bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Transcriptomic changes resulting from

2 STK$%B overexpression identifies pathways

3 potentially relevant to essential tremor

4 Calwing Liao.,0, Faezeh Sarayloo.,0, Veikko Vuokila0, Daniel Rochefort0, Fulya Akçimen.,0, 5 Simone Diamond0, Alexandre D. Laporte0, Dan Spiegelman0, Qin HeJ, Hélène Catoire0, Patrick A. 6 Dion0,O, Guy A. Rouleau.,0,O 7 8 .Department of Human Genetics, McGill University, Montréal, Quebec, Canada 9 0Montreal Neurological Institute, McGill University, Montréal, Quebec, Canada. 10 JDepartment of Biomedical Sciences, Université de Montréal, Montréal, Quebec, Canada 11 ODepartment of Neurology and Neurosurgery, McGill University, Montréal, Quebec, Canada 12 13 Corresponding Author: 14 Dr. Guy A. Rouleau, MD, PhD, FRCPC, OQ 15 Department of Neurology and Neurosurgery 16 McGill University 17 Montréal, Québec, Canada 18 HJA 0BO 19 E-mail: [email protected] 20 21 Keywords: STK$%B, essential tremor, transcriptome, FUS 22 Conflict of Interests: All authors report no conflict of interests. 23 Funding Sources: Canadian Institutes of Health Research 24 bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

25 Abstract 26 Essential tremor (ET) is a common movement disorder that has a high heritability. A number of 27 genetic studies have associated different and loci with ET, but few have investigated the 28 biology of any of these genes. STK$%B was significantly associated with ET in a large GWAS 29 study and was found to be overexpressed in ET cerebellar tissue. Here, we overexpressed STK$%B 30 in human cerebellar DAOY cells and used an RNA-Seq approach to identify differentially 31 expressed genes by comparing the transcriptome profile of these cells to the one of control DAOY 32 cells. Pathway and ontology enrichment identified axon guidance, olfactory signalling and 33 calcium-voltage channels as significant. Additionally, we show that overexpressing STK$%B 34 affects transcript levels of previously implicated ET genes such as FUS. Our results investigate the 35 effects of overexpressed STK$%B and suggest that it may be involved in relevant ET pathways and 36 genes.

37 Introduc;on 38 Essential tremor (ET) is one of the most common movement disorders and is typically 39 characterized by a kinetic tremor in the hand or arms.. The severity tends to increase with age and 40 may involve different regions such as the head, voice or jaw0. Previous studies investigating the 41 histology of post-mortem tissue have pinpointed the cerebellum as a region of interest for ETJ. 42 Specifically, abnormalities with Purkinje cell axons and dendrites were found in ET brainsO. 43 Furthermore, the olivocerebellar circuitry has been implicated in ET pathology and many calcium 44 voltage channels are highly expressed in this circuitryj. 45 46 The genetic etiology of ET has remained largely elusive and most studies have focused on common 47 or rare variants and on looking for genetic overlap with other disordersk,l. Twin studies have shown 48 that ET has a concordance of km–mJ% in monozygotic twins and 0l–0m% in dizygotic twins, which 49 suggests that both genetic and environmental factors drive the onset and development of this 50 complex traitp. A recent genome-wide association study (GWAS) identified a significant in 51 STK$%B and found that ET patients overexpressed STK$%B in cerebellar tissue by comparison to 52 healthy controlsm. STK$%B is transcribed and translated into YANK0, a serine/threonine kinase, 53 that has not been well characterized. There have been several exome-wide studies that implicated 54 different genetic variants as causes of ET. The first ET-implicated gene found through exome bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

55 sequencing was the Fused in Sarcoma gene (FUS).s. However, it is unclear whether these “ET” 56 genes interact or have indirect effects on the expression of each other. 57 58 To understand the effects of overexpressed STK$%B and identify pathways potentially relevant to 59 ET, we overexpressed the gene in human cerebellar DAOY cells and compared transcriptomic 60 changes in overexpressed cells and empty-vector controls using RNA sequencing. Several 61 interesting pathways such as axon guidance, calcium ion transmembrane transport, and olfactory 62 transduction were significantly enriched after overexpression of STK$%B. We also identified 63 previously implicated ET genes whose expression is dysregulated through the overexpression of 64 STK$%B, suggesting that overexpressed STK$%B may have relevant downstream effects.

65 Results

66 Confirming overexpression of STK$%B 67 The STK$%B stable cell lines had higher RNA expression of STK$%B, which was detected by 68 reverse-transcriptase qPCR (Supplementary Figure .). RNA sequencing (RNA-Seq) also 69 confirmed that the stable cell lines have higher STK$%B RNA levels compared to controls; it was 70 the most significantly differentially expressed gene (Figure .–0). 71 72 Quality control of RNA-seq analyses 73 The QQ-plot for differentially expressed genes did not show stratification or potential biases 74 (Supplementary Figure 0). The M-A plot showed slight enrichment of downregulated genes 75 compared to upregulated genes (Supplementary Figure J). A principal component (PC) plot 76 showed separation of controls and STK$%B cell lines (Figure J). Additionally, the dendrogram with 77 heatmap shows that controls and STK$%B cell lines are distinct (Supplementary Figure O). The 78 mean-variance plot shows the shrinkage under the sleuth model (Supplementary Figure j). 79 80 Overexpression of STK$%B affects expression of previously iden;fied ET-associated genes 81 A total of J,lmO genes were found to be differentially expressed with a q-value |s.j| and q-value

85 CACNA-A (q-value = s.sJs, β= s.ls0). Several pathways and gene ontologies were enriched for 86 differentially expressed genes (DEGs) due to the overexpression of STK$%B, including olfactory 87 transduction (P=0.kpE-Jm), axon guidance (P=m.jsE-Jk), and calcium ion transmembrane 88 transport (P=J.s0E-J.) (Table .). 89 90 Gene network analyses 91 To identify which cluster of DEGs drove the significant pathways, gene network analysis based 92 on co-expression was done and identified .O different clusters within the differentially expressed 93 genes (Figure O). Analysis of the top J largest clusters for pathway enrichments found several 94 similar pathways in different databases including axon guidance (P<..smE-.0) in cluster . and 95 calcium signalling pathway in cluster J (0.skE-sj).

96 Discussion 97 The genetic etiology of ET is complex and likely explained by a combination of copy number 98 variants, rare variants, gene-gene interactions and common variant drivers. One of the most 99 significantly enriched pathways we identified was olfactory signalling and transduction. Previous 100 reports have shown conflicting evidence about olfactory loss in ET..,.0. It may be that ET patients 101 with dysregulated olfactory signalling have overexpressed STK$%B, which may contribute to the 102 subset of ET patients with olfactory loss. 103 104 Interestingly, FUS was amongst the genes dysregulated when STK$%B was overexpressed. In the 105 exome familial ET study that identified FUS, we also found reduced mRNA levels for FUS.s. 106 Similarly, the expression of FUS is lower when STK$%B is overexpressed, suggesting an indirect 107 relationship between the two genes. Additionally, two calcium voltage-gated channel genes, 108 CACNA-C AND CACNA-A were overexpressed. Enrichment analyses found calcium ion 109 transmembrane transport to be highly significant in GO Processes, suggesting that STK$%B may 110 play a role upstream of these genes. In the olivocerebellar circuitry, a system implicated in ET, is 111 enriched for both of these genesj,.J. CACNA-A has been shown to be predominantly expressed in 112 Purkinje cells, a cell type relevant to ET.J. Another enriched pathway was axon guidance. A 113 previous study identified the TENM0 missense variant segregating within ET families.O. TENM0 114 is a regulator of axon guidance and myelination and TenmO knockout mice showed an ET bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

115 phenotype, suggesting that axon guidance is important in ET.O. Similarly, overexpressed STK$%B 116 could be dysregulating important pathways relevant to axon guidance. 117 118 By sub-stratifying the overexpressed genes into different clusters that are co-expressed, several 119 interesting pathways involving the cardiovascular system were identified. Pathways such as right 120 ventricular cardiomyopathy in KEGG and angiogenesis in GO Processes were found among the 121 top five significant pathways. In certain ET patients, beta-blockers can reduce tremor magnitude 122 and frequency. Beta-blockers lower blood pressure and are used to treat irregular heart rhythm and 123 other cardiovascular phenotypes. A common treatment for ET is propranolol, a beta-blocker. This 124 was a drug developed for cardiovascular health that affects adrenergic activity, but still reduces 125 tremor in ET individuals. This could suggest that overexpressed STK$%B affects cardiovascular 126 health, which may in turn affect or lead to the ET phenotype or that STK$%B may be pleiotropic 127 and affect the nervous and cardiovascular system differently. 128 129 Due to the heterogeneity of ET, it is likely that not all ET-affected individuals have overexpression 130 of STK$%B. One limitation of this study is that DAOY cells are a cancerous cell line and may not 131 be the ideal model for understanding the transcriptome of Purkinje cells. Future studies on the 132 effects of STK$%B overexpression could use induced pluripotent stem cells differentiated into cells 133 with Purkinje cells properties; such cells would likely have less biological noise. However, our 134 findings support the idea that STK$%B is a gene of interest for a subset of ET cases, and further 135 investigations into how STK$%B may interact with other genes are warranted. 136

137 Methods and Materials

138 Cell Culture and plasmid construc;on 139 The DAOY cell line was cultured in Eagle’s Minimum Essential Medium (EMEM) with .s% fetal

140 bovine serum (FBS) at Jl degrees Celsius and j% CO0 with the Glico Pen-Strep-Glutamine 141 cocktail at .X. Cells were passaged every 0 days at ps-ms% confluence and at the same time. The 142 cDNA of STK$%B (NM_ss.Jsksp0..) was inserted into a pcDNAJ..(+) vector containing a 143 neomycin resistance gene, used as a selectable marker. This transcript was picked because it had 144 the highest expression in the cerebellum based on GTEx jJ vl. The plasmid was transformed and bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

145 expanded in XL.s-Gold Escherichia coli strain (Stratagene). The cDNA of the plasmid was sent 146 for Sanger sequencing to validate the gene sequence. 147 148 Transfec;on and deriving stable cell lines 149 The cells were transfected for Op hours with p ug of DNA of using the jetPRIME transfection 150 reagent (Polyplus). A green fluorescent (GFP) control vector was used to assess and 151 estimate transfection efficiency and optimize transfection parameters. Stable cell lines were 152 established by subjecting transfected cells to GO.p antibiotic at . µg/mL for .s days and maintained 153 at a concentration of Oss µg/mL for an additional 0 weeks. A kill curve was done to determine the 154 ideal antibiotic selection. In parallel, empty pcDNAJ..(+) vector control lines were also grown and 155 underwent antibiotic selection. 156 157 RNA extrac;on and sequencing 158 RNA was extracted using the RNeasy Mini Kit (Qiagen). The RNA concentration was measured 159 using the Synergy HO microplate reader. RNA for the top three overexpressed STK$%B cell lines 160 and three empty-vector controls was sent to Macrogen Inc. for sequencing. RT-qPCR was used to 161 confirm overexpression in the cell liens compared to controls. Library preparation was done with 162 the TruSeq Stranded Total RNA Kit (Ilumina) with Ribo-Zero depletion. Sequencing was done on 163 the NovaSeq ksss at .jsbp paired-end reads with a total of 0ssM reads. Samples were randomized 164 for cell lysis, RNA extraction, Ribo-Zero depletion, library preparation and sequencing to account 165 for potential batch effects. 166 167 Data processing and quality control 168 The FASTQ files were pseudo-aligned with Salmon using the Ensembl vmO annotation of the 169 .k. The parameters of Salmon included the following: 0ss bootstraps with mapping 170 validation, GC bias correction, sequencing bias correction, O range factorization bins, a minimum 171 score fraction of s.mj and VB optimization with a VB prior of .e-j. The estimated counts were 172 analyzed using sleuth to identify DEGs.l. A likelihood ratio test was used to identify differentially

173 expressed transcripts and a Wald test was used to get β values (log0(x + s.j)). Additionally, 174 transcript-level aggregation was done against the Ensembl vmO annotation to determine gene-level 175 differential expression. P-values were corrected using the Benjamini-Hochberg procedure to bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

176 account for false discovery rate (FDR). Q-value (p-values corrected for FDR) significance was set 177 for an FDR of |s.j| were 182 analyzed for pathway enrichments. Network clustering was done by grouping genes based on co- 183 expression. Pathway analyses were also conducted with Gene Network 0.s for the clusters using a 184 Wilcoxin test. 185

186 Acknowledgments 187 This work was supported by a Canadian Institutes of Health Research Foundation Scheme grant 188 (#JJ0ml.). G.A.R. holds a Canada Research Chair in Genetics of the Nervous System and the 189 Wilder Penfield Chair in Neurosciences. C.L. is a recipient of the Frederick Banting and Charles 190 Best Canada Graduate Scholarship from the Canadian Institutes of Health Research (CIHR). 191

192 Data availability statement 193 Any data is available upon request of the corresponding author. 194 195 References 196 .. Elble, R. J. What is Essential Tremor? Curr. Neurol. Neurosci. Rep. :;, JjJ (0s.J). 197 0. Haubenberger, D. & Hallett, M. Essential Tremor. N. Engl. J. Med. ;=>, .ps0–.p.s (0s.p). 198 J. Gironell, A. The GABA Hypothesis in Essential Tremor: Lights and Shadows. Tremor 199 Other Hyperkinet. Mov. (N. Y). ?, 0jO (0s.O). 200 O. Axelrad, J. E. et al. Reduced Purkinje cell number in essential tremor: a postmortem 201 study. Arch. Neurol. @A, .s.–l (0ssp). 202 j. Schmouth, J.-F., Dion, P. A. & Rouleau, G. A. Genetics of essential tremor: From 203 phenotype to genes, insights from both human and mouse studies. Prog. Neurobiol. ::B– 204 :DE, .–.m (0s.O). 205 k. Liao, C. et al. Investigating the association and causal relationship between restless legs 206 syndrome and essential tremor. Parkinsonism Relat. Disord. (0s.p). 207 doi:.s..s.k/j.parkreldis.0s.p..s.s00 208 l. Houle, G. et al. transmembrane protein O is not a cause for essential tremor in a 209 Canadian population. Mov. Disord. ;D, 0m0–0mj (0s.l). 210 p. Tanner, C. M. et al. Essential tremor in twins: an assessment of genetic vs environmental 211 determinants of etiology. Neurology A=, .Jpm–m. (0ss.). bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

212 m. Müller, S. H. et al. Genome-wide association study in essential tremor identifies three new 213 loci. Brain :;B, J.kJ–J.km (0s.k). 214 .s. Merner, N. D. et al. Exome Sequencing Identifies FUS Mutations as a Cause of Essential 215 Tremor. Am. J. Hum. Genet. B:, J.J–J.m (0s.0). 216 ... Applegate, L. M. & Louis, E. D. Essential tremor: Mild olfactory dysfunction in a 217 cerebellar disorder. Parkinsonism Relat. Disord. ::, Jmm–Os0 (0ssj). 218 .0. Shah, M., Muhammed, N., Findley, L. J. & Hawkes, C. H. Olfactory tests in the diagnosis 219 of essential tremor. Parkinsonism Relat. Disord. :?, jkJ–jkp (0ssp). 220 .J. Mori, Y. et al. Primary structure and functional expression from complementary DNA of a 221 brain calcium channel. Nature ;AE, Jmp–Os0 (.mm.). 222 .O. Hor, H. et al. Missense mutations in TENMO , a regulator of axon guidance and central 223 myelination, cause essential tremor. Hum. Mol. Genet. D?, jkll–jkpk (0s.j). 224 .j. Kim, J., Basak, J. M. & Holtzman, D. M. The role of apolipoprotein E in Alzheimer’s 225 disease. @;, 0pl–JsJ (0ssm). 226 .k. Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast 227 and bias-aware quantification of transcript expression. Nat. Methods :?, O.l–O.m (0s.l). 228 .l. Pimentel, H., Bray, N. L., Puente, S., Melsted, P. & Pachter, L. Differential analysis of 229 RNA-seq incorporating quantification uncertainty. Nat. Methods :?, kpl–kms (0s.l). 230

231 Data availability statement 232 C.L. conducted the experiments, analyses and drafted the manuscript. F.S., V.V., D.R., F.A., S.D., 233 Q.H., H.C. helped with experiments. A.D.L. and D.S. helped with bioinformatic analyses. 234 P.A.D., G.A.R oversaw the study and drafting the manuscript.

235 Figures and Tables ENSG00000152953

7

condition 6 control tpm overexpression

5

4 control overexpression

S34574 S34575 S34576 S34571 S34572 S34573 236 sample 237 Figure :. The RNA expression of STK$%B overexpressed cells compared to empty-vector 238 controls based on RNA sequencing data. Error bars show the technical variance determined with 239 0ss bootstraps. Abbreviations: TPM, transcript per million. bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

250 STK32B

200

150 significant

FALSE TRUE log10(qval) 100 −

50

0

−8 −4 0 4 240 beta_value 241 Figure D. Volcano plot of comparing overexpressed STK$%B to empty-vector controls. 242 Differentially expressed genes (q < s.sj) are shown in red. Beta value was determined with a 243 Wald’s test.

S34576 S34572 S34571

25000

0 condition a control PC2 a overexpression S34574

−25000

S34575 −50000 S34573

−2e+05 −1e+05 0e+00 1e+05 2e+05 244 PC1 245 Figure ;. Principle component analysis plot of RNA sequencing data of STK$%B 246 overexpressed cells and empty-vector controls. Overexpressed cells shown in blue and controls 247 shown in red. bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

PDSS1 KIF1A

ZNF469

ZNF587B IFRD1 DEGS1

CGB5 L3HYPDH LHX1 RFX8 MMP24 ZNF286B PLD6 HIST1H2AL

LFNG ADM5

WNT7B CREB3L1

JPH1 SHROOM1 BAIAP2L2 ADCYAP1 GAPDHP14 PKD1P1 MICAL3 SYNC IL18BP

PRR22

MID1 VARS HOXA3 TMEM158

SLC43A3 CRACR2A FHOD3 DOK3 FLG

TANC2 ETS2 CALB2 ANKLE1 ODC1

ACKR3 KCNC4

HOXA9 ADAMTS16 CHML MOK P2RY11

STC2 PSMD2 CNTF UHRF1 WISP1 CCIN CARD9 MYPN IGF1R HK2 ATP2B4 TRIM58 COL6A2

ADAM12

EPB41L4B LOX SALL3 ANKS1B SLC2A1 ANKRD33B TNFRSF21 PTPRF GAL SYNJ2 SMG1P2 TUFT1 SRGAP1 DIRAS3 KCNJ14 COL6A1 NRG1 ANXA8 NALCN

FJX1 MAP3K14 PAWR SLC7A5 KITLG MET CCDC80 KCTD9 AC068946.1 RBM3

COL5A3 NAV3 WNK4 NT5E DOCK5 MYO19 LCAT AJUBA ALPK2 DAPK1 CALD1 TMEM249 OTUD6B AMOTL2 ANPEP KIFC3 CAV1 DCBLD2 PTPN14 THBS1 MPP3 NEGR1 DNER SLC7A1 OR2T8 NEXN FLNC KCNH1 CTGF ZNF488 INHBA CARD10 NPPB SHISA2 CAV2 CYR61 RPS3AP26

HIST3H2A KRT80 MFSD2A

JUN EPHA2 EDN1 KCNMA1 SLC7A5P1 ITGBL1 TAGLN KIRREL3 JPH2 NTF3 RASAL2 GADD45B LASP1 KLF2 MYOF NRP1 SBDSP1 ARL14 AKAP12 PTHLH CNN1 MGLL ZIC5 AXL SRF SERPINE1 ANXA8L1 CARS AMTN AMIGO2 MYLK SLC7A2 PLAUR PRKCA CPA4 CLCF1 KCNQ5 TRBC2 PRKAG2 ROS1 FOSL1 WNT5B TGFB2 LIF GLIPR1 GREM1 ANXA3 HBEGF WNT5A SIPA1L2 SFXN2 SMTN GDF5 PLAC8

SPOCD1 ARC FHL2 RUSC2 L1CAM S100A2 PGM5P2 IL11 TENM2 GDNF MEST ANKRD13A CAPN2 EZR KCNN4 ARTN DDX12P

MICAL2 PDCD1LG2 LAMC2 NF2 FOSL2 ARL4C

HIST1H2AE TNS4 PAGE1 ZYX TERT HIST1H2BG NT5DC3 MYH9 UCN2 JAG1 SENP3-EIF4A1 KRT75 COL7A1 HNRNPA1P33 FOXC2

VGF ZNF699 CDH13 PABPC1 FOXD1 CDH5 BRPF1 SGK1 MLPH MAT2A ADAMTS15 TFAP2C CEMIP LY6G5B SRSF6 AP1S3 FGF5 PABPC1L

GNG4 LIME1 KCNN3 CTAG2 MPP4 ABL2 SIN3B ZFC3H1 GTF2H2 FAM86B3P CAPRIN2

RAB39A MICA RGS4 PLEC PIEZO1

KRI1 ENTPD6 DDA1 HIC1 MYO9B UAP1

HHIP ATP13A1 AEN SRRT POLR1E HTR1D HGS CUZD1 CDK5R1 SMG1P3 ZNF598 MAMDC4 ZNF628 KLF16

PLXNA2 SPTBN5 CTNNAL1 DPP9 SLC19A1 NOL8 RNF126 AATF AMH ARFGAP1 TRMT1 CCDC190 TCOF1 GATAD2A BRI3BP NOP56 PRR12 WASH5P HIST1H3A RTEL1-TNFRSF6B MYBBP1A CLUH HCFC1 RBM14 HIST1H2AB F3 CASP2 NOP58 ATAD3B MSANTD3 FBRSL1 BEND3 WDR90 PUM3 IPO4 DOT1L SRM

POLR1A RRP15 XPO6 GNL3 TMEM201 ITGB3 CD3EAP DHX37 PSG5 RBM28 CAMTA2 UFSP1 CHTF18 GPATCH4 PPRC1 CGB8 NOL6 ATXN2L ASAP1 FHDC1 POLR2A KLF7 WDR43 TRMT61A PUS7 DHX34 UTP20 ANKRD52 HEATR3 DDX21 PUS1

NOM1 PLEKHG4 RRP12 NOP2 SRRM2 PNO1 WDR4 POLE UTP15 FHOD1 XRCC2 PKMYT1 SPDL1 KISS1 ARID3B USP36 ZNF367 CTPS1 ISM1 DDX11 CDC25A FANCA AGAP6 FUS MSTO1 SEMA7A HNRNPD MCM10 PA2G4 GCLM ANLN

CLSPN

CCNE2 HIST1H2AH TIPIN SLC5A6 ZNF326 MYLK2

IGHMBP2 DUSP7

CEP152 MYBL1 DGKI

SLC25A19

STK32B CEP170P1 HIST1H2BL

AFG3L1P ZFP36L2

DND1 FLVCR1 DHRS9 DDX24 HIST1H2BE AC007040.2 248 249 Figure ?. Gene network of differentially expressed genes based on co-expression of RNA 250 sequencing data for ;:,?BB samples. Each unique colour represents a cluster. 251 252 253 254 255 256 257 258 259 260 bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

261 Table '. and pathway analyses of differentially expressed genes. Gene Set ID Gene Set Name P Database KEGG_OLFACTORY_TRANSDUCTION Olfactory transduction C.EFE-HI KEGG_PATHWAYS_IN_CANCER Pathways in cancer C.IEE-CF KEGG_FOCAL_ADHESION Focal adhesion P.PIE-CE KEGG KEGG_BLADDER_CANCER Bladder cancer E.RHE-CR KEGG_ECM_RECEPTOR_INTERACTION Ecm- interaction F.HFE-CR GO:WWWPERW fibrillar center P.PHE-CI GO:WWHPWPC extracellular matrix F.RZE-CZ GO GO:WWWPZCR stress fiber P.C[E-CE Cellular GO:WWWRICR focal adhesion C.FCE-CH GO:WWPRECI [.WCE-CC REACTOME:R-HSA-HFPZRH Olfactory Signaling Pathway H.CCE-[E REACTOME:R-HSA-[PFRRR G alpha (s) signalling events P.PIE-CI REACTOME:R-HSA-HWWWPZW Syndecan interactions P.RHE-CZ Reactome Assembly of collagen fibrils and other multimeric REACTOME:R-HSA-CWCCWIW [.HPE-CR structures REACTOME:R-HSA-P[Z[CIW Collagen formation C.HRE-C[ detection of chemical stimulus involved in sensory GO:WWRWIPP P.WWE-[[ perception of smell GO:WWWZ[PP axon guidance I.RWE-HE Go GO:WW[FP[E positive regulation of fibroblast proliferation P.HPE-HH Processes GO:WWZWRFF calcium ion transmembrane transport H.WCE-HP detection of chemical stimulus involved in sensory GO:WWRWIWZ H.P[E-HP perception 262 263 264 bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

265 Table =. Gene ontology and pathway analyses of differentially expressed gene for the three largest clusters based on co- 266 expression. Gene Set Name P Database Cluster Regulation of actin cytoskeleton 1.03E-13 Pathways in cancer 1.23E-13 Axon guidance 1.09E-12 KEGG Focal adhesion 4.96E-12 Arrhythmogenic right ventricular cardiomyopathy (ARVC) 4.63E-09 fibrillar center 1.13E-29 extracellular matrix 8.57E-27 stress fiber 1.24E-26 GO Cellular focal adhesion 2.82E-23 actin cytoskeleton 4.02E-22 actin binding 3.66E-15 laminin binding 2.37E-13 1 insulin-like growth factor binding 9.72E-13 GO Function collagen binding 3.73E-12 protein heterodimerization activity 5.54E-12 cell migration involved in sprouting angiogenesis 7.07E-18 cell migration 2.94E-16 inactivation of MAPK activity 3.31E-15 GO Processes cytoskeleton organization 6.34E-15 negative regulation of cell migration 9.12E-15 Cell-extracellular matrix interactions 7.16E-15 Signal Transduction 1.98E-13 Reactome Non-integrin membrane-ECM interactions 2.58E-12 Signaling by Rho GTPases 1.84E-11 bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

EPHA-mediated growth cone collapse 2.30E-11 Rna polymerase 4.57E-13 Pyrimidine metabolism 1.37E-12 Alzheimer's disease 3.55E-11 KEGG Purine metabolism 4.11E-11 Gnrh signaling pathway 1.84E-09 nucleolus 5.96E-19 preribosome, large subunit precursor 2.21E-18 small-subunit processome 5.13E-18 GO Cellular DNA-directed RNA polymerase III complex 5.10E-15 DNA-directed RNA polymerase I complex 1.85E-14 pseudouridine synthase activity 7.75E-16 RNA binding 1.25E-15 snoRNA binding 4.90E-14 GO Function 2 rRNA binding 3.55E-13 ATP-dependent RNA helicase activity 5.23E-13 RNA secondary structure unwinding 1.57E-16 tRNA modification 7.73E-16 positive regulation of gene expression, epigenetic 3.09E-15 GO Processes ribosomal large subunit biogenesis 3.17E-15 maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S 3.32E-15 rRNA, LSU-rRNA) rRNA modification in the nucleus and cytosol 3.38E-18 tRNA processing 1.65E-17 tRNA modification in the nucleus and cytosol 1.66E-16 Reactome rRNA processing 9.91E-15 tRNA processing in the nucleus 1.69E-14 RIG-I-like receptor signaling pathway 1.24E-10 KEGG bioRxiv preprint doi: https://doi.org/10.1101/552901; this version posted May 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Cytosolic DNA-sensing pathway 1.92E-07 O-glycan biosynthesis 1.163E-06

1.36893E- Focal adhesion 06 2.06986E- Calcium signaling pathway 05 caveola 2.00E-08 proteinaceous extracellular matrix 7.41E-08 postsynaptic membrane 2.45E-06 GO Cellular

microfibril 4.99E-06 extracellular matrix 1.12E-05 3 type I interferon receptor binding 3.64E-07 binding 5.89E-07 scavenger receptor activity 6.23E-07 GO Function calcium-dependent phospholipid binding 9.44E-07 RNA binding 2.46E-06 response to mechanical stimulus 8.42E-09 lung development 1.55E-08 negative regulation of neuron apoptotic process 5.29E-08 GO Processes cellular response to hypoxia 7.29E-08 regulation of type I interferon-mediated signaling pathway 1.11E-07 O-glycosylation of TSR domain-containing 3.73E-10 Defective B3GALTL causes Peters-plus syndrome (PpS) 5.15E-10 DDX58/IFIH1-mediated induction of interferon-alpha/beta 1.29E-09 Reactome O-linked glycosylation 4.91E-08 Diseases associated with O-glycosylation of proteins 1.04E-07 267 268