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1 Drosophila models of pathogenic copy-number variant genes show global and

2 non-neuronal defects during development

3 Short title: Non-neuronal defects of fly homologs of CNV genes

4 Tanzeen Yusuff1,4, Matthew Jensen1,4, Sneha Yennawar1,4, Lucilla Pizzo1, Siddharth

5 Karthikeyan1, Dagny J. Gould1, Avik Sarker1, Yurika Matsui1,2, Janani Iyer1, Zhi-Chun Lai1,2,

6 and Santhosh Girirajan1,3*

8 1. Department of Biochemistry and Molecular Biology, Pennsylvania State University,

9 University Park, PA 16802

10 2. Department of Biology, Pennsylvania State University, University Park, PA 16802

11 3. Department of Anthropology, Pennsylvania State University, University Park, PA 16802

12 4 contributed equally to work

14 *Correspondence:

15 Santhosh Girirajan, MBBS, PhD

16 205A Life Sciences Building

17 Pennsylvania State University

18 University Park, PA 16802

19 E-mail: [email protected]

20 Phone: 814-865-0674

22 ABSTRACT

23 While rare pathogenic copy-number variants (CNVs) are associated with both neuronal and non-

24 neuronal phenotypes, functional studies evaluating these regions have focused on the molecular

25 basis of neuronal defects. We report a systematic functional analysis of non-neuronal defects for

26 homologs of 59 genes within ten CNVs and 20 neurodevelopmental genes in Drosophila. Using

27 wing-specific knockdown of 136 RNA interference lines, we identified qualitative and

28 quantitative phenotypes in 72/79 homologs, including six lines with lethality and 21 lines with

29 severe wing defects. Assessment of 66 lines for tissue-specific effects showed no correlation

30 between the severity of wing and eye-specific defects. We observed disruptions in cell

31 proliferation and apoptosis in larval wing discs for 23/27 homologs, and altered Wnt, Hedgehog

32 and Notch signaling for 9/14 homologs, including AATF/Aatf, PPP4C/Pp4-19C, and

33 KIF11/Klp61F. These findings were further validated with differences in human tissue-specific

34 expression and network connectivity of CNV genes. Our findings suggest that multiple genes

35 within each CNV differentially affect both global and tissue-specific developmental processes

36 within conserved pathways, and that their roles are not restricted to neuronal functions.

37 INTRODUCTION

38 Rare copy-number variants (CNV), or deletions and duplications in the genome, are associated

39 with neurodevelopmental disorders such as autism, intellectual disability (ID), and schizophrenia

40 [1,2]. While dosage alteration of CNV regions contribute predominantly to defects in nervous

41 system development, several CNV-associated disorders also lead to early developmental features

42 involving other organ systems [3,4], including cardiac defects [5,6], kidney malformations[7],

43 craniofacial features [3], and skeletal abnormalities [8]. For example, the 1q21.1 deletion causes

44 variable expression of multiple neuronal and non-neuronal phenotypes, including developmental

45 delay, autism, and schizophrenia as well as craniofacial features, cataracts, cardiac defects, and

46 skeletal abnormalities [9–11]. Additionally, while the 7q11.23 deletion associated with

47 Williams-Beuren syndrome (WBS) causes neuropsychiatric and behavioral features, other non-

48 neuronal phenotypes, including supravalvular aortic stenosis, auditory defects, hypertension,

49 diabetes mellitus, and musculoskeletal and connective tissue anomalies, are also observed among

50 the deletion carriers [12].

51 Despite the notable prevalence of non-neuronal phenotypes in CNV carriers, functional

52 studies of CNV genes have primarily focused on detailed assessments of neuronal and behavioral

53 phenotypes in model systems. For example, mouse models for the 16p11.2 deletion exhibited

54 post-natal lethality, reduced brain size and neural progenitor cell count, motor and habituation

55 defects, synaptic defects, and behavioral defects [13–15]. Similarly, mouse models for the 3q29

56 deletion showed decreased weight and brain size, increased locomotor activity and startle

57 response, and decreased spatial learning and memory [16,17]. However, fewer studies have

58 focused on detailed evaluation of non-neuronal phenotypes in functional models of CNV

59 disorders. For example, Arbogast and colleagues evaluated obesity and metabolic changes in

60 16p11.2 deletion mice, which showed reduced weight and impaired adipogenesis [18]. While

61 Haller and colleagues showed that mice with knockdown of MAZ, a gene within the 16p11.2

62 deletion region, contribute to the genitourinary defects observed in individuals with the deletion

63 [19], mouse studies on other homologs of 16p11.2 genes, including TAOK2, KCTD13, and

64 MAPK3, have only focused on assessing neuronal defects [20–24]. Furthermore, Dickinson and

65 colleagues reported a high-throughput analysis of essential genes in mice and identified both

66 neuronal and non-neuronal phenotypes for individual gene knockouts, including more than 400

67 genes that lead to lethality [25]. While these efforts aided in implicating novel genes with human

68 disease, our understanding of how genes associated with neurodevelopmental disorders

69 contribute towards non-neuronal phenotypes is still limited. Therefore, a large-scale analysis of

70 non-neuronal phenotypes is necessary to identify specific candidate genes within CNV regions

71 and associated biological mechanisms that contribute towards these phenotypes.

72 Drosophila melanogaster is an excellent model system to evaluate homologs of

73 neurodevelopmental genes, as many developmental processes and signaling pathways are

74 conserved between humans and flies [26]. In fact, over 75% of human disease genes have

75 homologs in Drosophila, including many genes involved in cellular signaling processes [27,28].

76 We recently examined the contributions of individual Drosophila homologs of 28 genes within

77 the 16p11.2 and 3q29 deletion regions towards specific neurodevelopmental phenotypes,

78 including rough eye phenotypes and defects in climbing ability, axon targeting, neuromuscular

79 junction, and dendritic arborization [29,30]. While these findings implicated multiple genes

80 within each CNV region towards conserved cellular processes in neuronal tissues, the conserved

81 role of these genes in non-neuronal tissues is not well understood. The Drosophila wing is an

82 effective model system to evaluate such developmental defects, as key components of conserved

83 signaling pathways, such as Notch, epidermal growth factor receptor (EGFR), Hegdehog, and

84 Wnt pathways, were identified using fly wing models [31–37]. Although fly wing phenotypes

85 cannot be directly translated to human phenotypes, defects observed in fly wings can be used to

86 assess how homologs of human disease genes alter conserved cellular and developmental

87 mechanisms. For example, Wu and colleagues showed that overexpression of the Drosophila

88 homolog for UBE3A, associated with Angelman syndrome, leads to abnormal wing and eye

89 morphology defects [38]. Furthermore, Drosophila mutant screens for developmental

90 phenotypes, including wing defects, were used to identify conserved genes for several human

91 genetic diseases, including Charcot-Marie-Tooth disease and syndromic microcephaly [39].

92 Kochinke and colleagues also recently performed a large-scale screening of ID-associated genes,

93 and found an enrichment of wing trichome density and missing vein phenotypes in ID genes

94 compared to control gene sets [40]. Hence, the fly wing provides a model system that is ideal for

95 evaluating the contributions of individual homologs of CNV genes towards cellular and

96 developmental defects.

97 In this study, we tested tissue-specific and cellular phenotypes of 79 fly homologs of

98 human genes within ten pathogenic CNV regions and genes associated with neurodevelopmental

99 disorders. Particularly, we used the adult fly wing to evaluate phenotypes in a non-neuronal

100 tissue, and observed a wide range of robust qualitative and quantitative wing phenotypes among

101 the 136 RNA interference (RNAi) lines tested in our study, including size defects, ectopic and

102 missing veins, severe wrinkling, and lethality. Further analysis of cellular phenotypes revealed

103 disruptions in conserved developmental processes in the larval imaginal wing disc, including

104 altered levels of cell proliferation and apoptosis as well as altered expression patterns in the Wnt,

105 Hedgehog, and Notch signaling pathways. However, we found no correlation in the severity of

106 phenotypes observed with wing and eye-specific knockdown. Our findings were further

107 supported by differences in expression patterns and network connectivity of human CNV genes

108 across different tissues. Our analysis emphasizes the importance of multiple genes within each

109 CNV region towards both global and tissue-specific developmental processes.

110 RESULTS

111 Knockdown of fly homologs of CNV genes contribute to a range of wing defects

112 Using an RNAi based analysis driven by the bxMS1096-GAL4 wing-specific driver, we tested a

113 total of 136 RNAi lines for 59 homologs of genes within pathogenic CNV regions (chromosomal

114 locations 1q21.1, 3q29, 7q11.23, 15q11.2, 15q13.3, 16p11.2, distal 16p11.2, 16p12.1, 16p13.11,

115 and 17q12) and 20 homologs of genes associated with neurodevelopmental disorders (Supp.

116 Data 1). Fly homologs of these genes were identified using the DIOPT orthology prediction tool

117 [41]. We list both the human gene name and the fly gene name for each tested gene as HUMAN

118 GENE/Fly gene (i.e. KCTD13/CG10465) as well as the human CNV region for context at first

119 instance. We scored 20-25 adult wings for five distinct wing phenotypes in each non-lethal

120 RNAi line, including wrinkled wing, discoloration, ectopic veins, missing veins, and bristle

121 planar polarity phenotypes (Fig. 1A; Supp. Data 2). We first categorized each wing phenotype

122 based on their severity and performed k-means clustering analysis to categorize each RNAi line

123 by their overall phenotype severity (Fig. 1B-C). We observed four clusters of RNAi lines: 75

124 lines with no observable qualitative phenotypes (55.2%), 24 lines with mild phenotypes (17.7%),

125 10 lines with moderate phenotypes (7.4%), 21 lines with severe phenotypes (15.4%), and 6 lines

126 with lethal phenotypes (4.4%), including ACACA/ACC within 17q12, DLG1/dlg1 within 3q29,

127 and STX1A/Syx1A within 7q11.23 (Fig. 1B-D; Supp. Data 2). We observed severe wrinkled

128 wing phenotypes for 13/79 fly homologs, including PPP4C/Pp4-19C within 16p11.2,

129 ATXN2L/Atx2 within distal 16p11.2, AATF/Aatf within 17q12, and MFI2/Tsf2 within 3q29 (Fig.

130 2A-B, Supp. Data 3). Interestingly, seven out of ten CNV regions contained at least one

131 homolog that showed lethality or severe wing phenotypes, and five CNV regions (3q29, 16p11.2,

132 distal 16p11.2, 16p12.1, and 17q12) had multiple homologs showing lethality or severe wing

133 phenotypes (Fig. 2A, Supp. Data 3). For example, RNAi lines for both UQCRC2/UQCR-C2 and

134 POLR3E/Sin within 16p12.1 showed lethality. Within the 3q29 region, NCBP2/Cbp20 and

135 MFI2/Tsf2 showed severe phenotypes while DLG1/dlg1 showed lethality. In contrast, 12/20

136 known neurodevelopmental genes showed no observable wing phenotypes, suggesting that these

137 genes could be responsible for neuronal-specific phenotypes (Fig. 2B, Supp. Data 3). We note

138 that 18/79 fly homologs showed discordant phenotypes between two or more RNAi lines for the

139 same gene, which could be due to differences in expression of the RNAi construct among these

140 lines (Supp. Data 3).

141 Certain qualitative phenotypes exhibited higher frequency in males compared to females.

142 For example, discoloration (87 lines in males compared to 56 lines in females; p=1.315×10-4,

143 two-tailed Fisher’s exact test) and missing vein phenotypes (92 lines in males compared to 29

144 lines in females; p=2.848×10-16, two-tailed Fisher’s exact test) at any degree of severity were

145 more commonly observed in males than females (Supp. Data 2). In particular, 25/92 lines in

146 males (compared to 1/29 in females) showed a total loss of the anterior crossvein (ACV) (Supp.

147 Data 2). We further identified 17 RNAi lines that were lethal in males with wing-specific

148 knockdown of fly homologs. While higher frequencies of wing phenotypes in males could be

149 due to a sex-specific bias of developmental phenotypes, the increased severity we observed in

150 males is most likely due to a stronger RNAi knockdown caused by an X-linked dosage

151 compensation, as the bxMS1096-GAL4 driver is inserted on the fly X chromosome [42,43].

152 Next, we measured the total adult wing area and the lengths of six veins (longitudinal L2,

153 L3, L4, L5, ACV, and posterior crossvein or PCV) in the adult wing for each of the tested RNAi

154 lines that did not show lethality (or severe wrinkled phenotypes for vein length measurements)

155 (Fig. 3A). Overall, we identified significant wing measurement changes for 89 RNAi lines

156 compared to controls, which included lines that did not have an observable qualitative wing

157 phenotype (Fig. 1D). A summary of L3 vein lengths is presented in Fig. 3B, and the

158 measurements for the remaining five veins are presented in Supp. Figure 1 and Supp. Data. 2.

159 We found that 33/61 of the homologs (54%) showed significant changes in L3 vein length,

160 including 20 homologs with longer vein lengths and 13 homologs with shorter vein lengths

161 (Supp. Data 3). Additionally, 41/74 of the fly homologs (55%) showed changes in wing area

162 (Supp. Data 3), including 36 homologs which showed smaller wing areas and five homologs

163 showed larger wing areas compared to controls (Supp. Data 3). For example, both homologs of

164 genes within 1q21.1 region, BCL9/lgs and FMO5/Fmo-2, showed decreased wing area and vein

165 length, potentially mirroring the reduced body length phenotype observed in mouse models of

166 the deletion [44] (Fig. 3B-C). In addition, PAK2/Pak within 3q29, TBX1/org-1 within 22q11.2,

167 autism-associated CHD8/kis, and microcephaly-associated ASPM/asp also showed smaller wing

168 areas and vein lengths (Fig. 3B-C). In contrast, TRPM1/Trpm within 15q13.3 and the cell

169 proliferation gene PTEN/Pten [45] both showed larger wing areas and vein lengths (Fig. 3B-C).

170 Furthermore, we identified eight homologs that showed no qualitative wing phenotypes but had

171 significant changes in wing areas and vein lengths (Supp. Data 3), including CCDC101/Sgf29 in

172 distal 16p11.2, FMO5/Fmo-2, TRPM1/Trpm, DHRS11/CG9150 in 17q12, and NSUN5/Nsun5 in

173 7q11.23 (Fig. 3B-C; Supp. Data 3). These results indicate that homologs of certain CNV genes

174 may influence variations in size without causing adverse wing phenotypes, and may be

175 specifically implicated towards cellular growth mechanisms.

176

177

178

179 Homologs of CNV genes show global and tissue-specific effects during development

180 We previously showed that many of the same fly homologs of CNV genes that showed wing

181 defects in the current study also contributed towards neuronal phenotypes in the fly eye [29,30],

182 suggesting a role for these genes in global development. We therefore performed ubiquitous and

183 eye-specific knockdown of fly homologs to assess tissue-specific effects in comparison to the

184 wing phenotypes. First, we used the da-GAL4 driver at 25°C to drive ubiquitous knockdown of

185 RNAi lines for 31 homologs of CNV genes, including 19 that were previously published [29,30],

186 and observed complete or partial lethality at larval and pupal stages with knockdown of 10/31

187 homologs (32.3%) (Fig. 4A). Lethal phenotypes have also been documented for 43/130

188 knockout mouse models of individual CNV genes as well as for the entire deletion (Supp. Data

189 4). For example, mouse models heterozygous for the 16p11.2 deletion showed partial neonatal

190 lethality, while knockout mouse models of four individual genes within the 16p11.2 region,

191 including Ppp4C-/- and Kif22-/-, showed embryonic lethality [13,46,47]. In our study, the

192 DLG1/dlg1 line that showed lethality with wing-specific knockdown also exhibited larval

193 lethality with ubiquitous knockdown, indicating its role in global development (Fig. 4A). In

194 addition, six homologs that showed severe wing phenotypes also showed larval or pupal lethality

195 with ubiquitous knockdown, including ALDOA/Ald and PPP4C/Pp4-19C within 16p11.2 and

196 ATXN2L/Atx2 and TUFM/mEFTu1 within distal 16p11.2 (Fig. 4A). The remaining homologs

197 that showed lethality with ubiquitous knockdown showed at least a mild qualitative or

198 quantitative wing phenotype.

199 We next compared the phenotypes observed with wing-specific knockdown of fly

200 homologs to their corresponding eye-specific knockdowns to evaluate tissue-specific effects. To

201 quantitatively assess the phenotypic severity of cellular defects with eye-specific knockdown of

202 fly homologs, we developed a tool called Flynotyper [48] that determines the degree of

203 disorganization among the ommatidia in the adult eye. We analyzed phenotypic scores obtained

204 from Flynotyper for 66 RNAi lines of 45 fly homologs, including from previously-published

205 datasets [29,30,48]. We found that 37/45 homologs (82.2%) exhibited both eye and wing-

206 specific defects (Fig. 4B, Supp. Fig. 2, Supp. Data 5). Two homologs with significant eye

207 phenotypes did not show any wing phenotypes, including SPNS1/spin within distal 16p11.2 and

208 microcephaly-associated SLC25A19/Tpc1 [49], while five homologs only showed wing-specific

209 phenotypes, including CDIPT/Pis and YPEL3/CG15309 within 16p11.2, FBXO45/Fsn and

210 OSTalpha/CG6836 within 3q29, and UQCRC2/UQCR-C2 (Fig. 4B, Supp. Fig. 2). In particular,

211 UQCRC2/UQCR-C2 showed lethality with wing-specific knockdown, suggesting potential

212 tissue-specific effects of this gene in non-neuronal cells (Fig. 4B). While most homologs

213 contributed towards both eye and wing-specific phenotypes, we observed a wide range of

214 severity in eye phenotypes that did not correlate with the severity of quantitative or qualitative

215 wing phenotypes (Fig. 4C). For example, TUFM/mEFTu1 showed a severe wing phenotype but

216 only a mild increase in eye phenotypic score, while SH2B1/Lnk, also within the distal 16p11.2

217 region, showed severe rough eye phenotypes but only a mild increase in wing size (Fig. 4D).

218 Similarly, BCL9/lgs also showed opposing tissue-specific effects with mild qualitative wing

219 phenotype and severe eye phenotype, suggesting that the role of these homologs towards

220 development differs across tissue types.

221

222 CNV genes show variable expression across different tissues in flies and humans

223 To assess how expression levels of CNV genes vary across different tissues, we first examined

224 the expression patterns of fly homologs in larval and adult tissues using the FlyAtlas Anatomical

225 Microarray dataset [50]. We found that 76/77 homologs with available data were expressed in at

226 least one larval and adult tissue (Supp. Fig. 3, Supp. Data 6). In general, we did not observe a

227 correlation between wing phenotype severity and expression patterns of homologs in larval or

228 adult tissues (Fig. 5A). For example, 58/77 homologs (75.3%) showed ubiquitous larval

229 expression, including both fly homologs that showed no qualitative wing phenotypes, such as

230 KCTD13/CG10465 within 16p11.2 and FBXO45/Fsn, and those with severe wing phenotypes,

231 such as PPP4C/Pp4-19C and NCBP2/Cbp20 (Fig. 5A, Supp. Fig. 3). Furthermore, 30/39

232 homologs (76.9%) that showed eye phenotypes also had ubiquitous larval expression, providing

233 further support to the observation that genes causing neuronal phenotypes may also contribute to

234 developmental phenotypes in other tissues (Supp. Data 5). Of note, 9/77 homologs (11.7%) did

235 not have any expression in the larval central nervous system, including FMO5/Fmo-2,

236 BDH1/CG8888 within 3q29, and TBX6/Doc2 within 16p11.2 (Fig. 5A, Supp. Fig. 3). However,

237 we observed wing phenotypes for 8/9 of these homologs, suggesting that they may contribute to

238 tissue-specific phenotypes outside of the nervous system. Except for the epilepsy-associated

239 SCN1A/para [51], which was exclusively expressed in both the larval central nervous system

240 (CNS) and adult brain tissues, other tested neurodevelopmental genes were also expressed in

241 non-neuronal tissues (Fig. 5A).

242 We further used the GTEx Consortium dataset [52] to examine tissue-specific expression

243 of 150 human CNV and known neurodevelopmental genes across six tissues including brain,

244 heart, kidney, lung, liver, and muscle. We found 121 genes that were expressed in at least one

245 adult tissue, including 49 genes (32.7%) that showed ubiquitous expression across all six tissues

246 (Supp. Data 6). Of the 112 genes expressed in non-neuronal tissues, 34 did not have any

247 neuronal expression, including TBX1, FMO5 and GJA5 within 1q21.1, and ATP2A1 within distal

248 16p11.2 (Fig. 5B, Supp. Data 6). FMO5 and TBX1 also showed non-neuronal expression in

249 Drosophila tissues, suggesting that their tissue-specific expression is highly conserved (Fig. 5A).

250 Other genes showing ubiquitous expression also had preferentially high expression for specific

251 non-neuronal tissues, including ALDOA and UQCRC2 for muscle and heart (Fig. 5B). In

252 contrast, we found nine genes that were expressed only in the adult brain, including FAM57B

253 and DOC2A within 16p11.2, as well as SCN1A, which showed similar CNS-only expression in

254 Drosophila tissues (Fig. 5B, Supp. Data 6).

255

256 Knockdown of fly homologs of CNV genes lead to disruption of cellular processes

257 The disruption of basic cellular processes in neuronal cells, such as cell proliferation and

258 apoptosis, have been implicated in neurodevelopmental disorders [53–55]. We previously

259 identified defects in cell proliferation among photoreceptors neurons in larval eye discs with

260 knockdown of 16p11.2 homologs, as well as increased apoptosis with knockdown of a subset of

261 3q29 homologs [29,30]. Here, we explored how these basic cellular processes are altered in non-

262 neuronal cells, specifically in the developing wing disc, with knockdown of homologs of CNV

263 genes. We targeted 27 fly homologs that showed a range of adult wing phenotypes for changes in

264 cell proliferation and apoptosis, using anti-phospho-Histone H3 Ser10 (pH3) and anti-

265 Drosophila caspase-1 (dcp1), respectively, in the third instar larval wing discs. We identified

266 23/27 homologs that showed significant increases in apoptotic cells compared to controls,

267 including seven homologs, such as PPP4C/Pp4-19C, ATXN2L/Atx2, and AATF/Aatf , which

268 showed dcp1 staining across the entire larval wing pouch (Fig. 6A-B, Supp. Figs. 4-5, Supp.

269 Data 7). In addition, 16/27 genes showed decreased levels of proliferation, including eight

270 homologs which also showed apoptosis defects, such as CYFIP1/Sra-1 within 15q11.2,

271 SH2B1/Lnk, and the microcephaly gene KIF11/Klp61F (Fig. 6A and 6C, Supp. Figs. 4-5, Supp.

272 Data 7). All six of the tested homologs with severe adult wing phenotypes showed both

273 increased apoptosis and decreased proliferation (Supp. Data 7). Similarly, 3/4 homologs of

274 genes showing lethality with wing-specific knockdown also showed defects in apoptosis or

275 proliferation, with the exception of ACACA/ACC (Supp. Figure 4, Supp. Data 7). As bxMS1096-

276 GAL4 is located on the X-chromosome, we expected to see more severe defects in males

277 compared with females with knockdown of homologs due to the X-linked dosage compensation

278 [42,43]. However, knockdown of 3/11 tested homologs with sex-specific differences in adult

279 wing phenotypes, including BCL9/lgs, CYFIP1/Sra-1, and DNAJC30/CG11035 within 7q11.23,

280 showed significantly decreased levels of cell proliferation in females but no change for males

281 compared to their respective controls, suggesting a sex-specific effect of these genes for cell

282 proliferation (Supp. Fig. 5, Supp. Data 7). Overall, our results suggest that cell proliferation and

283 apoptosis are disrupted by reduced expression of homologs of CNV genes in both neuronal and

284 non-neuronal tissues.

285

286 Knockdown of homologs of CNV genes disrupt conserved signaling pathways

287 Several conserved signaling pathways that are active in a spatial and temporal manner in the

288 larval wing disc, such as Wnt, Hedgehog, BMP, and Notch signaling, regulate the anterior-

289 posterior (A/P) and dorsal-ventral (D/V) boundaries to determine accurate morphology and vein

290 patterning in the adult wing [36,37,56–58]. For example, Wnt and Notch signaling pathways

291 both act along the D/V boundary to determine cell fate [59,60], while Hedgehog signaling is

292 dependent upon expression of both engrailed in the posterior compartment and patched along the

293 A/P border [61,62]. Furthermore, O’Roak and colleagues showed that genes identified from de

294 novo mutations in patients with autism are linked to β-catenin/Wnt pathway [63]. In addition,

295 familial loss-of-function mutations in the human hedgehog signaling pathway gene PTCH1 are

296 implicated in basal cell nevus syndrome, leading to basal cell carcinoma [64,65].

297 Based on adult wing phenotypes and disruptions to cellular processes, we next tested

298 whether knockdown of 14 fly homologs disrupt conserved signaling pathways in the third instar

299 larval wing disc (Supp. Data 7). In particular, we evaluated the role of Wnt, Hedgehog, and

300 Notch signaling pathways by testing the expression patterns of four key proteins within these

301 pathways, including wingless (Wnt), patched (Hedgehog), engrailed (Hedgehog), and delta

302 (Notch). We found that 9/14 homologs, including 8/10 homologs showing severe wing

303 phenotypes or lethality, exhibited disruptions in at least one signaling pathway. For example, five

304 homologs with severe or lethal phenotypes showed disruptions of all four signaling pathways,

305 including AATF/Aatf, NCBP2/Cbp20, POLR3E/Sin, PPP4C/Pp4-19C, and KIF11/Klp61F (Fig.

306 7, Supp. Data 7). Our observations are in concordance with previous findings by Swarup and

307 colleagues, who showed that PPP4C/Pp4-19C is a candidate regulator of Wnt and Notch

308 signaling pathways in Drosophila larval wing discs [66]. Furthermore, two genes from the 3q29

309 region, DLG1/dlg1 and MFI2/Tsf2, showed altered expression patterns for delta and patched but

310 not for engrailed, indicating that they selectively interact with the Hedgehog as well as Notch

311 signaling pathway (Supp. Fig. 6). In fact, Six and colleagues showed that Dlg1 directly binds to

312 the PDZ-binding domain of Delta1 [67]. In contrast, ACACA/ACC and UQCRC2/UQCR-C2

313 showed no changes in expression patterns for any of the four signaling proteins tested,

314 suggesting that the observed lethality could be due to other cellular mechanisms (Supp. Fig. 6).

315 We conclude that several homologs disrupt the expression of key proteins in signaling pathways

316 in the developing larval wing discs, potentially accounting for the developmental defects

317 observed in the adult wings.

318

319 Connectivity patterns of CNV genes vary across human tissue-specific networks

320 We examined patterns of connectivity for the nine candidate genes, based on the disruptions of

321 signaling pathways identified in the developing Drosophila wing discs, within the context of

322 human brain, heart, and kidney-specific gene interaction networks [68]. These tissue-specific

323 networks were constructed using Bayesian classifier-generated probabilities for pairwise genetic

324 interactions based on co-expression data [68]. We calculated the lengths of the shortest paths

325 between each candidate gene and 267 Wnt, Notch, and Hedgehog pathway genes in each

326 network as a proxy for connectivity (Supp. Data 8). In all three networks, each of the candidate

327 genes were connected to a majority of the tested signaling pathway genes (Fig. 8A, Supp. Fig.

328 7). Interestingly, we observed a higher connectivity (i.e. shorter path distances) between

329 candidate genes and Wnt and Hedgehog pathway genes in the brain-specific network compared

330 to the heart and kidney-specific networks (Fig. 8B). We further identified enrichments for genes

331 involved in specific biological processes among the connector genes that were located in the

332 shortest paths within neuronal and non-neuronal tissue-specific networks (Fig. 8C, Supp. Data

333 8). For example, axon-dendrite transport, dopaminergic signaling, and signal transduction

334 functions were enriched among connector genes only for the brain-specific network, while

335 organelle organization and protein ubiquitination were enriched among connector genes only for

336 kidney and heart networks (Fig. 8C). However, several core biological processes, such as cell

337 cycle, protein metabolism, transcriptional regulation, and RNA processing/splicing, were

338 enriched among connector genes within all three tissue-specific networks (Fig. 8C). Our analysis

339 highlights that human CNV genes potentially interact with developmental signaling pathways in

340 a ubiquitous manner, but may affect different biological processes in neuronal and non-neuronal

341 tissues.

342 DISCUSSION

343 We used the Drosophila wing as a model to assess how homologs of key CNV genes contribute

344 towards non-neuronal phenotypes. We tested fly homologs of 79 genes and identified multiple

345 homologs within each CNV region that exhibited strong phenotypes indicative of developmental

346 disruptions. Several themes have emerged from our study highlighting the importance of fly

347 homologs of CNV genes towards both global and tissue-specific phenotypes.

348 First, we found that fly homologs of CNV genes contribute towards developmental

349 phenotypes through ubiquitous roles in neuronal and non-neuronal tissues. Although we did not

350 study models for the entire CNV, nearly all individual fly homologs of CNV genes contribute to

351 wing-specific developmental defects. It is likely that these genes may also contribute to

352 additional phenotypes in other tissues that we did not assess. In fact, a subset of these homologs

353 also showed early lethality with ubiquitous knockdown in addition to severe or lethal wing-

354 specific phenotypes. However, we found no correlation between the severity of the eye and wing

355 phenotypes, suggesting tissue-specific effects of these homologs towards developmental defects.

356 In contrast, fly homologs of known neurodevelopmental genes generally showed milder wing

357 phenotypes compared with eye phenotypes, indicating a more neuronal role for these genes.

358 While our study only examined a subset of CNV genes with Drosophila homologs, phenotypic

359 data from knockout mouse models also support a global developmental role for individual CNV

360 genes. In fact, 44/130 knockout models of CNV genes within the Mouse Genome Informatics

361 (MGI) database [69] exhibited non-neuronal phenotypes, including 20 homologs of CNV genes

362 that showed both neuronal and non-neuronal phenotypes (Supp. Data 4). For example, knockout

363 mouse models of Dlg1-/- show defects in dendritic growth and branching in the developing

364 nervous system, in addition to craniofacial features and multiple kidney and urinary tract defects

365 [70–73]. Furthermore, Chapman and colleagues showed that knockout of Tbx6-/- caused defects

366 in mesodermal and neuronal differentiation early in development, leading to abnormal vascular,

367 tail bud, and neural tube morphology [74]. These observations further support our findings that

368 most fly homologs of CNV genes have a global role in development that could account for the

369 observed non-neuronal defects.

370 Second, based on tissue-specific phenotypes, we identified fly homologs of CNV genes

371 that are key regulators of conserved cellular processes important for development. For example,

372 9/10 homologs with severe or lethal adult wing phenotypes also exhibited defects in cell

373 proliferation and apoptosis during development. In fact, we found concordance between cellular

374 processes affected by wing and eye-specific knockdown of homologs of genes within 16p11.2

375 and 3q29 regions, including decreased proliferation for MAPK3/rl and increased apoptosis for

376 NCBP2/Cbp20 and DLG1/dlg1 [29,30]. While eye-specific knockdown of BDH1/CG8888

377 showed decreased cell proliferation in larval eye discs [30], we found increased cell proliferation

378 with wing-specific knockdown, suggesting a tissue-specific effect for this gene. Notably, at least

379 one fly homolog per CNV region showed defects in cell proliferation or apoptosis, suggesting

380 these cellular processes are important for development in both neuronal and non-neuronal

381 tissues. For example, ATXN2L/Atx2, SH2B1/Lnk, and CCDC101/Sgf29 each showed decreased

382 proliferation and increased apoptosis, suggesting a potential shared cellular mechanism for

383 several genes within the distal 16p11.2 deletion. Furthermore, a subset of these genes also

384 disrupted multiple signaling pathways, indicating a potential role for these homologs as key

385 regulators of developmental processes. We specifically identified five homologs whose

386 knockdown caused disruptions of Wnt, Notch, and hedgehog signaling pathways. Each of these

387 genes have important roles in cell cycle regulation, apoptosis, transcription, or RNA processing,

388 based on Gene Ontology annotations [75,76]. In fact, we found that the RNA transport protein

389 NCBP2/Cbp20 [77], which we recently identified as a key modifier gene for the 3q29 deletion

390 [30], interfaced with all three signaling pathways. Furthermore, AATF disrupts apoptosis and

391 promotes cell cycle progression through displacement of HDAC1 [78–80], while PPP4C

392 promotes spindle organization at the centromeres during mitosis [81]. While we only evaluated

393 the role of these genes towards development in a single fly tissue, our additional analysis of

394 human gene interaction networks showed strong connectivity between the CNV genes and

395 signaling pathways in multiple neuronal and non-neuronal human tissues. In fact, cell cycle

396 genes were enriched among the connector genes in all three tissue-specific networks, further

397 emphasizing the role of cell cycle processes towards developmental phenotypes. Notably, we

398 also observed certain biological processes enriched among connector genes that were specific to

399 neuronal or non-neuronal tissues, indicating that genes within CNV regions may affect different

400 biological processes in a tissue-specific manner.

401 Overall, we show that fly homologs of most CNV genes contribute towards global

402 developmental phenotypes, although exactly how they contribute toward such phenotypes varies

403 between neuronal and non-neuronal tissues. Previous functional studies for CNV disorders have

404 focused primarily on identifying candidate genes for the observed neuronal phenotypes. In this

405 study, we identified several homologs of CNV genes that are responsible for non-neuronal

406 defects, as well as novel associations between these homologs and conserved biological

407 processes and pathways. We therefore propose that multiple genes within each CNV region

408 differentially disrupt conserved cellular pathways and biological processes in neuronal versus

409 non-neuronal tissues during development (Fig. 9). These results are in line with a multigenic

410 model for CNV disorders, as opposed to models where individual causative genes are

411 responsible for specific phenotypes [29,30,82]. Our study further exemplifies the utility of

412 evaluating non-neuronal phenotypes in addition to neuronal phenotypes in functional models of

413 individual genes and CNV regions associated with developmental disorders, including future

414 studies in mammalian or cellular model systems. Further studies exploring how CNV genes

415 interact with each other and with other developmental pathways could more fully explain the

416 conserved mechanisms underlying global developmental defects and identify potential

417 therapeutic targets for these disorders.

418

419 MATERIALS AND METHODS

420 Fly stocks and genetics

421 We tested 59 Drosophila homologs for 130 human genes that span across 10 pathogenic CNV

422 regions associated with neurodevelopmental disorders (1q21.1, 3q29, 7q11.23, 15q11.2, 15q13.3,

423 16p11.2, distal 16p11.2, 16p12.1, 16p13.11, and 17q12) [83] (Supp. Data 1). In addition, we

424 evaluated fly homologs of 20 human genes known to be in involved in neurodevelopmental

425 disorders [48,84] (Supp. Data 1). These include genes involved in beta-catenin signaling

426 pathway (5 genes), core genes implicated in neurodevelopmental disorders (8 genes), and genes

427 associated with microcephaly (7 genes) [85]. We used the DRSC Integrative Ortholog Prediction

428 Tool (DIOPT, v.7.1) to identify the fly homologs for each human gene [41] (Supp. Data 1).

429 To knockdown individual genes in specific tissues, we used RNA interference (RNAi)

430 and the UAS-GAL4 system (Fig. 1A), a well-established tool that allows for tissue-specific

431 expression of a gene of interest [86]. RNAi lines were obtained from Vienna Drosophila

432 Resource Center (VDRC) that include both GD and KK lines. We tested a total of 136 lines in

433 our final data analysis (Supp. Data 9), after eliminating KK lines with additional insertion that

434 drives the overexpression of the Tiptop (tio) transcription factor [87,88]. A complete list of stock

435 numbers and full genotypes for all RNAi lines used in this study is presented in Supp. Data 9.

436 We used the bxMS1096-GAL4/FM7c;;UAS-Dicer2/TM6B driver for wing-specific knockdown and

437 w1118;GMR-GAL4;UAS-Dicer2 driver (Claire Thomas, Penn State University) for eye-specific

438 knockdown of RNAi lines. Ubiquitous knockdown experiments were performed using the w;da-

439 GAL4;+ driver (Scott Selleck, Penn State University). For all experiments, we used appropriate

440 GD (w1118, VDRC# 60000) or KK (y,w1118; P{attP,y+,w3`}, VDRC# 60100) lines as controls to

441 compare against lines with knockdown of individual homologs. All fly lines were reared on

442 standard yeast Drosophila medium at room temperature. All crosses were set and maintained at

443 25°C, except for the eye knockdown experiments which were maintained at 30°C.

444

445 Phenotypic analysis of adult wing images

446 Adult progeny were isolated from crosses between RNAi lines and bxMS1096-GAL4 driver shortly

447 after eclosion, and kept at 25°C until day 2-5 (Fig. 1A). At that point, the progeny were frozen at

448 -80°C, and were then moved to -20°C prior to imaging and storage. Approximately 20-25

449 progeny, both male and female, were collected for each RNAi line tested. The adult wings were

450 plucked from frozen flies and mounted on a glass slide. The slides were covered with a coverslip

451 and sealed using clear nail polish. Adult wing images were captured using a Zeiss Discovery

452 V20 stereoscope (Zeiss, Thornwood, NY, USA), with a ProgRes Speed XT Core 3 camera and

453 CapturePro v.2.8.8 software (Jenoptik AG, Jena, Germany) at 40X magnification.

454 For each non-lethal RNAi line, we scored the adult wing images for five qualitative

455 phenotypes, including wrinkled wing, discoloration, missing veins, ectopic veins, and bristle

456 planar polarity defects, on a scale of 1 (no phenotype) to 5 (lethal) (Fig. 2C). Lines showing

457 severely wrinkled wings or lethality were scored as 4 (severe) or 5 (lethal) for all five

458 phenotypes. We calculated the frequency of each phenotypic score (i.e. mild bristle polarity,

459 moderate discoloration) across all of the wing images for each line (Fig. 2A-B), and then

460 performed k-means clustering of these values to generate five clusters for overall wing

461 phenotypes (Fig. 1C). For quantitative analysis of wing phenotypes, we used the Fiji ImageJ

462 software [89] to calculate the wing area using the Measure Area tool, and calculated the lengths

463 of longitudinal veins L2, L3, L4, and L5 as well as the anterior and posterior crossveins (ACV

464 and PCV), by tracing individual veins using the Segmented Line tool (Fig. 3A, Supp. Data 2).

465 We determined discordant homologs when RNAi lines for the same homologs showed

466 inconsistent wing phenotypes. For each homolog with multiple RNAi lines, we checked

467 discordance among RNAi lines for no phenotype versus any qualitative or quantitative

468 phenotypes, followed by discordance for small or large wing measurement phenotypes (Supp.

469 Data 3).

470

471 Phenotypic analysis of adult eye images

472 We crossed RNAi lines with GMR-GAL4 to achieve eye-specific knockdown of homologs of

473 CNV and known neurodevelopmental genes. Adult 2-3-day old female progenies from the

474 crosses were collected, immobilized by freezing at -80°C, and then moved to -20°C prior to

475 imaging and storage. Flies were mounted on Blu-tac (Bostik Inc, Wauwatosa, WI, USA) and

476 imaged using an Olympus BX53 compound microscope with LMPLan N 20X air objective using

477 a DP73 c-mount camera at 0.5X magnification (Olympus Corporation, Tokyo, Japan). CellSens

478 Dimension software (Olympus Corporation, Tokyo, Japan) was used to capture the eye images,

479 which were then stacked using the Zerene Stacker software (Zerene Systems LLC, Richland,

480 WA, USA). All eye images presented in this study are maximum projections of 20 consecutive

481 optical z-sections, at a z-step size of 12.1μm. Finally. we used our computational method called

482 Flynotyper (https://flynotyper.sourceforge.net) to quantify the degree of rough eye phenotypes

483 present due to knockdown of homologs of CNV or neurodevelopmental genes [48]. Flynotyper

484 scores for homologs of 16p11.2 and 3q29, as well as select core neurodevelopmental genes, were

485 derived from our previous studies [29,30,48].

486

487

488 Immunohistochemistry

489 Wing imaginal discs from third instar larvae were dissected in 1X PBS. The tissues were fixed

490 using 4% paraformaldehyde and blocked using 1% bovine serum albumin (BSA). The wing discs

491 were incubated with primary antibodies using appropriate dilutions overnight at 4°C. We used

492 the following primary antibodies: mouse monoclonal anti-pHistone3 (S10) (1:100 dilutions, Cell

493 Signaling 9706L), rabbit polyclonal anti-cleaved Drosophila Dcp1 (Asp216) (1:100 dilutions,

494 Cell Signaling 9578S), mouse monoclonal anti-Wingless (1:200 dilutions, DSHB, 4D4), mouse

495 monoclonal anti-Patched (1:50 dilutions, DSHB, Drosophila Ptc/APA1), mouse monoclonal

496 anti-Engrailed (1:50 dilutions, DSHB, 4D9), and mouse monoclonal anti-Delta (1:50 dilutions,

497 DSHB, C594.9B). Following incubation with primary antibodies, the wing discs were washed

498 and incubated with secondary antibodies at 1:200 dilution for two hours at room temperature.

499 We used the following secondary antibodies: Alexa Fluor 647 dye goat anti-mouse (A21235,

500 Molecular Probes by Invitrogen/Life Technologies), Alexa Fluor 568 dye goat anti-rabbit

501 (A11036, Molecular Probes by Invitrogen/Life Technologies), and Alexa Fluor 568 dye goat

502 anti-mouse (A11031, Molecular Probes by Invitrogen/Life Technologies). All washes and

503 antibody dilutions were made using 0.3% PBS with Triton-X.

504 Third instar larvae wing imaginal discs were mounted in Prolong Gold antifade reagent

505 with DAPI (Thermo Fisher Scientiﬁc, P36930) for imaging using an Olympus Fluoview FV1000

506 laser scanning confocal microscope (Olympus America, Lake Success, NY). Images were

507 acquired using FV10-ASW 2.1 software (Olympus, Waltham, MA, USA). Composite z-stack

508 images were analyzed using the Fiji ImageJ software [89]. To calculate the number of pH3

509 positive cells within the wing pouch area of the wing discs, we used the AnalyzeParticles

510 function in ImageJ, while manual counting was used to quantify Dcp1 positive cells. We note

511 that cell proliferation and apoptosis staining for NCBP2/Cbp20, DLG1/dlg1, BDH1/CG8888, and

512 FBXO45/Fsn were previously published[30].

513

514 Statistical analysis

515 Significance for the wing area and vein length measurements, cell counts for proliferation and

516 apoptosis, and Flynotyper scores were compared to appropriate GD or KK controls using one-

517 tailed or two-tailed Mann-Whitney tests. P-values for each set of experiments were corrected for

518 multiple testing using Benjamini-Hochberg correction. All statistical and clustering analysis was

519 performed using R v.3.6.1 (R Center for Statistical Computing, Vienna, Austria). Details for the

520 statistical tests performed for each dataset are provided in Supp. Data 10.

521

522 Expression data analysis

523 We obtained tissue-specific expression data for fly homologs of CNV genes from the FlyAtlas

524 Anatomical Microarray dataset [50]. Raw FPKM (fragments per kilobase of transcript per

525 million reads) expression values for each tissue were categorized as follows: <10, no expression;

526 10-100, low expression; 100-500, moderate expression; 500-1000, high expression; and >1000,

527 very high expression (Supp. Data 6). The median expression among midgut, hindgut,

528 Malpighian tube, and (for adult only) crop tissues was used to represent the overall gut

529 expression. We similarly obtained human tissue-specific expression data for CNV genes from

530 the GTEx Consortium v.1.2 RNA-Seq datasets [52]. Median TPM (transcripts per million reads)

531 expression values for each tissue were categorized as follows: <3, no expression; 3-10, low

532 expression; 10-25, moderate expression; 25-100, high expression; and >100, very high

533 expression (Supp. Data 6). The median expression among all brain and heart sub-tissues was

534 used to represent brain and heart expression, while the median expression among all colon,

535 esophagus, small intestine, and stomach sub-tissues was used to represent digestive tract

536 expression. Preferential gene expression for a particular tissue within the GTEx dataset was

537 determined if the expression values for that tissue were greater than the third quartile of all tissue

538 expression values for that gene, plus 1.5 times the interquartile range. Venn diagrams were

539 generated using the Venny webtool (http://bioinfogp.cnb.csic.es/tools/venny) (Supp. Fig. 3).

540

541 Network analysis

542 We obtained human tissue-specific gene interaction networks for brain, heart, and kidney tissues

543 from the GIANT network database [68] within HumanBase (https://hb.flatironinstitute.org).

544 These networks were built by training a Bayesian classifier based on tissue-specific gene co-

545 expression datasets, which then assigned a posterior probability for interactions between each

546 pair of genes within the genome for a particular tissue. We downloaded the “Top edge” version

547 of each tissue-specific network, and extracted all gene pairs with posterior probabilities >0.2 to

548 create sub-networks containing the top ~0.5% tissue-specific interactions. Next, we identified the

549 shortest paths in each sub-network between human CNV genes whose fly homologs disrupted

550 signaling pathways in the larval wing disc and human genes within each disrupted pathway,

551 using the inverse of the posterior probability as weights for each edge in the network. Gene sets

552 from the human Notch (KEGG:map04330), Wnt (KEGG:map04310) and Hedgehog pathways

553 (KEGG:map04340) were curated from the Kyoto Encyclopedia of Genes and Genomes (KEGG)

554 pathway database [90]. Using the NetworkX Python package [91], we calculated the shortest

555 distance between each CNV gene and pathway gene, and identified connecting genes that were

556 within each of the shortest paths for the three tissue-specific networks. We further tested for

557 enrichment of Gene Ontology (GO) terms (PantherDB GO-Slim) among the connector genes

558 using the PantherDB Gene List Analysis tool [92]. Lists of the shortest paths and connector

559 genes in each tissue-specific network, as well as enriched GO terms for sets of connector genes,

560 are provided in Supp. Data 8. Gene networks were visualized using Cytoscape v.3.7.2 [93]

561 using an edge-weighted spring embedded layout.

562

563 DATA AVAILABILITY

564 All data supporting the findings of this study are available within the paper and its

565 supplementary information files.

566

567 CODE AVAILABILITY

568 All source code for data analysis in this manuscript, including scripts for k-means clustering of

569 fly phenotypes and network connectivity of CNV and developmental pathway genes, are

570 available on the Girirajan lab GitHub page at https://github.com/girirajanlab/CNV_wing_project.

571

572 ACKNOWLEDGEMENTS

573 The authors thank Drs. Scott Selleck and Claire Thomas for providing fly lines for the

574 experiments, and members of the Girirajan Lab for their helpful discussions and comments on

575 the manuscript. This work was supported by NIH R01-GM121907 and resources from the Huck

576 Institutes of the Life Sciences to S.G., and NIH T32-GM102057 to M.J.

577

578 CONTRIBUTIONS

579 T.Y., M.J., S.Y., and S.G. designed the study. T.Y., S.Y., L.P., S.K., D.J.G., A.S., Y.M., J.I., and

580 Z.C.L. performed the functional experiments. T.Y. and M.J. performed the expression and

581 network experiments. T.Y., M.J., and S.G. analyzed the data and wrote the manuscript with input

582 from all authors.

583

584 COMPETING OF INTERESTS

585 The authors declare that they have no competing interests.

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859 FIGURE LEGENDS

860 Figure 1. Targeted analysis to identify global developmental phenotypes with knockdown

861 of homologs of CNV genes. (A) Strategy for identifying non-neuronal phenotypes and

862 underlying cellular mechanisms for homologs of CNV and known neurodevelopmental genes

863 using the fly wing as a model system. We evaluated 59 Drosophila homologs of genes within 10

864 CNV regions and 20 known neurodevelopmental genes (79 total homologs). Using the UAS-

865 GAL4 system with wing-specific bxMS1096 driver, we knocked down 136 individual RNAi lines

866 for the CNV and neurodevelopmental homologs, and evaluated qualitative and quantitative

867 phenotypes. We next clustered RNAi lines based on severity of qualitative phenotypes, and

868 compared adult wing phenotypes to phenotypes observed with ubiquitous and eye-specific

869 knockdown of homologs. Furthermore, we evaluated underlying cellular mechanisms for the

870 observed wing-specific phenotypes, and examined the connectivity patterns of candidate

871 homologs for developmental phenotypes in multiple human tissue-specific networks. (B)

872 Representative brightfield images of adult wing phenotype severity observed with knockdown of

873 homologs of CNV genes, based on clustering analysis, are shown. (C) Heatmap with k-means

874 clustering of qualitative phenotypes in adult female wings across 136 RNAi lines is shown. The

875 color of each cell represents the frequency of individual fly wings (n=20-25 adult wings) for

876 each RNAi line (x-axis) that show a specific severity (no phenotype, mild, moderate, severe,

877 lethal) for the five qualitative phenotypes assessed (y-axis; wrinkled wings, ectopic veins,

878 missing veins, discoloration, bristle planar polarity), as detailed in Supp. Data 2. Based on these

879 data, we identified clusters for no phenotype (n=75 lines), mild (n=24 lines), moderate (n=10

880 lines), severe (n=21 lines), and lethal (n=6 lines). (D) Summary table of qualitative and

881 quantitative adult wing phenotypes for all tested RNAi lines of homologs of CNV and

882 neurodevelopmental genes. Quantitative phenotype totals do not include lethal RNAi lines for

883 both area and vein length. In addition, L3 vein length totals do not include severe RNAi lines.

884

885 Figure 2. Qualitative adult wing phenotypes of Drosophila homologs of CNV and

886 neurodevelopmental genes. Heatmaps representing the five qualitative adult wing phenotypes

887 for all 136 RNAi lines, with (A) all 59 tested homologs for 10 CNV regions and (B) 20

888 homologs for neurodevelopmental genes (β-catenin, core neurodevelopmental genes, and

889 microcephaly genes), are shown. The color of each cell represents the frequency of each of the

890 five qualitative phenotypes by severity (wrinkled wings, WR; ectopic veins, EV; missing veins,

891 MV; discoloration, DC; bristle planar polarity, BP), ranging from no phenotype to lethal. (C)

892 Representative brightfield images of adult fly wings (scale bar = 500µm) with wing-specific

893 knockdown of homologs of CNV and neurodevelopmental genes showing the five assessed

894 qualitative phenotypes, including discoloration, wrinkled wings, bristle polarity, ectopic veins,

895 and missing veins are shown. The panels in the bxMS1096-GAL4 control and C6836KK112485 images

896 highlight bristle planar polarity phenotypes for the representative images. Black arrowheads

897 highlight ectopic veins and white arrowheads highlight missing veins. Genotypes for the images

898 are: w1118/bxMS1096-GAL4;+; UAS-Dicer2/+, w1118/bxMS1096-GAL4;UAS-RphGD7330 RNAi/+;UAS-

899 Dicer2/+, w1118/bxMS1096-GAL4;UAS-CG15528KK107736 RNAi/+; UAS-Dicer2/+, w1118/bxMS1096-

900 GAL4;UAS-CG6836KK112485 RNAi/+; UAS-Dicer2/+, w1118/bxMS1096-GAL4;+;UAS-

901 CG14182GD2738 RNAi/UAS-Dicer2, and w1118/bxMS1096-GAL4;UAS-kisKK100890 RNAi/+; UAS-

902 Dicer2/+.

903

904 Figure 3. Quantitative adult wing phenotypes of Drosophila homologs of CNV and

905 neurodevelopmental genes. (A) Representative brightfield images of adult fly wings (scale bar

906 = 500µm) with wing-specific knockdown of homologs of CNV and neurodevelopmental genes

907 with size defects are shown. The bxMS1096-GAL4 control image highlights the six veins, including

908 longitudinal veins L2, L3, L4, and L5 as well as the anterior and posterior crossveins (ACV and

909 PCV), that were measured for quantitative analysis. The dotted line in the control image

910 represents the total wing area calculated for each RNAi line. Genotypes for the images are:

911 w1118/bxMS1096-GAL4;+; UAS-Dicer2/+, w1118/bxMS1096-GAL4;UAS-Fmo-2KK109203 RNAi/+; UAS-

912 Dicer2/+, and w1118/bxMS1096-GAL4;+;UAS-TrpmGD4541 RNAi/UAS-Dicer2. (B) Boxplot of L3

913 vein lengths for knockdown of select homologs in adult fly wings (n = 9-91, *p < 0.05, two-

914 tailed Mann–Whitney test with Benjamini-Hochberg correction). Vein measurements for all

915 other longitudinal veins and crossveins (ACV and PCV) for these lines are represented in Supp

916 Fig. 2. (C) Boxplot of wing areas for knockdown of select homologs in adult fly wings (n = 9-

917 91, *p < 0.05, two-tailed Mann–Whitney test with Benjamini-Hochberg correction). All boxplots

918 indicate median (center line), 25th and 75th percentiles (bounds of box), and minimum and

919 maximum (whiskers), with red dotted lines representing the control median.

920

921 Figure 4. Comparison of wing-specific, eye-specific, and ubiquitous knockdown of

922 homologs of CNV and known neurodevelopmental genes. (A) Heatmap with the penetrance

923 of phenotypes with ubiquitous knockdown (da–GAL4) of select homologs of CNV genes,

924 compared to their adult wing-specific (bxMS1096–GAL4) phenotypic severity is shown. (B)

925 Boxplots of Flynotyper-derived phenotypic scores for adult eyes with eye-specific knockdown

926 (GMR-GAL4) of select homologs of CNV and neurodevelopmental genes, normalized as fold-

927 change (FC) to control values (n = 7–40, *p < 0.05, one-tailed Mann–Whitney test with

928 Benjamini-Hochberg correction). The boxplots are arranged by severity of adult wing

929 phenotypes observed for each RNAi line, while the Flynotyper phenotypic scores are categorized

930 into four severity categories: no change (0–1.1 FC), mild (1.1–1.5 FC), moderate (1.5–2.0 FC),

931 and severe (>2.0 FC). (C) Boxplot showing the average eye phenotypic scores for 66 RNAi lines

932 of select homologs of CNV and neurodevelopmental genes, normalized as fold-change (FC) to

933 control values, by wing phenotypic category (n=4–30 RNAi lines per group). We did not observe

934 any significant changes in eye phenotype severity across the five wing phenotypic categories

935 (Kruskal-Wallis rank sum test, p=0.567, df = 5, χ2 = 3.881). Examples of average eye phenotypic

936 scores for RNAi lines with no phenotype (paraGD3392_1), mild (rlKK115768), and lethal (dlg1GD4689)

937 wing phenotype severity are highlighted in the graph. All boxplots indicate median (center line),

938 25th and 75th percentiles (bounds of box), and minimum and maximum (whiskers), with red

939 dotted lines representing the control median. (D) Representative brightfield adult eye (scale

940 bar = 100 µm) and adult wing (scale bar = 500µm) images with tissue-specific knockdown of

941 homologs of CNV genes are shown. Genotypes for the eye images are: w1118;GMR-GAL4/+;

942 UAS-Dicer2/+, w1118;GMR-GAL4/UAS-LnkKK105731 RNAi; UAS-Dicer2/+, w1118;GMR-

943 GAL4/UAS-mEFTu1GD16961 RNAi; UAS-Dicer2/+. Genotypes for the wing images are:

944 w1118/bxMS1096-GAL4;+; UAS-Dicer2/+, w1118/bxMS1096-GAL4; UAS-LnkKK105731 RNAi/+; UAS-

945 Dicer2/+, and w1118/bxMS1096-GAL4; UAS-mEFTu1GD16961 RNAi/+; UAS-Dicer2/+.

946

947 Figure 5. Expression patterns of Drosophila homologs and human CNV and

948 neurodevelopmental genes across multiple tissues. (A) Heatmap with expression of fly

949 homologs of select CNV and neurodevelopmental genes in multiple Drosophila larval and adult

950 tissues, derived from the FlyAtlas Anatomical Microarray dataset, compared with adult wing

951 phenotype severity, is shown. Expression values are grouped into no expression (<10 fragments

952 per kilobase of transcript per million reads, or FPKM), low (10–100 FPKM), moderate (100–500

953 FPKM), high (500–1000 FPKM), and very high (>1000 FPKM) expression categories. (B)

954 Heatmap with expression of select human CNV and neurodevelopmental genes in multiple adult

955 tissues, derived from the Genotype-Tissue Expression (GTEx) dataset v.1.2, is shown.

956 Expression values are grouped into no expression (<3 transcripts per million reads, or TPM), low

957 (3–10 TPM), moderate (10–25 TPM), high (25–100 TPM), and very high (>100 TPM)

958 expression categories. X symbols denote preferential expression in a particular tissue (see

959 Methods). Expression data for all CNV and neurodevelopmental genes are provided in Supp.

960 Data 6.

961

962 Figure 6. Drosophila homologs of CNV and neurodevelopmental genes show altered levels

963 of apoptosis and proliferation. (A) Larval imaginal wing discs (scale bar = 50 µm) stained with

964 nuclear marker DAPI, apoptosis marker dcp1, and cell proliferation marker pH3 illustrate altered

965 levels of apoptosis and cell proliferation due to wing-specific knockdown of select fly homologs

966 of CNV genes. We quantified the number of stained cells within the wing pouch of the wing disc

967 (white box), which becomes the adult wing. Additional representative images of select homologs

968 are presented in Supp Fig. 5. Genotypes for the wing images are: w1118/bxMS1096-GAL4;+; UAS-

969 Dicer2/+, w1118/bxMS1096-GAL4;+; UAS-AatfGD7229 RNAi/UAS-Dicer2, w1118/bxMS1096-GAL4;UAS-

970 Pp4-19CGD9561/+; UAS-Dicer2/+, w1118/bxMS1096-GAL4;+; UAS-Atx2GD11562 RNAi/UAS-Dicer2,

971 and w1118/bxMS1096-GAL4;+; UAS-SinGD7027 RNAi/UAS-Dicer2. (B) Box plot of dcp1-positive

972 cells in larval wing discs with knockdown of select fly homologs of CNV and

973 neurodevelopmental genes, normalized to controls (n = 7–18, *p < 0.05, two-tailed Mann–

974 Whitney test with Benjamini-Hochberg correction). We note that several RNAi lines showed

975 severe dcp1 staining across the entire wing disc and could not be quantified. The number of dcp1

976 positive cells were calculated manually. (C) Box plot of pH3-positive cells in the larval wing

977 discs with knockdown of select fly homologs of CNV and neurodevelopmental genes,

978 normalized to controls (n = 6–18, *p < 0.05, two-tailed Mann–Whitney test with Benjamini-

979 Hochberg correction). The number of pH3 positive cells were calculated using the

980 AnalyzeParticles function in ImageJ. All boxplots indicate median (center line), 25th and 75th

981 percentiles (bounds of box), and minimum and maximum (whiskers), with red dotted lines

982 representing the control median.

983

984 Figure 7. Candidate Drosophila homologs of genes within CNV regions interact with

985 conserved signaling pathways. Larval imaginal wing discs (scale bar = 50 µm) stained with (A)

986 wingless, (B) patched, (C) engrailed, and (D) delta illustrate disrupted expression patterns for

987 proteins located within the Wnt (wingless), Hedgehog (patched and engrailed), and Notch (delta)

988 signaling pathways due to wing-specific knockdown of select fly homologs of CNV and

989 neurodevelopmental genes. Dotted yellow boxes represent expected expression patterns for

990 signaling proteins in bxMS1096-GAL4 control images. White arrowheads and dotted white boxes

991 highlight disruptions in expression patterns of signaling proteins with knockdown of CNV or

992 neurodevelopmental genes. Additional representative images of select homologs are presented in

993 Supp Fig. 7. Genotypes for the wing images are: w1118/bxMS1096-GAL4;+; UAS-Dicer2/+,

994 w1118/bxMS1096-GAL4;UAS-Pp4-19CGD9561/+; UAS-Dicer2/+, w1118/bxMS1096-GAL4;UAS-

995 Cbp20KK109448/+; UAS-Dicer2/+, w1118/bxMS1096-GAL4;+; UAS-SinGD7027 RNAi/UAS-Dicer2,

996 w1118/bxMS1096-GAL4;+; UAS-AatfGD7229 RNAi/UAS-Dicer2, and w1118/bxMS1096-GAL4;UAS-

997 Klp61FGD14149/+; UAS-Dicer2/+.

998

999 Figure 8. Connectivity of human CNV genes with conserved signaling pathway genes in

1000 human tissue-specific networks. (A) Representative diagrams of eight human CNV and

1001 neurodevelopmental genes whose fly homologs disrupt the Notch signaling pathway and 57

1002 human Notch signaling genes within kidney, heart, and brain-specific gene interaction networks

1003 are shown. Yellow nodes represent CNV and neurodevelopmental genes, pink nodes represent

1004 Notch signaling pathway genes, and green nodes represent connector genes within the shortest

1005 paths between CNV and Notch pathway genes. (B) Violin plots showing the average

1006 connectivity (i.e. inverse of shortest path lengths) of CNV genes to genes in Hedgehog, Notch,

1007 and Wnt signaling pathways across the tested tissue-specific networks (n=322–810 pairwise

1008 interactions, *p < 0.05, two-tailed Welch’s t-test with Benjamini-Hochberg correction). (C)

1009 Table showing enriched clusters of Gene Ontology (GO) Biological Process terms for connector

1010 genes observed for each signaling pathway in the three tested tissue-specific networks,

1011 categorized by enrichments in ubiquitous, neuronal, and non-neuronal tissues (p<0.05, Fisher’s

1012 Exact test with Benjamini-Hochberg correction).

1013

1014 Figure 9. A multigenic model for neuronal and non-neuronal phenotypes associated with

1015 pathogenic CNVs. Schematic of a multigenic model for neuronal and non-neuronal phenotypes

1016 associated with pathogenic CNVs. While a subset of genes within CNV regions contribute

1017 towards tissue-specific phenotypes, a majority of genes contribute towards both neuronal and

1018 non-neuronal phenotypes through disruption of developmental signaling pathways and global

1019 biological processes.

1020 SUPPLEMENTARY LEGENDS

1021 Supplementary Figure 1. Quantitative vein length phenotypes for select Drosophila

1022 homologs of CNV and neurodevelopmental genes. Boxplots of longitudinal veins (A) L2, (B)

1023 L4, (C) L5, and (D) anterior crossvein (ACV) and (E) posterior crossvein (PCV) lengths for

1024 knockdown of select homologs in adult fly wings (n = 9-91, *p < 0.05, two-tailed Mann–

1025 Whitney test with Benjamini-Hochberg correction). All boxplots indicate median (center line),

1026 25th and 75th percentiles (bounds of box), and minimum and maximum (whiskers), with red

1027 dotted lines representing the control median.

1028

1029 Supplementary Figure 2. Comparisons of eye-specific and wing-specific knockdowns for

1030 select Drosophila homologs of CNV and neurodevelopmental genes. Boxplots of Flynotyper-

1031 derived phenotypic scores for 66 tested adult eyes with eye-specific knockdown (GMR-GAL4) of

1032 select homologs of CNV and neurodevelopmental genes, normalized as fold-change (FC) to

1033 control values (n = 1–40, *p < 0.05, one-tailed Mann–Whitney test with Benjamini-Hochberg

1034 correction). RNAi lines that do not show any observable qualitative adult wing phenotypes,

1035 including lines that show wing measurement phenotypes, are represented in (A), and RNAi lines

1036 with observable mild to lethal qualitative wing phenotypes are represented in (B). All boxplots

1037 indicate median (center line), 25th and 75th percentiles (bounds of box), and minimum and

1038 maximum (whiskers), with red dotted lines representing the control median.

1039

1040 Supplementary Figure 3. Expression of Drosophila homologs of CNV and

1041 neurodevelopmental genes in larval and adult tissues. Venn diagrams representing the

1042 number of 76/77 tested fly homologs for CNV and neurodevelopmental genes expressed (>10

1043 fragments per kilobase of transcript per million reads, or FPKM) in (A) larval (central nervous

1044 system or CNS, gut, trachea, and fat body) and (B) adult tissues (brain, gut, heart and fat body),

1045 are shown.

1046

1047 Supplementary Figure 4. Additional Drosophila homologs of CNV and neurodevelopmental

1048 genes show altered levels of cell proliferation and apoptosis. Larval imaginal wing discs

1049 (scale bar = 50 µm) stained with nuclear marker DAPI, apoptosis marker dcp1, and cell

1050 proliferation marker pH3 illustrate altered levels of cell proliferation and apoptosis due to wing-

1051 specific knockdown of select fly homologs of CNV genes. We examined changes in the number

1052 of stained cells within the wing pouch of the wing disc (white box), which becomes the adult

1053 wing. Genotypes for the wing images are: w1118/bxMS1096-GAL4;+; UAS-Dicer2/+, w1118/bxMS1096-

1054 GAL4;UAS-Cbp20KK109448/+; UAS-Dicer2/+, w1118/bxMS1096-GAL4;+; UAS-dlg1GD4689

1055 RNAi/UAS-Dicer2, w1118/bxMS1096-GAL4;UAS-CG8888GD3777/+; UAS-Dicer2/+, w1118/bxMS1096-

1056 GAL4;+; UAS-UQCR-C2GD11238 RNAi/UAS-Dicer2, w1118/bxMS1096-GAL4;+; UAS-ACCGD3482

1057 RNAi/UAS-Dicer2, w1118/bxMS1096-GAL4;UAS-Klp61FGD14149/+; UAS-Dicer2/+, and

1058 w1118/bxMS1096-GAL4;UAS-RphGD7330 RNAi/+;UAS-Dicer2/+.

1059

1060 Supplementary Figure 5. Select female and male Drosophila homologs of CNV and

1061 neurodevelopmental genes show altered levels of cell proliferation and apoptosis. (A) Larval

1062 imaginal wing discs (scale bar = 50 µm) stained with nuclear marker DAPI, apoptosis marker

1063 dcp1, and cell proliferation marker pH3 illustrate altered levels of cell proliferation and apoptosis

1064 due to wing-specific knockdown of select fly homologs of CNV genes in females and males. We

1065 examined changes in the number of stained cells within the wing pouch of the wing disc (white

1066 box), which becomes the adult wing. Genotypes for the wing images are: w1118/bxMS1096-GAL4;+;

1067 UAS-Dicer2/+, w1118/bxMS1096-GAL4;+; UAS-lgsGD1241/UAS-Dicer2, w1118/bxMS1096-GAL4;+;

1068 UAS-Sra-1GD11477/UAS-Dicer2, and w1118/bxMS1096-GAL4;UAS-CG11035KK101201 RNAi+; UAS-

1069 Dicer2/+. (B) Box plot of dcp1-positive cells in larval wing discs with knockdown of select fly

1070 homologs of CNV and neurodevelopmental genes, normalized to controls (n = 9–13, *p < 0.05,

1071 two-tailed Mann–Whitney test with Benjamini-Hochberg correction). The number of dcp1

1072 positive cells were calculated manually. (C) Box plot of pH3-positive cells in the larval wing

1073 discs with knockdown of select fly homologs of CNV and neurodevelopmental genes,

1074 normalized to controls (n = 9–13, *p < 0.05, two-tailed Mann–Whitney test with Benjamini-

1075 Hochberg correction). The number of pH3 positive cells were calculated using the

1076 AnalyzeParticles function in ImageJ. All boxplots indicate median (center line), 25th and 75th

1077 percentiles (bounds of box), and minimum and maximum (whiskers), with red dotted lines

1078 representing the control median.

1079

1080 Supplementary Figure 6. Additional Drosophila homologs of genes within CNV regions

1081 interact with conserved signaling pathways to induce developmental phenotypes. Larval

1082 imaginal wing discs (scale bar = 50 µm) stained with (A) wingless, (B) patched, (C) engrailed,

1083 and (D) delta illustrate disrupted expression patterns for proteins located within the Wnt

1084 (wingless), Hedgehog (patched and engrailed), and Notch (delta) signaling pathways due to

1085 wing-specific knockdown of additional fly homologs of CNV and neurodevelopmental genes.

1086 Dotted yellow boxes represent expected expression patterns for signaling proteins in bxMS1096-

1087 GAL4 control images. White arrowheads and dotted white boxes highlight disruptions in

1088 expression patterns of signaling proteins with knockdown of CNV or neurodevelopmental genes.

1089 Genotypes for the wing images are: w1118/bxMS1096-GAL4;+; UAS-Dicer2/+, w1118/bxMS1096-

1090 GAL4;+; UAS-dlg1GD4689 RNAi/UAS-Dicer2, w1118/bxMS1096-GAL4;+; UAS-Tsf2GD2442

1091 RNAi/UAS-Dicer2, w1118/bxMS1096-GAL4;+; UAS-Atx2GD11562 RNAi/UAS-Dicer2, w1118/bxMS1096-

1092 GAL4;+; UAS-UQCR-C2GD11238 RNAi/UAS-Dicer2, w1118/bxMS1096-GAL4;+; UAS-nudEGD15226

1093 RNAi/UAS-Dicer2, and w1118/bxMS1096-GAL4;+; UAS-ACCGD3482 RNAi/UAS-Dicer2.

1094

1095 Supplementary Figure 7. Tissue-specific network diagrams showing connectivity of human

1096 CNV genes with conserved signaling pathway genes. Representative network diagrams of nine

1097 human CNV and neurodevelopmental genes whose fly homologs disrupt the (A) Wnt and (B)

1098 Hedgehog signaling pathway and 162 human Wnt and 46 human Hedgehog signaling genes

1099 within kidney, heart, and brain-specific gene interaction networks are shown. Yellow nodes

1100 represent CNV and neurodevelopmental genes, pink nodes represent Notch signaling pathway

1101 genes, and green nodes represent connector genes within the shortest paths between CNV and

1102 Notch pathway genes.

1103

1104 Supplementary Data 1 (Excel file). Drosophila homologs of human CNV and

1105 neurodevelopmental genes as determined using DIOPT v.7.1.

1106

1107 Supplementary Data 2 (Excel file). Qualitative and quantitative adult wing phenotypic data for

1108 Drosophila homologs of human CNV and neurodevelopmental genes. This file shows the raw

1109 frequencies of severity for the five qualitative wing phenotypes and average areas and vein

1110 lengths for all 136 female and male tested RNAi lines. In addition, this file also includes k-means

1111 clustering analysis for the female RNAi lines.

1112

1113 Supplementary Data 3 (Excel file). Summary of adult wing qualitative and quantitative

1114 phenotypes by Drosophila homologs. This file summarizes qualitative k-means clustering and

1115 longitudinal L3 vein length and wing area changes for all 136 RNAi lines by fly homologs. We

1116 define discordant homologs when RNAi lines for the same homologs showed inconsistent wing

1117 phenotypes. For each homolog with multiple RNAi lines, we checked discordance among RNAi

1118 lines for no phenotype versus any qualitative or quantitative phenotypes, followed by

1119 discordance for small or large quantitative phenotypes.

1120

1121 Supplementary Data 4 (Excel file). Phenotypes of mouse knockdown models for homologs of

1122 CNV genes. This file lists lethality and neuronal and non-neuronal phenotypes, categorized using

1123 top-level Mammalian Phenotype Ontology terms, for knockdown models of 130 mouse

1124 homologs of CNV genes derived from the Mouse Genome Informatics (MGI) database.

1125

1126 Supplementary Data 5 (Excel file). Summary of eye-specific and wing-specific phenotypes for

1127 fly homologs. This file summarizes eye-specific and wing-specific phenotypes by severity

1128 category for 66 RNAi lines by fly homologs of CNV and neurodevelopmental genes. Eye

1129 phenotype severity is defined by Flynotyper phenotypic scores with fold-change (FC)

1130 normalization to control as follows: no change (0–1.1 FC), mild (1.1–1.5 FC), moderate (1.5–2.0

1131 FC), and severe (>2.0 FC). Wing phenotype severity is defined by k-means clustering for

1132 qualitative phenotypes and quantitative size changes as listed in Supp. Data 3.

1133

1134 Supplementary Data 6 (Excel file). Tissue-specific expression of Drosophila homologs and

1135 human CNV and neurodevelopmental genes. This file lists expression values across multiple fly

1136 and human tissues for all 79 Drosophila homologs and 150 human genes. Fly expression data

1137 (fragments per kilobase of transcript per million reads, or FPKM) was derived from the FlyAtlas

1138 Anatomical Microarray dataset, and human expression data (transcripts per million reads, or

1139 TPM) was derived from the Genotype-Tissue Expression (GTEx) dataset v.1.2.

1140

1141 Supplementary Data 7 (Excel file). Summary of immunostaining of the larval imaginal wing

1142 discs. This file summarizes changes in apoptosis (27 homologs), cell proliferation (27

1143 homologs), and Wnt, Hedgehog, and Notch signaling pathway proteins (14 homologs), along

1144 with qualitative and quantitative adult wing phenotypes (as listed in Supp. Data 2.), for female

1145 and male fly homologs.

1146

1147 Supplementary Data 8 (Excel file). Tissue-specific network connectivity for candidate CNV

1148 genes and signaling pathway genes. This file lists the shortest path lengths between nine

1149 candidate CNV genes and 265 genes within Wnt, Hedgehog, and Notch signaling pathways for

1150 heart, kidney, and brain-specific gene interaction networks, along with the connector genes that

1151 are within the shortest paths. Enriched Gene Ontology (GO) Biological Process, Cellular

1152 Component, and Molecular Function terms for sets of connector genes for each signaling

1153 pathway in each tissue-specific networks are also represented.

1154

1155 Supplementary Data 9 (Excel file). List of Drosophila stocks used for experiments, including

1156 stock numbers and genotypes.

1157

1158 Supplementary Data 10 (Excel file). Statistics for all experimental data. This file shows all

1159 statistical information (sample size, mean/median/standard deviation of datasets, test statistics, p-

1160 values, degrees of freedom, confidence intervals, and Benjamini-Hochberg FDR corrections) for

1161 all data. Statistical information for Kruskal-Wallis test includes factors, degrees of freedom, test

1162 statistics, and post-hoc pairwise Wilcoxon tests with Benjamini-Hochberg correction.

Figure 1

A Experimental strategy B Phenotypic analysis of adult wings in Drosophila × bxMS1096-GAL4 control No phenotype Mild Moderate Severe Wing-specific driver UAS-RNAi bxMS1096-GAL4 (79 genes)

C K-means clustering of qualitative wing data Phenotyping of adult wings Qualitative analysis Quantitative analysis No observable phenotype Mild Moderate Severe Lethal Frequency (n=75) (n=24) (n=10) (n=21) (n=6) • Wrinkled wings • Longitudinal veins 1.0 • Ectopic veins (L2,L3,L4,L5) 0.8 • Missing veins • Crossveins (ACV, PCV) • Discoloration • Wing area 0.6 Bristle planar polarity • 0.4

0.2 Tissue-specific effects Phenotypic severity 0.0 • Compare to ubiquitous knockdown 136 Drosophila RNAi lines • Compare to eye-specific knockdown • Gene expression across multiple tissues (Human and flies) D Summary of qualitative and quantitative adult wing phenotypes

Cellular processes and Qualitative phenotypes Quantitative phenotypes Fly homologs RNAi lines developmental pathways No pheno. Mild Moderate Severe Lethal Wing area L3 vein length • Apoptosis CNV genes • Cell proliferation (15q11.2, 15q13.3, 16p11.2, Distal 16p11.2, 16p12.1, 16p13.11, 17q12, 1q21.1, 3q29, 7q11.23) 95 45 21 7 16 6 65 53 • Disruptions in signaling pathways Known neurodevelopmental genes • Human tissue-specific network analysis (β-catenin, core genes, microcephaly) 41 30 3 3 5 0 24 21 (Brain, heart, kidney) Total 136 7524 10 21 6 89 74 bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 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 4.0 International license.

Figure 2 A Qualitative wing phenotypes of homologs of CNV genes 15q11.2 15q13.3 16p11.2 Distal 16p11.2 16p12.1 16p13.3 17q12 1q21.1 3q29 7q11.23 BP MV DC WR Lethal EV BP MV DC WR Severe EV BP MV DC

Mod. WR EV BP MV DC Mild WR EV BP MV DC WR EV No pheno. GD4689 GD7229 GD3061 GD9561 GD6674 GD9689 GD2249 GD1241 GD6991 GD6343 GD3482 GD9362 GD4372 GD2442 GD9322 GD9260 GD7330 GD6560 GD9986 GD7027 GD15691 GD16961 GD11699 GD564_1 GD11803 GD11383 GD11562 GD564_2 GD15226 GD17096 GD13375 GD14217 GD10067 KK104454 KK105731 KK102281 KK108714 KK100134 KK108281 KK101874 KK101201 KK108049 KK107404 KK107824 KK102573 KK108623 KK100453 KK104813 KK109203 KK102092 KK108812 KK108732 KK104762 KK115768 KK109448 KK107680 KK102370 KK108740 KK101936 KK105756 KK109046 KK108466 KK107736 KK116285 KK107305 KK112485 KK110317 KK109207 KK107577 KK108227 KK109717 KK108387 KK101457 GD8318_1 GD4541_1 GD1138_1 GD2738_1 GD3777_1 GD9586_1 GD1266_1 GD2856_1 GD8318_2 GD2738_2 GD4541_2 GD1266_2 GD2856_2 GD1138_2 GD3777_2 GD9586_2 GD11477_1 GD14152_1 GD11238_1 GD14152_2 GD11477_2 GD11238_2 ke r l lgs Sin ke Lnk Pa1 Aatf lgs Cen Rph dlg1 Tsf2 Pis Fsn Sin ACC Lnk shu Lim1 app Pak app Trpm coro Pa1 Ald1 Atx2 Aatf spin dlg1 Pcyt1 Ald1 Marf1 spict nudE a−1 a−1 Lim1 MRP nudE Doc2 Nsun5 Trpm Trpm Sgf29 Pcyt1 Marf1 Syx1A Syx1A Sgf29 Nsun5 Ada2a Nsun5 PIG−Z Cbp20 LIMK1 LIMK1 PIG−X Sr Sr Fmo−2 CG9150 Fmo−2 Fmo−2 PIG−Wb mEFTu1 CG5589 Pp4−19C mEFTu1 CG2059 CG15528 CG5589 CG8204 CG6836 CG8888 CG8888 Grip128 Grip128 BCL7−li α 5 nAChR CG15309 CG11035 CG17841 CG10465 CG15528 CG14411 CG14182 CG11035 CG14411 CG11035 CG14182 α 5 nAChR α 5 nAChR CLIP−190 BCL7−li UQCR−C2 UQCR−C2 UQCR−C2 B Qualitative wing phenotypes of homologs C Representative qualitative phenotypes of neurodevelopmental genes in the adult wing Frequency β-catenin genes Core genes Microcephaly bxMS1096-GAL4 RphGD7330 BP 1.0 MV DC WR Lethal EV BP 0.8 MV DC

WR Discoloration (DC) Severe EV 0.6 BP KK112485 CG14182GD2738_1 MV CG6836 DC

Mod. WR EV 0.4 BP MV DC Mild WR EV

0.2 Ectopic vein (EV) BP Bristle polarity (BP) MV DC CG15528KK107736 kisKK100890 WR EV

No pheno. 0.0 GD39 GD15 GD2619 GD9974 GD1372 GD6852 GD2905 GD2535 rk GD450_1 GD14481 GD11734 GD10101 GD450_2 GD13500 GD12537 KK108009 KK108534 KK109063 KK101542 KK100878 KK109278 KK100890 KK102545 KK107895 KK101537 KK108197 GD3392_1 GD1375_1 GD6121_1 GD9502_1 GD3392_3 GD3392_2 GD6121_2 GD9502_2 GD1375_2 GD14451_1 GD14383_1 GD14149_1 GD14383_2 GD14451_2 GD14149_2 Eph rk arm kis Eph rk rk Wrinkled (WR) Wrinkled Eph Gat Tpc1 asp asp arm Pten para Pten para para para Nrx−1 Sas−4 Missing vein (MV) org−1 Ube3a Ube3a Sas−4 Prosap MCPH1 Cadps Cadps Scs β G Nrx−1 Nrx−1 Prosap MCPH1 Grip163 Grip163 Klp61F Klp61F Cep135 Cep135 bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 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 4.0 International license.

Figure 3 A Representative quantitative phenotypes in the adult wing Smaller area/L3 vein length Larger area/L3 vein length L5

PCV L4 ACV L3

L2 bxMS1096-GAL4 Fmo-2KK109203 TrpmGD4541_2

B L3 vein lengths for select homologs C Wing area for select homologs epha ly epha ly gen es -catenin -catenin q21 .1 5q13 .3 q21 .1 5q13 .3 q2 9 q11 .23 6p11 .2 q2 9 q11 .23 6p11 .2 6p1 3.11 5q11 .2 5q11 .2 1 3 7 1 1 17q12 β Core Genes Microc 1 3 7 1 1 1 β Core Microc 1 1 17q12 Distal 16p11.2 Distal 16p11.2 2500 20 * * ** * * * * * * *

) 15 * * 2 * * * * * * 2000 * * * * * * * * * µm * 5 * * * * * * * 10 * *

Le ngth ( μ m) *

1500 Area (×10 * 5 * * * *

1000 0 124 1 656 0 124 1 656 0 AL 4 AL 4 1156 2 1138 3 1709 6 1006 7 1375 _1 3777 _1 9586 _1 4541 _2 4541 _2 1375 _1 GD GD GD GD -G 14149 _2 11477 _2 -G 11477 _2 14451 _2 GD GD GD GD KK 1092 03 KK 1094 48 KK 1018 74 KK 1087 32 KK 1057 31 KK 1157 68 KK 1087 14 KK 1014 57 KK 1004 53 KK 1092 78 KK 1015 37 KK 1081 97 KK 1008 90 KK 1092 03 KK 1124 85 KK 1018 74 KK 1087 32 KK 1057 31 KK 1025 73 KK 1157 68 KK 1087 14 KK 1084 66 KK 1092 78 KK 1015 37 KK 1081 97 KK 1008 90 GD GD GD GD GD GD GD GD lgs GD GD lgs rl rl kis kis Fsn 91 50 91 50 Atx2 Pa k asp Ln k Pa k asp Ln k Aa tf Pten Pten 109 6 MS 20 59 bp 20 109 6 MS 68 36 20 59 82 04 org-1 88 MRP Sg f29 org-1 Trpm Trpm un 5 Ns 153 09 178 41 153 09 Fmo-2 p6 1F Fmo-2 C Sra-1 LIMK1 CG Nrx-1 Sra-1 CG Prosap Prosap bx bx CG CG CG CG Kl CG CG CG CG bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 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 4.0 International license.

Figure 4

A Ubiquitous knockdown B Eye versus wing phenotypes

No wing Wing measurement Mild wing Mod. wing Sev. wing phenotype phenotype phenotype phenotype phenotype Lethal 3 * * * * * * Severe * * * * * Fly RNAi line LarvalPupal lethalityWing lethality Nodefects gross defectsWing phenotypeCNV region sco re (FC) 2 * * * Moderate Sra−1GD11477_2 ns

KK102281 15q11.2 yp ic ns spict Mild Grip128GD14152_1 1 CG14411GD8318_2 GD9986 15q13.3 Trpm phen ot No change Ald1KK107680 KK109207 Pis Eye 0 coroGD14217 GD7330 290 5 261 9 124 1 273 8 733 0 468 9

Rph 1350 0 1696 1 KK102573 GD GD GD GD GD GD

CG17841 GD GD KK107577 KK105756 16p11.2 KK 1048 13 KK 1057 56 KK 1057 31 KK 102573 KK 1157 68 KK 1094 48 KK 1088 12 rl CG10465 lgs Rph KK115768 dlg1 Tpc1 Nrx-1 GMR-GAL4 Lnk Pten

rl spin 141 82

GD9561 bp 20 Sgf29

Pp4−19C 104 65 C KK108623 CR -C2 mEFTu1 Doc2 CG CG17841 KK108714 CG CG15309 UQ Atx2GD11562 Sgf29KK107577 C Severity of eye D Representative eye and wing images LnkKK105731 Distal spinKK104813 16p11.2 versus wing phenotype GMR-GAL4 bxMS1096-GAL4 mEFTu1GD16961 3 lgsGD1241 ns KK109203 1q21.1 Fmo−2 KK115768 CG8888GD3777_1 rl dlg1GD4689 FsnGD11383 2 dlg1GD4689 Control Tsf2GD2442 Cbp20KK109448 3q29 sc ore (FC) CG6836KK112485 PakKK101874

KK109717 Average e ye

PIG−X no typic 1 KK107404 PIG−Z GD3392_1

para KK105731 Ubiquitous Wing ph e Lnk Ubiquitous (da-GAL4) Wing-specific MS1096 0 Full penetrance (bx -GAL4)

Partial penetance Lethal Mild

No phenotype Le thal GD16961 Severe Se vere ode rate

Moderate he notype M Mild phenotype

Wing size changes Measurement No p

No phenotype mEFTu1 Wing phenotypic severity Figure 5 Larval Adult CNV regions bioRxiv preprint not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailable Drosophila Drosophila (FlyAtlas) (FlyAtlas) Muscle Kidney Heart Brain Lung Liver Wing phenotype B A doi:

CYFIP1 15q11.2 https://doi.org/10.1101/855338 Fat body Fat body Expression patternsofflyhomologs ofCNVgenesacrossmultipleflytissues

NIPA2 Trachea CHRNA7

15q13.3 Heart Brain

X X TRPM1 CNS Gut Gut

X ALD O A

X CORO1A DOC2A FAM57B Expression patternsofCNVgenesacrossmultiplehumantissues KCTD13 16p11.2 MRP MAPK3 Cen

X X PPP4C spin

QPRT under a

asp ; this versionpostedDecember6,2019. X YPEL3 Cadps ATP2A1 Sgf29 ATXN2L CC-BY 4.0Internationallicense SH2B1 Distal 16p11.2 CG9150 SPNS1 Fsn TUFM CG10465 CCDC101 Lim1 CDR2 Nrx−1 16p12.1 PIG−X

X X POLR3E Pten

X X X X UQCRC2 ABCC6 para MYH11 16p13.11 Lnk DHRS11 Trpm AATF Ube3a DDX52 17q12 Fmo−2 . LHX1 CG2059 The copyrightholderforthispreprint(whichwas

X X ACACA lgs HNF1B CG8888 BCL9 1q21.1 CLIP−190 X X X FMO5 CG11035

X GJA5 BDH1 CG17841 DLG1 LIMK1 FBXO45 rl MFI2 org−1 NCBP2 3q29 CG15309 PAK2 BCL7−like Sra−1 X PIGX kis

X TFRC ABHD11 Rph BCL7B Pak CLIP2 Aatf DNAJC30 7q11.23 X Atx2 ELN nAChRα5

X LIMK1 arm

X X STX1A CG5589 CLDN3 Klp61F GTF2IRD1 Tsf2 CTNNB1 β-catenin Cbp20 NRXN1 Pp4−19C

X PTEN TBX1 mEFTu1 CADPS2 UQCR−C2 Core genes ACC

X CHD8 Sin

X SCN1A UBE3A dlg1

ASPM Microcephaly Expression Wing phenotype KIF11 Mild No phenotype No expression Low Moderate High Very high Wing sizechanges Moderate Severe Lethal Expression No expression Low Moderate High Very high Figure 6 A SinGD7027 Atx2GD11562 Pp4-19CGD9561 AatfGD7229 bxMS1096-GAL4 DAPI Cellular processesinlarvalwingdiscs bioRxiv preprint not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailable doi: https://doi.org/10.1101/855338 dcp1 ppoi Cellproliferation Apoptosis Increased Increased Increased Increased pH3 under a ; this versionpostedDecember6,2019. CC-BY 4.0Internationallicense Decreased Decreased Decreased Decreased

Normalized pH3 Normalized dcp1 C B positive cells positive cells 0.0 0.5 1.0 1.5 0 1 2 3 4 5 . The copyrightholderforthispreprint(whichwas MS1096 bx -GAL4 (GD) bxMS1096-GAL4 (GD) MS1096 bx -GAL4 (KK) MS1096 * bx -GAL4 (KK) armKK102545 * KK109448 KK115768 * Cbp20 rl GD11562 * Atx2 KK105756 ns GD9561 * CG10465

Pp4-19C ns *

Quantification ofproliferatingcells GD3482 GD1241 ACC

lgs Quantification ofapoptoticcells * GD7229 GD11383 ns Aatf Fsn KK108197 * org-1 GD4689 ns LnkKK105731 * dlg1 * GD7330 KK101201 * Rph CG11035 KK107577 * Sgf29 KK107577 *

* Sgf29 Sra-1GD11477_2 *

* GD1241 rkKK108009 lgs * Klp61FGD14149_2 KK108009 * GD7027 * rk

Sin GD11477_2 * rl KK115768 * Sra-1 * CG11035KK101201 KK100890 *

ns kis KK109203 Fmo-2 GD11238_1 * ns UQCR-C2 UQCR-C2GD11238_1 ns KK108197 * CG15309 KK108714 org-1 KK102573 ns CG17841 KK108714 * sns ns CG15309 CG10465 KK105756 *

ns KK102573 ACCGD3482 CG17841

GD15226 KK109203 * nudE Fmo-2 KK100890 ns

kis KK105731 * ns Lnk FsnGD11383 * * GD3777_1 CG8888GD3777_1 CG8888 dlg1GD4689 * bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 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 4.0 International license.

Figure 7 Disruption of signaling pathways in larval wing discs bxMS1096-GAL4 Pp4-19CGD9561 Cbp20KK109448 SinGD7027 AatfGD7229 Klp61FGD14149_2 A Wnt wingless

B patched

C Hedgehog engrailed

D delta Notch bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 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 4.0 International license.

Figure 8 A Human CNV genes interact with Notch signaling pathway genes in multiple tissues Kidney Heart Brain

CTNNB1

DTX4

TCF3

CTNNA1 NDE1

GSK3B

SOCS5 ARF1

RBM39 MTA1

PHLDA1

MAML2

NR3C1

ATXN1L PAFAH1B1

IRF9

NUMBL

TIMP3

FOXO1

NDRG1 NCK1 ABL1

WBP11 PML NUMBL

SMAD3 KAT2A OS9 NOTCH2NL

CD44 DTX3L

HNRNPU

BZW1 CREB1

PSME3

TNS1

HES1 VIM

PSEN1

CANX TRIP6

ATXN1L

NRIP1

COL4A2 KAT2B

SIRT1

INPP5D

CIR1 PLSCR1

TMX1 MCL1

COPS5

TRIM27

TAB2 PTEN LYN

CDKN1B PDCD4

UBN1 HMGN4

TRIP10

PRR11

APP

EZR UBE2K MR1

HEY1 DVL1 FEZ1 SLK

TGIF1

STAT2

DLL4

HSPA4

DLL1 PSMC2

ATP2B4

NCOA3

KARS

RANBP2

CSNK1E PSMA1

RELA HLTF

CTDSP2 CREBBP

FMR1 EP300

NAB1 PPP2CA

PSMC6

CASK TM4SF1 HSP90AA1

LUC7L3

SMAD4

SMARCC1 CDC27

PRKD2

H2AFX

CEP170 PSMA1

CAMSAP2 FYN TLE2 KCTD12

CTCF LAPTM4B AZIN1

NARS NRIP1 UBR2 DAG1

PUF60

KAT6A UBE2D3 BUB3

NOTCH3

ATP2B4

TMED10

GFPT1 MORC3

NCOA3 VIM

LEPROTL1

SLC39A6

BHLHE40

FLNA NFKBIA NCOR1 RB1 NPR1 MAML2 NUMB CTBP1 ZNF146 PTDSS1 GRN PSEN2

MYO1B DDX1 DNAJB6

YY1

CSE1L COPS3

PTP4A2

BRCA1 TP53

QKI RBM10 SFSWAP PSENEN C1D

RAN

MKI67

DLL1 UTP6 P4HB PPP1R2

E2F1 DNAJA1 SRSF2

FNDC3B RAD54B LSM14A

BAZ2B HEYL SOCS5 CIR1 DPYSL2 CDK1 RANBP1

TNPO1 RBPJ

NOTCH2 NCSTN

DEK CTBP2 XPO1

PRPF3 CENPA PAXIP1

KAT2B ATXN1 MAML1 NOTCH1 SYNCRIP SRSF10

CCT5

DVL3 NCK1 NCOR2 DAZAP2 MFNG

CTBP2 SSRP1 SIRT1 PSMD14

CCT6A NAB1 SNW1

CIB1

JAG1

PA2G4

NOTCH2

APH1B DDX23 UBE2D2 ABCE1 FLNA SSR1 TRIOBP PUM2 CBX3

PAPOLA SSBP2 AKAP13 ATXN1 SET

GFPT1 PSMD14

DTX2 HDAC1

TUBB4B DVL2

RAB32 RBPMS

USP1 RASA1 PIK3CA ZC3H11A

PPP4R1

HFE RBBP8

SNW1

TLE4 CSNK1D

HES1 RHOXF2 RNF11

SIRT1 COPS2 KHSRP DKC1 PPP6C

BHLHE40

KIAA0355

NCBP2 NUP153 OAT ATF2 TPM4

NUP98 ABI1 IER2 RAB21 DDX39A NUP98 DTX2 AKT3 UBR5

PRPF4 DHX15

KHDRBS1 COPS3

NOTCH3

SNRPG QKI BCL6 HDAC2

FLAD1 ATP2B4

NARS ADAM10 TCF7L2

TLE1 HSPA4 NUMB HACD2 TLE4

ADAM17

RHOXF2 CDC20 PFN1 TGFB2 P4HA1 PARP1 DHX15 ITGB4

NOTCH1 MAD2L1 ARHGAP5

LAMP1 SRSF1 CCT5 DLG1 ILF3 SNRPD3

XRCC5 POLE2 ATP6V0B USP15 UBE2G1 NCK1

MTF2 KMT2A

PRPF40A MCM7 YES1 ARL1 COIL

RBPJ

PAXIP1 IL6ST

TLE4 UBR5

SNRPD1 EGFR SLC19A1

SAP30 SYT7

MAML2 TAGLN2 CYBRD1

CCT5 HDAC2

KDM2A ABL1 USP1

MAPRE1 SRSF3 HEY1

DAZAP2

XRCC6

PIK3R1 KDM5B

SERBP1 PPP1CA ADAM17 CDKN1A SMARCA4 EIF3A

RBPJ PRKDC PAPOLA CDC25A STK24 CTBP2

MAML1 RBPMS VEGFA EP300

IL6ST

MYC TLE1 HNRNPUL1 COIL DDR1 RAC1 MRPL3 PTBP1

LAMP1 TLE3 PSMD13 SMARCC1 CBFB CAVIN1

GSK3B DLG1 PJA2 NOTCH2 PTBP1

CCNA2 POLR3E

APH1A CDKN1A DEK

RB1CC1

CTNNA1 CLIC1 PPP4R1

JAG2 AATF NDE1 RSBN1

PAICS TOPBP1

CREBBP CALD1 WDR77

PUM2 SRSF2 BCAP31 EWSR1

GNA11 SNW1

EWSR1

LEPROTL1 TACC1 PTPN11 TUBG1 SMARCA5 NUDC

TLE2

NRP1

CKAP5

CREBBP PPP2R5C XRCC5 EP300

MRPL3 MAD2L1 SNRPG HDAC1 NRP2 TRIO GNB2 MAML1 NCBP2 NCOA3 SCARB2

AZIN1

RELA FYN ACTN1 JAG2 AATF PTBP1

HSPE1

SEC24A DKC1 DHX15

PDCD11 ACTL6A CTNNA1 HMOX2 YY1 TOPBP1 ATXN1

DDX1 TK1 CDC20 DNAJB6 USP1 XRCC6 NCBP2

DDR1 VARS UBR5

NUMB KIF11 ID4

JAG1 DLL1 UBE2N CCNB1

CIR1 CTBP1 RANBP9

TMED10 HDAC2

DNAJB6 CUL4B PRKDC RNPS1

ATP6V0D1

SERINC3 CSE1L TMEM109

UPF1

PA2G4 GMEB2

MCM5 TP53

JAG1 HSPA13 IL6ST GNA11

APLP2

PARP1 CCT7 SRSF2 CTBP1 TRIM2

ADAM17 CENPA PSEN1 SSRP1 DLG1

NCBP1 SEPT10 TPD52L2

BUB3 UBE2D4 TRIP13 CCT3 RELA ERBB2

ZMYM4 NOTCH1 DTX1 TRIP13 CCT3

SMAD4 PA2G4 SRP19 PDGFRA EIF3B STK24 ILF3

KAT2B BCS1L CCT7

ATIC TRA2B FLNA YWHAZ TPP1 PRPF3

GTSE1

YWHAZ

RUNX1 STAT3

MTF2 RAC1

HRAS HEY1 NUP62 MTDH PNN

EIF3A CCT3

KHDRBS1 PPP4C HEYL HSPA13 AGPAT2 RANBP9 CCT2 SUGP2 SYNCRIP GNB1 TTC3

TMEM97

TOPBP1

GMPS DKC1 NCSTN MAPRE1 ATP1B1

MORC3 PSME3 NCOR1

NUP85 POLR3E CDKN2C NCOR2 AZIN1 KDM5B

PPP1CA EIF2S1 DLL4 TIMP3

ATIC

EIF4G1

CREB1

UBE2D3

HDAC1 KDELR3

KDELR1 USP34

TPP1 DEK POLR3E HSP90AA1 KLC1 ATXN1L MAD2L1 TLE1

DAZAP2

SMARCA4 XRCC6 RFC3 KIF11 AIMP2

SELENOT

PRIM1

DIMT1 HNRNPA3 UBE2M GNB2 NDE1

CDK4 TROAP PARP1 EIF3B UBE2N HGS

CCNB1

UBE2D2 RNASEH2A C15orf39 DVL2 RNF11

GFPT1

CCNF

DLL3

PPP4C TRIM2 RFNG

TRIP13 CASK CDKN2C VARS CEP126 MSH6

CCT6A NUMBL PAICS

PSENEN

CANX

ACOT7 HRAS

SMARCA4 FUBP1 TMED10 SAP30

UBE2K DTX3 DVL3 HES1 CDK4 SRSF1 DDR1 RAD21 ILF3 SART3 TOMM40

CSE1L KAT2A

UBQLN1

APH1B

PKP4

TLE3 HRAS

BRCA1 RBBP7

AATF RNF126 DTX4

WDR77 PRPF3 UBQLN1 CCT7

ADRM1 HEY2

KAT2A CD151

BRCA1 RB1 PSEN2

UBE2D3 RNF138 NOTCH3 CTDSP2

CSNK1E HAP1

FNDC3B

ARFGAP1

HNRNPUL1

RNPS1 SSRP1 CSNK1D

POLE2

ADAM10

AGPAT2

GNB2 PPP4C RIN1 MBNL2 ILF2 TRIM27 UBE2D2

RUVBL2 NSD2 ILF2 CDC20 UPF3A TRIP6

VARS ANK3

CASP2 FKBP4 TCF3 PSEN2 BRD4

ESR1

PTPRK

GNA11 TMPO RAC1 RB1 BAG6 SFRP1 DVL1 KIF11 TLE3

DVL3 APH1B APH1A

DMWD CDK13 PPP2R1A SMAD3 HGS

UBE2H SPEN

PPP1CC CIB1

RANBP9 HSPA4 ARHGDIA

JAG2 MTUS1

NCOR2 TMBIM6 CSNK1G2 TAL1 PSEN1 PPP1CA SNRPA

TOP2A

DMWD MCM7 CXXC1

BOP1 NCSTN TRA2B UBE2K

TUBG1 BRF1 NONO RNPS1 GNB1 UBE2D4 RNF11 MCM5 NUDC

RFNG YWHAG

CUL4B JPT1 ALCAM APH1A

UBE2M

PBK

UBE2D4 CCNA2

HSPG2 UBE2M PLK1

CIB1

NPTXR

DTX3L

STRN4 RBPMS

ERBB4

CDK1 PSMB3

MTA1

EPHB4 DTX3

DTX1 TLE2

ATP6V0B

ADGRL1 SRP19 CTSC PSMB3

LTBP2

PTCRA BOP1

PPP2R1A TPX2 TOMM20

CCNA2

EWSR1 GRK2 PPP4R1

PSENEN MTA1 AKT2

UBE2H

PTCRA E2F4 ZDHHC17

RECQL5 ATP6V0C LAMP1 MCM4

HGS MFI2 SYNCRIP CASP8

JAK3 ATIC DRD2

TRIM27

USP19 E2F1

CCNF RARB ARL4D PLSCR1

RNF126

ARPC1A

DTX3L

MYCBP

ATP6V0C NFIB

TAOK2 GSK3B

RAC2 GTSE1 FEN1

MYO9B PHB

SNTA1

TP53

ARHGDIA RAD50

DTX3 ASXL1

MLF2

ATP6V0D1 MKI67

DTX4 CSNK1E

OTUB1

NOTCH4

VBP1

RCN2 TMBIM6 E2F1

EMP3

PAX8

CYP2A6

FTH1 ATN1

NCKIPSD

RFNG CASP10 DTX1

PLK1 TOMM40 DVL2 PSME2

RUNX2

EPS8L1

DDX11

BTRC CDC25B UBC

DVL1

FGF18

CDKN3 RAD54B BAG6

ARHGEF1

FZR1

MCRS1

NTRK3

GSTM5 FKBP8

MFNG FOXK1

DGKZ

GRK6 VAMP2

RHOXF2 DTX2

RBPJL

COL11A2 LYN

RAC2

HGH1

GET4

DNAJC4 LFNG

MAML3

HEYL

GSK3A

MFNG

PAPPA2

ESR2

CCDC9

BCAN

INPP5D

LFNG CNV/neurodevelopmental gene Signaling pathway gene Connector gene B Average connectivity of CNV genes to C GO term enrichment for connector genes in signaling pathways genes in tissue-specific networks

Hedgehog Notch Wnt en t ns 0.6 0.6 0.6 bo lism ns ub iquitination

* pa ir ne acetylation on se to stimulus Signaling on se to stress lle organizationane lle

* an developm * pathway Tissue Org Protein folding Protein Proteolysis Histo Org Signal trans du ction Dopaminergic signaling Axon-dendrite transport Cell cycle re DNA Chromatin organization Protein localization Protein meta Regulation of transcription 0.4 * 0.4 0.4 Resp Resp processing/splicingRNA Brain Hedgehog Heart path length path length path length Kidney

Brain 0.2 0.2 0.2 Notch Heart Kidney 1/shortest 1/shortest 1/shortest Brain Wnt Heart 0.0 0.0 0.0 Kidney KidneyHeart Brain KidneyHeart Brain KidneyHeart Brain Ubiquitous GO enrichments Neuronal GO Non-neuronal enrichments GO enrichments bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 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 4.0 International license.

Figure 9

CNV region

( Gene A Gene B Gene C Gene D Gene E Gene F )

Developmental pathways Wnt, Notch, Hedgehog

Biological processes Signal transduction Cell cycle RNA processing Axonal transport Apoptosis Protein degradation

Neuronal phenotypes Non-neuronal phenotypes Autism Cardiac defects Epilepsy Kidney defects Intellectual disability Craniofacial features Micro/macrocephaly Musculoskeletal defects bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 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 4.0 International license.

Supp. Figure 1 A L2 vein length in adult wing B L4 vein length in adult wing C L5 vein length in adult wing 11 .2 11 .2 11 .2 epha ly epha ly epha ly 16 p 16 p 16 p -catenin -catenin -catenin 5q13 .3 q21 .1 q21 .1 5q13 .3 q21 .1 5q13 .3 6p11 .2 q2 9 q11 .23 q2 9 q11 .23 6p11 .2 6p11 .2 q2 9 q11 .23 5q11 .2 5q11 .2 5q11 .2 1 1 β 1 3 7 Distal 17q12 Core Genes Microc 1 3 7 1 Distal 1 17q12 β Core Genes Microc 1 17q12 Core Genes Microc 1 3 7 1 β 1 1 1 Distal 2000 2000 1500 * * * * ns * * * * * * * ns * * * * * * * * * * * * * * * * * * * * * 1500 * * * * 1500 * * * * * * * * * * * * * * * * * * * * 1000 * * * * * * * 1000 1000 * Le ngth ( μm) Le ngth ( μm) Le ngth ( μm)

500 500 500

0 0 0 AL 4 AL 4 AL 4 124 1 656 0 124 1 124 1 656 0 656 0 1138 3 1709 6 1006 7 1138 3 1138 3 1709 6 1709 6 -G 1006 7 1006 7 -G -G 1375 _1 4541 _2 1375 _1 1375 _1 4541 _2 4541 _2 GD GD GD GD GD GD 14451 _2 11477 _2 11477 _2 14451 _2 14451 _2 11477 _2 GD GD GD GD GD GD GD GD GD KK 1087 14 KK 1057 31 KK 1018 74 KK 1025 73 KK 1092 03 KK 1087 32 KK 1157 68 KK 1092 78 KK 1008 90 KK 1084 66 KK 1124 85 KK 1015 37 KK 1081 97 KK 1087 14 KK 1057 31 KK 1087 14 KK 1018 74 KK 1025 73 KK 1092 03 KK 1057 31 KK 1025 73 KK 1018 74 KK 1092 03 KK 1087 32 KK 1157 68 KK 1157 68 KK 1092 78 KK 1087 32 KK 1008 90 KK 1092 78 KK 1008 90 KK 1084 66 KK 1084 66 KK 1124 85 KK 1124 85 KK 1015 37 KK 1015 37 KK 1081 97 KK 1081 97 GD GD GD GD GD GD lgs lgs lgs rl rl rl GD GD GD GD GD GD Fsn 91 50 Fsn Fsn 91 50 91 50 kis kis kis asp Ln k asp asp Ln k Ln k Pa k 109 6 MS Pa k Pa k 109 6 MS 109 6 MS Pten Pten Pten 20 59 Sg f29 org-1 82 04 68 36 20 59 20 59 Sg f29 Sg f29 82 04 org-1 org-1 82 04 68 36 68 36 Trpm Trpm un 5 Ns Trpm un 5 Ns un 5 Ns 178 41 178 41 178 41 CG 153 09 CG CG 153 09 153 09 Fmo-2 Fmo-2 Fmo-2 bx Nrx-1 Sra-1 bx bx Nrx-1 Nrx-1 Sra-1 Sra-1 Prosap Prosap Pros ap CG CG CG CG CG CG CG CG CG CG CG CG CG CG CG D ACV length in adult wing E PCV length in adult wing 11 .2 11 .2 epha ly epha ly 16 p 16 p -catenin -catenin q21 .1 5q13 .3 q21 .1 5q13 .3 6p11 .2 q2 9 q11 .23 6p11 .2 q2 9 q11 .23 5q11 .2 5q11 .2 1 1 1 17q12 β Core Genes Microc 1 3 7 1 1 17q12 β Core Genes Microc 3 7 1 1 Distal Distal 250 100 * * ns ns 200 ns * 80 ns * ns * * ns ns * * * * ns ns * * * * * * ns * * ns * * * * * * * * 60 * * 150 * * * *

100 Le ngth ( μm) 40 Le ngth ( μm)

20 50 Missing ACV 0 0 _1 AL 4 AL 4 124 1 656 0 124 1 656 0 1138 3 1709 6 1138 3 1006 7 1709 6 1006 7 -G -G 1375 4541 _2 1375 _1 4541 _2 GD GD GD GD 14451 _2 11477 _2 14451 _2 11477 _2 D GD GD GD GD GD GD KK 1087 14 KK 1057 31 KK 1025 73 KK 1018 74 KK 1092 03 KK 1157 68 KK 1087 32 KK 1092 78 KK 1008 90 KK 1084 66 KK 1124 85 KK 1087 14 KK 1057 31 KK 1025 73 KK 1015 37 KK 1081 97 KK 1018 74 KK 1092 03 KK 1157 68 KK 1087 32 KK 1092 78 KK 1008 90 KK 1084 66 KK 1124 85 KK 1015 37 KK 1081 97 G GD GD GD lgs lgs rl rl GD GD GD GD Fsn Fsn 91 50 91 50 kis kis asp asp Ln k Ln k Pa k Pa k 109 6 MS 109 6 MS Pten Pten 20 59 Sg f29 org-1 82 04 68 36 20 59 Sg f29 org-1 82 04 68 36 Trpm Trpm un 5 Ns un 5 Ns 178 41 178 41 CG CG 153 09 153 09 Fmo-2 Fmo-2 bx bx Nrx-1 Sra-1 Nrx-1 Sra-1 Prosap Prosap CG CG CG CG CG CG CG CG CG CG Eye phenotypic score (FC) 0 1 2 3 B A Supp. Figure2 bioRxiv preprint not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailable

GMR-GAL4

ns Eye phenotypic score (FC) CG15309KK108714 0 1 2 3 Mild phenotype rkKK108009 *

GD3777_1 *

CG8888 GMR-GAL4 doi: ns No phenotype KK109203 * Fmo-2 CenGD9689 https://doi.org/10.1101/855338

GD14152_1 * Grip128 rkGD14383_2 * KK102573 *

CG17841 * Tpc1GD2905 KK110317 *

Pa1 GD3392_2 *

* para Sra-1GD11477_2 GD4541_1 * * Trpm rlKK115768 *

* GD1266_2 lgsGD1241 Fmo-2 Grip128GD14152_2 *

spinKK104813 *

Eye phenotypic score (FC) under a ; 0 1 2 3 this versionpostedDecember6,2019. Moderate CC-BY 4.0Internationallicense GMR-GAL4 Eye phenotypic score (FC) KK101874 *

Pak 0 1 2 3

GD14383_1 * rk GMR-GAL4 phenotype ns No observable phenotype GD11477_1 * CG6836KK112485

Sra-1 Observable phenotype ns PisKK109207 * CG14182 GD2738_1 ns FsnGD11383 * * KK109717 RphGD7330 PIG-X * coroGD14217 . The copyrightholderforthispreprint(whichwas CG14182GD2738_2 *

GD3392_1 * Eye phenotypic score (FC) para * 0 1 2 3 Ube3aGD450_1 * EphGD39 Wing measurement phenotype GMR-GAL4 * Severe GD9986 GD10101 ns Trpm Prosap GD10067 * GD11238_1 ns Sgf29

UQCR-C2 * paraKK108534 GD16961 *

mEFTu1 * phenotype Pa1GD6674 KK107305 *

mEFTu1 * Sgf29KK107577 KK109448 * Cbp20 * Doc2KK108623 *

KK101936 * Sin PtenKK109278 *

KK102545 * arm Fmo-2GD1266_1 *

GD9561 * Pp4-19C PIG-Z KK107404 * GD11562 * Atx2 LnkGD9362

TrpmGD4541_2 * * Ald1GD11803

KK108227 * Eye phenotypic score (FC) app GD2619 * 0 1 2 3 Nrx-1 spictKK102281 * Lethal phenotype GMR-GAL4 ProsapKK101537 *

CadpsGD9502_1 * ns

UQCR-C2 KK108812 KK105756 * CG10465

KK105731 * * Lnk UQCR-C2GD11238_2 CadpsGD9502_2 * * * GD13500 SinGD7027 Pten

dlg1GD4689 * bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 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 4.0 International license.

Supp. Figure 3

A Larval Drosophila expression B Adult Drosophila expression

Gut Trachea Gut Heart

1 1 1 1

2 2 0 2 2 1 2 2 4 1 2 2 1 2

58 59 0 0 0 1 CNS 1 2 Fat Body Brain 0 1 Fat Body 1 2 bioRxiv preprint not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailable Supp. Figure4 CG8888GD3777 dlg1GD4689 Cbp20KK109448 beadexMS1096-GAL4 DAPI doi: https://doi.org/10.1101/855338 dcp1 under a ; this versionpostedDecember6,2019. Increased Increased Increased CC-BY 4.0Internationallicense Cellular processesinlarvalwingdiscs pH3 Decreased Increased Increased . The copyrightholderforthispreprint(whichwas

RphGD7330 Klp61FGD14149_2 ACCGD3482 UQCR-C2GD11238_1 No change Increased Increased Increased No change No change Decreased Decreased CG11035KK101201 Sra-1GD11477_2 lgsGD1241 bxMS1096-GAL4 DAPI A Supp. Figure5 bioRxiv preprint not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmadeavailable Cellular processesinfemaleandmalelarvalwingdiscs dcp1 doi: https://doi.org/10.1101/855338 Female Increased Increased Increased pH3 Decreased Decreased Decreased under a ; this versionpostedDecember6,2019. CC-BY 4.0Internationallicense . The copyrightholderforthispreprint(whichwas Male Increased Increased Increased No change No change No change B C

Normalized pH3 positive cells Cell proliferationinlarvalwingdiscs Normalized pH3 positive cells 0.0 0.5 1.0 1.5 0 1 2 3 4 Apoptosis inlarvalwingdiscs

MS1096 bx MS1096-GAL4 (GD) bx -GAL4 (GD)

MS1096 bx MS1096-GAL4 (KK) Female

bx -GAL4 (KK) Female *

* GD1241 lgsGD1241 lgs

GD11477_2 * Sra-1GD11477_2 * Sra-1

KK101201 * CG11035 KK101201 * CG11035

MS1096 bx MS1096-GAL4 (GD) bx -GAL4 (GD) MS1096 bx MS1096-GAL4 (KK) bx -GAL4 (KK) Male Male *

ns GD1241 GD1241 lgs lgs *

ns GD11477_2 GD11477_2 Sra-1 Sra-1 * ns CG11035 KK101201 CG11035 KK101201 bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 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 4.0 International license.

Supp. Figure 6 Disruption of signaling pathways in larval wing discs

bxMS1096-GAL4 dlg1GD4689 Tsf2GD2442 Atx2GD11562 UQCR-C2GD11238_1 nudEGD15226 ACCGD3482 A Wnt wingless

B patched

C Hedgehog engrailed

Supp. Figure 7 A Human CNV genes interact with Wnt signaling pathway genes in multiple tissues Kidney Heart Brain

SFRP4

CTNNBIP1 WNT10A

FZD8

TCF7

RNF43

WNT5B NFATC2

CEP126

BRF1

CHD3

CAPNS1

JUP

FXYD5

CALM3

NCOA2

ARVCF

SFRP5

AFF2

WIF1

NFATC2

E2F4 CER1 FGFR3

NFATC1

PRICKLE1

ADGRL1

TRIM28 TCF7

TNK2

POLR1B ZNF160

AGRN

VIM

INVS RRBP1 NKD2

DBN1

DHX9

IQGAP1

RUNX1T1 AKAP8L DLX4

ARHGAP5 LAMA4 CTNNBIP1

CYLD

PPP2R1A

PTCRA

APPBP2 GNB2

SGSM2

PDCD5 FGFR1 DVL1

GTF3C1

SS18

TSPAN4

ARHGAP1

MTA1

TBK1

LRP5 SHANK2

PAX8 ZNF207

GTF2H1

DVL1 MINK1

TUBB4B RAD23A GPC5 CAMK2A BCS1L

CYP2A6 PSMA1

FEZ1

GAPDH EPS8

CHD8

CHD4 XPOT

CSNK1D ARHGDIA PRRC2A RAC2

PLAUR AP2S1 RAC2 CCNA2 DLG3

CER1

SFRP2 GLP1R

GTSE1

EIF4G1 SQSTM1 EMP3

MAP1LC3B PAXIP1

PLOD3 7-Mar

PRPF3

PLOD3 TUBB2A SNRPF

CAMK2D

SAC3D1 CDC25A E2F1

TAF9B FEN1 TGFA LEF1 OGG1

GNAI3 DNAJC4 PLCB2 NCBP1 DMWD DAG1 FUT6

CTCF SOX17 WBP11

KLHDC3 DVL3 MSH2 MUC3A

FMR1

ACACA NIFK TCF3 PLCB3

CAMK2B PRPF8 PRKDC CTSC PPP3R1 CCNF

BRCA1

PLOD3 RNASEH2A

FZD2 JUP

COL6A2 MCRS1 ARHGDIB PRKACA

MAPK10 BRD2 NPC2 BCCIP CNBP PLEKHA3 SF3B4 WDR5

CHD8 VARS

PIK3R1 AGPAT2 REL PDIA4

PAFAH1B1 PPIF

STAT3

FEN1 TAL1 GTF3C1 SUPT16H TK1 BAD WNT3

SMARCD1

CSNK2A2

NCK1 B4GALT2

MAPK8

VANGL1

NFATC4 ICMT

TMPO SNRPD3 HNRNPUL1

TOMM20 NONO RHOA WNT8B EIF3H

PPP4R1 EIF3H MBD4

NPM1 BAMBI ACP1 WNT16

TIA1 ARHGDIA FZD8 PABPN1

UBAP2L GSE1 FLII

BAMBI DMWD GNB2

CSNK1E VIM TFDP1 TLE2 SNRPA ARF5 YES1 FOSL1 AMD1

MKI67 DVL2

DVL2 NTRK3

RAD54B GNA11

PPP2R5D CSNK2A3 ESR1 MAPRE1 CHEK1 RNPS1 SSR1 COPS6 HNRNPAB EGFR RELA

PRKCA KARS

MMP7 RAC2 DAD1

CSNK1E SSRP1 MTERF3 ODC1

MOB4 RAC1 SUGP2 PRMT5

CALM1 VARS CASP8 PPIG

SFPQ

PIK3CA

EXOSC8 VEZF1 PSMD14 CSNK2A3

H2AFX CTCF CPSF1 RPA1 CBY1 ZNRF3 DVL3 PPP1CA MCM10 VARS DKK4 UBE2M PRKACG NOTUM GSK3A VANGL1 LRRFIP1

DDX18 SCARB1 GAS6

YY1 PRKCB

RANBP9 CYP2A6 ALDOB CAMK2D

PTBP1 CSNK2B SMARCA4 EMP3 NUP85 CALM1 GMFB

AIMP2 CSNK1D FZR1

CBFB SRSF10 NFATC1 CBY1

ATP6V0B E2F4 MKRN1 PRDX1 PA2G4 RHOA CSNK2A2 CTNND1

OGT

DTL

MCM5 SFPQ DNAJB6

TPX2 CSNK2B TLE3 SRSF3 EIF3M ILF3 NOL11 H2AFX

PA2G4 SERPINF1 TMPO SERBP1 ZNF148 SET PLSCR4

FZD2 RUVBL2 AURKA SMARCC1 PRKDC FOSL1 SUPT16H FZD4 MRPL12 WNT2B LGR4 ESR1 PPP4C SART3

SMAD2 COL4A3BP

PRICKLE4 TRA2B EP300 ZMYND11 PPARD CRK MCM4

BOP1

ITGB3BP MCM7

USP1 PRPF4 BUB3 CDC20 FZD5 IPO7

DAG1 SNX1 FZD9 ATIC USP1 PLSCR1 HDAC2

GNA11

FZD4 TOP1 MYCBP TMX1

EIF2S1 FZD5 WNT6 RUVBL1 CCT6A

UBC MCM7 RUVBL2

BTRC DVL2 ENSA

MCM5 RRS1 PRICKLE3 IKBKG CD44 HDAC1

NFATC4 FLAD1 SYNCRIP ABCE1 CHUK CSNK2A1 GNL3

CSNK2A2

GNA11 COL4A3BP BAMBI LRP6 AASDHPPT

ICMT PPP3R1 GTF3C2 ATP5C1 NFATC3 CCNA2 RUNX1 CAMK2A GLO1

PRPF3

ACTR1A DNAJB6 RAC1 KPNA2 MCM10 RHOXF2 CSE1L RBBP8 COL4A2 CHD8 PKP4 MCAM CTBP1 OCIAD2 GPC4

BRCA1 RNPS1 HSPA4 CCNA2 PSME3 RAC1 UBC AATF

MRPL42 MAPK9

MCM5 COL4A1 WDR77 CDK1 CCND3 PPARD PRIM1 NUP153 ACTN1 CAMK2G WDR77 SMARCA4 SYT7 VBP1 DKC1 RUNX2

SMAD2

CCT2

ARIH1 FZD6

MSH2 PPP2R1A

MYC TP53 OCIAD2 CDKN1A HGH1 PPP6C UBA2 PSMD14

CCNF

WNT1

YTHDF3 RBM10 HNRNPA3 SMC4 HMGA2 NCOR2 PSMB3 CDK4 TLE2 HSPA8 SMARCA4 EIF3H PARP1 UBE2N CCND3 ZNF148 JAG2 CSNK2B RYK MAPRE1 PFKP CREBBP EFEMP2 XRCC5 CSNK1G2

PAPOLA ARHGEF40 JPT1 YWHAZ SSRP1 EWSR1

ATP5C1 PLCB1 CCT5 PTTG1IP CACYBP HDAC2 DDX23 UBE2D3 MYC MRPL3

MSH2 COL4A2 DUT PPP3CC NCOR1

KLC1 SNRPD1 SERPINH1 ACTL6A

PRPF40A PRPF3 CSNK2A1 SNX1 PPP3CA PRSS23 TOP2A NECTIN1 ATF2 KIF11 VBP1 CHRD TP53 TIA1

WNT5A TBL1XR1 TP53 PKMYT1 TCF3 SLC2A1 PRKACA CCNB1 JUN CAMK2G PRKACB PSMD3 PRICKLE4 MAZ FBXW11 TRA2B HRAS PSMC6 BIRC5 SYNCRIP

PLCB4 JUN

AURKA

WNT10A CASP10

NUDC SPTBN1 PPP3CB ACTR3 AATF SLC25A10 CCT6A RNPS1 WNT10B CCND3 PLCB4 NCL CACYBP CCND2 WWP1

EGFR FNDC3B

PSEN2 TBL1XR1 CBY1

CCDC9 MRPS18B MORC3

SLC39A6 ILF2 LEF1 SENP2 TLE2 PSMA1 CDK4 WNT5A ATN1 CDC25A CLPTM1 PRPF40A NCBP2 RBX1 SMAD4 PARP1 KPNB1 SNRNP40 QKI

CSNK2A1 WNT6 YWHAZ MAP3K7 DEK CTBP2 SRSF1

PALLD PSEN1 DTL

PFN1 ARID1A CCT7 AATF EIF3B

ZNRF3 ATIC DDX11 DAZAP2 CCND1 PUM2

TCF7L1 UBAP2L UBE2M PRICKLE1 EP300 HNRNPU TFAP2A MAP3K7

CAND1 SSRP1 OPA1 PFDN2 NOLC1 ILF3 SENP2

CCNB1 WNT3A KIF11 TOP2A CCND2 CAMK2G APC BUB3 SOCS5 KARS CDC25C CDKN2C KLC1

PPP4R1 CAMK2B

PPP2CA PORCN PPP3CC CCT5 WDR5 TNPO1 RANBP2 EFEMP2 KARS CDKN1A CTBP1

CCT3 LRP6

CCND1 NRIP1

PSMA4 FZD1 OTUB1 POLR3E PPP3CA SLC16A1

XPO1 NCBP2 PSMB3 CSNK1A1

NFATC3 PRKACB CSNK1E

SNTA1 SRSF10 RUVBL1 PPP4C CUL1

RFNG CREBBP

RUVBL1

CDKN2C TCF7L1 NLK SFN HSPA4 MAPK10

GNB1 WNT7A

ACP1 LAMC1 TBL1X PSME3 RBX1 GNL3 PTPN11 TCF7L2

DEK ACRV1 UBQLN1 PAPOLA PLK1

PAFAH1B1 TOMM20 C1QBP XRCC5 APC2 UCK2

HSP90AA1 RRS1 CCT2 TYMS

SMAD3 HNRNPR SUMO1 NR3C1 TACR1 RECQL5 TLE3 SIAH1 TUBG1 SKP1 TCF7L2 NCBP2 PRKCG FBXW11 NFATC3 UBE2V2 PPP4C MRPL3 BUB3 CCT7 AZIN1 NLK FZD4 FZD7

YWHAZ RBM39 RFC5 HDAC1 HSPA13

NSD2 ATXN1 NUP62 SIAH1 DAAM1

PLSCR1 SKP1 MAPK10 NUP98

SSBP2 SRSF2 CTBP2 CTNNB1 DKC1 RACGAP1 KHDRBS1 NUDC PRKCA UBE2D2 PLSCR3 SOD1 PRKACB CTBP1 HSPE1 SMAD2

MAPK8 TBL1X RBPMS TRIP13 JAG2

CTPS1 FZD7 CREBBP RANBP2 PTTG1IP NOL9 PPP3CB FBLN1 KIF2C TLE1 KIF11 PARP1 IL6ST

LGR4 CBX3 DEK NFATC1 SFRP1 RAD21

TBL1X RBX1 CUL1 CCT6A KHDRBS1 PPP3CA SOX2

NCOA1 SKP1 HSP90AA1 EP300 SIAH1 HMGB2 PAICS FAM107A TOP1

PRKCA CSNK1A1 PTBP1 CUL1 TCF7L2 WNT11 HGS

EFNB2 ILF3 NIPSNAP2 UBE2N ATP5G1 FZD2 DVL1 CSE1L TLE4 PSMB3 LAMC1 DVL3 ROCK2 BOP1

RBPMS SCUBE3 APC FRAT2 DDX1

HNRNPA3 PAM

MAPK9 STK24

RAN DAAM2 NDUFS6

YWHAB

SMAD4 SERPINF1 SKP2 CSNK1A1 SMC3 CDK4 YY1 CDK1 PPP3CB EWSR1 ALCAM DHX15 SFRP2 NOL11 LRP5 CTBP2 GSK3B DDX18 POLE2 CTPS1 SCP2 SRC RYK HRAS G3BP1 CD151 UBQLN1 MTERF3 MYC

ROCK2

RHOA NUP98 DIP2C YWHAG

HDAC2 DAZAP2 CDC20 SRSF10 VSNL1 PNN PPP2CA ILF2 DNM1L WDR77 ROCK2 FBXW11 CCT3 CCT3 REEP5 TLE1 FKBP8 ROR1

PAFAH1B1 IRAK1

HMGB3 ROR1 SFRP1 RIN1 ADRM1 SNRPG CCT5 HOMER3 CAMSAP2 EIF3B MAPRE1 PSMC6 TCF3 UBE2N DTNB

CTNNB1 VBP1 TBL1XR1

LEF1 AZIN1 HSPA9 PAICS LAMP1 RELA CAND1 KLC1 PPARD PHLDA1 ATXN1 CDH2 AHSA1 TLE4 PRICKLE3 ELOC CDC20 CALU MAD2L1

BTRC

DUT UBE2G1 ATP6V0D1 TLE4

PA2G4 PAEP RSL1D1 MAPK9

GPC4 AURKA FZD1

C1S KCNJ4 PPP2R5E SLC39A6

PRICKLE4 PICK1 ATP5C1 APLP2 SMC4 PPP2CA PPP4R1 CACYBP UPF1

GABARAP

TRIP13 QKI HMGB2 SRRM1 WNT2 CTNND2

CSE1L CAV1 PLCB1 AZIN1 SMAD4 FZD6

TBK1 MAP3K1 RAE1 SRP19 NAB1

CANX LRP5 CSNK2A3

DNAJA1 PNN CCND1 APPBP2

USP1 CANX GAS1

BRD3 POLR3E EFNB3

JUN

RRM2 UBE2D3

PSEN1

ATP2C1 AIMP1

RAD23A CEBPG CBX3 RAD23A STOM UBE2D2

DNAJB6 PRICKLE1 SLC16A1 ATF2 SKP2 TCF7L1 AXIN1 CTNNB1 CLU EWSR1 STRN3 COPS5 DAAM2 MAP3K7 EIF3A

DIMT1 XRCC6 CSNK1G2 DKC1 POLR3E BTRC WNT4 NSD2 CDC27 RYK YWHAG SRSF2 PRMT1

SFRP1 NADK UBE2M

KIF2A CAMK2D

CCND2

CDC27 KIF2C CDC27 TIMP3 ISOC2

CAV1

ANGPTL2 TLE1 PPP3CC

PRPF40A FZD7 ATP6V0C FYN APC

SSR1 SFRP4

MRPL3 ACP1 RAC3

TOMM34

CALU HSPA4 SFRP4

FKBP4 RELA MARS

HSPA13

DKK1 DAAM1 PSD3 FUBP1 TSR1 DDX1 CBX1

XRCC5 NCKAP1

PUM2 DAAM1 PLOD2 RANBP9 ARHGDIA APPBP2 CDC25A

ATF2

COIL JMJD1C CIB1 DHX9 GAK SRP19 SQSTM1 RSL1D1 CEP57 RRS1

LAPTM4B BRD2

ANXA1 TIA1 DLG3 PRSS23

TFDP1 RFC3 QKI IDE CCHCR1 SYNCRIP TAF9 PPP3R1 COL4A2 GSK3B CYLD

ACTR1A NAB1 OPA1 CANX

REV3L

UBQLN1

HNRNPH1 ARIH1 CTNNA1 PRICKLE2 HIF1A NCKIPSD SMC3 PFDN2 EIF3B TFDP2 ATN1

SPTBN1 FTSJ1 MTERF3 NKD1

PSMA1 LRP6

CEBPB RNF43 DNAJA1 GSN NOXA1 GSK3B DNAJC10 MTA1

PTEN REL CKAP5 SUMO2 WNT5B AIMP2 RAB11FIP2 MLF2

WNT5A TRIP6

ERBB3

UPF3A ADSL

ROR1 BZW1 CD44 TLE3

LGR5

PTPN11 ZHX2

YTHDF3 FZD6 NUP85 NFIB TRIP13 AR

AXIN2

ANAPC11

BRWD1 TOPBP1

FZD1 FOSL1 NOL11

PSEN1 EPS8 SUMO1 MTMR4

AXIN1 TK1

MAPK8

PNN NIPBL SF3A2 TM4SF1 FN1

HNRNPDL ARL1

ENAH AKAP8L POLE2 MOB4

ATP2B1 PRKCG

NUP153 HSPA8 GNB2

NTRK2 PRKACA

CXXC4 VANGL2

PPP1R13L PDLIM7 COBLL1 CD164 TRIM27

TM4SF1

ANXA4 FRAT2

GPC4 ARF4 YY1

RANBP9

AXIN2

ASB13 OAT

SFRP2 PLAG1

TACC1 PLCB4

RANBP2 ATP13A3

CDC26 LMNA

WISP1 NUP98

FILIP1L PWP1

BCL11A TBCD IPO5 AMD1 TGFA

VANGL1

KDELR3

PRKCB TUBB6 MAP1LC3B ATXN1

WNT3A

EGFR CDH11 EIF3J JUP

FERMT2

PLCB1 NAV3

SERBP1

NRXN1

MPZL1 SQSTM1 NLK

ZNRF3

ATP5G1

BCL7A

ROR2

PTS CYR61 ASB13 FZD5 PML BAIAP2 ZMYM4 ATR UBR5

CDH11 ARF4 IMMT

RCN2 GRB2

LTBP2

LGR4 CTNNA1 PAXIP1

TUBB2A COIL INVS APC2 SENP2 TRIM27 PTK2

HIF1AN

UCHL1

SERPINF1 CYBA VIM

NR3C1

FBLN1 FRAT2

MCL1

IL6ST

FZD3

WNT5B

ATIC

TNPO1

JUNB

DKK2 CELF2 FRAT1

GPRC5A HIF1AN

CDC26

PAM ITGA2 UBE2I AXIN2

WNT3A

DKK1

CTNND2

CTNNBIP1 VAMP2

GABARAPL2

AXIN1

SH3BGRL

DKK1

RER1

RAB11FIP2

PLCB2

CA12

TTC3 NRP2

TPD52L1

INVS MYCBP2

MMP7 FRAT1

ANAPC11

NBEA

NFATC2

WISP1

FZD10

PRKCB

NKD2

NCF1

RSPO3

FZD8

APC2

MMP7 B Human CNV genes interact with Hedgehog signaling pathway genes in multiple tissues Kidney Heart Brain

PKNOX1

NIPBL

GRK3

NKTR PTCH1

PLOD2

DHX15

AASDHPPT

LUC7L3

PSMC6

ADAM10

ATP2B1 DNAJC10

FMR1 VIM

GRK2

BZW1

CBX1

IMMT EML4

TROVE2

PRPF4B

PWP1

PRR11

HNRNPR

PPP2CA

PIK3CA

SPAG9

ZMYND11

POU2F1

SRSF1

ZNF146

MAP4K5

NCK1 DDX1

GAS1

CSNK1G3

KPNA6

CBX3

E2F4

CSNK1A1

DEK PSMA1

FZD7

KIF3A

ARHGAP5

DICER1 PRKACB

E2F1 PIK3R1

CCT6A SMG1 SS18 PAPOLA

CSNK1G1

ABLIM1 PAXIP1 HSPH1 NDE1

CCT5

EPS8

CALCOCO2 CEP350

SPOP

PUM2

PNN

TNPO1

CBFB

BRAP

RMI1 CCND2

SPOPL

DVL2

ARL1

CCNB1 SMURF2 CDKN1A

TRIP13 RTN4

GPR161

SRSF10 GTF2H1

SPOP

UBE2G2

PRPF40A

NDRG1 JPT1

HSPA4 FLRT2

MAP3K7

FEN1 SMARCA2 TCF3

SYNCRIP

CDK1

RAN DCBLD2

SF3B1

NFATC2IP CBX3 USP1

BRD1

ARHGDIA NCOA2

YWHAG

GNL3 CAND1

TAF9

FYN

CCT2 TACC1 CHD8

UBQLN1 POLR3E SPOPL

CEP350 SMC4

IMMT DDHD1 CDC25A CASK

BIRC5

UBE2I CUL1 KIF3A NCBP2 CKAP5

NUDC CCND1 DUT EIF3A

CSNK1E NUP153 SCYL2

CNOT1 MARCH6

ITCH RANGAP1

ARHGAP5 RAD54B

PTBP1

RACGAP1

UBE2V2

TRIM27 UBE2I KMT2A UBN1

CTCF

CUL4B

RNPS1 RTN3 UCK2 ACTL6A

GLI2 PRKACB KDELR3 RAD54B

DAB2 TRIM2

PPP2R5E SUMO1

ENSA

MGRN1 GSK3B

CSE1L CSNK1D LAMC1 CCNA2 DAZAP2

WDR77 TTC3 DLG1 PAFAH1B1 ATP2B4 MKI67

CDK4 KARS CCNB1 BRWD1 SKP2

CCNB1

AIMP2

PPP2R1A

ETF1

GNB2 SLC39A6

ASXL1 PRIM1 TNPO1

CDK6

WDFY3 TPM1 TMPO SHANK2 EZR

BTRC PKP4

HNRNPU COPS6 RXRB

CSNK1G3 RASA1 MYC

BRCA1 MEN1 AURKA YES1 DKC1 DUT BUB3 ERC1

ATP6V0D1

NOVA1

PRKACB

CDK1

PA2G4 MTF2 ALCAM

XRCC5 NSD2 EIF3B AGO2

DNAJC10 DEK

TARDBP ATXN1 RACGAP1 TRA2B SON

USP1 PPP4R1 MGRN1 ADAM10 CDK1

MYLK GNAQ HSPA13 CSNK1G2 KIF11 ANXA1 BIRC5 RAN CDK4 CUL1 SMURF1 IGFBP5 CSNK1G1

C18orf25

ILF3 TOMM20 CCT5 SLC39A6 CAPRIN1 PPP2CA CSNK1G2 KIF11

CCND2

HMGB1 WDR77 FGFR1 HMGB2

HNRNPAB CAND1

MRPL42

RRM2 SMAD2 CDC20

CCT5 UBE2D3

HNRNPU SRSF10 NDE1

AZIN1 CUL4B SMARCC1 OAT NCOA3

MTUS1 TMX1 SMARCC1

RANBP2 SSRP1

SMAD3 DKC1

PAICS PSMD14 MCM4 TRIM28

BOC CCND1 DIP2C

HDAC1 GRB2 COPS5 HNRNPAB

ZDHHC17

PA2G4

SUGP2

CSNK1D

PLSCR1 GTSE1

ZNF263 ATIC PRKACA SMARCA4

RUNX1 DNAJB6 CCNA2 SMAD4 BRCA1 AP3B1

CHD4 SMURF2 PSME3 PSMD14 PPP4C UBA2

CCT6A

GLP1R ILF3

SKP2 ATP2B4 ILF3

CDK4

HMOX2

HSPA4 HERPUD1

AATF CCT3 NCKIPSD BOP1

CSNK1E TACC3

CSNK1A1

CCNA2 POLR3E RAE1 CHD3 AATF

EWSR1

HDAC1 OTUB1 MAPK1

PPP6C

TMPO BUB3 HMGB2

BCL2L11 DNAJB6 LTBP2 SPOP SRSF10 BAG6 HHIP

ADAMTSL3 RANBP9

RELA EWSR1

UBE2M

E2F1

BMPR1A KDM5C CDKN1A NCBP2 CSNK1A1 DLG1 PIK3R1

CSE1L SSRP1 SMARCD1 USP7 CDH11 ADGRL1 CCT7

HSPA4 CDON FBL RAB2A TMX1 SMURF1 BOP1 CDC20

YWHAB

NRXN2

HNRNPA3

STOM PRPF40A

UBE2M PAICS

CDC27 SMARCC2

SGSM2 ADRM1

MRPL3 AATF NOLC1

ARRB2 BRD4 UBE2D3

PTCH1

CCT6A GFPT1 SSRP1 RUNX2 PPP2CA KIAA0368 TXNDC9 CPSF6

PSMD3

RELA

CREBBP

EVC ATIC TCOF1 CSNK2A1

UBE2I USP1

GNA11 BARD1 NUDC PSME3 WDR77 RASA1 XRCC6 GOLGA2 SMARCA4

NUDC KIF11 ARRB2

ZNF764

MCM7 EIF3A TUBG1

ARID1A

CUL1

TNFAIP1

CCND2

GLI1 ERBB3

HMGXB4 RANBP9 ESRRA

HAP1

IL6ST POLR3E PPP4C CSNK1D BAG6

CCT3 KIF3A PRKD2

ARRB2 SMARCC2 PAPOLA CCND1

ESR1

UBR5

SMAD2 PTBP1

BOP1 NCBP2

UBE2M ARRB1 ADCY1 UPF3A HMGN4

ATXN2L CSNK1G2 EIF3B

GLI3 HGS

BCL2

E2F1

NRP2 ILF2

CTNNA1

TOMM20 PSMC6

SETDB1 MAZ

CLPTM1 RAD21

AIMP1 CCT2 RNPS1

HSPA13

CASK EPHB4

GSK3B

SYNCRIP

CCNF

RELA ATIC

H2AFZ

ARHGDIA CSNK1E

AZIN1 BCAP31

PAX8 BAIAP2 NTRK3

AIMP2 CSNK1G3

DHH VARS GTSE1

BCL2 EZH2

HNRNPA3 SNTA1 FZD7

TMCC1 CAND1 EGFR

ATRX

DBN1 FBXW11 THRA E2F4 TOMM20 PPP4C SKI RAN

ARRB1 NOL11

MGRN1

IRF3 MTA1

CCNF SEC62

BAX

SAP18 SF3B1 SUFU DNAJB6

AZIN1

DVL2

SMURF2

MYCBP NDE1 TNPO1

DDX1

GAS1

SMURF1 ATN1 ARFGAP1 PRKACA TUBB3 DLGAP4

E2F4 ELK1 EWSR1 DLG1 TAF9

GNB2

GSK3B

NECTIN1

MEGF8 DKC1

IHH

MAPK10

TFDP2 PAEP BNIP3L

LAMA4 MINK1

SUFU

SAP18 DMPK GPI EEF1E1 PPP2R5E

COPS2 LRP2

AP2S1

MYO9B BCL2

GRK2 PTP4A3

RB1

SRSF3 SCYL2 GLI3 SOX13

TCF3

CTNNA1 ARRB1

SMO

FKBP8 AGPAT1

CUL4B

TMBIM6 ATF1 TRIP13 VARS PPP2R1A MFI2 MRPL42 CYP2A6 RNPS1

STK35

PKMYT1 PTK7

HRAS

AURKA RET

CACTIN

AGO2

GRK2

PTPN11

FZR1

PSMC2 EPS8 TIMP3

IL6ST

SPOPL

SHH

ATP2B4 PRKACA MEF2D PLOD3 GSK3A NPTXR

COL6A1 EFEMP2

ZNF446

DMWD GRK6

FLYWCH1 COIL

OTUB1

RANBP9

GPR161 GNB2 DNAJC4 DAZAP2 RCN3 STRN4 MFAP1

CD151

RPL7L1 UBN1

MID1IP1

DBN1 RIN1

NUCB1

PFN1

ARHGEF40 BNIP3L

BTRC

SFTPC

SMAD3

ZBTB43

PTCH2

SSBP2

GLI2

GAS1 UBQLN1

FXYD5

ZNF263

PPP1CA

COL6A2 HGS

RARB TAOK2 ADD3

BAD ADAM10

PLA2G6

GRK3 RBPMS EDA

HOXB5 RERE

JAG1 ATN1

LAMB2

MUC3A

CSNK1G1 FBXL18

BCL2L11

UBE2L3

SFRP1

PRKACG

AGPAT2

IGF1R DRD2

ATXN1

SUFU

DIP2C

JAK3

GPR161 ACRV1

HRH3 FLNA

MID1

PLSCR1

MCL1

CDON

FZD7

COL4A2

SVIL

SLC17A7

ZNF764

MEGF8

GLI3

BOC PTCH1 CNV/neurodevelopmental gene Signaling pathway gene Connector gene