary file 4
Gene Ontology (GO) over representation analysis of differential expression results for different models (Full, Male, Female).
Ontology indicates the source of the term set (MF = Molecular Function, BP = Biological Processes, CC = Cellular Component). GO terms are identified by GO:ID number and term description. GeneRatio is the ratio of differentially expressed genes associated with the term to the total number of differentially expressed genes tested. BgRatio is the ratio of background genes associated with the term to the total number of genes in the background set. RichFactor is the ratio of observed differentially expressed genes to expected genes for the term (GeneRatio ÷ BgRatio). Fold enrichment of differentially expressed genes for the term relative to background expectation. The zscore, or standardized enrichment score reflecting deviation from expected overlap. Significance is indicated by the p-value from a Fisher’s exact test, multiple-testing–corrected p-value (p.adjust) using a Benjamini–Hochberg false discovery rate correction, and false discovery rate (q-value). geneID shows the differentially expressed genes in the GO term. DE indicates direction of differential expression in the model. Model indicates the differential expression model (Full = SVA-corrected full dataset, Male = male-only differential expression, Female = female-only differential expression).
- https://cdn.elifesciences.org/articles/109104/elife-109104-supp4-v1.csv
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Supplementary file 5
Gene ontology (GO) enrichment analysis using fgsea of differential expression results for different models (Full, Male, Female).
Ontology indicates the source of the term set (MF = Molecular Function, BP = Biological Processes, CC = Cellular Component). GO terms are identified by GO:ID number and term description. SetSize indicates the number of genes from the gene set that are present in the ranked gene list used for GSEA. EnrichmentScore is the raw gene set enrichment score reflecting the degree to which genes in the set are overrepresented at the top or bottom of the ranked list. The normalized enrichment score (NES), which accounts for gene set size and allows comparison across gene sets. Significance is indicated by the p-value from a Fisher’s exact test, multiple-testing–corrected p-value (p.adjust) using a Benjamini–Hochberg false discovery rate correction, and false discovery rate (q value). Rank position at which the maximum enrichment score is observed in the ranked gene list. The leading edge is the subset of genes from the gene set that contribute most strongly to the enrichment signal. Core enrichment, an indicator or list specifying genes belonging to the leading-edge subset. Model indicates the differential expression model (Full = SVA-corrected full dataset, Male = male-only differential expression, Female = female-only differential expression).
- https://cdn.elifesciences.org/articles/109104/elife-109104-supp5-v1.csv
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Supplementary file 6
MAJIQ-based alternative splicing analysis of Wac mutants.
Local splicing variations (LSVs) are reported per gene and junction. Genes are annotated by Ensembl gene ID. Splicing effect size is quantified as the mean change in percent spliced in (mean ΔPSI) per LSV junction. Statistical confidence is reported as the posterior probability that the splicing event is changing (probability_changing) or non-changing (probability_non_changing). Mean PSI values are provided separately for wild-type (WT_mean_psi) and heterozygous mutants (Het_mean_psi). Additional structural annotations include the number of junctions and exons per LSV, genomic coordinates of junctions, and intron retention coordinates where applicable.
- https://cdn.elifesciences.org/articles/109104/elife-109104-supp6-v1.tsv
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MDAR checklist
- https://cdn.elifesciences.org/articles/109104/elife-109104-mdarchecklist1-v1.pdf
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Complementary vertebrate Wac models exhibit phenotypes relevant to DeSanto-Shinawi Syndrome
eLife 14:RP109104.
https://doi.org/10.7554/eLife.109104.3
