Somatic Mutations in Renal Cyst Epithelium in Autosomal Dominant Polycystic Kidney Disease

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Adrian Y. Tan,1,2 Tuo Zhang,2 Alber Michaeel,1 Jon Blumenfeld,3,4 Genyan Liu,1 Wanying Zhang,1 Zhengmao Zhang,1 Yi Zhu,1 Lior Rennert,5 Che Martin,1 Jenny Xiang,2 Steven P. Salvatore,1 Brian D. Robinson,1 Sandip Kapur,6 Stephanie Donahue,4 Warren O. Bobb,4 and Hanna Rennert1

1Departments of Pathology and Laboratory Medicine, 2Microbiology and Immunology, 3Medicine, and 6Surgery, Weill Cornell Medicine, New York, New York; 4The Rogosin Institute, NewYork-Presbyterian Hospital/Weill Cornell Medicine, New York, New York; and 5Department of Public Health Sciences, Clemson University, Clemson, South Carolina

ABSTRACT Background Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy caused by mutations in PKD1 and PKD2 that is characterized by renal tubular epithelial cell proliferation and progressive CKD. Although the molecular mechanisms involved in cystogenesis are not established, concurrent inactivating constitutional and somatic mutations in ADPKD genes in cyst epithelium have been proposed as a cellular recessive mechanism. Methods We characterized, by whole-exome sequencing (WES) and long-range PCR techniques, the somatic mutations in PKD1 and PKD2 genes in renal epithelial cells from 83 kidney cysts obtained from nine patients with ADPKD, for whom a constitutional mutation in PKD1 or PKD2 was identified. Results Complete sequencing data by long-range PCR and WES was available for 63 and 65 cysts, respectively. Private somatic mutations of PKD1 or PKD2 were identified in all patients and in 90% of the cysts analyzed; 90% of these mutations were truncating, splice site, or in-frame variations predicted to be pathogenic mutations. No trans-heterozygous mutations of PKD1 or PKD2 genes were identified. Copy number changes of PKD1 ranging from 151 bp to 28 kb were observed in 12% of the cysts. WES also identified significant mutations in 53 non-PKD1/2 genes, including other ciliopathy genes and cancer-related genes. Conclusions These findings support a cellular recessive mechanism for cyst formation in ADPKD caused primarily by inactivating constitutional and somatic mutations of PKD1 or PKD2 in kidney cyst epithelium. The potential interactions of these genes with other ciliopathy- and cancer-related genes to influence ADPKD severity merits further evaluation.

J Am Soc Nephrol 29: 2139–2156, 2018. doi: https://doi.org/10.1681/ASN.2017080878

Autosomal dominant polycystic kidney disease mechanism of cyst formation in ADPKD, disease (ADPKD) is clinically and genetically heteroge- severity has been attributed to the affected PKD neous, caused by mutations in PKD1 or PKD2, gene locus and the timing and type of somatic and is the most common inherited CKD.1 In 7%– 10% of individuals with ADPKD, the pathogenic variant of a PKD gene is not detected, suggesting Received August 15, 2017. Accepted June 5, 2018. other genetic causes including hypomorphic alleles, Published online ahead of print. Publication date available at 2 mosaicism, or rare mutations of other genes. In- www.jasn.org. activating, somatic mutations of PKD1/2 genes that Correspondence: Dr. Hanna Rennert, Department of Pathology occur in renal epithelial cells, together with a con- and Laboratory Medicine, Weill Cornell Medicine, 525 East 68 current, constitutional mutation of PKD1 or PKD2, Street, F544, New York, NY 10065. Email: [email protected]. cause clonal proliferation of tubular epithelium edu and cyst formation.3 In this cellular recessive Copyright © 2018 by the American Society of Nephrology

J Am Soc Nephrol 29: 2139–2156, 2018 ISSN : 1046-6673/2908-2139 2139 BASIC RESEARCH www.jasn.org

PKD gene mutations. Kidney cyst formation also occurs when Significance Statement there is a deletion of chromosomal regions of the corresponding PKD1 or PKD2 allele, termed loss of heterozygosity Autosomal dominant polycystic kidney disease (ADPKD) is a cili- (LOH).4–8 These mechanisms have also been identified in opathy caused by mutations in PKD1 and PKD2, characterized by malignant neoplasms, suggesting that ADPKD is a benign renal epithelial cell proliferation and progressive CKD. Among the genetic mechanisms proposed for cystogenesis are inactivating 9,10 neoplastic process. constitutional and somatic mutations in PKD1/2 genes, and PKD1/2 Recent studies, utilizing more technically advanced methods, gene dosage effects. This article describes the identification, by concluded that modestly reduced levels of PKD1/2 gene expres- genomic sequencing methods, of the high prevalence of inactivating sion (i.e., haploinsufficiency), rather than PKD1/2 gene inactiva- somatic mutations in PKD1/2 and non-PKD1/2 genes in epithelial tion, caused a spectrum of cystic renal phenotypes, ranging from cells obtained from kidney cysts in patients with ADPKD. The findings support a primary cellular recessive mechanism for cyst few, small cysts with relatively well compensated CKD, to mark- formation in ADPKD caused by mutations in these genes in the – edly enlarged kidneys with early onset ESRD.3,11 14 Although the renal epithelium. Potential interactions of PKD1/2 genes with prevalence of PKD gene haploinsufficiency in the ADPKD pop- other ciliopathy- and cancer-related genes that might influence ulation has not been established, it appears to be low.15 ADPKD severity is proposed. Primary cilia are microtubule-based, nonmotile projections of epithelial cells thatextendintothe lumenof renaltubules and other (357,999 exons, .20,000 genes) of renal cyst epithelia and 16 structures. Integral membrane proteins, including polycystin 1 peripheral blood lymphocytes (PBL) DNA was performed us- (PC1, TRPP1) and polycystin 2 (PC2, TRPP2), encoded by PKD1 ing the Agilent HaloPlex Target Enrichment and massive par- 17 and PKD2, respectively, localize to these cilia. Mammalian cil- allel sequencing (Illumina, San Diego, CA). Data analysis of iopathies are inherited disorders characterized by various pheno- the cyst and matched constitutional DNA was performed for types, including renal cyst formation. They are caused by simultaneous detection of single nucleotide variants (SNVs), mutations in genes that encode proteins associated with cilia indels, and copy number variations (CNVs) as previously de- fi 18 and traf cking pathways. The potential interactions of PKD1/ scribed.22,23 Schematics of WES data analysis pipeline is 2 genes with non-PKD1/2 genes, including other ciliopathy genes, shown is Supplemental Figure 2. The pathogenic potential fi have not been well de ned. of missense variants was evaluated using the computational Given the complex ADPKD phenotype, and multiple factors analysis tool Combined Annotation–Dependent Depletion affecting cyst development, we characterized the prevalence and (CADD; http://cadd.gs.washington.edu/). A CADD value of diversity of somatic PKD1/2 gene mutations arising in renal cyst $15 suggests the variant is likely pathogenic.24 Significantly epithelium and investigated whether somatic mutations in non- mutated WES variants were further filtered using a 30% ADPKD genes occur that might contribute to the pathogenesis of variant allele frequency (VAF) cutoff and a population allele kidney cysts. We report a comprehensive genomic analysis of so- frequency of 5% in The Exome Aggregation Consortium matic mutations, using whole-exome sequencing (WES) and (ExAC) database (http://exac.broadinstitute.org/). Sanger sequencing, in cyst epithelial cells that were isolated after The significance of mutated genes and pathways was ana- native nephrectomy in patients with ADPKD for whom consti- lyzed with the Mutational Significance In Cancer(MuSiC) suite fi tutional mutations in PKD1/2 were identi ed.Inthisstudy,in- of tools (http://gmt.genome.wustl.edu/packages/genome- activating somatic mutations in PKD1/2 genes occurred in 90% of music/index.html) using default settings, accounting for kidney cysts. We also characterized somatic variants in non- gene background mutation rates. Potential splice-site effects PKD1/2 genes (e.g., ciliopathy, cancer) from these cysts, including were evaluated using splicing prediction tools.20 Acustom fl those associated with other ciliopathies, which might in uence analysis pipeline analyzed constitutional mutations in 211 cili- cyst formation and disease severity in ADPKD. opathy and 565 cancer genes (http://cancer.sanger.ac.uk/census/; T. Zhang et al., unpublished data). Pathway analysis was performed by Reactome (https://reactome.org/). Statistical METHODS analysis to study the association between somatic variant and cyst size was performed using a linear mixed effects model Patients with ADPKD were enrolled in this study if they were with the R software environment (https://cran.r-project.org/ scheduled for a living donor kidney transplant, and removal of web/packages/nlme/index.html). Additional details are one or both native kidneys at the New York-Presbyterian Hos- provided in Supplemental Material. pital/Weill Cornell Medicine campus. Study eligibility was determined by the transplant surgeon (S.K.) before enrollment of each participant and informed consent was obtained (Supple- RESULTS mental Figure 1). The protocol was approved by the Weill Cornell Medicine Institutional Review Board. Cyst epithelial Clinical Cohort cells were isolated according to standard procedures.5,19 Nine unrelated patients with ADPKD were enrolled. All but PKD1/2 genetic analysis was performed by long-range PCR one had unilateral nephrectomy during preemptive, living do- next-generation sequencing and Sanger sequencing.20,21 WES nor transplantation; one underwent bilateral nephrectomy in

2140 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2139–2156, 2018 www.jasn.org BASIC RESEARCH

Table 1. ADPKD patient cohort clinical and pathologic characteristics Age at Left/Right Kidney Size, No. of Cysts Diameter Range Patient Sex Age, yr Weight, g Diagnosis, yr Kidney L3W3H, cm Genotypeda of Cysts, cm BS9001424 F 73 48 Left 520 1637376 2–6 Right 530 15310374 PJ9001456 F 41 34 Left 3460 27.531638.5 9 1.2–6 Right 2740 27.531538.5 3 DR9001565 M 59 26 Left 4020 35318312 6 1.5–8 Right 3999 31317314 5 CD001574 M 58 27 Left 1770 24311.3311 4 1.5–6 Right 1680 23.5311.3311 4 BJA001578 M 52 39 Left 4560 30.7315.2314.5 4 1.5–10 Right 4010 31317313.5 6 CMJ0001593 M 59 53 Left 1680 24313310 5 2–10 Right 1500 23312.738.3 3 2–8 RP9001591 F 54 29 Left 2340 25.6314.5313 5 1.5–5 Right 2300 23.5315310 2 4–12 JS9001595 F 56 32 Left 1670 24311.538.2 4 2.5–7 Right 1900 2731439.3 4 3–8 BH9002280 M 62 33 Left 6800 41.5325312 5 4–12 Right 8900 43.5331313 4 6.5–20 L3W3H,length,width,andheight;F,female;M,male. aGenotyping performed as described in Figure 1. preparation for future kidney transplantation (Supplemental Detection of PKD1 and PKD2 Somatic Mutations in Figure 1). Each kidney contained innumerable cysts; 237 cysts Renal Cyst Epithelia were collected, with an average of 13 cysts per kidney. The Using a combination of WES and LR-PCR based methods, median cyst diameter was 3 cm (range: 1–20 cm; Table 1). A PKD1/2 genes were sequenced in epithelial cell DNA isolated total of 83 cysts were randomly selected for sequencing, where from 63 kidney cysts. Constitutional mutations were detected sufﬁcient DNA was available (Figure 1). There was no differ- in all PBL samples; PKD1 mutations were detected in 89% ence in size of the cysts analyzed and those not analyzed (eight out of nine participants); the remaining patient had (P=0.94). amutationinPKD2 (Supplemental Table 1). Of these

Figure 1. PKD1 and PKD2 mutation analysis was successfully performed using DNA obtained from renal cyst epithelia. Schematic repre- sentation of 83 cysts (nine patients) that were subjected to ADPKD gene mutation analysis, broken into subsets by sequencing method is shown. Of the 83 specimens, complete genotyping data by long-range PCR (LR-PCR) Sanger/next generation sequencing and whole exome sequencing (WES) was available for 63 and 65 cysts, respectively. A total of 46 specimens were successfully sequenced by both methods. Mutation detection rates for each sequencing group are denoted in the corresponding boxes. DQNS, DNA quantity not sufﬁcient.

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Table 2. Summary of somatic mutations in PKD genes by cyst for nine patients RESEARCH BASIC Exon/ Mutation Patienta/Gene Cyst Constitutional Mutation Exon Somatic Mutation Mutation Type Intron Designation . ora fteAeia oit fNephrology of Society American the of Journal BS9001424/PKD1 L1 c.4745 G C, p.Trp1582Ser Exon 15 ND L2 (CADD=26.3) c.4798dup, p.Thr1600Asnfs*15 Exon 15 Frameshift Pathogenic L3 c.12216_12217del, p.Thr4073Profs*83 Exon 45 Frameshift Pathogenic L4 c.348_352del, p.Asn116Lysfs*2 Exon 3 Frameshift Pathogenic L5 c.3225del, p.Pro1076Argfs*28 Exon14 Frameshift Pathogenic www.jasn.org L6 c.348_352del, p.Asn116Lysfs*2 Exon 3 Frameshift Pathogenic R1 c.11888G.A, p.Trp3963* Exon 43 Nonsense Pathogenic R2 c.5395C.T, p.Gln1799* Exon 15 Nonsense Pathogenic R4 c.3162–2A.C IVS 11 Splicing Pathogenic R5 c.4745 G.C,p.Trp1582Ser (LOH) Exon 15 LOH Likely pathogenic PJ9001456/PKD1 L1 c.6364delG, p.Val2122Lysfs*3 Exon 15 ND L4 c.6364delG, p.Val2122Lysfs*3(LOH) Exon 15 LOH Pathogenic L5 c.348_352del, p.Asn116Lysfs*2 Exon 3 Frameshift Pathogenic L6 c.8016+1del Exon 21 Splicing Pathogenic L7 c.12006_12010del, p.Gln4004Alafs*151 Exon 44 Frameshift Pathogenic L10 c.7655_7658 delinsTTG, p.Ala2552Valfs*68 Exon 19 Frameshift Pathogenic L11 c.3745del, p.Asp1249Thrfs*24 Exon 15 Frameshift Pathogenic L12 c.6364delG, p.Val2122Lysfs*3(LOH) Exon 15 LOH Pathogenic L15 c.1284_1292del, p.Trp429_Gln431del Exon 6 In-frame deletion Likely pathogenic R4 c.3831_3847del, p.Ser1278Glyfs*17 Exon 15 Frameshift Pathogenic R7 c.12004–2_12019del (splice_acceptor) Exon 44 Splicing Pathogenic R16 ND DR9001565/PKD1 L3 c.9504C.G, p.Phe3168Leu Exon 27 c.9504C.G,p.Phe3168Leu (LOH) Exon 27 LOH Pathogenic L5 (CADD=27) c.8935_8937delTTC, p.Phe2979del Exon 24 In-frame deletion Likely pathogenic L7 c.5395C.T, p.Gln1799* Exon 15 Nonsense Pathogenic R8 c.2157del, p.His719Glnfs*66 Exon 11 Frameshift Pathogenic R9 c.1789del, p.Gln597Argfs*188 Exon 9 Frameshift Pathogenic c.4916dup, p.Gly1640Argfs*18 Exon 15 Frameshift Pathogenic R10 c.5180del, p.Pro1727Argfs*32 Exon 15 Frameshift Pathogenic R13 c.12711C.A, p.Tyr4237* Exon 46 Nonsense Pathogenic CDS001574/PKD1 L2 c.10084del, p.Gln3362Serfs*35 Exon 31 c.10314_10315insGCTGGCA, Exon 33b Frameshift Pathogenic mScNephrol Soc Am J p.Arg3439Alafs*34 L9 c.348_352del, p.Asn116Lysfs*2 Exon 3 Frameshift Pathogenic R6 c.11817G.A, p.Trp3939* Exon 43 Nonsense Pathogenic R9 c.11884_11912del, p.Gln3962Alafs*5 Exon 43 Frameshift Pathogenic R10 c.10084del, p.Gln3362Serfs*35 (LOH) Exon 31 LOH Pathogenic 29: 2139 – 16 2018 2156, mScNephrol Soc Am J Table 2. Continued

Exon/ Mutation Patienta/Gene Cyst Constitutional Mutation Exon Somatic Mutation Mutation Type Intron Designation BJA001578/PKD1 L3 c.3929_3930del, Exon 43 c.6558_6568del, p.Trp2187Serfs*71 Exon 15 Frameshift Pathogenic 29: L4 p.Asp1310Glyfs*120 c.10948delC, p.His3650Thrfs*34 Exon 37 Frameshift Pathogenic

2139 L8 c.10319del, p.Gly3440Alafs*33/CNV* Exon 33 Frameshift Pathogenic

– R2 ND 16 08Mtto adcp nAPDb WES by ADPKD in Landscape Mutation 2018 2156, R4 c.1551_1560dup, p.Leu521* Exon 7 Frameshift Pathogenic R10 c.412C.T, p.Arg138* Exon 4 Nonsense Pathogenic R11 c.3929_3930del, p.Asp1310Glyfs*120 (LOH) Exon 45 LOH Pathogenic R13 ND R16 c.12707T.A, p.Val4236Asp Exon 46 Missense Likely pathogenic (CADD=21.4) CMJ001593/PKD1 L2 c.10406_10407insG, Exon 34 c.6994_7000dup, p.Val2334Glyfs*88 Exon 16 Frameshift Pathogenic L3 p.Asp3469Glufs*2 c.6384C.A, p.Asn2128Lys Exon 15 Missense Likely pathogenic (CADD=26.7) L10 c.10977del, p.Glu3660Lysfs*24 Exon 37 Frameshift Pathogenic L11 c.8815G.T, p.Glu2939* Exon 24 Nonsense Pathogenic R3 c.11817G.A, p.Trp3939* Exon 43 Nonsense Pathogenic R9 c.11817G.A, p.Trp3939* Exon 43 Nonsense Pathogenic RP9001591/PKD1 L2 c.G10549T, p.Glu3517* Exon 35 c.8996_8997insG, p.Phe2999Leufs*70 Exon 25 Frameshift Pathogenic L11 c.G10549T, p.Glu3517* (LOH) Exon 35 LOH Pathogenic R17 c.1005delC, p.Val336Cysfs*129 Exon 5 Frameshift Pathogenic JS9001595/PKD1 R2 c.6743_6744dup, Exon 15 c.10220+2T.G IVS 35 Splicing Pathogenic R10 p.Val2249Metfs*2 c.3184C.T, p.Gln1062* Exon 14 Nonsense Pathogenic BH9002280/PKD2 L1 c.923del, p.Phe308Serfs*9 Exon 4 c.2409delA, p.Ser804Valfs*40 Exon 13 Frameshift Pathogenic L2 c.242 C.A, p.Ser81* Exon 1 Nonsense Pathogenic L5 c.962 T.A, p.Ile321Lys (CADD=24.6) Exon 4 Missense Likely pathogenic L6 c.1843G.A, p.Ala615Thr (CADD=33.0) Exon 8 Missense Likely pathogenic L9 c.1258A.T, p.Arg420Trp (CADD=27.7) Exon 5 Missense Likely pathogenic R1 ND R2 c.1392A.G, p.=c Exon 6 Splicing Likely pathogenic R7 c.1366 C.T, p.Gln456* Exon 6 Nonsense Pathogenic www.jasn.org R8 c.2682del, p.Gly895Valfs*14 Exon 15 Frameshift Pathogenic ND, mutation not detected; L, Left kidney; R, Right kidney. LOH. loss of heterozygosity. aProbable pathogenic mutation as evaluated by CADD. A CADD value of $15 suggests the variant is likely pathogenic (http://cadd.gs.washington.edu/). bPKD1 exon 33 single-copy region. cMutation (c.1392A.G, p.=) predicted to create a new donor splice site using splice site prediction tools (ESEfinder3.0, NNSplice and Human splicing finder3.1) with default settings. AI RESEARCH BASIC 2143 BASIC RESEARCH www.jasn.org mutations, six (67%) were truncating and two were missense cysts of patients with constitutional mutations in PKD1 and variants predicted to be likely pathogenic (CADD score $15). PKD2, respectively. The vast majority (90%) were truncating, Pathogenic somatic mutations in PKD1/2 genes were iden- splice site, and in-frame variations predicted to be pathogenic; tified in all patients and in 90% of cysts (57 out of 63; Supple- five (10%) were missense, likely pathogenic variants (Figure mental Table 2, Table 2). Long-range PCR Sanger sequencing 2B). Seven of the 49 PKD1 mutation-positive cysts (14%) were confirmed all somatic mutations identified by WES. Muta- homozygous for constitutional PKD1 mutations, consistent tions were distributed across PKD1 and PKD2 (Figure 2A). with LOH (Figure 2C, Supplemental Figure 3, Table 2 for PKD1 and PKD2 somatic mutations were limited to PKD1 and representative LOH events identified by long-range PCR PKD2 carriers, respectively. There were 51 unique pathogenic/ Sanger/next-generation sequencing and WES). Mean alter- likely pathogenic somatic mutations detected in 49 and eight nate allele frequencies were 0.48 and 0.96 for the heterozygous

Figure 2. Unique pathogenic somatic mutations in PKD1 and PKD2 were identiﬁed in renal cyst epithelia. (A) Prevalence of PKD1 (n=37) and PKD2 (n=8) unique somatic mutations detected in 57 renal cyst DNA samples from patients with ADPKD. Loss of heterozygosity (LOH) mutations not shown. (B) Distribution of SNV and indel mutations in PKD1/2 genes in renal cyst epithelium from all nine patients with ADPKD. Alterations are colored according to mutation type. (C) PKD1 LOH mutations in renal cyst epithelia. Blood and renal cyst DNA heterozygous and homozygous for c.4745 G.C, Trp1582Ser, respectively, detected by long-range PCR (LR-PCR) Sanger sequencing (top panel). Blood and renal cyst DNAs heterozygous and homozygous for c.6364delG, p.Val2122fs, respectively, detected by LR-PCR next-generation sequencing (NGS; bottom panel). PKD1 NGS reads are piled up and are shown in Integrative Genomics Viewer. Allelic total coverage and variant allele frequency (VAF) are displayed for each sample type.

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Figure 2. Continued.

(constitutional) and homozygous (somatic) mutations, re- of a chromosome arm), and at least one CNV change per cyst spectively (Supplemental Table 3). Most (77%; 33 out of 43) was found. Of these, 1057 (31.7%) were copy number gains PKD1 somatic mutations were located in its duplicated region and 2276 (68.3%) were losses. The mutation load varied sig- (exons 1–33); the remaining mutations were located in its niﬁcantly, with a median of 49 and an interquartile range single-copy region. Several cysts in different patients shared (IQR) of 81-31=50 mutations per cyst (range: 12–171; the same PKD1 truncating mutations (c.5395C.T, Figure 3A). Per patient, a median of 65 and an IQR of p.Gln1799*; c.348_352del, p.Asn116Lysfs*2; c.11817G.A, 70-39=31 occurred for SNV/indels (range: 25–167), while a p.Trp3939*), suggesting a hotspot in PKD1. median of 38 and an IQR of 47.5-32=15.5 for CNVs (range: 31–71; Figure 3B). Of the SNV/indel mutations, 247 (7.6%) Characterization by WES of Somatic Mutations in Renal were truncating (frameshift, nonsense, splicing), whereas Cyst Epithelia 1942 (59%) were missense variants (Figure 3C, Supplemen- A total of 74 DNA samples (cyst, n=65; PBL, n=9) were ana- tal Figure 4, Supplemental Table 5). SNV transition/ lyzed by WES, with an average coverage of 89.5368.93;more transversion ratio was with a median of 0.35 and an IQR of than 95% of the exome was covered $103 (Supplemental 0.67-0.16=0.51 (range: 0.06–2.14; Figure 3D). Table 4). Somatic changes were identiﬁed in all 56 PKD1 Patient BS9001424 was incidentally found to have a papil- and nine PKD2 cysts analyzed. There were a total of 3263 lary renal cell carcinoma in the resected kidney, which was not somatic SNV/indels (2490 genes) and 3333 CNV changes in detected preoperatively.22 The mutation load for this patient 2456 genes. All CNVs were focal (i.e., encompassing ,25% (153 mutations per cyst) was higher than the median mutation

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Figure 3. WES of renal cyst epithelia DNA identified a wide spectrum of somatic alterations in all samples. (A) Distribution of mutation (SNV, indel, and CNV) load identified by WES by cyst and by PKD1/2 genes (n=65). (B) The frequency of mutation alterations in cyst epithelia varied significantly among the patients. Alterations are classified by mutation type. SNV/indel and CNV distribution are shown in the top and bottom panels, respectively. (C) Frequency of all somatic SNV substitutions and indel mutations detected in 65 renal cyst epithelia samples from nine patients with ADPKD. Alterations are colored according to mutation type. (D) Transition (Ti) to transversion (Tv) plot of SNVs detected. IGR, intergenic region; UTR, untranslated region. load (65 mutations per cyst) for the cohort (Figure 3B). This prevalence $30% were identified in 16% (nine out of 65) of patient demonstrated a constitutional heterozygous mutation cysts analyzed. in ATM (NM_000051.3: c.6228del; p.Leu2077Phefs*5), WES analysis of the principal pathogenic or likely patho- potentially accounting for the high background mutation genic mutations (n=46) ranked by variant prevalence identi- rate, and a somatic mutation in MET. No other patient fied among all cysts and alteration type (i.e., SNV/indel, CNV) had a constitutional mutation identified in ATM or other in 65 cysts showed that the majority of genes harbored one cancer genes. variation; rare exceptions included PKD1 and SEC24D,in Age was not associated with prevalence of somatic variants which cysts harboring two variations per gene were found. (P=0.20) for the cohort. However, in a multivariate analysis PKD1 and PKD2 were among the most commonly mutated of patients aged .50 years, age was significantly associated with a genes (Figure 4, A and B). higher variant count (P=0.005; Supplemental Material). The average coverage of PKD1 was 323 (613 for PKD1 Overall, 1785 variations (1477 genes) with VAF$5% were nonduplicated exons) and 723 for PKD2. Lower mean cov- classified as pathogenic or likely pathogenic mutations (Sup- erage (,303) occurred in the duplicated region of PKD1,in plemental Table 6). Of these, 1473 (83%) were missense PKD1 exons 42–43, and PKD2 exon 1 and exon 8. Mean cov- mutations (CADD score $15) and 312 (17%) were truncating erage across all exons in PKD1 and PKD2 was similar for the or likely pathogenic in-frame indels. Only 248 variations constitutional and somatic DNA (Supplemental Figure 6). (175 missense and 73 truncating or in-frame indels) in 191 Solely on the basis of WES analysis, PKD1 somatic mutations genes were present at a VAF $20%, suggesting they occurred were identified in only 43% (29 out of 56) of cysts harboring earlier in cyst development than mutations with lower VAFs PKD1 constitutional mutations (Supplemental Table 3), sig- (Supplemental Table 7 and Supplemental Figure 5). At least nificantly below the PKD1 detection rate by LR-PCR based one significant pathogenic or likely pathogenic SNV/indel sequencing (91%; Supplemental Table 2). All PKD1 mutations change was found in every cyst. A summary of the principal undetected by WES, which were identified by long-range PCR variants as classified by their potential effect on protein func- sequencing, were located in its duplicated region, likely re- tion and VAF ($30%) is presented in Table 3. No variants with flecting the low coverage by WES in this region. By contrast,

2146 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2139–2156, 2018 mScNephrol Soc Am J Table 3. Pathogenic and probable pathogenic mutations with VAF$30%, by cyst (n=103) Read CADD Pathway rs#/COSMIC Patient Cyst Gene Chr HGVSc HGVSp VAF P Value Depth Score (Reactome) (ExAC%) BJA001578 A23 PKD1 chr16 c.1560_1561ins p.Leu521Ter 10 0.8 VxPx cargo-targeting to 0.006 TAACACCGAC cilium 29: B13 B3GNT3 chr19 c.156_157insCCCCCC p.Pro52_Pro53insProPro 28 0.61 O-linked glycosylation 0.000 2139 of mucins

– .

16 08Mtto adcp nAPDb WES by ADPKD in Landscape Mutation 2018 2156, B16 PKD1 chr16 c.6485G A p.Arg2162Gln 753 0.35 25.8 VxPx cargo-targeting to 0.009 cilium B16 SMARCAD1 chr4 c.2572T.C p.Phe858Leu 66 0.48 23 * * B14 STK19 chr6 c.328G.T p.Asp110Tyr 42 0.31 19.46 * * A23 C4orf21 chr4 c.3919C.A p.His1307Asn 15 0.4 28.3 * * A24 PKD1 chr16 c.3929_3930del p.Asp1310Glyfs*120 (LOH) 12 1.00 VxPx cargo-targeting to 0.006 cilium BS9001424 A3 PKD1 chr16 c.11888G.A p.Trp3963Ter 18 0.56 41 VxPx cargo-targeting to 0.039 cilium A3 PSME4 chr2 c.4821_4832delCCCA p.Pro1608_Asn1611del 20 0.5 * * GTGGAAAA A3 WARS2 chr1 c.404G.T p.Arg135Leu 46 0.48 35 Mitochondrial tRNA 0.039 rs147962776 (0.03) aminoacylation A3 PAPD7 chr5 c.1371_1373delGTC p.Met457_Ser458delinsIle 204 0.48 * * A3 ITGA7 chr12 c.386G.A p.Arg129Gln 91 0.46 19.41 Laminin interactions 0.06 rs376167554 (0.002) A3 NCOR1 chr17 c.5506G.A p.Val1836Met 66 0.39 24.9 NR1D1 (REV-ERBA) 0.006 represses gene expression A3 USP51 chrX c.663_666delTGGG p.Ser221ArgfsTer22 32 0.59 * * A3 INSIG2 chr2 c.83T.G p.Leu28Trp 61 0.57 26.5 * * A3 NAA15 chr4 c.175delA p.Ile59SerfsTer34 48 0.56 * * rs375319596 (NA) A3 C1orf74 chr1 c.23C.A p.Ser8Ter 101 0.53 36 * * A3 BCOR chrX c.4690A.G p.Arg1564Gly 76 0.51 31 * * A3 EMX2 chr10 c.651A.T p.Glu217Asp 56 0.48 19.56 * * A3 ELP3 chr8 c.1486–1G.T 77 0.48 21.1 * * A3 SIDT1 chr3 c.2269G.A p.Ala757Thr 17 0.47 34 * * COSM201721

A3 RAB23 chr6 c.398A.T p.Asn133Ile 110 0.46 22.8 * * www.jasn.org A3 ZBTB40 chr1 c.435_436delAG p.Gln145HisfsTer7 47 0.45 * * A3 DMXL1 chr5 c.4099C.A p.His1367Asn 52 0.44 15.66 * * A3 CRYZ chr1 c.411_414delTCGA p.Tyr137Ter 143 0.4 * * A3 QRICH2 chr17 c.4226C.T p.Ala1409Val 43 0.33 21.4 * * A2 NOX5 chr15 c.844A.C p.Ser282Arg 25 0.32 23.9 * * rs150003957 (1.67) AI RESEARCH BASIC COSM964378 CDS001574 A21 PKD1 chr16 c.11817G.A p.Trp3939Ter 27 0.56 39 VxPx cargo-targeting to 0.006 cilium B7 AKT2 chr19 c.1016T.G p.Val339Gly 13 0.31 27.1 PKB-mediated events 0.001 2147 2148

Table 3. Continued RESEARCH BASIC

Read CADD Pathway rs#/COSMIC Patient Cyst Gene Chr HGVSc HGVSp VAF P Value Depth Score (Reactome) (ExAC%) ora fteAeia oit fNephrology of Society American the of Journal B12 PKD1 chr16 c.10084del p.Gln3362Serfs*35 (LOH) 304 0.96 VxPx cargo-targeting to 0.006 cilium B12 SNX8 chr7 c.910C.T p.Gln304Ter 66 0.44 36 * . A20 SPTY2D1 chr11 c.2044C G p.Leu682Val 46 0.3 20.8 * * www.jasn.org B7 WDFY3 chr4 c.2039A.C p.Gln680Pro 13 0.38 16.39 * * B7 SON chr21 c.331C.A p.His111Asn 32 0.38 23.3 * * B8 PRR12 chr19 c.5284G.A p.Gly1762Arg 86 0.37 19.09 * * B9 LRCH1 chr13 c.705G.T p.Leu235Phe 65 0.31 28.5 * * B10 TINAGL1 chr1 c.961C.T p.Arg321Cys 64 0.38 * * CMJ001593 A30 PARP14 chr3 c.2768C.A p.Ser923Tyr 79 0.32 26 Nicotinamide salvaging 0.006 A30 ZG16 chr16 c.157C.T p.Arg53Trp 64 0.31 30 * * A30 ANKK1 chr11 c.754C.T p.Gln252Ter 52 0.31 26.8 * * A31 CCT4 chr2 c.814A.T p.Met272Leu 27 0.56 25.9 Folding of actin by CCT/ 0.003 TriC A31 PNISR chr6 c.1651T.C p.Ser551Pro 12 0.67 22.9 * * A31 EVI5 chr1 c.1327A.T p.Ile443Phe 41 0.44 25.1 * * A33 MVP chr16 c.1807G.A p.Val603Ile 51 0.45 26 * * A33 CWF19L2 chr11 c.1880G.T p.Gly627Val 84 0.4 23 * * A34 SMARCAD1 chr4 c.2572T.C p.Phe858Leu 22 0.41 23 * * DR9001565 A15 PKD1a Chr16 c.9504C.G p.Phe3168Leu (LOH) 66 0.88 VxPx cargo-targeting to 0.006 cilium A15 C1QL2 chr2 c.590G.T p.Cys197Phe 10 0.3 31 * * A16 THRAP3 chr1 c.2701C.T p.Arg901Ter 9 0.33 41 Transcriptional 0.008 regulation of white adipocyte differentiation A17 DOCK4 chr7 c.4286_4287delAA p.Lys1429ArgfsTer3 124 0.39 Megakaryocyte 0.03 development and platelet production A17 CASC5 chr15 c.4886C.T p.Thr1629Ile 29 0.38 17.62 Nucleosome assembly 0.01

mScNephrol Soc Am J A18 PCDHGB6 chr5 c.1415C.A p.Ala472Glu 14 0.3 20.8 * * B3 ACSS1 chr20 c.500G.A p.Arg167His 12 0.42 31 Ethanol oxidation 0.001 B4 PKD1 chr16 c.6282G.T p.Trp2094Cys 9 0.33 26.8 VxPx cargo-targeting to 0.006 cilium B4 KIF17 chr1 c.3002G.C p.Arg1001Pro 15 0.33 34 Intraﬂagellar transport COSM182818 B5 PADI4 chr1 c.979G.C p.Ala327Pro 69 0.3 23.8 Chromatin modifying 0.04 rs145819522 (0.15) 29: enzymes 2139 B5 WRNIP1 chr6 c.1120G.A p.Val374Met 80 0.33 32 * * – 16 2018 2156, mScNephrol Soc Am J Table 3. Continued

Read CADD Pathway rs#/COSMIC Patient Cyst Gene Chr HGVSc HGVSp VAF P Value Depth Score (Reactome) (ExAC%) B6 UBE3C chr7 c.1332G.T p.Arg444Ser 16 0.31 23.2 Antigen processing: 0.03 29: ubiquitination and

2139 proteasome

– degradation 16 08Mtto adcp nAPDb WES by ADPKD in Landscape Mutation 2018 2156, JS9001595 B26 LILRB5 chr19 c.1652A.C p.Gln551Pro 17 0.35 16.26 Immunoregulatory 0.03 interactions: lymphoid and a nonlymphoid cell B27 PNPLA6 chr19 c.2966+2T.G 17 0.35 Glycerophospholipid 0.002 catabolism B27 ZNF518B chr4 c.641A.C p.Glu214Ala 45 0.96 27.7 * * B27 MYOM3 chr1 c.3751A.C p.Lys1251Gln 20 0.3 23.2 * * B29 PCSK5 chr9 c.1312G.T p.Val438Leu 13 0.31 26.8 NGF processing 0.001 B31 PKD1 chr16 c.10220+2T.G 39 0.46 VxPx cargo-targeting to 0.008 cilium B31 KCNC3 chr19 c.*81A.C 16 0.44 Voltage-gated 0.02 potassium channels B32 CRCP chr7 c.145–1G.T 19 0.32 RNA polymerase III 0.003 chain elongation B33 PKD1 chr16 c.3184C.T p.Gln1062Ter 27 0.63 35 VxPx cargo-targeting to 0.006 cilium B33 ANGPTL3 chr1 c.124G.C p.Asp42His 73 0.4 28.4 Assembly of active LPL 0.005 rs199772471 (0.02) and LIPC lipase complexes BH9002280 B34 PKD2 chr4 c.2409delA p.Ser804ValfsTer40 80 0.3 VxPx cargo-targeting to 0.004 cilium B35 CEP97 chr3 c.1142A.T p.Asp381Val 33 0.3 23.2 Anchoring of the basal 0.009 body to the plasma membrane L2 PKD2 chr4 c.242C.A p.Ser81Ter 14 0.57 36 VxPx cargo-targeting to 0.004 www.jasn.org cilium L5 PRKCI chr3 c.280G.C p.Glu94Gln 35 0.34 24.3 p75NTR recruits 0.003 signaling complexes L5 SCAF1 chr19 c.619_620ins p.Ser207_ 12 0.33 * * CCCCCCCCC Pro208insProProPro

R2 PKD2 chr4 c.1392A.G p.= 32 0.34 VxPx cargo-targeting to 0.004 RESEARCH BASIC cilium R7 PKD2 chr4 c.1366C.T p.Gln456Ter 98 0.38 42 VxPx cargo-targeting to 0.004 cilium B36 NXNL1 chr19 c.577C.G p.Arg193Gly 9 0.44 23.2 * * 2149 R8 YBX3 chr12 c.40_42delACC p.Thr14del 16 0.31 * * 2150

Table 3. Continued RESEARCH BASIC

Read CADD Pathway rs#/COSMIC Patient Cyst Gene Chr HGVSc HGVSp VAF P Value Depth Score (Reactome) (ExAC%) ora fteAeia oit fNephrology of Society American the of Journal L5 SCAF1 chr19 c.619_620ins p.Ser207_ 12 0.33 * * CCCCCCCCC Pro208insProProPro PJ9001456 A10 PKD1a chr16 c.7655_7658 delinsTTG p.Ala2552Valfs*68 779 0.32 VxPx cargo-targeting to 0.006

cilium www.jasn.org A11 MIB2 chr1 c.2092G.A p.Glu698Lys 10 0.3 22.8 Signaling by NOTCH1 0.003 HD Domain Mutants in Cancer A7 PKD1a chr16 c.6364delG p.Val2122Lysfs*3 (LOH) 804 0.92 VxPx cargo-targeting to 0.008 cilium A7 SVIL chr10 c.4121T.G p.Leu1374Arg 10 0.3 25.8 * * A8 PKD1a chr16 c.348_352del p.Asn116Lysfs*2 793 0.3 VxPx cargo-targeting to 0.006 cilium A9 PKD1 chr16 c.8016+1delG 12 0.5 VxPx cargo-targeting to 0.006 cilium B1 SLC14A2 chr18 c.2548A.C p.Asn850His 19 0.32 22.2 Transport -bile salts, 0.03 organic acids, metal ions, amine compounds B1 URGCP chr7 c.173A.C p.Asn58Thr 14 0.36 26.9 * * B1 DNAH5 chr5 c.5335G.T p.Val1779Phe 28 0.32 27.9 * * B2 EIF3A chr10 c.330G.T p.Gln110His 33 0.36 23.5 Formation of the ternary 0.01 complex, subsequently 43S complex B2 RGS1 chr1 c.553G.T p.Asp185Tyr 59 0.32 33 Ga(i) signaling events 0.004 RP9001591 B18 PKD1 chr16 c.8996_8997insG p.Phe2999LeufsTer70 64 0.33 VxPx cargo-targeting to 0.006 cilium B18 ATP2B3 chrX c.2866G.A p.Gly956Arg 92 0.3 27 Reduction of cytosolic 0.003 Ca++ levels B20 CACNA2D1 chr7 c.668G.T p.Trp223Leu 12 0.42 35 Phase 2 – plateau phase 0.00 COSM747982

mScNephrol Soc Am J B20 PRRC2B chr9 c.1997A.C p.Gln666Pro 11 0.36 25.6 * * B21 PKD1a chr16 c.G10549T p.Glu3517* (LOH) 140 0.97 VxPx cargo-targeting to 0.006 cilium B21 KIF26A chr14 c.5624C.T p.Pro1875Leu 75 0.39 22 Kinesins 0.01 rs200461988 (0.05) B21 KISS1R chr19 c.828_832delGGGCC p.Trp276CysfsTer28 72 0.35 Peptide ligand-binding 0.03 receptors 29: B22 SMARCA2 chr9 c.667_675del p.Gln226_Gln228del 111 0.36 RUNX1 interacts with 0.003 2139 CAGCAGCAG cofactors whose

– precise effect on 16 2018 2156, RUNX1 not known www.jasn.org BASIC RESEARCH

WES was 100% sensitive for detecting PKD2 somatic muta- ﬁ

ed tions identi ed by long-range PCR sequencing (Table 2). ﬁ We analyzed 211 genes associated with other ciliopathy disorders (Supplemental Table 8) and 565 cancer genes. At least (ExAC%)

cantly over- one pathogenic mutation in a ciliopathy or cancer gene was rs#/COSMIC ﬁ identiﬁed in 49% (32 out of 65) and 77% (50 out of 65) of all cysts, respectively. Of the 1477 genes harboring a pathogenic

15 suggests the variant is or likely pathogenic SNV or indel mutation, 2.0% (29 out of $ * * * *

Value 1477) were associated with a ciliopathy, whereas 5.6% (83 out 0.0267 rs149119701 (0.002) P of 1477) were cancer-related (Supplemental Tables 9 and 10). Among the 2456 genes demonstrating CNV changes (Supplemental Table 11), 36 (1.5%) were associated with ciliopathies, whereas 91 (3.7%) were related to cancer. Among * * * * the top 64 genes harboring four or more CNV changes (Figure

Pathway 4B), one (1.6%) was related to ADPKD and cilia dysfunction (Reactome) agellar transport 0.01 COSM182818

ﬂ (PKD1), and four (6.2%) were related to cancer (IKZF1, clathrin-mediated endocytosis BCL3, RANBP2,andDUSP4). Copy number changes of PKD1 were observed in 12% of the cysts. PKD1 copy number ed with David (https://david.ncifcrf.gov/). Only signi

ﬁ gains ranging from 151 bp to 28 kb were seen in seven Score CADD separate cysts obtained from three patients (Supplemental Figures7,SupplementalTable12).Forthesinglecyst

VAF (patient BJA001578/cystL8) harboring both PKD1/2 copy number changes, the PKD1 amplified region was approxi- 16 0.31 25.6 22 0.36 29.329 Cargo recognition for 0.52 7616 0.4111 0.38 23 0.36 34 31 Intra mately 20 kb long, spanning exons 5 through 46, whereas Read Depth the PKD2 amplification was approximately 70 kb long, including the entire gene. Further computational analysis with MuSiC identified 53 significantly mutated genes (Supplemental Table 13). Of these, ltered using a 30% VAF cutoff and a population allele frequency of 5% (http://exac.broadinstitute.org/).

ﬁ 7.5% (four out of 53) were related to ciliopathy disorders (PKD1, PKD2, CC2D2A,andDNAH5) and 9.4% (ﬁve out of

HGVSp 53) were related to cancer (RECQL, P2RY8, DNMT3A, SMARCA2,andSMARCAD1). Assessment of constitutional variants in ciliopathy genes identified unique heterozygous p.Arg500Trp p.Asp153AlafsTer7 p.Arg308Cys p.Arg1001Pro p.Glu29Ala p.Gln666Pro pathogenic mutations in CENPF and MYO7A (patient PJ9001456), and CC2D2A and TTC21B (patient CMJ001593) (Supplemental Table 14). No pathogenic constitutional mutation in a non-PKD1/2 ciliopathy gene was identified in BH9002280; this patient had the largest total kidney weight C T C T . . . HGVSc of the cohort (15.7 kg), despite the relatively young age and C . . cantly mutated WES variants were further PKD2 constitutional mutation. Pathway analysis of the most fi

ATGGGGAC significantly mutated genes with VAF $20% by Reactome, on , the probability that overlap between the queried variant and pathway occurs by chance, were reported. Cysts/variants for which no pathways were identi the basis of their biologic function (Table 4), indicated involve- i.e. ment in membrane trafficking, cytoskeleton filament assembly, chr1 c.3002G chr1 c.922C chr7 c.86A chr2 c.458_465del chr9 c.1997A chr14 c.1498C 0.05), and chromatin organization. , values (

P DISCUSSION SELE KIF17 STON2 PRRC2B ABCA13 ATAD2B Inactivating somatic mutations of a PKD gene, which lead to loss of function of the corresponding polycystin, cause clonal B24 B23 B24 B23 B24 B23 expansion of kidney cyst epithelium and cyst formation in Continued ADPKD. However, earlier studies reported a relatively low –

genotyping performed by long-range PCR next-generation sequencing. prevalence (20% 70%) of pathogenic somatic mutations, challenging the primacy of this mechanism in the pathogen- Patient Cyst Gene Chr

PKD1 3 likely pathogenic (http://cadd.gs.washington.edu/). Signi Table 3. The pathogenic potential of missense variants was evaluated using the computational analysis tool CADD, which enables scoring of deleteriousness of SNVs and indels. A CADD value of a Variants were further subjected for pathway analysis to identify overrepresented pathways using Reactome (https://reactome.org/) and veri represented pathways with low are indicated by *. Chr, chromosome;Somatic HGVSc, Mutations the In Human Cancer; Genome ExAC, Variation The Society Exome coding Aggregation sequence name; Consortium; HGVSp, tRNA, the transfer Human ribonucleic Genome Variation acid. Society protein sequence name; rs#/COSMIC, Catalogue Of esis of ADPKD. Methodologic limitations in some of those

J Am Soc Nephrol 29: 2139–2156, 2018 Mutation Landscape in ADPKD by WES 2151 BASIC RESEARCH www.jasn.org

Figure 4. WES analysis detected significant somatic alterations in commonly mutated genes. WES analysis of the most significantly mutated (pathogenic and likely pathogenic) genes (n=46) ranked by the frequency of all detected (A) SNV/indel and (B) CNV alterations found in the gene among all cysts (n=65). Only genes with three or more SNV/indel and six or more CNV, respectively, are presented. Segments with a log2 ratio (LogR) between renal cyst and PBL (constitutional) read count .0.6 were designated as gains and segments with a log2 value ,20.6 were categorized as losses. Results are grouped by cyst and by patient. Gene data are displayed by patient, indicated by the colored boxes at the bottom of each figure (patients BS9001424, PJ9001456, DR9001565, CDS001574, BJA001578, CMJ001593, RP9001591, JS9001595, and BH9002280). studies included the required detection of large chromosomal this study, comprehensive genomic analysis of renal cyst ep- deletions, and in others by the structural complexity of PKD1, ithelium, whereby the exome from individual cysts was particularly in its duplicated region, which limited the detec- screened by WES and long-range PCR and mutations were tion of smaller, inactivating somatic mutations.21,25–27 In confirmed by Sanger sequencing, found inactivating somatic

2152 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2139–2156, 2018 mScNephrol Soc Am J Table 4. Significantly altered pathways in cyst epithelia by Reactome (genes with VAF$0.2) Entities Entities Entities Pathway Identifier Pathway Name Submitted Entities Found Founda Totala P Value R-HSA-390471 Association of TriC/CCT with 4 40 0.004 USP11;KIF13A;TCP1;CCT4 target proteins during 29: biosynthesis 2139 R-HSA-5620920 Cargo trafficking to the periciliary 4 55 0.01 TCP1;PKD2;PKD1;CCT4 –

16 08Mtto adcp nAPDb WES by ADPKD in Landscape Mutation 2018 2156, membrane R-HSA-5617833 Cilium Assembly 8 207 0.02 DYNC2H1;CEP164;TCP1;CEP97;PKD2;KIF17;PKD1;CCT4 R-HSA-1852241 Organelle biogenesis and 11 334 0.02 DYNC2H1;NCOR1;CEP164;TCP1;TFAM;CEP97;PKD2;KIF17;PKD1;CCT4 maintenance R-HSA-390450 Folding of actin by CCT/TriC 2 13 0.02 TCP1;CCT4 R-HSA-4839726 Chromatin organization 9 257 0.02 KDM6B;NCOR1;TRRAP;DNMT3A;EP400;ELP3;PADI4;ARID1A;SMARCA2 R-HSA-3247509 Chromatin modifying enzymes 9 257 0.02 KDM6B;NCOR1;TRRAP;DNMT3A;EP400;ELP3;PADI4;ARID1A;SMARCA2 R-HSA-5576892 Phase 0 – rapid depolarization 3 47 0.04 CACNA2D1;SCN4A R-HSA-163282 Mitochondrial transcription 1 3 0.05 TFAM initiation R-HSA-4085023 Defective GFPT1 causes CMSTA1 1 3 0.05 GFPT1 R-HSA-73780 RNA polymerase III chain 2 22 0.05 CRCP;POLR3E elongation R-HSA-2408550 Metabolism of ingested H2SeO4 2 22 0.05 GSR;PAPSS2 and H2SeO3 into H2Se R-HSA-1655829 Regulation of cholesterol 4 86 0.05 INSIG2;SC5D;SEC24D biosynthesis by SREBP (SREBF) R-HSA-73980 RNA polymerase III transcription 2 23 0.05 CRCP;POLR3E termination R-HSA-3214858 RMTs methylate histone arginines 3 53 0.05 DNMT3A;ARID1A;SMARCA2 R-HSA-5620922 BBSome-mediated cargo- 2 24 0.06 TCP1;CCT4 targeting to cilium R-HSA-5620916 VxPx cargo-targeting to cilium 2 25 0.06 PKD2;PKD1 Pathway analysis was performed by Reactome (https://reactome.org/). aEntities, proteins with an accession sequence in UniPort and Ensemble databases. www.jasn.org AI RESEARCH BASIC 2153 BASIC RESEARCH www.jasn.org mutations of PKD1/2 genes in every patient, encompassing cyst L8) with a somatic mutation in PKD1. The significance of 90% of all cysts. This is consistent with the constitutional these findings is unclear. One consideration is that PKD gene mutation detection rate for ADPKD. In our cohort, PKD1 amplification can contribute to cyst development, although our and PKD2 were the most frequently mutated genes in cyst data are insufficient to support this conclusion. epithelia. The higher somatic mutation detection rate in our In this study, utilizing WES, we found a median of 65 so- study reflects the extensive and sensitive screening methods matic variations per cyst (0.022 mutations per megabase) and a utilized.4,5,7 transition/transversion ratio of 0.35, supporting the preference A key finding of our study is that approximately 90% of of transversion mutations. The quantity and ratio of these the PKD1/2 gene somatic mutations were truncating in- variations were overall lower than values reported for various frame deletions and splicing mutations, implicating them as cancers.35–37 Although common mechanisms can promote drivers for cyst development. One example is the rare silent cell proliferation in ADPKD and in cancer, studies associating change p.R464R (patient BH9002280), predicted to create a ADPKD with cancer are conflicting because of methodologic new cryptic donor site and aberrant splicing of PKD2.Splicing limitations. Their interpretations are confounded by the gen- defects caused by exonic mutations have been implicated in erally increased risk of renal cell cancer in CKD and the pau- the pathogenesis in ADPKD.28 No trans-heterozygous muta- city of prospective, adequately controlled clinical trials tions of PKD1/2 genes were identified, possibly reflecting the evaluating cancer in ADPKD.38,39 For example, the report small cohort. of a lower risk of cancer in patients with or without ADPKD This study was limited to patients in whom constitutional after kidney transplantation may have been confounded by the mutations of PKD1/2 genes were identified; as expected, 77% higher rate of malignancy identified before transplantation in of these were truncating. Altogether, the high prevalence of ADPKD as a consequence of more frequent radiographic concurrent truncating constitutional and somatic inactivation evaluation of complex kidney cysts, clinical assessment of of PKD1/2 genes, leading to null/null alleles and kidney cyst various symptoms (e.g., gross hematuria, pain), and nephrec- development, strongly supports a primary role for the cellular tomy that may have alerted clinicians to previously undetected recessive model of cyst formation.29 Although we could not malignancies.40 These findings have not been generalized to determine variant orientation (i.e., in-cis versus in-trans), pre- patients with ADPKD who have not undergone kidney vious studies using cloning techniques demonstrated that transplantation.35–37 PKD1/2 constitutional and somatic mutations occurred We determined that approximately 5% of the most prevalent in-trans.4,5,7,30 mutated genes are designated as cancer genes, including a regu- PKD1/2 gene haploinsufficiency caused by inadequate lators of gene methylation (i.e., DNMT3A) and chromatin re- gene dosage is an alternative mechanism for ADPKD.3,13,31 modeling (i.e., SMARCA2). However, we found no association In a mouse ortholog of homozygous mutation of Pkd1 alleles of mutation rate and disease severity. We detected an association in which aberrant splicing of Pkd1 yielded an ADPKD phe- of mutation count with older age, consistent with another report notype, 13%–20% of the homozygotes had normally spliced showing that the frequency of Phosphoribosyltransferase Pkd1 transcripts. The few heterozygotes that survived more (HRPT) gene mutations in 6-thioguanine (TG)-resistant renal than 1 year had Pkd1 expression levels approximately 40%– cortical epithelial cells from donors increase exponentially 50% of wild type, suggesting that modestly reduced levels of with donor age.41 The oldest patient in our cohort, who had PC1 sufficiently maintained renal function and prolonged the highest mutation load and a constitutional mutation of survival.11 Patients with ADPKD who were homozygous or ATM, which is a cell-cycle checkpoint kinase that regulates compound heterozygous for hypomorphic alleles reportedly various downstream proteins (e.g., p53, BRCA1), also had an had severe disease phenotypes, whereas milder disease occurred incidental finding of papillary renal cell carcinoma caused in heterozygotes with hypomorphic alleles.12 Although missense by a somatic mutation in MET.22 However, a somatic muta- mutations can provide sufficient gene activity to attenuate cyst tion of PKD1 was not identified in the tumor cells, and a growth3,32 no PKD1/2 missense/missense allele combination was somatic MET mutation was not identified in other, nonma- identified in our study other than LOH in two cysts with likely lignant renal cyst epithelium in this patient. Although our pathogenic constitutional mutations (patients BS9001424 and findings do not suggest a common mechanism for ADPKD DR9001565). Although we did not measure PC1 levels, the vast and renal cell cancer, this study was not designed to address majority of somatic mutations were truncating, and thus had this issue. negligible PKD1/2 gene expression. We identified pathogenic somatic mutations in genes asso- PKD gene overexpression in orthologous models can cause ciated with other ciliopathies, (i.e., CC2D2A, DNAH5), which kidney cyst development.33 In our study, numerous cysts from were among the most prevalent in this cohort, occurring in three patients had PKD1/2 copy number gains. Although large approximately 30% of all cysts. Mutations in CC2D2A cause constitutional rearrangements in PKD1/2 have previously Meckel syndrome and Joubert syndrome, both associated with been described,34 to our knowledge, this is the first report of kidney cysts, whereas mutations in DNAH5 account for somatic copy number gains in PKD1/2. Interestingly, amplifica- primary ciliary dyskinesia, an autosomal recessive disorder tion of both PKD1/2 genes occurred in one patient (BJA001578, involving abnormalities of motile cilia.

2154 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2139–2156, 2018 www.jasn.org BASIC RESEARCH

Our study was not designed to assess the potential role of REFERENCES variants in non-PKD1/2 genes in the pathogenesis of ADPKD. However, the finding of at least one pathogenic mutation of a 1. Chapman AB, Devuyst O, Eckardt KU, Gansevoort RT, Harris T, Horie S, non-PKD gene in all the evaluable cysts suggests that these et al.: Autosomal-dominant polycystic kidney disease (ADPKD): Exec- variants might influence cyst development. It is well estab- utive summary from a kidney disease: Improving global outcomes (KDIGO) controversies conference. Kidney Int 88: 17–27, 2015 lished that cyst generation involves activation of prolifera- 2. 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Watnick TJ, Torres VE, Gandolph MA, Qian F, Onuchic LF, Klinger KW, ing constitutional and somatic mutations in PKD1/2 genes, et al.: Somatic mutation in individual liver cysts supports a two-hit and confirmatory Sanger sequencing of PKD1/2 gene muta- model of cystogenesis in autosomal dominant polycystic kidney disease. Mol Cell 2: 247–251, 1998 tions detected by WES. To our knowledge, this is the most 5. Pei Y, Watnick T, He N, Wang K, Liang Y, Parfrey P, et al.: Somatic PKD2 comprehensive study of WES in the renal epithelium of pa- mutations in individual kidney and liver cysts support a “two-hit” model tients with ADPKD. Limitations of the study include low sen- of cystogenesis in type 2 autosomal dominant polycystic kidney dis- sitivity of the WES method to detect mutations in the ease. J Am Soc Nephrol 10: 1524–1529, 1999 duplicated region of PKD1,insufficient cyst DNA material 6. 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2156 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2139–2156, 2018 SUPPLEMENTAL INFORMATION

Somatic Mutations in Renal Cyst Epithelium in Autosomal Dominant Polycystic Kidney Disease

Adrian Y. Tan1,2, Tuo Zhang2, Alber Michaeel1 , Jon Blumenfeld3,4, Genyan Liu1, Wanying Zhang1, Zhengmao Zhang1, Yi Zhu1, Lior Rennert5, Che Martin1, Jenny Xiang2, Steven P. Salvatore1, Brian D. Robinson1, Sandip Kapur6, Stephanie Donahue4, Warren O. Bobb4, and *Hanna Rennert1

Departments of Pathology and Laboratory Medicine1, Microbiology2, Medicine3, and Surgery6, Weill Cornell Medicine, The Rogosin Institute4, New York, NY, Department of Biostatistics, University of Pennsylvania5, Philadelphia, PA5

Table of Contents SUPPLEMENTAL FIGURES ...... 3 Figure S1. Schematic diagram of the ADPKD mutation analysis study protocol ...... 3 Figure S2. Blood and renal cyst DNAs heterozygous and homozygous for c.6364delG, p.V2122fs, respectively, detected by whole exome sequencing (WES) analysis ...... 5 Figure S3. Distribution of somatic changes by alteration type and by cyst across all patients. Alterations are colored according to mutation type ...... 8 Figure S4. PKD1 and PKD2 gene coverage by Exon ...... 9 Figure S5. Copy number variations (CNVs) in PKD1 and PKD2 ...... 10 Figure S6. Illustration of the data analysis pipelines ...... 14 Figure S7. Distribution of significant somatic alterations in patients’ cysts samples .... 15 SUPPLEMENTAL TABLES ...... 17 Table S1. Summary of constitutional mutations in study cohort (n=9) ...... 17 Table S2. PKD gene sequence analysis summary statistics of cysts analyzed ...... 18 Table S3. Summary of constitutional and somatic mutations in PKD genes detected by LR-PCR sequencing and WES in individual renal cysts by subject (N=9) ...... 19 Table S4. WES analysis quality metrics (n=55) ...... 23 Table S5. Classification of somatic alterations (SNV/indels) detected by WES in cyst renal epithelia (N=65) ...... 25

1 Table S6. Pathogenic and probably pathogenic mutations by variant allele frequency26 Table S7. Significantly pathogenic (truncation/indels) and probably pathogenic (CADD score>15) gene variations with VAF >20% among the renal cyst samples ...... 28 Table S8. List of genes associated with ciliopathy disorders (N=211) ...... 36 Table S9. Gene variations in principal ciliopathy genes ...... 37 Table S10. Gene variations in principal cancer genes ...... 38 Table S11. Copy number alterations (CNAs) in most commonly altered (>4 CNV) genes among the cyst samples...... 41 Table S12. Copy number variation in PKD genes by renal cyst ...... 49 Table S13. WES analysis results of significantly mutated genes (n=53) in renal cyst epithelium by MuSiC ...... 50 Table S14. Constitutional pathogenic mutations in principal ciliopathy genes ...... 53 SUPPLEMENTAL STATISTICAL ANALYSIS: Analysis of somatic variant counts by patient age ...... 54 I. Analysis of variant counts (N=3,263 variations) in study subjects (N=9) ...... 54 II. Analysis of variant counts (N=1,784 variations classified as pathogenic or likely pathogenic) in study subjects (N=9) ...... 56 SUPPLEMENTAL METHODS ...... 58 Study Subjects ...... 58 Tissue processing and epithelial cell isolation ...... 58 Sanger Sequencing ...... 59 Long-range PCR (LR-PCR) Next Generation Sequencing (NGS) ...... 59 Whole Exome Sequencing (WES) and Data Analysis ...... 59 Significantly mutated genes and pathway analysis ...... 60 Droplet Digital PCR ...... 62 Statistical Analysis ...... 62 REFERENCES ...... 63

SUPPLEMENTAL FIGURES Supplemental Figure 1

Figure S1. Schematic diagram of the ADPKD mutation analysis study protocol. The study population comprised nine patients with ADPKD scheduled for a living kidney transplant and native kidney removal at the NYPH/WCM campus. The clinical eligibility for nephrectomy was determined by the transplant surgeon prior to enrollment. Prospective study subjects were identified during the pre-transplant evaluation period, following by eligibility assessment and obtaining of informed consent from all subjects. After surgery and pathologist’s gross-examination, renal cysts were identified and cyst criteria recorded followed by isolation of the cyst epithelia according to standard procedure (see Methods). Isolated cells were collected by centrifugation and stored at -80°C for further analyses.

4 Supplemental Figure 2 a.

5 c.

6 Figure S2. Blood and renal cyst DNAs heterozygous and homozygous for c.6364delG, p.V2122fs, respectively, detected by whole exome sequencing (WES) analysis. PKD1 WES reads are piled up and are shown in Integrative Genomics Viewer (IGV). The mutation total coverage and variant allele frequency (VAF) are displayed for each sample.

WES analysis of PBL constitutional DNA shows no coverage at the mutation site, but PKD1 Analysis results of renal cyst DNA clearly show LOH for selected SNPs (see table below for coverage and VAF data) at flanking exonic regions of PKD. Shown here are PKD1 exons 23 through 46.

Germline DNA Cyst DNA (A15) PKD1 Coverage Coverage chr16 location Exon/Intron Nomenclature depth VAF depth VAF chr16 2152651 Intron24 g.215651A>G 69 57% 58 88% chr16 2140680 Exon44 p.Ile4044Val/c.12130A>G 71 52% 70 84% chr16 2140454 Exon45 p.Ala4091Ala/c.12273A>G 87 59% 69 91%

7 Supplemental Figure 3

Figure S3. Distribution of somatic changes by alteration type and by cyst across all patients. Alterations are colored according to mutation type.

8 Supplemental Figure 4

a. PKD1 and PKD2 gene coverage in periperal blood lymphocytes (PBL)

b. PKD1 and PKD2 gene coverage in renal cyst epithelia

Figure S4. PKD1 and PKD2 gene coverage by Exon using (a) constitutional peripheral blood lymphocytes (n=18) and (b) renal cyst epithelia cells (n=65). HMW gDNA. PBL, peripheral blood lymphocytes.

9 Supplemental Figure 5

Figure S5. Copy number variations (CNVs) in PKD1 and PKD2. Shown here are plots of CNV analysis identified in selected cyst samples (Table 3 in the manuscript) presented as Log2 Ratio of fold-change at target PKD gene regions. Such alterations can typically affect a single gene to thousands of genes.

13 Supplemental Figure 6

Figure S6. Illustration of the data analysis pipelines. Listed are the analysis steps and the corresponding software/application programs involved.

14 Supplemental Figure 7 a.

15 Figure S7. Distribution of significant somatic alterations in patients’ cysts samples. (a) Distribution of missense (CADD>15) and truncation/inframe indels in each cyst by mutation count (a), and (b) mutation count variant allele frequency (VAF). Only mutation with VAF >20% are shown. Missesne and truncation/indel alterations are colored in blue and orange, accordingly.

16 SUPPLEMENTAL TABLES

Supplemental Table 1. Summary of constitutional mutations in study cohort (n=9) Mutation Mutation Case ID Gene Exon Constitutional mutation type designation c.4745 G>C, p.W1582S Probable BS9001424 PKD1 Exon 15 Missense (CADD=26.3) pathogenic PJ9001456 PKD1 Exon 15 c.6364delG, p.V2122fs*3 Frameshift Pathogenic c.9504C>G, Probable DR9001565 PKD1 Exon 27 Missense p.F3168L(CADD=27) pathogenic c.10084delC, CDS001574 PKD1 Exon 31 Frameshift Pathogenic p.Q3362fs*35 c.3929_3930het_delAC, BJA001578 PKD1 Exon 15 Frameshift Pathogenic P.D1310Gfs*120

c.10406_10407insG, CMJ001593 PKD1 Exon 43 Frameshift Pathogenic p.E3469fs*2

RP9001591 PKD1 Exon 35 c.G10549T, p.E3517X Nonsense Pathogenic

c.6743_6744dupA, JS9001595 PKD1 Exon 15 Frameshift Pathogenic p.N2248Kfs*14 c.923het_delT, BH9002280 PKD2 Exon 4 Frameshift Pathogenic p.F308Sfs*9

Supplemental Table 2. PKD gene sequence analysis summary statistics of cysts analyzed Sequence Mutation- Mutation- LR-NGS/ Overall Mutated Analysis positive positive cysts Patient ID WES Sanger mutation gene (Total) cysts (LR-PCR sequencing detection rate (WES) sequencing) BS9001424 PKD1 10 2 1 10 9 90% PJ9001456 PKD1 12 8 3 12 10 83% DR9001565 PKD1 11 9 3 7 7 100% CDS001574 PKD1 8 7 3 5 5 100% BJA001578 PKD1 10 7 2 9 7 78% CMJ001593 PKD1 8 8 4 6 6 100% RP9001591 PKD1 7 7 2 3 3 100% JS9001595 PKD1 8 8 2 2 2 100% BH9002280 PKD2 9 9 9 9 8 89% Total 83 65 29 63 57 90%

Supplemental Table 3. Summary of constitutional and somatic mutations in PKD1/2 genes detected by LR-PCR sequencing and WES in individual renal cysts by subject (N=9)

Detected Case Cyst WES Cyst Constitutional Somatic mutation Exon/ Cov Exon by WES VAF ID/Gene ID ID diam mutation (LR-PCR Sanger) Intron Depth (Yes/No) c.4745 BS9001424 *L1 5 Exon 15 ND __ NT G>C,p.W1582S c.4745 c.4798dup , PKD1 *L2 1 Exon 15 Exon 15 NT G>C,p.W1582S p.T1600Asnfs*15 c.4745 c.12216_12217del, *L3 2 Exon 15 Exon 45 NT G>C,p.W1582S p.Thr4073Profs*83 c.4745 c.348_352del , L4 A2 2.5 Exon 15 Exon 3 No G>C,p.W1582S p.Asn116Lysfs*2 c.4745 c.3225del , *L5 1.5 Exon 15 Exon14 NT G>C,p.W1582S p.Pro1076Argfs*28 c.4745 c.348_352del , *L6 2 Exon 15 Exon 3 NT G>C,p.W1582S p.Asn116Lysfs*2 c.4745 c.11888G>A, R1 A3 3 Exon 15 Exon 43 Yes 18 0.56 G>C,p.W1582S p.Trp3963* c.4745 c.5395C>T, *R2 2 Exon 15 Exon 15 NT G>C,p.W1582S p.Gln1799* c.4745 *R4 2.5 Exon 15 c.3162-2A>C IVS 11 NT G>C,p.W1582S c.4745 c.4745 G>C,p.W1582S *R5 1.5 Exon 15 Exon 15 NT G>C,p.W1582S (LOH) c.6364delG:p.V2122f PJ9001456 L1 4 Exon 15 ND __ NT s c.6364delG:p.V2122fs - PKD1 L4 A7 4 c.6364delG:p.V2122fs Exon 15 Exon 15 No ^(804) (LOH) 0.92 c.348_352del, - L5 A8 5.5 c.6364delG:p.V2122fs Exon 15 Exon 3 No ^(793) p.Asn116Lysfs*2 0.29

c.12006_12010del, L6 A9 4 c.6364delG:p.V2122fs Exon 15 Exon 44 No p.Gln4004Alafs*151

12 0.5

L7 3 c.6364delG:p.V2122fs Exon 15 c.8016+1del Exon 21 Yes ^(316 - x) 0.42 c.7655_7658 ^(779 - L10 A10 5 c.6364delG:p.V2122fs Exon 15 delinsTTG, Exon 19 No x) 0.32 p.Ala2552Valfs*68 c.3745del, ^(671 - L11 A11 2.5 c.6364delG:p.V2122fs Exon 15 Exon 15 No p.Asp1249Thrfs*24 x) 0.23 c.6364delG:p.V2122fs ^(638 L12 2.5 c.6364delG:p.V2122fs Exon 15 Exon 15 NT -0.9 (LOH) x)

c.1284_1292del, ^(748 *L15 7 c.6364delG:p.V2122fs Exon 15 Exon 6 NT -0.3 p.Trp429_Gln431del x)

c.3831_3847del, R4 B1 3.1 c.6364delG:p.V2122fs Exon 15 Exon 15 No p.Ser1278Glyfs*17 c.12004-2_12019del R7 B2 2.5 c.6364delG:p.V2122fs Exon 15 Exon 44 Yes 193 0.23 (splice_acceptor) c.6364delG:p.V2122f R16 A12 6 Exon 15 ND __ No s

DR9001565 L1 A14 8 c.9504C>G,p.F3168L Exon 27 NT __ No

19 #c.9504C>G,p.F3168L PKD1 *L3 A15 4 c.9504C>G,p.F3168L Exon 27 Exon 27 No (LOH) c.8935_8937delTTC, L5 B3 3.5 c.9504C>G,p.F3168L Exon 27 Exon 24 Yes 30 0.27 p.Phe2979del L6 B4 5 c.9504C>G,p.F3168L Exon 27 NT No c.5395C>T, L7 B5 2 c.9504C>G,p.F3168L Exon 27 Exon 15 Yes 41 0.34 p.Gln1799* L10 B6 3.5 c.9504C>G,p.F3168L Exon 27 NT No c.2157del, R8 A16 6 c.9504C>G,p.F3168L Exon 27 Exon 11 No p.His719Glnfs*66 c.1789del, R9 4 c.9504C>G,p.F3168L Exon 27 Exon 9 NT p.Gln597Argfs*188 c.4916dup, Exon 27 Exon 15 p.Gly1640Argfs*18 c.5180del, R10 3.5 c.9504C>G,p.F3168L Exon 27 Exon 15 NT p.Pro1727Argfs*32 c.12711C>A, R13 A17 3 c.9504C>G,p.F3168L Exon 27 Exon 46 Yes 39 0.28 p.Tyr4237* R14 A18 2.5 c.9504C>G,p.F3168L Exon 27 NT No c.10314_10315insGCT PKD1 c.10084del , CDS001574 L2 B7 4 Exon 22 GGCA, Exon 33 Yes 157 0.22 p.Gln3362Serfs*35 p.Arg3439Alafs*34 PKD1 c.10084del , PKD1 L4 B8 4 Exon 22 NT __ No p.Gln3362Serfs*35 PKD1 c.10084del , L6 B9 2 Exon 22 NT __ No p.Gln3362Serfs*35 PKD1 c.10084del , c.348_352del, *L9 A20 1.5 Exon 22 Exon 3 No p.Gln3362Serfs*35 p.Asn116Lysfs*2 PKD1 c.10084del , c.11887G>A, R6 A21 3 Exon 22 Exon 43 Yes 27 0.56 p.Gln3362Serfs*35 p.Trp3939* PKD1 c.10084del , R8 B10 1.5 Exon 22 NT __ No p.Gln3362Serfs*35 PKD1 c.10084del , c.11884_11912del, R9 2.5 Exon 22 Exon 43 NT p.Gln3362Serfs*35 p.Gln3962Alafs*5 c.10084del, PKD1 c.10084del , R10 B12 2.5 Exon 22 p.Gln3362Serfs*35 Exon 22 Yes p.Gln3362Serfs*35 (LOH) c.3929_3930del, c.6558_6568del, BJA001578 L3 B13 10 Exon 15 Exon 15 No p.Asp1310Glyfs*120 p.Trp2187Serfs*71 c.3929_3930del, c.10948delC, PKD1 L4 B14 4 Exon 15 Exon 37 Yes 118 0.15 p.Asp1310Glyfs*120 p.His3650Thrfs*34 c.3929_3930del, L7 B15 2 Exon 15 NT __ No p.Asp1310Glyfs*120 c.10319del, c.3929_3930del, L8 B16 5 Exon 15 p.Gly3440Alafs*33/ Exon 33 No p.Asp1310Glyfs*120 CNV* c.3929_3930del, R2 3 Exon 15 ND __ NT p.Asp1310Glyfs*120 c.3929_3930del, c.1551_1560dup, R4 A23 2 Exon 15 Exon 7 Yes 10 0.8 p.Asp1310Glyfs*120 p.Leu521* c.3929_3930del, R10 2.5 Exon 15 c.412C>T, p.ArgR138* Exon 4 NT p.Asp1310Glyfs*120 #c.3929_3930del, c.3929_3930del, R11 A24 3 Exon 15 p.Asp1310Glyfs*120 Exon 15 Yes 12 1 p.Asp1310Glyfs*120 (LOH)

20 c.3929_3930del, R13 A25 3 Exon 15 ND No p.Asp1310Glyfs*120 c.3929_3930del, c.12707T>A, R16 3 Exon 15 Exon 46 NT p.Asp1310Glyfs*120 p.Val4236Asp c.10406_10407insG, CMJ001593 L1 A27 10 Exon 34 NT __ No p.Asp3469Glufs*2 c.10406_10407insG, c.6994_7000dup, PKD1 L2 A28 4 Exon 34 Exon 16 No p.Asp3469Glufs*2 p.Val2334Glyfs*88 c.10406_10407insG, c.6384C>A, *L3 A29 4 Exon 34 Exon 15 No p.Asp3469Glufs*2 p.Asn2128Lys c.10406_10407insG, c.10977del, L10 A30 3.5 Exon 34 Exon 37 Yes 58 0.22 p.Asp3469Glufs*2 p.Glu3660Lysfs*24 c.10406_10407insG, c.8815G>T, L11 A31 3 Exon 34 Exon 24 Yes 46 0.22 p.Asp3469Glufs*2 p.Glu2939* c.10406_10407insG, R1 A32 8 Exon 34 NT __ No p.Asp3469Glufs*2 c.10406_10407insG, c.11887G>A, R3 A33 2 Exon 34 Exon 43 Yes 34 0.09 p.Asp3469Glufs*2 p.Trp3939* c.10406_10407insG, c.11887G>A, R9 A34 2 Exon 34 Exon 43 Yes 47 0.21 p.Asp3469Glufs*2 p.Trp3939* c.8996_8997insG, RP9001591 L2 B18 3 c.G10549T, p.E3517X Exon 35 Exon 25 Yes 64 0.33 p.Phe2999Leufs*70 PKD1 L9 B19 3 c.G10549T, p.E3517X Exon 35 NT __ No L10 B20 2 c.G10549T, p.E3517X Exon 35 NT __ No *c.G10549T, *L11 B21 2.5 c.G10549T, p.E3517X Exon 35 Exon 35 Yes 140 0.97 p.Glu3517* (LOH) L15 B22 1 c.G10549T, p.E3517X Exon 35 NT __ No R10 B23 10 c.G10549T: p.E3517X Exon 35 NT __ No c.G10549T , c.1005delC, R17 B24 7 Exon 35 Exon 5 Yes 38 0.21 p.E3517X p.Val336Cysfs*129 c.6743_6744dup, JS9001595 L1 B26 7 Exon 15 NT __ No p.Val2249Metfs*2 c.6743_6744dup, PKD1 L4 B27 7 Exon 15 NT __ No p.Val2249Metfs*2 c.6743_6744dup, L8 B28 3 Exon 15 NT __ No p.Val2249Metfs*2 c.6743_6744dup, L9 B29 1.5 Exon 15 NT __ No p.Val2249Metfs*2 c.6743_6744dup, R1 B30 5 Exon 15 NT __ No p.Val2249Metfs*2 c.6743_6744dup, c.10220+2T>G (splice R2 B31 4 Exon 15 IVS 5 Yes 39 0.46 p.Val2249Metfs*2 donor site) c.6743_6744dup, R9 B32 5 Exon 15 NT __ No p.Val2249Metfs*2 c.6743_6744dup, c.3184C>T, R10 B33 8 Exon 15 Exon 14 Yes 27 0.63 p.Val2249Metfs*2 p.Gln1062* c.923del , c.2409delA, BH9002280 L1 B34 4 Exon 4 Exon 13 Yes 80 0.3 p.Phe308Serfs*9 p.Ser804Valfs*40 c.923del , PKD2 L2 L2 5 Exon 4 c.242 C>A, p.Ser81* Exon 1 Yes 14 0.57 p.Phe308Serfs*9 c.923del , L5 L5 7 Exon 4 c.962 T>A, p.Ile321Lys Exon 4 Yes 23 0.17 p.Phe308Serfs*9 c.923del , c.1843G>A, L6 B35 4 Exon 4 Exon 8 Yes 22 0.18 p.Phe308Serfs*9 p.Ala615Thr c.923del , c.1258A>T, L9 B36 5 Exon 4 Exon 5 Yes 67 0.27 p.Phe308Serfs*9 p.Arg420Trp

21 c.923del , R1 R1 6.5 Exon 4 ND __ No p.Phe308Serfs*9 c.923del , R2 R2 6.5 Exon 4 +c.1392A>G, p.= Exon 6 Yes 32 0.34 p.Phe308Serfs*9 c.923del , R7 R7 7 Exon 4 c.1366 C>T, p.Gln456* Exon 6 Yes 146 0.38 p.Phe308Serfs*9 c.923del , c.2682del, R8 R8 5 Exon 4 Exon 15 Yes 55 0.11 p.Phe308Serfs*9 p.Gly895Valfs*14 *Tested by LR-PCR Sequencing (Sanger/NGS); NT, not tested; ND, mutation not detected (highlighted in bold); L, Left kidney; R, Right kidney; Cyst diam, Cyst diameter

^LR-PCR NGS Coverage depth and VAF (patient PJ9001456). VAF, variant allele frequency.

#Flanking genomic regions show LOH (patients DR9001565, cyst L3/A15; BJA001578, cyst R11/A24; RP9001591 cyst L11/B21).

+Mutation (c.1392A>G, p.= ) predicted to create a new donor splice site using splice site prediction tools (ESEfinder, NNSplice and Human splicing finder 3.1) with default settings as the following:

5' splicing site donor of human (U2 type); threshold: 6.67 Wt: score =0 Mut: score =6.72

NNSplice: 5' splicing site donor (score between: 0~1); threshold: 0.4 Wt: score =0 Mut: score =0.99

Human splicing finder (no threshold, but Ref score for both wt and mut sequence): Wt_5' Motif Ref score= 1.02 Mut_5' Motif Ref score= 9.2

Supplemental Table 4. WES analysis quality metrics (n=55)

Sample Captured % Captured Average Fraction Patient ID WES ID Total Reads Reads Reads Coverage Covered >=10X A2 59288140 46647670 79 79 95 BS9001424 A3 60948808 51182719 84 81 95 A7 61112704 51516100 84 81 94 A8 64692576 52908950 82 86 95 A9 57059740 48152933 84 76 94 A10 56696466 48222269 85 77 94 PJ9001456 A11 58105432 49809367 86 79 93 A12 53511126 44190577 83 73 93 B1 75218576 61014391 81 100 96 B2 69718392 56029475 80 92 96 A14 58622018 50119702 86 79 93 A15 57994028 49533195 85 79 93 A16 58038740 48101321 83 78 93 A17 57663748 49069555 85 78 94 DR9001565 A18 55774588 45270235 81 75 94 B3 68230676 54114712 79 88 96 B4 72135280 58102834 81 95 96 B5 78874860 63768230 81 104 96 B6 70553906 57460160 81 94 96 A20 55590492 46663285 84 74 94 A21 60246768 47784210 79 80 95 B7 72624574 58490167 81 95 96 CDS001574 B8 74513118 60092508 81 98 96 B9 68360776 57113523 84 91 96 B10 69178470 56933333 82 92 96 B12 74641886 61432923 82 98 96 A23 59382594 49963127 84 79 94 A24 59782360 46468366 78 80 94 A25 57531320 48098903 84 76 94 BJA001578 B13 75934774 59548796 78 99 96 B14 68692792 52338655 76 88 95 B15 73866112 59247067 80 97 96 B16 71212372 56576270 79 93 96 A27 60232948 50752932 84 80 95 CMJ0001593 A28 65011266 53044127 82 87 94 A29 69967054 54169683 77 92 95

23 A30 66966870 54225024 81 89 95 A31 64805090 52926697 82 86 94 A32 71051458 59647035 84 95 95 A33 66779892 55156803 83 89 94 A34 67611950 56878603 84 91 95 L2 62389122 50012518 80 81 95 L5 68866436 57674420 84 90 95 R1 66089250 55482615 84 87 95 R2 65830676 53939148 82 85 95 BH9002280 R7 67064636 55703092 83 88 95 R8 66508468 55034994 83 86 95 B34 75648008 61277253 81 101 97 B35 76221042 61680888 81 102 96 B36 71463126 58499640 82 96 96 B18 70125512 56105924 80 92 96 B19 74910468 60072054 80 99 96 B20 74945990 60396940 81 99 96 RP9001591 B21 73595282 59072274 80 97 96 B22 76493664 62258955 81 102 96 B23 72595400 58484269 81 96 96 B24 72419496 58365628 81 96 96 B26 74871118 60357216 81 99 96 B27 75409158 62195918 82 100 96 B28 75977794 61275216 81 100 96 B29 59530170 45746169 77 79 95 JS9001595 B30 77272994 62133223 80 102 96 B31 72114850 57032207 79 95 96 B32 77055686 62202623 81 102 96 B33 73808990 58468279 79 98 96

Average 67529724 55019045 81.6 89.5 95.2 SD 6989380 5241790 2.2 8.9 1.0

Supplemental Table 5. Classification of somatic alterations (SNV/indels) detected by WES in cyst renal epithelia (N=65) Alteration type Variant count Frameshift 58 Nonsense 130

Splice 59

Inframe indel 59 Missense 1942 Synonymous 555 UTR/Flank 119 Intron/IGR 272 RNA 69 Total 3263

Supplemental Table 6. Pathogenic and likely pathogenic mutations by variant allele frequency Truncate/ Missense Missense inframe Truncate/ Truncate/ Missense (20-29% (30-65% (5-19% inframe (20- inframe (30- Patient-ID Cyst ID (5-19% VAF) VAF) VAF) VAF) 29% VAF) 65% VAF) Total BH9002280 B34 17 4 3 1 1 26 B35 9 4 1 14 B36 14 3 1 1 19 L2 43 1 5 1 50 L5 25 1 5 1 1 33 R1 32 3 6 41 R2 47 5 2 54 R7 25 2 7 1 35 R8 35 1 10 1 47 BJA001578 A23 7 1 4 1 13 A24 2 2 A25 2 3 5 B13 24 3 3 1 31 B14 27 2 1 6 2 38 B15 19 3 2 2 26 B16 7 6 2 1 1 2 19 BS9001424 A2 78 2 18 98 A3 60 2 11 5 9 87 CDS001574 A20 8 1 1 10 A21 12 1 2 15 B10 15 4 1 1 2 2 25 B12 22 5 1 1 2 31 B7 11 5 3 2 5 26 B8 15 5 1 2 1 24 B9 8 1 1 3 3 1 17 CMJ0001593 A27 6 1 1 8 A28 35 2 10 47 A29 41 5 46 A30 34 2 2 6 1 1 46 A31 38 2 3 6 2 51 A32 47 2 6 55 A33 4 2 5 1 12 A34 36 2 1 5 1 45 DR9001565 A14 38 3 41 A15 23 2 1 4 1 31 A16 11 1 1 13 A17 16 1 2 2 1 22 A18 37 1 6 44 B3 5 1 1 7

26 B4 10 1 2 1 14 B5 13 2 2 4 1 22 B6 8 1 9 JS9001595 B26 7 1 3 11 B27 9 1 2 1 13 B28 14 4 18 B29 4 1 1 2 8 B30 8 1 2 1 12 B31 7 1 2 10 B32 17 1 2 2 22 B33 15 2 1 1 1 20 PJ9001456 A10 31 2 7 1 41 A11 29 1 4 1 35 A12 22 4 2 1 1 30 A7 9 1 5 1 1 17 A8 46 3 7 1 57 A9 48 1 16 2 67 B1 6 1 3 2 1 13 B2 19 1 2 2 2 1 27 RP9001591 B18 2 2 1 1 6 B19 6 2 2 10 B20 3 1 2 3 1 10 B21 6 4 1 2 1 14 B22 3 2 1 6 B23 13 1 2 4 1 21 B24 12 2 3 1 18 Grand Total 1302 109 62 227 37 48 1785

Total (VAF >5%)-1785

Missense-1473

Truncation/inframe indel-312

Total (VAF >20%)-248

Missesne- 175

Truncation/inframe indel- 73

Supplemental Table 7. Significantly pathogenic (truncation/indels) and probably pathogenic (CADD score>15) gene variations with VAF >20% among the renal cyst samples CADD Cyst Variant score(PH Patient ID# ID Gene CHR Variant classification HGVSc HGVSp coverage VAF RED) Existing variation

BJA001578 A23 PKD1 chr16 Frame_Shift_Ins c.1560_1561insTAACACCGAC p.Leu521Ter 10 0.80 NA BJA001578 A23 C4orf21 chr4 Missense_Mutation c.3919C>A p.His1307Asn 15 0.40 28.3 COSM145050 p.Pro52_Pro53insPr BJA001578 B13 B3GNT3 chr19 In_Frame_Ins c.156_157insCCCCCC oPro 28 0.61 NA BJA001578 B13 ACTR3 chr2 Missense_Mutation c.433G>T p.Ala145Ser 20 0.25 24.8 BJA001578 B13 ARID1A chr1 Missense_Mutation c.920G>T p.Gly307Val 8 0.25 22 BJA001578 B13 FCHSD2 chr11 Missense_Mutation c.2158C>A p.Pro720Thr 26 0.23 29 BJA001578 B13 LRCH1 chr13 Missense_Mutation c.711G>T p.Lys237Asn 51 0.20 27.4 BJA001578 B14 STK19 chr6 Missense_Mutation c.328G>T p.Asp110Tyr 42 0.31 19.46 BJA001578 B14 GSR chr8 Splice_Site c.422+1G>T 22 0.27 27.6 BJA001578 B14 TIAM2 chr6 Missense_Mutation c.494C>T p.Thr165Met 76 0.25 24 rs375863554 BJA001578 B14 HS2ST1 chr1 Nonsense_Mutation c.997C>T p.Arg333Ter 9 0.22 40 COSM912858 BJA001578 B14 NMI chr2 Missense_Mutation c.194C>A p.Pro65His 9 0.22 28.4 rs377654044 BJA001578 B15 KDM6B chr17 In_Frame_Del c.2253_2258delCACCAC p.Thr754_Thr755del 86 0.40 NA COSM985382 BJA001578 B15 RAB30 chr11 Missense_Mutation c.203A>C p.Gln68Pro 20 0.25 26.5 BJA001578 B15 ABTB1 chr3 Missense_Mutation c.112A>C p.Thr38Pro 20 0.25 25.9 BJA001578 B15 NF1 chr17 Missense_Mutation c.3245G>T p.Gly1082Val 44 0.23 29 BJA001578 B16 SMARCAD1 chr4 Missense_Mutation c.2572T>C p.Phe858Leu 66 0.48 23 BJA001578 B16 PKD1 chr16 Missense_Mutation c.6485G>A p.Arg2162Gln 753 0.35 25.8 rs377654044 BJA001578 B16 KDM6B chr17 In_Frame_Del c.2253_2258delCACCAC p.Thr754_Thr755del 76 0.33 NA COSM985382 BJA001578 B16 IGSF9B chr11 Missense_Mutation c.1427G>A p.Arg476His 87 0.28 28 BJA001578 B16 GFPT1 chr2 Splice_Site c.1010-2A>C 38 0.26 25.2 BJA001578 B16 GPR1 chr2 Missense_Mutation c.183G>T p.Trp61Cys 8 0.25 29.4 rs141816898 BJA001578 B16 CST2 chr20 Missense_Mutation c.232G>T p.Val78Leu 20 0.25 24.2 COSM1202533

BJA001578 B16 DNAH5 chr5 Missense_Mutation c.7009C>T p.Leu2337Phe 29 0.24 27.6 BJA001578 B16 EML3 chr11 Missense_Mutation c.2246A>C p.Glu749Ala 52 0.21 27.6

BJA001578 B16 NF1 chr17 Missense_Mutation c.3244G>T p.Gly1082Cys 29 0.21 29.8

28 rs150003957 BS9001424 A2 NOX5 chr15 Missense_Mutation c.844A>C p.Ser282Arg 24 0.29 23.9 COSM964378 BS9001424 A2 TIGD2 chr4 Missense_Mutation c.653C>A p.Thr218Lys 14 0.21 25.2

BS9001424 A3 USP51 chrX Frame_Shift_Del c.663_666delTGGG p.Ser221ArgfsTer22 32 0.59 NA BS9001424 A3 INSIG2 chr2 Missense_Mutation c.83T>G p.Leu28Trp 61 0.57 26.5

BS9001424 A3 NAA15 chr4 Frame_Shift_Del c.175delA p.Ile59SerfsTer34 48 0.56 NA rs375319596 BS9001424 A3 PKD1 chr16 Nonsense_Mutation c.11888G>A p.Trp3963Ter 18 0.56 41 BS9001424 A3 C1orf74 chr1 Nonsense_Mutation c.23C>A p.Ser8Ter 101 0.53 36 BS9001424 A3 BCOR chrX Missense_Mutation c.4690A>G p.Arg1564Gly 76 0.51 31

c.4821_4832delCCCAGTGGAAA p.Pro1608_Asn1611 BS9001424 A3 PSME4 chr2 In_Frame_Del A del 20 0.50 NA BS9001424 A3 EMX2 chr10 Missense_Mutation c.651A>T p.Glu217Asp 56 0.48 19.56 BS9001424 A3 ELP3 chr8 Splice_Site c.1486-1G>T 77 0.48 21.1 BS9001424 A3 WARS2 chr1 Missense_Mutation c.404G>T p.Arg135Leu 46 0.48 35 rs147962776 p.Met457_Ser458de BS9001424 A3 PAPD7 chr5 In_Frame_Del c.1371_1373delGTC linsIle 204 0.48 NA BS9001424 A3 SIDT1 chr3 Missense_Mutation c.2269G>A p.Ala757Thr 17 0.47 34 COSM201721 BS9001424 A3 RAB23 chr6 Missense_Mutation c.398A>T p.Asn133Ile 110 0.46 22.8 BS9001424 A3 ITGA7 chr12 Missense_Mutation c.386G>A p.Arg129Gln 91 0.46 19.41 rs376167554

BS9001424 A3 ZBTB40 chr1 Frame_Shift_Del c.435_436delAG p.Gln145HisfsTer7 47 0.45 NA BS9001424 A3 DMXL1 chr5 Missense_Mutation c.4099C>A p.His1367Asn 52 0.44 15.66

BS9001424 A3 CRYZ chr1 Frame_Shift_Del c.411_414delTCGA p.Tyr137Ter 143 0.40 NA BS9001424 A3 NCOR1 chr17 Missense_Mutation c.5506G>A p.Val1836Met 66 0.39 24.9 BS9001424 A3 QRICH2 chr17 Missense_Mutation c.4226C>T p.Ala1409Val 43 0.33 21.4 rs150003957 BS9001424 A3 NOX5 chr15 Missense_Mutation c.844A>C p.Ser282Arg 25 0.32 23.9 COSM964378 BS9001424 A3 MTBP chr8 Missense_Mutation c.365G>T p.Gly122Val 13 0.23 16.47 BS9001424 A3 SIGIRR chr11 Missense_Mutation c.1082C>A p.Pro361His 49 0.22 24.8 CDS001574 A20 SPTY2D1 chr11 Missense_Mutation c.2044C>G p.Leu682Val 46 0.30 20.8 CDS001574 A20 BCAR3 chr1 Nonsense_Mutation c.895C>T p.Arg299Ter 10 0.20 38 CDS001574 A21 PKD1 chr16 Nonsense_Mutation c.11817G>A p.Trp3939Ter 27 0.56 39 p.Val15_Leu16insLe rs10663835 CDS001574 A21 CNDP1 chr18 In_Frame_Ins c.43_44insTGC u 26 0.46 NA COSM307404 CDS001574 B10 TINAGL1 chr1 Missense_Mutation c.961C>T p.Arg321Cys 64 0.38 34 CDS001574 B10 OXR1 chr8 Splice_Site c.1960-1G>T 17 0.29 26

29 CDS001574 B10 WDFY3 chr4 Missense_Mutation c.2039A>C p.Gln680Pro 17 0.29 16.39 CDS001574 B10 CFH chr1 Missense_Mutation c.3029C>T p.Ala1010Val 92 0.29 17.6 CDS001574 B10 TRRAP chr7 Missense_Mutation c.2440C>G p.Pro814Ala 67 0.27 24.1 CDS001574 B10 PPIL6 chr6 Nonsense_Mutation c.244G>T p.Glu82Ter 19 0.26 37 CDS001574 B10 DNAH5 chr5 Missense_Mutation c.5340G>T p.Met1780Ile 29 0.21 16.41 CDS001574 B12 SNX8 chr7 Nonsense_Mutation c.910C>T p.Gln304Ter 66 0.44 36 CDS001574 B12 AKT2 chr19 Missense_Mutation c.1016T>G p.Val339Gly 14 0.29 27.1 CDS001574 B12 INTS9 chr8 Missense_Mutation c.269T>G p.Leu90Arg 33 0.24 27.4 CDS001574 B12 RAB30 chr11 Missense_Mutation c.203A>C p.Gln68Pro 25 0.24 26.5 CDS001574 B12 SLC14A2 chr18 Missense_Mutation c.2548A>C p.Asn850His 21 0.24 22.2 CDS001574 B12 UPF2 chr10 Nonsense_Mutation c.257C>A p.Ser86Ter 59 0.20 36 CDS001574 B12 PCDHA13 chr5 Missense_Mutation c.88C>A p.Gln30Lys 10 0.20 23.7 CDS001574 B7 WDFY3 chr4 Missense_Mutation c.2039A>C p.Gln680Pro 13 0.38 16.39 CDS001574 B7 SON chr21 Missense_Mutation c.331C>A p.His111Asn 32 0.38 23.3 CDS001574 B7 AKT2 chr19 Missense_Mutation c.1016T>G p.Val339Gly 13 0.31 27.1 CDS001574 B7 CCDC180 chr9 Splice_Site c.1921-1G>A 27 0.26 24.7 CDS001574 B7 MMP8 chr11 Nonsense_Mutation c.880G>T p.Glu294Ter 28 0.25 41 CDS001574 B7 ALDH5A1 chr6 Missense_Mutation c.815G>T p.Cys272Phe 20 0.25 27.3 rs75301270 CDS001574 B7 CEP164 chr11 Frame_Shift_Del c.337delA p.Lys116ArgfsTer22 25 0.24 NA COSM1351515 CDS001574 B7 GABRA6 chr5 Missense_Mutation c.850A>C p.Thr284Pro 21 0.24 26.9 CDS001574 B7 DROSHA chr5 Missense_Mutation c.3007A>C p.Ile1003Leu 17 0.24 23 COSM1186750 CDS001574 B7 INTS9 chr8 Missense_Mutation c.269T>G p.Leu90Arg 31 0.23 27.4 p.Arg3439AlafsTer3 CDS001574 B7 PKD1 chr16 Frame_Shift_Ins c.10314_10315insGCTGGCA 4 157 0.22 NA rs78912196 CDS001574 B7 TFAM chr10 Frame_Shift_Del c.432delA p.Glu148SerfsTer2 28 0.21 NA COSM1348365 CDS001574 B7 DGAT2 chr11 Missense_Mutation c.272T>G p.Leu91Arg 59 0.20 23.1 CDS001574 B8 PRR12 chr19 Missense_Mutation c.5284G>A p.Gly1762Arg 86 0.37 19.09 rs141816898 CDS001574 B8 CST2 chr20 Missense_Mutation c.232G>T p.Val78Leu 17 0.29 24.2 COSM1202533 CDS001574 B8 SEC24D chr4 Missense_Mutation c.1735A>C p.Ile579Leu 31 0.26 20.9 CDS001574 B8 SLC14A2 chr18 Missense_Mutation c.2548A>C p.Asn850His 16 0.25 22.2 CDS001574 B8 AMBRA1 chr11 Missense_Mutation c.143A>C p.Glu48Ala 17 0.24 27 CDS001574 B8 LRCH1 chr13 Missense_Mutation c.706G>T p.Val236Leu 56 0.23 29.1 CDS001574 B8 LRCH1 chr13 Missense_Mutation c.705G>T p.Leu235Phe 56 0.20 28.5 CDS001574 B9 LRCH1 chr13 Missense_Mutation c.705G>T p.Leu235Phe 65 0.31 28.5 CDS001574 B9 FAM155B chrX In_Frame_Del c.54_56delCTG p.Cys20del 26 0.27 NA rs374286243 CDS001574 B9 NUP155 chr5 Missense_Mutation c.1196C>A p.Ser399Tyr 14 0.21 29.6 COSM1218072 CDS001574 B9 TP53I11 chr11 Splice_Site c.335-2A>C 34 0.21 24.7

30 rs71436423 CMJ001593 A27 MEDAG chr13 Missense_Mutation c.68G>A p.Arg23His 15 0.27 28.5 COSM432293 CMJ001593 A28 BAI1 chr8 Missense_Mutation c.1004G>T p.Arg335Leu 8 0.25 23 CMJ001593 A28 DNMT3A chr2 Missense_Mutation c.1439T>G p.Val480Gly 50 0.22 27.4 rs200099128 CMJ001593 A30 PARP14 chr3 Missense_Mutation c.2768C>A p.Ser923Tyr 79 0.32 26 CMJ001593 A30 ZG16 chr16 Missense_Mutation c.157C>T p.Arg53Trp 64 0.31 30 CMJ001593 A30 ANKK1 chr11 Nonsense_Mutation c.754C>T p.Gln252Ter 52 0.31 26.8 CMJ001593 A30 ITGB1 chr10 Missense_Mutation c.1667A>G p.Asp556Gly 104 0.25 32 CMJ001593 A30 PKDCC chr2 Missense_Mutation c.911C>T p.Thr304Met 92 0.23 23.4 rs150069795 p.Ala3661ProfsTer2 CMJ001593 A30 PKD1 chr16 Frame_Shift_Del c.10980delA 3 58 0.22 NA CMJ001593 A31 PNISR chr6 Missense_Mutation c.1651T>C p.Ser551Pro 12 0.67 22.9 CMJ001593 A31 CCT4 chr2 Missense_Mutation c.814A>T p.Met272Leu 27 0.56 25.9 CMJ001593 A31 EVI5 chr1 Missense_Mutation c.1327A>T p.Ile443Phe 41 0.44 25.1 CMJ001593 A31 PTGER1 chr19 Missense_Mutation c.571G>T p.Gly191Cys 8 0.25 26.7 CMJ001593 A31 PTGER1 chr19 Missense_Mutation c.596G>T p.Arg199Leu 8 0.25 22.9 CMJ001593 A31 MN1 chr22 Nonsense_Mutation c.3029G>A p.Trp1010Ter 36 0.22 39 CMJ001593 A31 PKD1 chr16 Nonsense_Mutation c.8815G>T p.Glu2939Ter 46 0.22 39 CMJ001593 A32 MAGED2 chrX Missense_Mutation c.905G>T p.Arg302Leu 16 0.25 32 COSM191867 CMJ001593 A32 MCM4 chr8 Missense_Mutation c.2019C>A p.Ser673Arg 10 0.20 20.9 CMJ001593 A33 MVP chr16 Missense_Mutation c.1807G>A p.Val603Ile 51 0.45 26 CMJ001593 A33 CWF19L2 chr11 Missense_Mutation c.1880G>T p.Gly627Val 84 0.40 23 CMJ001593 A33 TSHZ1 chr18 Nonsense_Mutation c.1564C>T p.Gln522Ter 70 0.29 36 CMJ001593 A34 SMARCAD1 chr4 Missense_Mutation c.2572T>C p.Phe858Leu 22 0.41 23 CMJ001593 A34 DNMT3A chr2 Missense_Mutation c.1439T>G p.Val480Gly 42 0.24 27.4 rs200099128 CMJ001593 A34 PKD1 chr16 Nonsense_Mutation c.11817G>A p.Trp3939Ter 47 0.21 39 CMJ001593 A34 TCP1 chr6 Missense_Mutation c.269C>A p.Thr90Asn 15 0.20 27.7 DR9001565 A15 C1QL2 chr2 Missense_Mutation c.590G>T p.Cys197Phe 10 0.30 31 DR9001565 A15 FER chr5 Missense_Mutation c.2248C>T p.Leu750Phe 24 0.29 31 rs75301270 DR9001565 A15 CEP164 chr11 Frame_Shift_Del c.337delA p.Lys116ArgfsTer22 58 0.21 NA COSM1351515 DR9001565 A15 WIZ chr19 Missense_Mutation c.1112G>T p.Arg371Leu 10 0.20 23.2 DR9001565 A16 THRAP3 chr1 Nonsense_Mutation c.2701C>T p.Arg901Ter 9 0.33 41

DR9001565 A17 DOCK4 chr7 Frame_Shift_Del c.4286_4287delAA p.Lys1429ArgfsTer3 124 0.39 NA DR9001565 A17 CASC5 chr15 Missense_Mutation c.4886C>T p.Thr1629Ile 29 0.38 17.62 DR9001565 A17 PKD1 chr16 Nonsense_Mutation c.12711C>A p.Tyr4237Ter 39 0.28 38 CM003948 DR9001565 A18 PCDHGB6 chr5 Missense_Mutation c.1415C>A p.Ala472Glu 14 0.29 20.8 DR9001565 B3 ACSS1 chr20 Missense_Mutation c.500G>A p.Arg167His 12 0.42 31 DR9001565 B3 TIA1 chr2 Missense_Mutation c.199A>C p.Asn67His 20 0.25 26.5

31 DR9001565 B4 KIF17 chr1 Missense_Mutation c.3002G>C p.Arg1001Pro 15 0.33 34 COSM182818

DR9001565 B4 PKD1 chr16 Missense_Mutation c.6282G>T p.Trp2094Cys 9 0.33 26.8 DR9001565 B4 SEC24D chr4 Missense_Mutation c.1787G>T p.Arg596Ile 36 0.25 34 DR9001565 B4 KCNC3 chr19 3'UTR c.*81A>C 20 0.25 19.57 DR9001565 B5 WRNIP1 chr6 Missense_Mutation c.1120G>A p.Val374Met 80 0.33 32 DR9001565 B5 PADI4 chr1 Missense_Mutation c.979G>C p.Ala327Pro 69 0.30 23.8 rs145819522

DR9001565 B5 ODF3L2 chr19 Frame_Shift_Ins c.577_578insC p.Arg193ProfsTer8 31 0.26 NA DR9001565 B5 NOD2 chr16 Missense_Mutation c.1057T>A p.Cys353Ser 13 0.23 16.44 DR9001565 B5 RYR3 chr15 Missense_Mutation c.9831A>T p.Arg3277Ser 73 0.22 27.2 DR9001565 B6 UBE3C chr7 Missense_Mutation c.1332G>T p.Arg444Ser 16 0.31 23.2 JS9001595 B26 LILRB5 chr19 Missense_Mutation c.1652A>C p.Gln551Pro 17 0.35 16.26 JS9001595 B27 ZNF518B chr4 Missense_Mutation c.641A>C p.Glu214Ala 45 0.96 27.7 JS9001595 B27 PNPLA6 chr19 Splice_Site c.2966+2T>G 17 0.35 24.9 JS9001595 B27 MYOM3 chr1 Missense_Mutation c.3751A>C p.Lys1251Gln 20 0.30 23.2 JS9001595 B27 POLR3E chr16 Missense_Mutation c.1459C>T p.Arg487Trp 166 0.20 17.53 JS9001595 B28 EPG5 chr18 Missense_Mutation c.1906T>G p.Tyr636Asp 36 0.28 26.3 JS9001595 B28 XPNPEP2 chrX Missense_Mutation c.1100C>A p.Ala367Asp 11 0.27 18.56 JS9001595 B28 SC5D chr11 Missense_Mutation c.439T>G p.Tyr147Asp 48 0.25 31 JS9001595 B28 UBR7 chr14 Missense_Mutation c.246T>G p.Cys82Trp 39 0.23 27.1 JS9001595 B29 PCSK5 chr9 Missense_Mutation c.1312G>T p.Val438Leu 13 0.31 26.8 JS9001595 B29 PAPSS2 chr10 Missense_Mutation c.377C>A p.Ala126Glu 20 0.20 15.35 JS9001595 B30 CHD8 chr14 Missense_Mutation c.1478G>A p.Arg493Gln 10 0.20 22.3 JS9001595 B31 PKD1 chr16 Splice_Site c.10220+2T>G 39 0.46 23.2 JS9001595 B31 KCNC3 chr19 3'UTR c.*81A>C 16 0.44 19.57

JS9001595 B31 POTEJ chr2 Missense_Mutation c.3103C>T p.Arg1035Cys 9 0.22 31 JS9001595 B32 CRCP chr7 Splice_Site c.145-1G>T 19 0.32 23.7 JS9001595 B32 TOX3 chr16 Missense_Mutation c.1147A>G p.Ile383Val 289 0.23 20.8 JS9001595 B33 PKD1 chr16 Nonsense_Mutation c.3184C>T p.Gln1062Ter 27 0.63 35 JS9001595 B33 ANGPTL3 chr1 Missense_Mutation c.124G>C p.Asp42His 73 0.40 28.4 rs199772471 JS9001595 B33 LILRB5 chr19 Missense_Mutation c.1652A>C p.Gln551Pro 17 0.29 16.26 JS9001595 B33 PCSK5 chr9 Missense_Mutation c.1312G>T p.Val438Leu 23 0.22 26.8

BH9002280 B34 PKD2 chr4 Frame_Shift_Del c.2409delA p.Ser804ValfsTer40 80 0.30 NA BH9002280 B34 MYT1 chr20 Silent c.2538G>A p.= 78 0.26 15.84 BH9002280 B34 ZNF142 chr2 Missense_Mutation c.4499G>A p.Arg1500Gln 9 0.22 35 COSM214198 BH9002280 B34 MKNK2 chr19 Missense_Mutation c.434T>C p.Leu145Pro 36 0.22 27.5 BH9002280 B34 PRUNE2 chr9 Missense_Mutation c.9086G>T p.Ser3029Ile 29 0.21 32

32 BH9002280 B34 IGF1R chr15 Missense_Mutation c.311C>A p.Thr104Lys 10 0.20 32 BH9002280 B35 CEP97 chr3 Missense_Mutation c.1142A>T p.Asp381Val 33 0.30 23.2 rs373204728 BH9002280 B35 MYBPC3 chr11 Missense_Mutation c.917G>C p.Arg306Pro 23 0.26 21 COSM1475467

BH9002280 B35 MYO18A chr17 Missense_Mutation c.3613C>T p.Arg1205Cys 9 0.22 35 BH9002280 B35 PRRC2B chr9 Missense_Mutation c.1997A>C p.Gln666Pro 27 0.22 25.6 BH9002280 B35 LAMP1 chr13 Missense_Mutation c.430G>T p.Asp144Tyr 40 0.20 26.1 BH9002280 B36 NXNL1 chr19 Missense_Mutation c.577C>G p.Arg193Gly 9 0.44 23.2 BH9002280 B36 PKD2 chr4 Missense_Mutation c.1258A>T p.Arg420Trp 67 0.27 27.7 CM044694 BH9002280 B36 WDFY3 chr4 Missense_Mutation c.2029A>C p.Asn677His 16 0.25 24.3 BH9002280 B36 PIP5K1C chr19 Missense_Mutation c.1144G>A p.Ala382Thr 9 0.22 31 BH9002280 L2 PKD2 chr4 Nonsense_Mutation c.242C>A p.Ser81Ter 14 0.57 36 BH9002280 L2 DYNC2H1 chr11 Missense_Mutation c.1412C>T p.Ser471Phe 60 0.25 23.5 BH9002280 L5 PRKCI chr3 Missense_Mutation c.280G>C p.Glu94Gln 35 0.34 24.3

p.Ser207_Pro208ins BH9002280 L5 SCAF1 chr19 In_Frame_Ins c.619_620insCCCCCCCCC ProProPro 12 0.33 NA BH9002280 L5 MRM1 chr17 Splice_Site c.543-2A>G 62 0.29 24.7 BH9002280 R1 AK9 chr6 Missense_Mutation c.2479G>A p.Glu827Lys 80 0.23 35 BH9002280 R1 ZNF862 chr7 Missense_Mutation c.1852G>A p.Val618Met 68 0.22 17.02 BH9002280 R1 SNTB1 chr8 Missense_Mutation c.937G>A p.Gly313Arg 53 0.21 23.5 BH9002280 R2 PKD2 chr4 Silent c.11887G>A p.= 32 0.34 NA p.Leu754_Ser755de BH9002280 R2 GRID2IP chr7 In_Frame_Del c.2259_2264delGCTCAG l 29 0.24 NA

BH9002280 R2 FAM46A chr6 In_Frame_Del c.139_141delAGC p.Ser47del 52 0.21 NA BH9002280 R7 PKD2 chr4 Nonsense_Mutation c.1366C>T p.Gln456Ter 98 0.38 42 BH9002280 R7 OR4C46 chr11 Missense_Mutation c.493C>A p.Pro165Thr 23 0.22 22.8 BH9002280 R7 FAM155B chrX In_Frame_Del c.54_56delCTG p.Cys20del 28 0.21 NA rs374286243 BH9002280 R7 KLK15 chr19 Missense_Mutation c.245G>T p.Gly82Val 14 0.21 26.4 rs188149275

BH9002280 R8 YBX3 chr12 In_Frame_Del c.40_42delACC p.Thr14del 16 0.31 NA BH9002280 R8 SMC4 chr3 Missense_Mutation c.2113C>T p.Arg705Cys 13 0.23 26.4

PJ9001456 A10 PKD1 chr16 Frame_Shift_Del c.7655_7658 delinsTTG p.Ala2552Valfs*68 0.32 PJ9001456 A10 XAF1 chr17 Missense_Mutation c.614C>T p.Thr205Met 56 0.27 16.55 rs371250443 PJ9001456 A10 KLHL35 chr11 Missense_Mutation c.920G>T p.Gly307Val 12 0.25 26.8 PJ9001456 A11 MIB2 chr1 Missense_Mutation c.2092G>A p.Glu698Lys 10 0.30 22.8

33 PJ9001456 A11 PKD1 chr16 Frame_Shift_Del c.3745del p.Asp1249Thrfs*24 0.23

PJ9001456 A12 DNAH5 chr5 Missense_Mutation c.10220G>T p.Cys3407Phe 13 0.23 25.1 PJ9001456 A12 CCDC28A chr6 Missense_Mutation c.338A>T p.Asn113Ile 159 0.22 26.3 PJ9001456 A12 FAM83B chr6 Missense_Mutation c.2386G>A p.Ala796Thr 38 0.21 23.4 PJ9001456 A12 SCN4A chr17 Missense_Mutation c.1244C>T p.Thr415Ile 53 0.21 21.3 rs34914278

PJ9001456 A12 EP400 chr12 Frame_Shift_Ins c.102_103insCCCCCCCCCC p.Ser37ProfsTer31 35 0.20 NA rs199726033 PJ9001456 A7 SVIL chr10 Missense_Mutation c.4121T>G p.Leu1374Arg 10 0.30 25.8 COSM146919

PJ9001456 A7 EP400 chr12 Frame_Shift_Ins c.102_103insCCCCCCCCCC p.Ser37ProfsTer31 40 0.20 NA

PJ9001456 A8 PKD1 chr16 Frame_Shift_Del c.348_352del p.Asn116Lysfs*2 0.29 PJ9001456 A8 DHX38 chr16 Splice_Site c.3477+2T>G 18 0.28 25.8 COSM304954 PJ9001456 A8 ZBTB38 chr3 Missense_Mutation c.62C>A p.Ser21Tyr 13 0.23 25.8 PJ9001456 A8 TCP1 chr6 Missense_Mutation c.847G>A p.Ala283Thr 56 0.21 25.8 rs371340812 PJ9001456 A8 PLCH2 chr1 Missense_Mutation c.170G>T p.Arg57Leu 15 0.20 26.8 COSM381226 PJ9001456 A8 FAM83A chr8 Missense_Mutation c.619A>G p.Lys207Glu 87 0.20 23.9 PJ9001456 A9 PKD1 chr16 Splice_Site c.8016+1delG 12 0.50 NA PJ9001456 A9 TMPRSS6 chr22 Missense_Mutation c.1477G>T p.Ala493Ser 15 0.20 23 PJ9001456 B1 URGCP chr7 Missense_Mutation c.173A>C p.Asn58Thr 14 0.36 26.9 PJ9001456 B1 DNAH5 chr5 Missense_Mutation c.5335G>T p.Val1779Phe 28 0.32 27.9 PJ9001456 B1 SLC14A2 chr18 Missense_Mutation c.2548A>C p.Asn850His 19 0.32 22.2 PJ9001456 B1 ADAMTS14 chr10 Missense_Mutation c.1231C>A p.His411Asn 83 0.25 28.6 PJ9001456 B2 EIF3A chr10 Missense_Mutation c.330G>T p.Gln110His 33 0.36 23.5 PJ9001456 B2 RGS1 chr1 Missense_Mutation c.553G>T p.Asp185Tyr 59 0.32 33 PJ9001456 B2 DNAH5 chr5 Missense_Mutation c.5335G>T p.Val1779Phe 23 0.26 27.9

PJ9001456 B2 NKD2 chr5 In_Frame_Del c.1315_1317delCAC p.His439del 26 0.23 NA COSM1291992

c.12004- 2_12019delAGGCTGCCCAGCA PJ9001456 B2 PKD1 chr16 Splice_Site GCTAC 193 0.23 NA PJ9001456 B2 SRCIN1 chr17 Missense_Mutation c.3298A>C p.Lys1100Gln 41 0.20 26.4 p.Phe2999LeufsTer RP9001591 B18 PKD1 chr16 Frame_Shift_Ins c.8996_8997insG 70 64 0.33 NA RP9001591 B18 ATP2B3 chrX Missense_Mutation c.2866G>A p.Gly956Arg 92 0.30 27

34 RP9001591 B18 KIF13A chr6 Missense_Mutation c.3918A>C p.Glu1306Asp 22 0.23 24.4 RP9001591 B18 TNRC6B chr22 Missense_Mutation c.265C>T p.Arg89Trp 10 0.20 34 RP9001591 B19 EEF1D chr8 Missense_Mutation c.73C>T p.Arg25Trp 10 0.20 30 RP9001591 B19 MYOM3 chr1 Missense_Mutation c.3751A>C p.Lys1251Gln 20 0.20 23.2 RP9001591 B20 CACNA2D1 chr7 Missense_Mutation c.668G>T p.Trp223Leu 12 0.42 35 COSM747982 RP9001591 B20 PRRC2B chr9 Missense_Mutation c.1997A>C p.Gln666Pro 11 0.36 25.6 RP9001591 B20 PRLR chr5 Nonsense_Mutation c.713G>A p.Trp238Ter 14 0.29 38

RP9001591 B20 KIF13A chr6 Missense_Mutation c.3918A>C p.Glu1306Asp 21 0.24 24.4

RP9001591 B21 KIF26A chr14 Missense_Mutation c.5624C>T p.Pro1875Leu 75 0.39 22 rs200461988

RP9001591 B21 KISS1R chr19 Frame_Shift_Del c.828_832delGGGCC p.Trp276CysfsTer28 72 0.35 NA RP9001591 B21 ACIN1 chr14 Missense_Mutation c.3499C>T p.Arg1167Trp 8 0.25 34 RP9001591 B21 PSAT1 chr9 Missense_Mutation c.160A>C p.Asn54His 76 0.22 21.4 RP9001591 B21 ABCA13 chr7 Missense_Mutation c.86A>C p.Glu29Ala 18 0.22 31 RP9001591 B21 GPR20 chr8 Missense_Mutation c.709G>A p.Val237Met 18 0.22 19.88 p.Gln226_Gln228de RP9001591 B22 SMARCA2 chr9 In_Frame_Del c.667_675delCAGCAGCAG l 111 0.36 NA

RP9001591 B23 ATAD2B chr2 Frame_Shift_Del c.458_465delATGGGGAC p.Asp153AlafsTer7 29 0.52 NA RP9001591 B23 SELE chr1 Missense_Mutation c.922C>T p.Arg308Cys 76 0.41 23 RP9001591 B23 STON2 chr14 Missense_Mutation c.1498C>T p.Arg500Trp 22 0.36 29.3 rs149119701 RP9001591 B23 NSMAF chr8 Missense_Mutation c.1268A>T p.Tyr423Phe 19 0.21 29.7 RP9001591 B24 KIF17 chr1 Missense_Mutation c.3002G>C p.Arg1001Pro 16 0.38 34 COSM182818 RP9001591 B24 ABCA13 chr7 Missense_Mutation c.86A>C p.Glu29Ala 11 0.36 31 RP9001591 B24 PRRC2B chr9 Missense_Mutation c.1997A>C p.Gln666Pro 16 0.31 25.6 RP9001591 B24 RSPRY1 chr16 Missense_Mutation c.703A>C p.Lys235Gln 25 0.24 15.76 p.Val336CysfsTer12 RP9001591 B24 PKD1 chr16 Frame_Shift_Del c.1005delC 9 38 0.21 NA RP9001591 B24 COL9A3 chr20 Missense_Mutation c.467C>A p.Pro156His 10 0.20 24.9 The pathogenic potential of missense variants was evaluated using the computational analysis tool Combined Annotation–Dependent Depletion (CADD), which enables scoring of deleteriousness of SNVs and indels. A CADD value of >15 suggests the variant is likely pathogenic (http://cadd.gs.washington.edu/).

35 Supplemental Table 8. List of genes associated with ciliopathy disorders (N=211) CEP41 BBS5 HYDIN CE104 CLRN1 DNAAF1 DNAL1 CC28B CE41B TM231 LEFTY2 TOPORS ALMS1 DDX59 DYH11 CCD65 CRX NODAL WDR35 WDR60 TM216 CU002 TSC2 GDF1 TTC8 ZMY10 DRC1 C2D2A TCTN2 PACS1 BBS4 DYHC2 LBN TM138 NPHP5 TTC21B BBS2 FRITZ FTM DYXC1 DYNC2H1 CEP164 MKS1 F161A IF172 IFT27 BBS9 EVC2 B9D1 DAAF1 DYH5 C5orf42 ATXN10 7SEP KIF7 CCNO UROM TMEM216 ZNF423 TMEM67 DNAI1 CE41A CCD39 GPR98 XPNPEP3 PCDH15 NEK8 TRI32 CU059 SCNN1B SCNN1A

NPHP4 BBIP1 SDCG8 ACVR2B WDPCP

WDR19 PDE6D CC151 INPP5E TMEM138

INVS SPAG1 IF140 RPE65 RD3

EVC RSH4A RPGR1 SCNN1G CEP290

IFT43 POC1A XPP3 MYO7A TCTN1

PKD1 CC103 TT21B ARL13B CRELD1

RSPH9 CE290 CC114 LCA5 CRB1

OFD1 AR2BP OCRL AIPL1 USH1G

DNAI2 MRE11 TM237 C2orf71 TMEM231

GLIS2 CE042 ANKS6 NME8 TSC1

BBS1 TALD3 TILB NKX2-5 IMPDH1

IQCB1 K0556 ARMC4 DFNB31 FOXH1

BBS10 TMM17 TXND3 CDH23 TULP1

PKD2 AR13B LZTL1 RDH12 TRIM32

HYLS1 RSPH1 MCIN CCDC40 USH1C

RPGR CE164 IF122 DNAH11 SPATA7

PKHD1 KTU CEP83 RSPH4A DNAH5

BBS12 CE120 DAAF3 PTCH1 RPGRIP1

NPHP3 DAAF5 ATX10 KIF3A IFT88

IFT80 CB071 PTHB1 LRAT USH2A

B9D2 TECT1 TAPT1 ZIC3 CCDC39

NEK1 DCDC2 CENPF CCDC28B KIF3B

NPHP1 TECT3 INP5E TMEM237 GUCY2D

MKKS CCD40 C2CD3 UMOD CFTR

AHI1 CSPP1 KIF14 BRCC3 VHL

BBS7 RSPH3 TECT2 CC2D2A RPGRIP1L

MKS3 WDR34 GAS8 DNAAF2 KCNJ13

ARL6 MIPT3 ZN423 SDCCAG8 DNAAF3

36 Supplemental Table 9. Gene variations in principal ciliopathy genes

CADD Cyst Total ALT dbSNP Patient ID Gene Chr HGVSc HGVSp Score ID coverage VAF rs/COSMIC (PHRED)

JS9001595 B28 AHI1 chr6 c.1210A>C p.Met404Leu 39 0.15 20.7 CMJ0001593 A34 BRCC3 chrX c.286C>A p.Gln96Lys 74 0.05 24.3 DR9001565 A17 C5orf42 chr5 c.8960C>A p.Pro2987Gln 48 0.06 23.4 JS9001595 B26 CC2D2A chr4 c.4735T>G p.Tyr1579Asp 65 0.14 23.2 JS9001595 B27 CC2D2A chr4 c.4735T>G p.Tyr1579Asp 55 0.15 23.2 JS9001595 B29 CC2D2A chr4 c.4735T>G p.Tyr1579Asp 49 0.14 23.2 JS9001595 B30 CC2D2A chr4 c.4735T>G p.Tyr1579Asp 74 0.15 23.2 JS9001595 B31 CC2D2A chr4 c.4735T>G p.Tyr1579Asp 54 0.11 23.2 JS9001595 B32 CC2D2A chr4 c.4735T>G p.Tyr1579Asp 75 0.15 23.2 JS9001595 B33 CC2D2A chr4 c.4735T>G p.Tyr1579Asp 73 0.12 23.2 DR9001565 A18 CCDC28B chr1 c.562C>A p.Leu188Met 77 0.05 28.7 rs75301270 DR9001565 A15 CEP164 chr11 c.337delA p.Lys116ArgfsTer22 58 0.21 COSM1351515 rs75301270 CDS001574 B7 CEP164 chr11 c.337delA p.Lys116ArgfsTer22 25 0.24 COSM1351515 PJ9001456 A10 CRX chr19 c.202G>T p.Ala68Ser 47 0.06 24.4 PJ9001456 A7 CSPP1 chr8 c.853C>A p.Pro285Thr 28 0.11 22.5 PJ9001456 A10 DNAH11 chr7 c.3976C>A p.Leu1326Ile 24 0.13 COSM1088396 26 PJ9001456 A12 DNAH5 chr5 c.10220G>T p.Cys3407Phe 13 0.23 25.1 PJ9001456 B1 DNAH5 chr5 c.5335G>T p.Val1779Phe 28 0.32 27.9 PJ9001456 B1 DNAH5 chr5 c.5335G>T p.Val1779Phe 28 0.32 27.9 CDS001574 B10 DNAH5 chr5 c.5340G>T p.Met1780Ile 29 0.21 16.41 BJA001578 B16 DNAH5 chr5 c.7009C>T p.Leu2337Phe 29 0.24 27.6 PJ9001456 B2 DNAH5 chr5 c.5335G>T p.Val1779Phe 23 0.26 27.9 BH9002280 R8 DNAH5 chr5 c.12795C>A p.Asn4265Lys 28 0.11 25.1 BH9002280 L2 DYNC2H1 chr11 c.1412C>T p.Ser471Phe 60 0.25 23.5 PJ9001456 A9 GPR98 chr5 c.14135C>A p.Ala4712Asp 28 0.11 28.6 DR9001565 A14 IFT88 chr13 c.1519G>T p.Gly507Cys 26 0.12 31 PJ9001456 A8 KIF14 chr1 c.2604G>T p.Lys868Asn 46 0.07 25.1 BS9001424 A2 LEFTY2 chr1 c.37C>A p.Leu13Met 30 0.1 17.59 BS9001424 A2 NODAL chr10 c.1006C>A p.His336Asn 39 0.08 COSM1675318 28.1 PJ9001456 A10 PACS1 chr11 c.753C>A p.Tyr251Ter 43 0.07 35 PJ9001456 A9 PCDH15 chr10 c.4916C>A p.Thr1639Lys 40 0.1 COSM364266 20.5 PJ9001456 A9 7SEP chr7 c.113C>A p.Pro38Gln 46 0.07 31 CMJ0001593 A29 7SEP chr7 c.832G>T p.Gly278Cys 49 0.06 25.6 BH9002280 R2 TMEM67 chr8 c.1862C>A p.Ala621Glu 33 0.09 29.6 DR9001565 A18 TSC1 chr9 c.2148C>A p.Asn716Lys 48 0.06 29.3 DR9001565 A15 TSC2 chr16 c.5010C>A p.His1670Gln 36 0.08 rs376306544 22.3 BS9001424 A3 TTC8 chr14 c.1224+1G>T 48 0.06 26.1 CMJ0001593 A34 UMOD chr16 c.757G>T p.Gly253Cys 36 0.08 34 CMJ0001593 A29 USH2A chr1 c.9752G>T p.Cys3251Phe 62 0.06 34 CMJ0001593 A27 WDR19 chr4 c.2659G>T p.Asp887Tyr 27 0.11 25.8 BH9002280 R1 WDR19 chr4 c.784G>T p.Gly262Ter 41 0.07 34 BH9002280 L2 WDR34 chr9 c.670G>T p.Ala224Ser 73 0.05 19.47 PJ9001456 A10 ZIC3 chrX c.737C>A p.Pro246His 41 0.07 26.4

37 Supplemental Table 10. Gene variations in principal cancer genes

CADD Cyst Total ALT dbSNP Patient ID Gene Chr HGVSc HGVSp ID Cov VAF ID/COSMIC Score (PHRED)

rs201725154 BH9002280 R2 ABL1 chr9 c.1870_1872delAAG p.Lys624del 100 0.07 COSM1645213 PJ9001456 A8 AFF4 chr5 c.2519C>A p.Ser840Tyr 46 0.07 24.4 CMJ0001593 A32 AKAP9 chr7 c.6631C>A p.Gln2211Lys 22 0.18 27 CDS001574 B12 AKT2 chr19 c.1016T>G p.Val339Gly 14 0.29 27.1 CDS001574 B7 AKT2 chr19 c.1016T>G p.Val339Gly 13 0.31 27.1 CMJ0001593 A34 ALDH2 chr12 c.541C>A p.Gln181Lys 44 0.07 33 BJA001578 B13 ARID1A chr1 c.920G>T p.Gly307Val 8 0.25 22 BJA001578 B13 ARID2 chr12 c.4673C>A p.Ala1558Glu 18 0.17 27.5 RP9001591 B18 ATP2B3 chrX c.2866G>A p.Gly956Arg 92 0.3 27 BS9001424 A2 BCL3 chr19 c.783G>T p.Leu261Phe 37 0.08 23.1 BS9001424 A2 BCOR chrX c.4572C>A p.Asn1524Lys 42 0.07 32 BS9001424 A3 BCOR chrX c.4690A>G p.Arg1564Gly 76 0.51 31 BS9001424 A2 BRD4 chr19 c.3650C>A p.Ala1217Asp 34 0.09 23.9 DR9001565 B5 CANT1 chr17 c.1060G>A p.Asp354Asn 50 0.06 22.3 JS9001595 B27 CARD11 chr7 c.922G>A p.Asp308Asn 33 0.09 35 BS9001424 A2 CARS chr11 c.1396-1C>T 32 0.09 COSM926250 25.8 DR9001565 A17 CASC5 chr15 c.4886C>T p.Thr1629Ile 29 0.38 17.62 CMJ0001593 A31 CASC5 chr15 c.5858G>T p.Arg1953Leu 52 0.08 COSM1372707 14.46 CDS001574 B12 CHEK2 chr22 c.-3C>T 32 0.16 19.31 BJA001578 B15 CLTCL1 chr22 c.3533C>A p.Ala1178Asp 72 0.06 29.6 CDS001574 B12 COL1A1 chr17 c.3783C>A p.Asp1261Glu 46 0.07 23.8 BS9001424 A2 CREBBP chr16 c.6340G>T p.Gly2114Cys 36 0.08 rs139169188 14.95 BH9002280 L5 CRTC1 chr19 c.1899C>A p.Asp633Glu 47 0.06 28.8 CMJ0001593 A28 DNMT3A chr2 c.1439T>G p.Val480Gly 50 0.22 rs200099128 27.4 CMJ0001593 A29 DNMT3A chr2 c.1439T>G p.Val480Gly 41 0.15 rs200099128 27.4 CMJ0001593 A34 DNMT3A chr2 c.1439T>G p.Val480Gly 42 0.24 rs200099128 27.4 BH9002280 R2 EBF1 chr5 c.42C>A p.Ser14Arg 30 0.1 21 DR9001565 A15 ECT2L chr6 c.659C>A p.Pro220His 32 0.09 22.6 BS9001424 A2 FANCA chr16 c.2499C>A p.Cys833Ter 50 0.06 36 BH9002280 R1 FAT1 chr4 c.1063G>A p.Val355Met 32 0.09 20.8 JS9001595 B32 FBXW7 chr4 c.622A>T p.Thr208Ser 52 0.13 14.04 PJ9001456 A11 FGFR2 chr10 c.1265G>A p.Arg422His 40 0.08 34 BH9002280 L2 FGFR4 chr5 c.1391G>T p.Arg464Leu 36 0.08 34 PJ9001456 A10 FOXL2 chr3 c.315C>A p.Asn105Lys 48 0.06 29.3 DR9001565 B4 GNAS chr20 c.1993C>T p.Arg665Cys 45 0.11 25.6 RP9001591 B23 IDH1 chr2 c.433G>T p.Val145Phe 57 0.14 34 BH9002280 R7 KAT6B chr10 c.1075G>T p.Asp359Tyr 41 0.1 26.5 BJA001578 B13 KDR chr4 c.3274G>T p.Gly1092Cys 46 0.07 33 DR9001565 A14 KMT2C chr7 c.8559C>A p.Cys2853Ter 37 0.08 35 BH9002280 L2 KMT2C chr7 c.14274G>T p.Lys4758Asn 40 0.08 32 CMJ0001593 A30 LEF1 chr4 c.722+1C>T 30 0.1 COSM585200 26.1 BS9001424 A3 MAML2 chr11 c.781G>T p.Gly261Cys 49 0.06 24.7 BH9002280 R8 MDM2 chr12 c.*2475A>G 62 0.16 JS9001595 B28 MLH1 chr3 c.2164A>C p.Lys722Gln 113 0.07 22.4 JS9001595 B28 MLH1 chr3 c.2165A>C p.Lys722Thr 113 0.12 24.7 BJA001578 B14 MLH1 chr3 c.2165A>C p.Lys722Thr 85 0.09 24.7 BS9001424 A2 MLLT10 chr10 c.1464G>T p.Glu488Asp 36 0.08 COSM1286220 23.2

38 CMJ0001593 A31 MN1 chr22 c.3029G>A p.Trp1010Ter 36 0.22 39 DR9001565 A18 MSI2 chr17 c.57C>A p.Asp19Glu 18 0.17 23.1 CMJ0001593 A28 MYC chr8 c.397G>T p.Asp133Tyr 49 0.06 31 BS9001424 A3 NCOR1 chr17 c.5506G>A p.Val1836Met 66 0.39 24.9 CDS001574 B8 NF1 chr17 c.3244G>T p.Gly1082Cys 48 0.15 29.8 BJA001578 B15 NF1 chr17 c.3245G>T p.Gly1082Val 44 0.23 29 BJA001578 B16 NF1 chr17 c.3244G>T p.Gly1082Cys 29 0.21 29.8 PJ9001456 B2 NF1 chr17 c.3244G>T p.Gly1082Cys 38 0.18 29.8 BH9002280 B34 NFIB chr9 c.92G>T p.Trp31Leu 40 0.08 28.1 BJA001578 A24 NSD1 chr5 c.1034C>T p.Pro345Leu 49 0.06 33 JS9001595 B28 NT5C2 chr10 c.1460A>C p.Glu487Ala 28 0.11 29.6 BH9002280 L2 NT5C2 chr10 c.926C>A p.Thr309Asn 46 0.07 23.4 DR9001565 A14 NUMA1 chr11 c.4549G>T p.Val1517Phe 44 0.07 24.2 CDS001574 B12 P2RY8 chrX c.815A>C p.Tyr272Ser 119 0.08 25.1 CDS001574 B8 P2RY8 chrX c.815A>C p.Tyr272Ser 114 0.07 25.1 PJ9001456 B2 P2RY8 chrX c.815A>C p.Tyr272Ser 132 0.09 25.1 BH9002280 B35 P2RY8 chrX c.815A>C p.Tyr272Ser 118 0.07 25.1 CMJ0001593 A29 PAX7 chr1 c.892C>A p.Pro298Thr 42 0.07 19.04 PJ9001456 A8 PDE4DIP chr1 c.636+11C>T 55 0.07 5.573 PJ9001456 A8 PHOX2B chr4 c.224G>T p.Ser75Ile 65 0.06 18.27 CMJ0001593 A31 PICALM chr11 c.1602G>T p.Lys534Asn 44 0.07 24.6 CMJ0001593 A34 PICALM chr11 c.1804G>T p.Ala602Ser 46 0.07 16.67 BS9001424 A2 PIK3R1 chr5 c.1274T>A p.Leu425His 56 0.14 28.1 DR9001565 A14 POU5F1 chr6 c.670G>A p.Glu224Lys 45 0.07 23.7 CDS001574 B9 PPFIBP1 chr12 c.2515-3C>A 16 0.25 15.42 BJA001578 A23 PRCC chr1 c.406C>T p.Pro136Ser 61 0.07 rs11264542 18.1 PJ9001456 A8 PTPN11 chr12 c.349C>A p.Leu117Ile 26 0.12 COSM467817 24.4 PJ9001456 A10 RAD21 chr8 c.505G>T p.Glu169Ter 38 0.08 41 rs80184931 PJ9001456 B1 RANBP17 chr5 c.529G>T p.Ala177Ser 51 0.14 COSM249534 23.8 rs80184931 PJ9001456 B1 RANBP17 chr5 c.529G>T p.Ala177Ser 51 0.14 COSM249534 23.8 RP9001591 B20 RAP1GDS1 chr4 c.512-2A>T 37 0.14 22.9 PJ9001456 A7 RARA chr17 c.848C>A p.Thr283Asn 50 0.06 31 BH9002280 L5 RUNX1 chr21 c.1126C>A p.Arg376Ser 31 0.1 24.5 PJ9001456 A9 RUNX1T1 chr8 c.121+245C>T 46 0.07 15.59 JS9001595 B27 SETD2 chr3 c.1031G>T p.Ser344Ile 41 0.07 19.69 BS9001424 A2 SFPQ chr1 c.1094G>T p.Arg365Leu 42 0.1 34 BJA001578 B16 SMARCAD1 chr4 c.2572T>C p.Phe858Leu 66 0.48 23 CMJ001593 A34 SMARCAD1 chr4 c.2572T>C p.Phe858Leu 22 0.41 23

p.Gln226_Gln228 RP9001591 B22 SMARCA2 chr9 c.667_675delCAGCAGCAG 111 0.36 del

BH9002280 L2 SPEN chr1 c.676C>T p.Arg226Trp 42 0.07 34 BS9001424 A3 SS18 chr18 c.322G>T p.Gly108Ter 41 0.07 37 CDS001574 B10 STAG2 chrX c.2188G>T p.Val730Phe 39 0.18 25.1 RP9001591 B21 TET2 chr4 c.3901G>T p.Ala1301Ser 86 0.07 28.7 DR9001565 A16 THRAP3 chr1 c.2701C>T p.Arg901Ter 9 0.33 41 BJA001578 B16 TPM3 chr1 c.567-621T>C 63 0.1 15.12 CDS001574 B10 TRRAP chr7 c.2440C>G p.Pro814Ala 67 0.27 24.1 DR9001565 A18 TSC1 chr9 c.2148C>A p.Asn716Lys 48 0.06 29.3 DR9001565 A15 TSC2 chr16 c.5010C>A p.His1670Gln 36 0.08 rs376306544 22.3 BJA001578 B13 YWHAE chr17 c.623C>T p.Thr208Met 38 0.08 23.5 rs372909378 BJA001578 A23 ZFHX3 chr16 c.5221_5223delCAA p.Gln1741del 107 0.07 COSM973528

39 rs372909378 BH9002280 B36 ZFHX3 chr16 c.5221_5223delCAA p.Gln1741del 162 0.07 COSM973528 DR9001565 A18 ZRSR2 chrX c.684C>A p.Ser228Arg 22 0.14 rs372992384 25.5

40 Supplemental Table 11. Copy number alterations (CNAs) in most commonly altered (>4 CNV) genes among the cyst samples. Cyst Chr #Covered #Chr Start End Size Log2ratio Type Class p_value Gene(s) ID arm Genes A2 chr9 88967882 88968131 249 -1.6613 deletion focal 9q 8.90E-16 1 ZCCHC6 A8 chr8 29207565 29207762 197 1.8895 amplification focal 8p 4.59E-15 1 DUSP4 A9 chr8 29207565 29207760 195 1.6685 amplification focal 8p 3.68E-10 1 DUSP4 A10 chr17 80048915 80048992 77 2.116 amplification focal 17q 5.06E-10 1 FASN A12 chr8 29207565 29207762 197 1.5937 amplification focal 8p 4.93E-11 1 DUSP4 A12 chr9 34834124 34834503 379 -1.7457 deletion focal 9p 3.19E-13 1 FAM205B A14 chr2 54150092 54154655 4563 -0.8578 deletion focal 2p 1.62E-09 1 PSME4 A14 chr6 152451796 152451986 190 -1.2775 deletion focal 6q 7.94E-06 1 SYNE1 A14 chr9 140063932 140064192 260 -1.4343 deletion focal 9q 1.08E-07 1 LRRC26 A14 chr17 17699029 17699140 111 1.891 amplification focal 17p 3.66E-12 1 RAI1 A15 chr2 109400015 109400282 267 -1.0377 deletion focal 2q 3.29E-05 1 RANBP2 A15 chr2 179583358 179583585 227 1.1507 amplification focal 2q 7.54E-17 1 TTN A15 chr12 57594755 57594906 151 -1.4565 deletion focal 12q 9.11E-61 1 LRP1 A15 chr17 17699048 17699125 77 1.8025 amplification focal 17p 4.90E-11 1 RAI1 A16 chr1 228503564 228503856 292 0.9693 amplification focal 1q 8.46E-16 1 OBSCN A16 chr2 54152612 54153125 513 -1.1327 deletion focal 2p 4.43E-08 1 PSME4 A16 chr2 73151446 73151703 257 -1.704 deletion focal 2p 3.59E-13 1 EMX1 A16 chr2 179452530 179452662 132 -1.351 deletion focal 2q 8.27E-62 1 TTN A16 chr5 138729459 138729994 535 -1.0243 deletion focal 5q 6.95E-110 1 PROB1 A16 chr11 20404467 20404810 343 -1.096 deletion focal 11p 2.11E-07 1 HTATIP2 A16 chr12 57594755 57595001 246 -1.091 deletion focal 12q 8.03E-27 1 LRP1 A17 chr12 57594755 57595001 246 -1.2297 deletion focal 12q 4.68E-12 1 LRP1 A17 chr16 75018837 75033868 15031 0.9517 amplification focal 16q 8.12E-05 2 ZNRF1;WDR59 A17 chr17 17699034 17699145 111 1.4843 amplification focal 17p 2.06E-10 1 RAI1 A18 chr3 142775189 142839795 64606 1.0457 amplification focal 3q 7.94E-31 2 CHST2;U2SURP A18 chr8 124382010 124383222 1212 -0.8392 deletion focal 8q 2.49E-95 1 ATAD2 A18 chr12 57594755 57594906 151 -1.391 deletion focal 12q 3.04E-12 1 LRP1 A18 chr16 74808399 74908179 99780 1.0433 amplification focal 16q 2.03E-34 2 FA2H;WDR59 A18 chr17 2089896 2090209 313 0.773 amplification focal 17p 6.48E-58 1 SMG6 A18 chr17 17699029 17699125 96 1.7915 amplification focal 17p 1.63E-11 1 RAI1 A18 chr22 40058021 40058283 262 1.2817 amplification focal 22q 3.82E-20 1 CACNA1I A18 chr22 51158734 51158986 252 1.3375 amplification focal 22q 1.87E-07 1 SHANK3 A21 chr1 186649287 186649487 200 -1.4627 deletion focal 1q 3.13E-11 1 PTGS2 A21 chr4 117220797 117221011 214 -1.583 deletion focal 4q 3.42E-14 1 MIR1973 A23 chr16 2165656 2173558 7902 -1.017 deletion focal 16p 1.73E-10 1 PKD1 A24 chr4 117220797 117221011 214 -1.39 deletion focal 4q 8.93E-13 1 MIR1973 A24 chr9 34834370 34834503 133 -2.128 deletion focal 9p 3.71E-19 1 FAM205B A24 chr17 28548645 28548761 116 -1.751 deletion focal 17q 1.37E-11 1 SLC6A4 A24 chr22 37771163 37771232 69 -1.628 deletion focal 22q 6.29E-10 1 ELFN2

41 A25 chr8 29207516 29207762 246 1.4793 amplification focal 8p 7.15E-12 1 DUSP4 A25 chr11 6585470 6585688 218 -1.251 deletion focal 11p 9.73E-09 1 DNHD1 A27 chr5 176821167 176821225 58 -1.7185 deletion focal 5q 9.89E-11 1 SLC34A1 A27 chr8 145649607 145649666 59 -2.1955 deletion focal 8q 8.59E-19 1 VPS28 A28 chr5 176821171 176821225 54 -1.926 deletion focal 5q 2.72E-11 1 SLC34A1 A28 chr8 145639222 145639276 54 -2.335 deletion focal 8q 5.74E-17 1 SLC39A4 A28 chr8 145649607 145649666 59 -2.677 deletion focal 8q 1.65E-27 1 VPS28 A28 chr17 80048906 80049027 121 -2.2185 deletion focal 17q 1.44E-14 1 FASN A28 chr19 16435952 16436100 148 -1.982 deletion focal 19p 3.77E-12 1 KLF2 A28 chr22 37770507 37770863 356 -1.2957 deletion focal 22q 1.41E-08 1 ELFN2 A29 chr5 176821166 176821225 59 -1.4625 deletion focal 5q 6.69E-07 1 SLC34A1 A29 chr9 34833951 34834822 871 0.978 amplification focal 9p 5.64E-14 1 FAM205B A29 chr10 85981792 85981846 54 -1.522 deletion focal 10q 1.97E-18 1 LRIT2 A29 chr11 6541070 6541124 54 -1.608 deletion focal 11p 2.08E-10 1 DNHD1 A29 chr17 80048912 80048966 54 -1.816 deletion focal 17q 6.12E-12 1 FASN A30 chr5 132158646 132159102 456 -1.1692 deletion focal 5q 4.28E-13 1 SHROOM1 A30 chr7 100230963 100238353 7390 -1.593 deletion focal 7q 4.78E-15 1 TFR2 A30 chr7 156469165 156469217 52 -1.8185 deletion focal 7q 1.26E-11 1 RNF32 A30 chr8 124381366 124381470 104 -1.6855 deletion focal 8q 3.55E-10 1 ATAD2 A30 chr8 145639222 145639281 59 -2.034 deletion focal 8q 2.27E-16 1 SLC39A4 A30 chr17 80048912 80049034 122 -2.1185 deletion focal 17q 2.72E-17 1 FASN A31 chr1 186649287 186649472 185 -1.6845 deletion focal 1q 5.47E-11 1 PTGS2 A31 chr3 46414577 46414709 132 -1.6825 deletion focal 3p 8.32E-12 1 CCR5 A31 chr3 142839617 142839728 111 -2.1695 deletion focal 3q 8.61E-18 1 CHST2 A31 chr7 100230941 100238353 7412 -1.312 deletion focal 7q 2.93E-36 1 TFR2 A32 chr1 186649287 186649382 95 -1.8705 deletion focal 1q 4.37E-13 1 PTGS2 A32 chr2 73144998 73145197 199 -1.7115 deletion focal 2p 4.18E-12 1 EMX1 A32 chr2 109335979 109336107 128 -1.622 deletion focal 2q 1.26E-09 1 RANBP2 A32 chr7 156469165 156469217 52 -1.361 deletion focal 7q 8.89E-07 1 RNF32 A32 chr8 29207316 29207767 451 -1.1267 deletion focal 8p 8.67E-13 1 DUSP4 A32 chr14 100625826 100705839 80013 -1.3802 deletion focal 14q 4.21E-16 2 DEGS2;YY1 A32 chr16 74993396 75018951 25555 -1.1564 deletion focal 16q 4.88E-10 1 WDR59 A32 chr19 16435952 16436868 916 -1.8797 deletion focal 19p 1.42E-14 1 KLF2 A33 chr3 45988080 45988218 138 -2.362 deletion focal 3p 5.09E-23 1 CXCR6 A33 chr4 117220797 117221011 214 -1.7153 deletion focal 4q 2.40E-17 1 MIR1973 A33 chr7 156469165 156469217 52 -1.636 deletion focal 7q 1.54E-08 1 RNF32 A33 chr8 29207316 29207762 446 -1.5237 deletion focal 8p 3.40E-17 1 DUSP4 A33 chr8 145639227 145639281 54 -2.2075 deletion focal 8q 1.55E-18 1 SLC39A4 A33 chr8 145649607 145649666 59 -2.6625 deletion focal 8q 5.37E-23 1 VPS28 A33 chr9 34834370 34834822 452 1.5777 amplification focal 9p 1.10E-13 1 FAM205B A33 chr10 85981797 85981851 54 -1.868 deletion focal 10q 1.08E-12 1 LRIT2 A33 chr14 100705589 100705782 193 -1.4067 deletion focal 14q 3.08E-09 1 YY1 A33 chr16 2141697 2144251 2554 1.1213 amplification focal 16p 3.85E-47 1 PKD1

42 A33 chr17 80048912 80048992 80 -2.9085 deletion focal 17q 8.68E-46 1 FASN A34 chr1 186649287 186649482 195 -1.855 deletion focal 1q 1.00E-12 1 PTGS2 A34 chr3 142839617 142839763 146 -1.604 deletion focal 3q 3.06E-08 1 CHST2 A34 chr5 132158922 132159082 160 -1.69 deletion focal 5q 4.07E-09 1 SHROOM1 A34 chr7 100230965 100238353 7388 -1.522 deletion focal 7q 1.37E-11 1 TFR2 A34 chr7 156469165 156469217 52 -1.648 deletion focal 7q 5.53E-08 1 RNF32 A34 chr8 29207516 29207762 246 -1.7057 deletion focal 8p 5.37E-15 1 DUSP4 A34 chr8 145639222 145639281 59 -1.637 deletion focal 8q 1.07E-10 1 SLC39A4 A34 chr8 145649607 145649666 59 -1.8755 deletion focal 8q 6.42E-14 1 VPS28 A34 chr10 85981792 85981851 59 -1.6955 deletion focal 10q 3.32E-10 1 LRIT2 A34 chr14 100705689 100705772 83 -1.7135 deletion focal 14q 9.28E-10 1 YY1 A34 chr16 2141697 2150027 8330 3.5649 amplification focal 16p 1.89E-15 1 PKD1 A34 chr16 2155274 2159259 3985 0.8213 amplification focal 16p 4.40E-17 1 PKD1 A34 chr16 2164597 2166657 2060 2.5752 amplification focal 16p 4.69E-73 1 PKD1 A34 chr17 80048912 80049030 118 -2.3423 deletion focal 17q 1.58E-25 1 FASN A34 chr19 16435952 16436100 148 -2.2115 deletion focal 19p 1.19E-16 1 KLF2 B1 chr5 176825611 176827304 1693 1.1094 amplification focal 5q 2.44E-08 2 SLC34A1;PFN3 B1 chr6 152776662 152776782 120 -1.877 deletion focal 6q 2.06E-10 1 SYNE1 B1 chr16 74926407 74927812 1405 -1.4488 deletion focal 16q 5.33E-09 1 WDR59 B1 chr21 9825799 9826243 444 1.5545 amplification focal 21p 1.78E-10 2 MIR3648;MIR3687 B2 chr1 228509919 228509955 36 -1.9805 deletion focal 1q 2.71E-10 1 OBSCN B2 chr1 228553243 228553356 113 -2.108 deletion focal 1q 4.41E-12 1 OBSCN B2 chr3 45988518 45988785 267 -1.4422 deletion focal 3p 7.58E-08 1 CXCR6 B2 chr6 152776662 152776782 120 -2.4195 deletion focal 6q 2.23E-15 1 SYNE1 B2 chr7 48965090 48965240 150 -1.8115 deletion focal 7p 1.69E-08 1 CDC14C B2 chr7 150490214 150490333 119 -2.3025 deletion focal 7q 4.50E-13 1 TMEM176B B2 chr10 43701488 43701586 98 -2.6095 deletion focal 10q 9.40E-19 1 RASGEF1A B2 chr14 100625825 100706000 80175 1.2708 amplification focal 14q 1.22E-09 2 DEGS2;YY1 B2 chr21 9825804 9825862 58 1.91 amplification focal 21p 4.04E-07 1 MIR3648 B4 chr3 45988520 45988796 276 -1.65 deletion focal 3p 5.81E-09 1 CXCR6 B4 chr3 46415373 46449287 33914 1.2725 amplification focal 3p 1.06E-07 2 CCR5;CCRL2 B4 chr5 138729831 138730341 510 -1.614 deletion focal 5q 5.31E-48 1 PROB1 B4 chr7 100230939 100231088 149 1.4725 amplification focal 7q 3.03E-59 1 TFR2 B4 chr8 124382259 124382359 100 -2.4455 deletion focal 8q 2.43E-128 1 ATAD2 B4 chr10 43701488 43701586 98 -2.3825 deletion focal 10q 3.89E-13 1 RASGEF1A B4 chr11 47611995 47612271 276 -1.1743 deletion focal 11p 1.73E-72 1 C1QTNF4 B4 chr12 57600493 57601866 1373 -1.272 deletion focal 12q 2.26E-76 1 LRP1 B4 chr13 111935393 111935581 188 1.665 amplification focal 13q 2.35E-09 1 ARHGEF7 B4 chr19 16436032 16436100 68 2.0465 amplification focal 19p 3.65E-08 1 KLF2 B5 chr1 186649309 186649464 155 1.773 amplification focal 1q 9.28E-13 1 PTGS2 B5 chr3 45988343 45988796 453 -1.2036 deletion focal 3p 1.68E-08 1 CXCR6 B5 chr3 46415373 46449681 34308 1.2063 amplification focal 3p 3.13E-12 2 CCR5;CCRL2 B5 chr13 111935404 111935581 177 1.632 amplification focal 13q 7.08E-09 1 ARHGEF7

43 B5 chr19 16436032 16436102 70 2.1625 amplification focal 19p 3.84E-10 1 KLF2 B5 chr22 40058164 40058349 185 1.3883 amplification focal 22q 2.36E-06 1 CACNA1I B6 chr3 46415373 46449912 34539 1.1287 amplification focal 3p 1.95E-10 2 CCR5;CCRL2 B6 chr7 48965090 48965240 150 -2.249 deletion focal 7p 1.06E-32 1 CDC14C B6 chr10 43701488 43701586 98 -2.029 deletion focal 10q 4.73E-12 1 RASGEF1A B6 chr13 111935393 111935581 188 1.9473 amplification focal 13q 1.88E-12 1 ARHGEF7 B6 chr17 2139638 2147991 8353 1.1798 amplification focal 17p 2.18E-08 1 SMG6 B6 chr19 16436032 16436100 68 2.093 amplification focal 19p 2.72E-09 1 KLF2 B7 chr7 150490191 150490333 142 -1.997 deletion focal 7q 1.30E-11 1 TMEM176B B7 chr12 22354498 22354572 74 -1.728 deletion focal 12p 2.00E-08 1 ST8SIA1 B7 chr15 65942820 65942902 82 1.974 amplification focal 15q 2.47E-09 1 SLC24A1 B7 chr17 5307315 5307526 211 -1.5743 deletion focal 17p 2.03E-08 1 NUP88 B7 chr21 9825827 9825862 35 1.932 amplification focal 21p 1.97E-08 1 MIR3648 B7 chr22 39966697 39967018 321 1.3755 amplification focal 22q 2.34E-09 1 CACNA1I B8 chr7 150490214 150490333 119 -2.882 deletion focal 7q 3.33E-26 1 TMEM176B B8 chr12 22354498 22354588 90 -1.9393 deletion focal 12p 1.28E-15 1 ST8SIA1 B8 chr15 65942801 65942911 110 1.753 amplification focal 15q 6.07E-10 1 SLC24A1 B8 chr17 2139648 2139911 263 1.3837 amplification focal 17p 8.22E-07 1 SMG6 B8 chr17 5307315 5307526 211 -1.3473 deletion focal 17p 3.08E-07 1 NUP88 B8 chr21 9825827 9825862 35 1.8795 amplification focal 21p 1.14E-07 1 MIR3648 B9 chr2 54164337 54164601 264 -1.0807 deletion focal 2p 6.08E-12 1 PSME4 B9 chr5 138729829 138730341 512 -1.2277 deletion focal 5q 1.36E-35 1 PROB1 B9 chr7 48965090 48965240 150 -1.4005 deletion focal 7p 1.82E-168 1 CDC14C B9 chr7 127961182 127961515 333 -1.3228 deletion focal 7q 6.31E-07 1 RBM28 B9 chr8 124381918 124382359 441 -1.4767 deletion focal 8q 1.26E-10 1 ATAD2 B9 chr13 111935394 111935581 187 1.573 amplification focal 13q 2.92E-08 1 ARHGEF7 B9 chr15 65942751 65942911 160 1.573 amplification focal 15q 3.27E-13 1 SLC24A1 B9 chr17 1961193 1962167 974 0.9136 amplification focal 17p 6.34E-10 1 HIC1 B9 chr17 5307315 5307576 261 -1.5173 deletion focal 17p 1.14E-11 1 NUP88 B9 chr22 40075617 40075754 137 2.203 amplification focal 22q 5.86E-47 1 CACNA1I B10 chr5 82876236 82876333 97 -1.787 deletion focal 5q 1.35E-11 1 VCAN B10 chr7 127961182 127961499 317 -1.3065 deletion focal 7q 2.12E-09 1 RBM28 B10 chr7 150490191 150490333 142 -1.854 deletion focal 7q 6.22E-10 1 TMEM176B B10 chr11 720806 721194 388 -1.8205 deletion focal 11p 8.57E-13 1 EPS8L2 B10 chr12 22354498 22354572 74 -2.456 deletion focal 12p 3.35E-16 1 ST8SIA1 B10 chr15 65942801 65942911 110 1.7313 amplification focal 15q 2.22E-10 1 SLC24A1 B10 chr16 58608978 58612656 3678 -1.05 deletion focal 16q 2.32E-09 1 CNOT1 B10 chr17 2139648 2139911 263 1.4765 amplification focal 17p 4.90E-10 1 SMG6 B10 chr17 5307315 5307526 211 -1.5467 deletion focal 17p 3.76E-08 1 NUP88 B10 chr21 9825827 9825862 35 1.883 amplification focal 21p 2.39E-09 1 MIR3648 B12 chr4 117220749 117221011 262 -1.5763 deletion focal 4q 1.56E-11 1 MIR1973 B12 chr5 82876236 82876333 97 -1.963 deletion focal 5q 4.89E-12 1 VCAN B12 chr5 132161461 132161833 372 -1.2808 deletion focal 5q 6.15E-10 1 SHROOM1

44 B12 chr7 150490214 150490333 119 -1.9495 deletion focal 7q 6.69E-12 1 TMEM176B B12 chr10 43701420 43701586 166 -1.5267 deletion focal 10q 1.71E-10 1 RASGEF1A B12 chr12 22354498 22354572 74 -2.073 deletion focal 12p 2.70E-13 1 ST8SIA1 B12 chr21 9825827 9825862 35 1.78 amplification focal 21p 3.78E-08 1 MIR3648 B13 chr1 228553240 228553356 116 -1.917 deletion focal 1q 1.18E-09 1 OBSCN B13 chr6 152776662 152776782 120 -2.089 deletion focal 6q 5.14E-12 1 SYNE1 B13 chr7 48965090 48965240 150 -2.2745 deletion focal 7p 2.85E-13 1 CDC14C B13 chr10 43701488 43701586 98 -2.2395 deletion focal 10q 1.63E-13 1 RASGEF1A B13 chr17 1960150 1960251 101 -1.8065 deletion focal 17p 6.73E-07 1 HIC1 B13 chr17 5307315 5307526 211 -1.7193 deletion focal 17p 4.54E-09 1 NUP88 B14 chr7 150490214 150490333 119 -2.3325 deletion focal 7q 8.71E-15 1 TMEM176B B15 chr1 228553240 228553356 116 -1.85 deletion focal 1q 1.09E-09 1 OBSCN B15 chr5 82876236 82876333 97 -1.9095 deletion focal 5q 1.43E-16 1 VCAN B15 chr6 152776662 152776782 120 -1.854 deletion focal 6q 2.42E-10 1 SYNE1 B15 chr7 48965090 48965240 150 -2.166 deletion focal 7p 3.83E-09 1 CDC14C B15 chr7 50450128 50450270 142 -1.2557 deletion focal 7p 3.21E-06 1 IKZF1 B15 chr12 22354485 22354583 98 -1.3757 deletion focal 12p 6.46E-08 1 ST8SIA1 B16 chr5 82876236 82876333 97 -2.0305 deletion focal 5q 4.19E-11 1 VCAN B16 chr6 152776662 152776782 120 -2.2005 deletion focal 6q 2.40E-13 1 SYNE1 B16 chr7 48964824 48965240 416 -1.7197 deletion focal 7p 7.52E-12 1 CDC14C B16 chr12 22354485 22354588 103 -1.7763 deletion focal 12p 2.41E-13 1 ST8SIA1 B16 chr16 2139910 2168152 28242 3.13535 amplification focal 16p 1.82E-06 2 MIR1225;PKD1 B18 chr2 242743036 242743481 445 -1.6063 deletion focal 2q 6.53E-20 1 GAL3ST2 B18 chr5 132161561 132161833 272 -1.3735 deletion focal 5q 9.70E-19 1 SHROOM1 B18 chr13 20716327 20716453 126 -1.807 deletion focal 13q 4.88E-16 1 GJA3 B18 chr22 51158898 51159277 379 0.8507 amplification focal 22q 7.65E-05 1 SHANK3 B19 chr2 242742908 242743481 573 -1.8317 deletion focal 2q 1.05E-22 1 GAL3ST2 B19 chr3 52399006 52399077 71 1.666 amplification focal 3p 3.07E-14 1 DNAH1 B19 chr11 725632 726206 574 -1.049 deletion focal 11p 9.71E-11 1 EPS8L2 B19 chr13 20716231 20716803 572 -1.1435 deletion focal 13q 8.42E-19 1 GJA3 B19 chr17 28548645 28548761 116 -1.3615 deletion focal 17q 2.82E-09 1 SLC6A4 B19 chr19 45261486 45261686 200 1.6175 amplification focal 19q 6.62E-13 1 BCL3 B19 chr22 51158561 51159366 805 1.0255 amplification focal 22q 5.04E-11 1 SHANK3 B20 chr9 140063392 140063522 130 1.4865 amplification focal 9q 1.45E-11 1 LRRC26 B20 chr20 3641504 3641629 125 1.3345 amplification focal 20p 1.41E-09 1 GFRA4 B20 chr22 51158561 51159277 716 0.8748 amplification focal 22q 2.75E-14 1 SHANK3 B21 chr4 117220796 117220996 200 -1.8945 deletion focal 4q 1.07E-17 1 MIR1973 B21 chr5 132161461 132161833 372 -1.3123 deletion focal 5q 2.03E-15 1 SHROOM1 B21 chr5 138730521 138730809 288 0.8453 amplification focal 5q 7.00E-12 1 PROB1 B21 chr7 50467867 50468252 385 1.2358 amplification focal 7p 2.14E-18 1 IKZF1 B21 chr8 145638556 145639186 630 -0.6606 deletion focal 8q 5.50E-07 1 SLC39A4 B21 chr9 140063392 140063522 130 1.2487 amplification focal 9q 3.09E-11 1 LRRC26 B21 chr11 20404467 20409321 4854 -0.7895 deletion focal 11p 2.69E-06 2 PRMT3;HTATIP2

45 B21 chr17 1961819 1961905 86 -2.217 deletion focal 17p 1.24E-25 1 HIC1 B21 chr20 3641404 3641629 225 1.175 amplification focal 20p 4.43E-10 1 GFRA4 B21 chr22 51158596 51159277 681 1.04 amplification focal 22q 2.57E-11 1 SHANK3 B22 chr3 52399006 52399077 71 1.4835 amplification focal 3p 1.39E-11 1 DNAH1 B22 chr5 138730521 138730794 273 0.8583 amplification focal 5q 1.02E-16 1 PROB1 B22 chr9 140063392 140063523 131 1.137 amplification focal 9q 3.03E-09 1 LRRC26 B22 chr17 28548645 28548761 116 -1.315 deletion focal 17q 5.42E-16 1 SLC6A4 B22 chr20 3641404 3641629 225 0.9707 amplification focal 20p 1.06E-13 1 GFRA4 B22 chr22 51158561 51159277 716 0.9116 amplification focal 22q 2.03E-07 1 SHANK3 B23 chr1 6531079 6532607 1528 0.7618 amplification focal 1p 7.33E-08 1 PLEKHG5 B23 chr2 242742822 242743481 659 -1.6527 deletion focal 2q 2.03E-15 1 GAL3ST2 B23 chr3 52399006 52399077 71 1.635 amplification focal 3p 4.22E-13 1 DNAH1 B23 chr5 132161561 132161833 272 -1.3883 deletion focal 5q 3.23E-14 1 SHROOM1 B23 chr7 50467858 50468170 312 1.1407 amplification focal 7p 9.50E-12 1 IKZF1 B23 chr8 144917966 144918286 320 1.445 amplification focal 8q 1.73E-08 1 NRBP2 B23 chr13 20716327 20716583 256 -1.4507 deletion focal 13q 1.63E-14 1 GJA3 B23 chr19 45261486 45261686 200 1.7085 amplification focal 19q 8.97E-15 1 BCL3 B23 chr20 3641504 3641629 125 1.3705 amplification focal 20p 2.09E-09 1 GFRA4 B23 chr22 51158561 51159227 666 1.0244 amplification focal 22q 1.96E-17 1 SHANK3 B24 chr1 6531066 6531923 857 0.9837 amplification focal 1p 3.76E-15 1 PLEKHG5 B24 chr1 228520472 228520964 492 -0.7784 deletion focal 1q 8.62E-08 1 OBSCN B24 chr3 52399006 52399077 71 1.282 amplification focal 3p 1.66E-08 1 DNAH1 B24 chr7 50467867 50468243 376 1.0012 amplification focal 7p 2.98E-09 1 IKZF1 B24 chr17 28548645 28548761 116 -1.535 deletion focal 17q 1.81E-12 1 SLC6A4 B24 chr19 45261486 45261686 200 1.409 amplification focal 19q 7.47E-10 1 BCL3 B24 chr20 3641404 3641629 225 1.0893 amplification focal 20p 4.46E-09 1 GFRA4 B24 chr22 51158561 51159277 716 0.8182 amplification focal 22q 8.46E-08 1 SHANK3 B26 chr2 73151367 73151693 326 -1.2532 deletion focal 2p 3.03E-16 1 EMX1 B26 chr2 242742828 242743468 640 -1.3116 deletion focal 2q 2.33E-20 1 GAL3ST2 PRRT3- B26 chr3 9988735 9989235 500 0.8192 amplification focal 3p 7.14E-10 2 AS1;PRRT3 B26 chr5 132161189 132161561 372 -1.2353 deletion focal 5q 1.78E-11 1 SHROOM1 B26 chr11 725632 725797 165 -1.539 deletion focal 11p 6.76E-11 1 EPS8L2 B26 chr22 51158561 51159267 706 0.9688 amplification focal 22q 1.93E-07 1 SHANK3 B27 chr2 73151469 73151693 224 -1.9695 deletion focal 2p 1.63E-42 1 EMX1 PRRT3- B27 chr3 9988746 9989170 424 0.9142 amplification focal 3p 4.65E-12 2 AS1;PRRT3 B27 chr5 132161211 132161837 626 -0.901 deletion focal 5q 1.58E-12 1 SHROOM1 B27 chr7 50467975 50468336 361 0.9292 amplification focal 7p 1.89E-08 1 IKZF1 B27 chr11 6592269 6592647 378 0.9815 amplification focal 11p 2.72E-10 1 DNHD1 B27 chr13 20716324 20717024 700 -1.0808 deletion focal 13q 6.04E-28 1 GJA3 B27 chr20 3641497 3641627 130 1.2855 amplification focal 20p 2.96E-09 1 GFRA4 B28 chr1 6531535 6531723 188 1.0777 amplification focal 1p 6.98E-10 1 PLEKHG5 B28 chr11 725632 725795 163 -1.538 deletion focal 11p 1.59E-12 1 EPS8L2

46 B28 chr11 20404467 20404667 200 -1.2905 deletion focal 11p 8.98E-08 1 HTATIP2 B28 chr11 47611533 47612149 616 1.0485 amplification focal 11p 1.28E-11 1 C1QTNF4 B28 chr13 20716231 20716684 453 -1.6984 deletion focal 13q 3.89E-38 1 GJA3 B28 chr16 2140981 2141132 151 1.3995 amplification focal 16p 1.11E-10 1 PKD1 B28 chr22 51158561 51159267 706 0.9202 amplification focal 22q 6.36E-09 1 SHANK3 B29 chr11 725632 725797 165 -1.3175 deletion focal 11p 4.85E-08 1 EPS8L2 B29 chr16 58620511 58620706 195 -1.7075 deletion focal 16q 1.98E-14 1 CNOT1 B29 chr16 70366776 70366976 200 -1.3285 deletion focal 16q 4.07E-08 1 DDX19B B29 chr19 45261486 45261877 391 0.8518 amplification focal 19q 1.92E-08 1 BCL3 B30 chr2 54152718 54153229 511 -0.8728 deletion focal 2p 2.36E-09 1 PSME4 B30 chr5 132161208 132161837 629 -1.0965 deletion focal 5q 6.04E-19 1 SHROOM1 B30 chr7 50467960 50468156 196 1.196 amplification focal 7p 4.74E-09 1 IKZF1 B30 chr11 47611533 47612149 616 0.7858 amplification focal 11p 1.09E-11 1 C1QTNF4 B30 chr16 2141010 2141224 214 1.2497 amplification focal 16p 1.12E-13 1 PKD1 B30 chr16 58620364 58620706 342 -1.159 deletion focal 16q 3.53E-13 1 CNOT1 B30 chr22 51158561 51159257 696 0.9432 amplification focal 22q 1.55E-06 1 SHANK3 B31 chr1 6531535 6531723 188 1.314 amplification focal 1p 6.76E-14 1 PLEKHG5 PRRT3- B31 chr3 9988746 9989146 400 0.8335 amplification focal 3p 1.70E-07 2 AS1;PRRT3 B31 chr9 140063347 140063522 175 1.021 amplification focal 9q 8.48E-08 1 LRRC26 B31 chr16 2140994 2141224 230 1.2693 amplification focal 16p 2.94E-12 1 PKD1 B31 chr16 58620511 58620706 195 -1.9055 deletion focal 16q 3.45E-19 1 CNOT1 B32 chr1 6531535 6531723 188 1.5887 amplification focal 1p 4.06E-20 1 PLEKHG5 PRRT3- B32 chr3 9988737 9989137 400 0.799 amplification focal 3p 1.92E-10 2 AS1;PRRT3 B32 chr9 140063397 140063522 125 1.3035 amplification focal 9q 1.85E-08 1 LRRC26 B32 chr11 20404467 20404667 200 -1.342 deletion focal 11p 7.48E-10 1 HTATIP2 B32 chr11 47611533 47612149 616 1.0077 amplification focal 11p 6.21E-11 1 C1QTNF4 B32 chr17 1953499 1959727 6228 0.8245 amplification focal 17p 3.01E-07 2 MIR212;HIC1 B32 chr20 3641499 3641713 214 1.0373 amplification focal 20p 4.68E-08 1 GFRA4 B33 chr3 9988746 9988868 122 1.401 amplification focal 3p 3.06E-10 1 PRRT3 B33 chr5 132161211 132161837 626 -1.0332 deletion focal 5q 7.45E-16 1 SHROOM1 B33 chr9 140063362 140063522 160 1.231 amplification focal 9q 4.21E-12 1 LRRC26 B33 chr11 6592269 6592583 314 1.1078 amplification focal 11p 2.35E-16 1 DNHD1 B33 chr11 47611533 47611777 244 1.2553 amplification focal 11p 5.27E-13 1 C1QTNF4 B33 chr16 2141005 2141224 219 1.17 amplification focal 16p 1.55E-10 1 PKD1 B34 chr1 228561970 228562197 227 -1.417 deletion focal 1q 3.65E-07 1 OBSCN B34 chr2 179399126 179399195 69 2.0045 amplification focal 2q 2.60E-10 1 TTN B34 chr7 127953217 127953384 167 1.673 amplification focal 7q 4.60E-07 1 RBM28 B34 chr9 88967881 88968115 234 1.5827 amplification focal 9q 5.58E-10 1 ZCCHC6 B35 chr2 109380223 109380558 335 1.5542 amplification focal 2q 4.42E-12 1 RANBP2 B35 chr2 179399126 179399295 169 1.718 amplification focal 2q 3.04E-11 1 TTN B35 chr7 127953217 127953384 167 1.972 amplification focal 7q 2.36E-09 1 RBM28 B35 chr9 88967884 88968115 231 1.8347 amplification focal 9q 6.87E-13 1 ZCCHC6

47 B36 chr2 109380223 109380566 343 1.3542 amplification focal 2q 1.76E-08 1 RANBP2 B36 chr2 179399126 179399295 169 1.5203 amplification focal 2q 1.39E-08 1 TTN B36 chr7 127953217 127953384 167 1.885 amplification focal 7q 1.16E-08 1 RBM28 B36 chr9 88967877 88968115 238 1.5647 amplification focal 9q 2.51E-09 1 ZCCHC6 B36 chr10 85984601 85984842 241 1.471 amplification focal 10q 2.16E-08 1 LRIT2 L2 chr8 144919846 144919935 89 1.591 amplification focal 8q 1.03E-11 1 NRBP2 L5 chr2 54197623 54197858 235 -1.577 deletion focal 2p 1.59E-07 1 PSME4 L5 chr7 100238479 100238542 63 1.8115 amplification focal 7q 3.24E-09 1 TFR2 L5 chr8 144919854 144919930 76 2.301 amplification focal 8q 2.80E-19 1 NRBP2 L5 chr10 7769599 7769737 138 -1.83 deletion focal 10p 1.93E-07 1 ITIH2 R1 chr8 144919859 144919935 76 1.963 amplification focal 8q 9.28E-14 1 NRBP2 R1 chr16 70365719 70367074 1355 -1.1927 deletion focal 16q 3.50E-06 1 DDX19B R2 chr10 7769599 7769731 132 -2.22 deletion focal 10p 9.46E-12 1 ITIH2 R2 chr16 70365722 70365878 156 -2.0755 deletion focal 16q 2.84E-09 1 DDX19B R2 chr22 37770784 37770863 79 1.5575 amplification focal 22q 1.21E-06 1 ELFN2 R7 chr2 54197623 54197855 232 -1.9393 deletion focal 2p 4.32E-14 1 PSME4 R7 chr8 144919859 144919910 51 1.8225 amplification focal 8q 5.02E-09 1 NRBP2 R7 chr10 7769599 7769736 137 -2.1405 deletion focal 10p 4.49E-11 1 ITIH2 R7 chr16 70365714 70367558 1844 -1.1529 deletion focal 16q 7.01E-09 1 DDX19B R8 chr3 142841136 142841168 32 1.598 amplification focal 3q 1.12E-07 1 CHST2 R8 chr8 144919859 144919935 76 2.045 amplification focal 8q 4.19E-16 1 NRBP2 R8 chr10 7769599 7769736 137 -1.8335 deletion focal 10p 6.79E-08 1 ITIH2 R8 chr22 37770784 37770863 79 1.8855 amplification focal 22q 1.48E-09 1 ELFN2

Supplemental Table 12. Copy number variation in PKD genes by renal cyst *Log2 Chr Size Patient ID Cyst WES# Chr Type P value Gene Exon Ratio arm (bp)

JS9001595 B28 (L8) chr16 1.4 Amplification 16p 151 1.11E-10 PKD1 Ex43

JS9001595 B30 (R1) chr16 1.25 amplification 16p 214 1.12E-13 PKD1 Ex43

JS9001595 B31 (R2) chr16 1.27 amplification 16p 230 2.94E-12 PKD1 Ex43

JS9001595 B33 (R10) chr16 1.17 amplification 16p 219 1.55E-10 PKD1 Ex43

CM001593 A34 (R9) chr16 3.57 amplification 16p 8330 1.89E-15 PKD1 Ex29~Ex41

CM001593 A34 (R9) chr16 0.82 amplification 16p 3985 4.40E-17 PKD1 Ex15~Ex21

CM001593 A34 (R9) chr16 2.58 amplification 16p 2060 4.69E-73 PKD Ex8~Ex11

CM001593 A33 (R3) chr16 1.12 amplification 16p 2554 3.85E-47 PKD1 Ex35-Ex41

PKD1; BJA001578 B16 (L8) chr16 3.36 amplification 16p 28242 1.68E-29 EX5~EX46 MIR1225

BJA001578 B16 (L8) chr4 2.06 amplification 4q 67810 6.76E-169 PKD2 EX1-EX15

BJA001578 A23 (R4) chr16 -1.02 deletion 16p 7902 1.73E-10 PKD1 Ex2-Ex10

All CNV changes are focal. Segment with an average read count Log2 ratio >0.6 were designated as gains and segments with a log2 value < -0.6 were categorized as losses.

49 Supplemental Table 13. WES analysis results of significantly mutated genes (n=53) in renal cyst epithelium by MuSiC Tot Covd Muts Gene Indels SNVs FDR Protein family Protein Class Muts Bps pMbp 110762 PKD1 10 20 30 27.09 < 10-23 Polycystin-1 1 RECQL 0 9 9 136458 65.95 < 10-23 ATP-dependent DNA helicase Q1 DNA helicase(PC00011)

FAM46A 6 0 6 165776 36.19 6.65E-13 Protein FAM46A SEC24D 0 8 8 349918 22.86 4.08E-09 Protein transport protein Sec24D vesicle coat protein(PC00235)

G-protein Small G protein signaling SGSM1 0 7 7 253079 27.66 1.47E-08 modulator(PC00022);cysteine modulator 1 protease(PC00081) KIAA1217 0 8 8 518291 15.44 4.49E-08 Sickle tail protein homolog G-protein modulator(PC00022);ion channel(PC00133);membrane-bound PKD2 2 4 6 226448 26.5 1.63E-05 Polycystin-2 signaling molecule (PC00152); protease(PC00190)

Coiled-coil and C2 domain- CC2D2A 0 8 8 589344 13.57 1.63E-07 containing protein 2A

Tuberoinfundibular peptide of 39 PTH2 3 0 3 19187 156.4 1.10E-07 residues

membrane-bound signaling SEMA4C 0 5 5 172406 29 4.69E-08 Semaphorin-4C molecule(PC00152) P2RY8 0 4 4 78641 50.86 2.48E-07 P2Y purinoceptor 8 non-motor microtubule binding MAP kinase-interacting protein(PC00166);non-receptor MKNK2 0 4 4 105276 38 5.02E-06 serine/threonine-protein kinase 2 serine/threonine protein kinase(PC00167) SLC14A2 0 5 5 187786 26.63 3.83E-05 Urea transporter 2 hydrolase(PC00121);microtubule DNAH5 0 9 9 910125 9.89 0.000504 Dynein heavy chain 5, axonemal binding motor protein(PC00156) Coiled-coil domain-containing CCDC122 0 3 3 54214 55.34 0.000149 protein 122

Leucine-rich repeat and calponin LRCH1 0 5 5 169623 29.48 3.94E-05 homology domain-containing protein 1

50 TNN 0 4 4 271054 14.76 3.94E-05 Tenascin-N signaling molecule(PC00207) SERTA domain-containing protein SERTAD4 0 4 4 151849 26.34 0.001463 4

ALS2 0 5 5 596566 8.38 4.70E-05 Alsin Acyl-CoA:lysophosphatidylglycerol LPGAT1 0 3 3 88832 33.77 0.000399 acyltransferase 1

CST2 0 2 2 27452 72.85 0.001211 Cystatin-SA ribonucleoprotein(PC00201);transfer/c FRG1 0 3 3 59621 50.32 0.001463 Protein FRG1 arrier protein(PC00219) Histidine-rich carboxyl terminus HRCT1 1 1 2 19005 105.2 0.009061 protein 1

Vacuolar protein sorting- non-motor microtubule binding VPS4B 0 2 2 79449 25.17 0.001384 associated protein 4B protein(PC00166) Voltage-dependent calcium voltage-gated calcium CACNG7 0 2 2 81648 24.5 0.000915 channel gamma-7 subunit channel(PC00240) CNPY1 0 2 2 18915 105.7 0.00209 Protein canopy homolog 1 DCTN4 0 3 3 138712 21.63 0.000936 Dynactin subunit 4 Ras GTPase-activating-like protein IQGAP1 0 3 3 351205 8.54 0.004638 G-protein modulator(PC00022) IQGAP1

FAM155B 2 0 2 74222 26.95 0.001651 Transmembrane protein FAM155B

Lysosome-associated membrane membrane trafficking regulatory LAMP1 0 3 3 91360 32.84 0.014356 glycoprotein 1 protein(PC00151) FRAS1-related extracellular matrix FREM1 0 5 5 558999 8.94 0.029635 protein 1

rRNA methyltransferase 1, MRM1 0 2 2 69244 28.88 0.025275 mitochondrial

6-pyruvoyl tetrahydrobiopterin PTS 0 2 2 33871 59.05 0.00475 synthase

ESYT3 0 3 3 208710 14.37 0.001546 Extended synaptotagmin-3 Putative E3 ubiquitin-protein ligase UBR7 0 3 3 185315 16.19 0.001906 UBR7

cysteine CAD 0 4 4 627659 6.37 0.000654 CAD protein protease(PC00081);ligase(PC00142);

51 metalloprotease(PC00153);transferas e(PC00220)

Dehydrogenase/reductase SDR DHRS4 0 2 2 47853 41.79 0.004503 family member 4

actin and actin related ACTR3 0 3 3 95408 31.44 0.00475 Actin-related protein 3 protein(PC00039)

MAZ 0 3 3 269853 11.12 0.001546 Myc-associated zinc finger protein

Transmembrane channel-like TMC1 0 3 3 210577 14.25 0.002581 protein 1

Sodium/potassium-transporting NKAIN2 0 3 3 89860 33.39 0.042416 ATPase subunit beta-1-interacting protein 2

Probable global transcription SMARCA2 2 1 3 395769 7.58 0.014643 DNA helicase(PC00011) activator SNF2L2 WD repeat and FYVE domain- WDFY3 0 5 5 737275 6.78 0.012448 containing protein 3

DNA (cytosine-5)- DNMT3A 0 3 3 303234 9.89 0.002589 methyltransferase 3A

basic helix-loop-helix transcription NEUROD2 0 2 2 68730 29.1 0.073826 Neurogenic differentiation factor 2 factor(PC00055);nuclease(PC00170) OR5M1 0 2 2 54979 36.38 0.108772 Olfactory receptor 5M1

SWI/SNF-related matrix- associated actin-dependent SMARCAD1 0 2 2 223937 8.93 0.008335 DNA helicase(PC00011) regulator of chromatin subfamily A containing DEAD/H box 1

KIAA1377 0 3 3 290782 10.32 0.00732 Centrosomal protein of 126 kDa GJC2 1 1 2 55640 35.95 0.125793 Gap junction gamma-2 protein gap junction(PC00105) SPG7 0 3 3 347762 8.63 0.003873 Paraplegin metalloprotease(PC00153) ZFHX3 2 1 3 701642 4.28 0.015709 Zinc finger homeobox protein 3 RGS1 0 2 2 41636 48.04 0.036208 Regulator of G-protein signaling 1 G-protein modulator(PC00022)

Prostaglandin E2 receptor EP1 PTGER1 0 2 2 43242 46.25 0.022244 G-protein coupled receptor(PC00021) subtype FDR, false discovery rate.

Supplemental Table 14. Constitutional pathogenic mutations in principal ciliopathy genes

dbSNP Gene Category #Chr Position ID# AlleleFreq SnpEffEffect Gene SnpEffHGVS.c SnpEffHGVS.p PJ9001456 P.C. chr1 214822159 None 43/21 frameshift_variant CENPF c.7975_7976dupAG p.Ser2659fs PJ9001457 P.S chr11 76912556 None 34/26 frameshift_variant MYO7A c.4919delG p.Gly1640fs CMJ001593 P.C chr4 15589551 None 45/28 splice_donor_variant&intron_variant CC2D2A c.4179+1delG CMJ001595 P.S chr2 166810195 None 19/74 frameshift_variant&splice_region_variant TTC21B c.20_21insCG p.Lys7fs P.S., pathogenic inferred from snpEff 'HIGH' impact annotation; P.C., pathogenic inferred from ClinVar.

SUPPLEMENTAL STATISTICAL ANALYSIS

Analysis of somatic variant counts by patient age I. Analysis of variant counts (N=3,263 variations) in study subjects (N=9)

Below, we fit a linear mixed effects model, where the mixed effects consist of fixed and random effects. The fixed effects are diameter and age. The random effect is a random intercept for each subject. The model is; therefore:

Log(mutation countij) = β0 + β1*diameterij + β2*agei + γ0i + εij,

Where i=1,...,9 indicates the subject and j=1,...,mi indicates the # of observations per subject. Here γ0i ~ Normal(0,σγ) is the random effect for subject i, and εij ~ Normal(0,σε) is the residual error.

54 lme(log.mutation.count~diameter+age,random=~1|patient) Linear mixed-effects model fit by REML Random effects: Formula: ~1 | patient (Intercept) Residual StdDev: 0.477987 0.4896461

Fixed effects: log.mutation.count ~ diameter + age Value Std.Error DF t-value p-value (Intercept) 1.9914013 1.2904255 55 1.543213 0.1285 diameter 0.0385568 0.0277155 55 1.391165 0.1698 age 0.0314109 0.0224326 7 1.400234 0.2042 Correlation: (Intr) diamtr diameter -0.111 age -0.987 0.019

Standardized Within-Group Residuals: Min Q1 Med Q3 Max -2.5490576 -0.6135790 0.1578540 0.6405002 1.6287799

Number of Observations: 65 Number of Groups: 9 The reason age is not significant (p=0.2042) in the model above is because patient PJ9001456 (grey *) is very young (41) but has a very high mutation count. Therefore we do not see a linear increase in mutation count with age. If we were to restrict the analysis to all subjects over 50, then the effect of age would be very significant (p=0.005; see output below).

Linear mixed-effects model fit by REML Data: NULL AIC BIC logLik 108.0787 118.0236 -49.03933

Random effects: Formula: ~1 | patient (Intercept) Residual StdDev: 0.2104161 0.4903749

Fixed effects: log.mutation.count ~ diameter + age Value Std.Error DF t-value p-value (Intercept) -1.4649076 1.2212564 48 -1.199509 0.2362 diameter 0.0387396 0.0279791 48 1.384590 0.1726 age 0.0885528 0.0207249 6 4.272762 0.0052 Correlation: (Intr) diamtr diameter -0.137 age -0.992 0.037

Standardized Within-Group Residuals: Min Q1 Med Q3 Max -2.37587345 -0.46163074 -0.01423317 0.81118109 1.87578346

Number of Observations: 57 Number of Groups: 8

55 II. Analysis of variant counts (N=1,784 variations classified as pathogenic or likely pathogenic) in study subjects (N=9)

> out=lme(log.pathogenic.count~diameter+age,random=reStruct(~1|patient,pdClas s="pdSymm",REML=FALSE)) > summary(out) Linear mixed-effects model fit by REML Data: NULL AIC BIC logLik 170.2983 180.934 -80.14915

Random effects: Formula: ~1 | patient (Intercept) Residual StdDev: 0.3965993 0.7146579

Fixed effects: log.pathogenic.count ~ diameter + age Value Std.Error DF t-value p-value (Intercept) 2.5354821 1.2618525 55 2.0093331 0.0494 diameter -0.0037608 0.0401213 55 -0.0937364 0.9257

56 age 0.0058488 0.0219831 7 0.2660608 0.7979 Correlation: (Intr) diamtr diameter -0.147 age -0.982 0.010

Standardized Within-Group Residuals: Min Q1 Med Q3 Max -2.5909548 -0.6564159 0.2112497 0.7442148 1.4777303

Number of Observations: 65 Number of Groups: 9

When we remove patient PJ9001456, age is a significant predictor (higher age implies higher pathogenic count).

> out2=lme(log.pathogenic.count~diameter+age,random=reStruct(~1|patient,pdCla ss="pdSymm",REML=FALSE)) > summary(out2) Linear mixed-effects model fit by REML Data: NULL AIC BIC logLik 133.5011 143.446 -61.75055

Random effects: Formula: ~1 | patient (Intercept) Residual StdDev: 2.573239e-05 0.6503021

Fixed effects: log.pathogenic.count ~ diameter + age Value Std.Error DF t-value p-value (Intercept) -4.004220 1.2008126 48 -3.334592 0.0017 diameter 0.058036 0.0362775 48 1.599769 0.1162 age 0.112998 0.0204946 6 5.513526 0.0015 Correlation: (Intr) diamtr diameter -0.129 age -0.988 -0.005

Standardized Within-Group Residuals: Min Q1 Med Q3 Max -2.07998196 -0.64887401 0.08380959 0.72471385 1.99506941

Number of Observations: 57 Number of Groups: 8

57 SUPPLEMENTAL METHODS

Study Subjects

Patients enrolled in this study were previously diagnosed with ADPKD and were scheduled for a living donor kidney transplant, and removal of one, or both native kidneys at the NewYork-

Presbyterian Hospital/Weill Cornell Medicine campus (NYPH-WCM). Subjects were identified during a preoperative evaluation that occurred 7-10 days before transplant surgery

(Supplementary Figure 1). The transplant surgeon (S.K.) determined whether there were clinical indications for nephrectomy prior to enrollment of each subject. If nephrectomy was planned, and the subject was eligible for enrollment, then informed consent was obtained. The protocol was approved by the WCM IRB.

Tissue processing and epithelial cell isolation

Following nephrectomy, the kidney(s) were immediately placed on ice and underwent gross examination

(S.S., B.R.). Thereafter, 5-20 cysts per kidney were processed; cyst size and volume were recorded, and cyst epithelial cells were isolated according to standard procedures 1. Each cyst cavity was washed several times with phosphate buffered solution (PBS) and then incubated with PBS/EDTA to remove epithelial cells from the basement membrane. Epithelial cells were collected by centrifugation and stored at -80°C. DNA was isolated from the cyst epithelial cells and peripheral blood leukocytes (PBL) using the Gentra Puregene DNA extraction kit (Qiagen, Germantown, MD).

58 Sanger Sequencing

PKD1 (NM_000296.2) and PKD2 (NM_000297.2) were analyzed by Sanger sequencing as previously described 2. To avoid amplification of PKD1 pseudogenes, PKD1 was amplified using long-range PCR

(LR-PCR) primers anchored either in the rare mismatched region within the human homologs, or in the single-copy region of PKD1 (GeneAmp High Fidelity PCR System, ABI, Foster City, CA). Sanger sequencing data were analyzed by Mutation Surveyor software vs.4.0 (Soft Genetics, State College, PA).

Long-range PCR (LR-PCR) Next Generation Sequencing (NGS)

PKD gene analysis using paired-end NGS was performed by multiplexing individually bar-coded LR-

PCR libraries analyzing them in one MiSeq system flow cell (Illumina). Sequencing data were analyzed using a laboratory-developed computational pipeline as described by Tan et al. 3.

Whole Exome Sequencing (WES) and Data Analysis

WES (357,999 exons, >20,000 genes) analyses of renal cyst epithelia and PBL specimens were performed using the HaloPlex Target Enrichment System (Agilent Technologies) and massive parallel sequencing on the Illumina system (San Diego, CA). Somatic and constitutional mutations were analyzed in matched cyst and PBL samples, respectively, and co-analyzed according to established procedures. Briefly, 225 ng of genomic DNA was digested using restriction enzymes, purified and assessed for quality using the Bioanalyzer. The resulting DNA was then hybridized to HaloPlex probes coupled to Illumina sequence motifs and sample barcodes, followed by solid phase capture. Biotinylated

DNA-probe hybrids were then ligated and amplified by PCR. The resulting exome DNA library was purified using the Agencourt AMPure XP beads (Beckman Coulter, Inc., Brea, CA) and sequenced on the

Illumina HiSeq 2500 platform (100 bp paired-end reads). The total number of reads and captured reads

59 among these specimens were 67.5 M7.0 M and 55.0 M5.2 M (81.6%  2.2%), respectively.

WES data analyses of the cyst and matched germline were performed for simultaneous detection of somatic single nucleotide variation (SNV), indels, and copy number variations (CNVs) as previously described 4. Germline variants were eliminated from the somatic variant list using an FDR corrected

Fisher exact test. For identification of somatic mutations, demultiplexed reads from Illumina sequencers were mapped against the GRC37/hg19 human reference genome (vs.19) using Burrows-Wheeler Aligner

(BWA 0.7.8) (http://bio-bwa.sourceforge.net/) with the recommended standard settings and preprocessed with SAMtools 0.1.19. GATK (Version 2.8) (https://www.broadinstitute.org/gatk/) for local realignment and base quality score recalibration of the mapped reads. SNVs were called by MuTect (Version 1.1.4)

(https://www.broadinstitute.org/cancer/cga/mutect) with the default criteria and filters. Small indels were detected by GATK. Mutations were filtered by variant allele frequency (VAF) where the mutation had to be present in the cyst with minimum total coverage of 8x and a VAF >5% and/or a min of 2 reads.

Somatic CNVs were called with VarScan2 (http://varscan.sourceforge.net). Raw copy number regions with chromosome, start position, stop position, and log2 value were processed with DNAcopy package

(http://www.bioconductor.org/packages/release/bioc/html/DNAcopy.html) to produce segmented calls delineated by significant change-points of at least three standard deviations (SD). Segments with a log2 value >0.6 were designated as gains and segments with a log2 value < -0.6 were categorized as losses.

Significantly mutated genes and pathway analysis

The pathogenic potential of missense variants was evaluated using the computational analysis tool Combined Annotation–Dependent Depletion (CADD), which enables scoring of deleteriousness of SNVs as well as indel variants. A CADD value of >15 suggests the variant is

60 potentially pathogenic 5 (http://cadd.gs.washington.edu/). Potential splice-site effects were evaluated using splice site prediction tools ESEfinder 3.0 (http://krainer01.cshl.edu/cgi- bin/tools/ESE3/esefinder.cgi?process=home), Human splicing finder 3.1

(http://www.umd.be/HSF3/) and NNSplice (https://omictools.com/nnsplice-tool) with default settings for all missense, synonymous and intronic changes. Pathogenic and likely pathogenic variants were further filtered by VAF (>30%) and subjected for pathway analysis to identify overrepresented pathways using Reactome (https://reactome.org/) and verified with David

(https://david.ncifcrf.gov/). Only significantly overrepresented pathways with low p-values

(<0.05); probability that overlap between the queried variant and pathway occur by chance, were reported. Existing variations with a population allele frequency >5% in ExAC

(http://exac.broadinstitute.org/) were filtered out as well.

The significance of mutated genes and pathways was analyzed with the Mutational Significance

In Cancer (MuSiC) suite of tools (http://gmt.genome.wustl.edu/packages/genome-music/index.html) with the default settings. The MuSiC algorithm calculates the mutation number per gene and compares it to the background mutation rate, applying a Q-score metric and ranking the mutation significance. This algorithm assigns mutations to seven categories: AT transition, AT transversion, CG transition, CG transversion, CpG transition, CpG transversion and indel, and then uses statistical methods based on convolution, the hypergeometric distribution, and likelihood to combine the category-specific binomials to obtain overall P or q values.

61 Droplet Digital PCR

PKD1 and PKD2 CNV testing was performed by droplet digital PCR (ddPCR), using 24ng DNA, 1.2 μL of each PrimePCR (Cat#10031276, custom designed PKD1 and PKD2 CNV assay) and the Copy

Number Reference Assay RPP30 (67 bp, UniqueAssayID: dHsaCP2500350), labeled with FAM and

HEX dyes, respectively, 12 μL of the ddPCR™ Supermix (no dUTPs) and 1.2 μL of diluted HaeIII or

HingIII enzyme (New England Biolabs, Ipswich, MA) in a total reaction volume of 24 μL, according to the manufacturer’s instructions (PrimePCR ddPCR CNV Assay, Bio-Rad, Hercules, CA). Droplets were generated in a QX200 droplet generator (Bio-Rad). PCR was performed in duplicate in a C1000 Touch

PCR thermal cycler, and the results were read in a QX200 droplet reader (BioRad).

Statistical Analysis

Each parameter is expressed as the mean ± SD. Two-tailed Student t-test was used to compare normally distributed variables, otherwise, a nonparametric test was applied (i.e., Wilcoxon Rank

Sum test). The association between somatic variant count and cyst size was analyzed using linear mixed effects model with log (pathogenic count) as the outcome and age and diameters as predictors. The log transform of pathogenic count was performed in order to satisfy normality assumptions. A random intercept was included for each subject in order to adjust for within subject correlation. These analyses were performed using the "lme" function within the "nlme" package in the R software environment (https://cran.r- project.org/web/packages/nlme/index.html).

62 REFERENCES

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63 SIGNIFICANCE STATEMENT

Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy caused by mutations in PKD1 and PKD2, characterized by renal epithelial cell proliferation and progressive CKD. Among the genetic mechanisms proposed for cystogenesis are inactivating constitutional and somatic mutations in PKD1/2 genes, and PKD1/2 gene dosage effects. This article describes the identification, by genomic sequencing methods, of the high prevalence of inactivating somatic mutations in PKD1/2 and non- PKD1/2 genes in epithelial cells obtained from kidney cysts in patients with ADPKD. The findings support a primary cellular recessive mechanism for cyst formation in ADPKD caused by mutations in these genes in the renal epithelium. Potential interactions of PKD1/2 genes with other ciliopathy- and cancer-related genes that might influence ADPKD severity is proposed.