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Deletion in the Cobalamin Synthetase W Domain–Containing 1 Is associated with Congenital Anomalies of the Kidney and Urinary Tract

Shoichiro Kanda,1,2 Masaki Ohmuraya,3 Hiroyuki Akagawa,4 Shigeru Horita,5 Yasuhiro Yoshida,1 Naoto Kaneko,2 Noriko Sugawara,2 Kiyonobu Ishizuka,2 Kenichiro Miura,2 Yutaka Harita,1 Toshiyuki Yamamoto,4,6 Akira Oka ,1 Kimi Araki,7 Toru Furukawa,4,8 and Motoshi Hattori2

1Department of Pediatrics, The University of Tokyo, Tokyo, Japan; 2Department of Pediatric Nephrology, 5Department of Pathology, Kidney Center, School of Medicine, and 6Institute of Medical Genetics, Tokyo Women’s Medical University, Tokyo, Japan; 3Department of Genetics, Hyogo College of Medicine, Hyogo, Japan; 4Tokyo Women’s Medical University Institute for Integrated Medical Sciences, Tokyo, Japan; 7Division of Developmental Genetics, Institute of Resource Development and Analysis, Kumamoto University, Kumamoto, Japan; and 8Department of Investigative Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan

ABSTRACT Background Researchers have identified about 40 with mutations that result in the most common cause of CKD in children, congenital anomalies of the kidney and urinary tract (CAKUT), but approximately 85% of patients with CAKUT lack mutations in these genes. The anomalies that comprise CAKUT are clinically heterogenous, and thought to be caused by disturbances at different points in kidney development. However, identification of novel CAKUT- causing genes remains difficult because of their variable expressivity, incomplete penetrance, and heterogeneity. Methods We investigated two generations of a family that included two siblings with CAKUT. Although the parents and another child were healthy, the two affected siblings presented the same manifestations, unilateral renal agenesis and contralateral renal hypoplasia. To search for a novel causative gene of CAKUT, we performed whole-exome and whole-genome sequencing of DNA from the family members. We also generated two lines of genetically modified mice with a gene deletion present only in the affected siblings, and performed immunohistochemical and phenotypic analyses of these mice. Results We found that the affected siblings, but not healthy family members, had a homozygous deletion in the Cobalamin Synthetase W Domain–Containing Protein 1 (CBWD1) gene. Whole-genome sequencing uncovered genomic breakpoints, which involved exon 1 of CBWD1, harboring the initiating codon. Immunohistochemical analysis revealed high expression of Cbwd1 in the nuclei of the ureteric bud cells in the developing kidneys. Cbwd1-deficient mice showed CAKUT phenotypes, including hydronephrosis, hydroureters, and duplicated ureters. Conclusions The identification of a deletion in CBWD1 gene in two siblings with CAKUT implies a role for CBWD1 in the etiology of some cases of CAKUT.

JASN 31: 139–147, 2020. doi: https://doi.org/10.1681/ASN.2019040398

Received April 21, 2019. Accepted October 2, 2019. Correspondence: Dr. Shoichiro Kanda, Department of Pediat- rics, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo Published online ahead of print. Publication date available at 113-8655, Japan. Email: [email protected] www.jasn.org. Copyright © 2020 by the American Society of Nephrology

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Congenital anomalies of the kidney and urinary tract Significance Statement (CAKUT) are the most common cause of ESKD in children.1–4 CAKUT are clinically heterogeneous. These disorders Most patients with congenital anomalies of the kidney and urinary encompass a large variety of anatomic malformations that tract (CAKUT), the leading cause of pediatric ESKD, do not have range from abnormal phenotypes of the renal parenchyma, mutations in any of the approximately 40 CAKUT-causing genes that have been identified to date. The authors studied a family with such as renal agenesis and renal hypoplasia, to those of two siblings with CAKUT that appeared to be caused by an auto- the collecting system, such as vesicoureteral reflux and dupli- somal recessive mutation in an as-yet unidentified gene. Using cated ureter.5 The diverse manifestations of CAKUT are whole-exome and whole-genome sequencing, they found that the thought to be caused by disturbances at different points in affected children but not healthy family members had a homozy- kidney development.6,7 gous deletion in the Cobalamin Synthetase W Domain–Containing Protein 1 (CBWD1) gene. They also demonstrated in mice that The kidney develops through a multistage process. After Cbwd1 protein was expressed in the ureteric bud cells, and that budding from the nephric duct, the ureteric bud cells invade Cbwd1-deficient mice showed CAKUT. These findings suggest a the metanephric mesenchyme cells, followed by reciprocally role for CBWD1 in CAKUT etiology. inductive interactions between these two precursors.5 Metanephric mesenchyme differentiates into nephrons P16–009) and were carried out in accordance with the ap- (glomerular, proximal tubular, and distal tubular epithelium), proved guidelines. and further branching of the ureteric bud forms the kidney collecting system (collecting tubular and ureter epithelium). Whole-Exome Sequencing Our understanding of the mechanisms involved in nephro- Genomic DNA collected from family members was used for genesis is mostly derived from mouse models.8,9 whole-exome sequencing (WES). DNA was extracted using a The pathogenesis of CAKUT is multifactorial. Genetic, en- QIAamp DNA Blood Maxi kit (Qiagen, Hilden, Germany). vironmental, and epigenetic factors are known to be involved A fragment library was constructed from the extracted in its development. Several lines of evidence indicate that DNA using the AB Library Builder System (Life Technologies, CAKUT can also be caused by mutations in single genes. Carlsbad, CA). Constructed libraries were subjected to whole- The mutation is familial (autosomal dominant or recessive) exome enrichment using a TargetSeq Target Enrichment Kit or de novo. Approximately 40 genes causative of (Life Technologies). The prepared exome libraries were se- CAKUT have been identified.5,10,11 However, approximately quenced using the massively parallel deep sequencer 5500xl 85% of patients with CAKUT do not possess mutations in any SOLiD System (Life Technologies) by the paired-end sequenc- of them.12,13 Therefore, it is likely that there are additional ing method. Data were analyzed using LifeScope software (Life as-yet unidentified genes causative of CAKUT. Technologies) with mapping on the Refer- Despite the great advances of next-generation sequencing ence, GRCh37/hg19 (The Genome Reference Consortium; technology, identification of novel CAKUT-causing genes re- http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/ mains difficult because of their variable expressivity, incom- index.shtml). All procedures were performed in accordance plete penetrance, and heterogeneity.14,15 Therefore, it with the manufacturers’ instructions. Obtained data were an- remains a challenge to identify new components of the path- notated and stringently filtered to exclude false variation ogenesis of human CAKUT. calls using our previously described programs developed Here, we investigated two generations of a family, including in house.16 Summary of sequence data are shown in Supple- two siblings with CAKUT, which resulted in identification of a mental Table 1. homozygous deletion in Cobalamin Synthetase W Domain– Containing Protein 1 (CBWD1) only in the family members Validating PCR for CBWD1 Deletion with CAKUT. We showed that Cbwd1 was localized in the Validating PCR reactions were performed using KOD FX Neo ureteric bud. We also constructed Cbwd1-deficient mice in accordance with the manufacturer’sinstructions(TOYOBO showing anomalies of the urinary collecting system. Collectively, Co. Ltd., Tokyo, Japan). All amplifications were performed our results indicate a role for CBWD1 in CAKUTetiology. for 40 cycles using 100 ng of genomic DNA with the in- dicated primers and annealing temperature as follows: paired primers of Chr9_177574_F, 59-TGTAGTAGAAGC- METHODS CAAAAGAACAGC-39, and Chr9_179736_R, 59-GCT- TTTACGTTAGGAATGAC-39, at 60°C; Chr9_163253_F, This study was approved by the Central Ethics Board of Tokyo 59-GTGTTCAGGTAAATAAAGTAAGAC-39, and Chr9_164850_R, Women’s Medical University. All family members provided 59-AGGTGGACAGCTTTAGACGC-39, at 60°C. written informed consent to participate in the study. Array Comparative Genomic Hybridization Animals Extracted genomic DNA was used as a template. Genomic All animal experiments were approved by the institutional copy number aberrations were analyzed using the SurePrint committees of the University of Tokyo (approval number G3 Hmn CGH 60 k Oligo Microarray (Agilent Technologies,

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Santa Clara, CA), in accordance with previous reports.17 incubation with Protein Block (Genostaff, Tokyo, Japan) and Briefly, 250 ng each of target and reference DNA was digested avidin/biotin blocking kit (Vector, Burlingame, CA). The sec- with the restriction enzymes, AluIandRsaI. Cy-5 (target) or tions were incubated with anti-CBWD1, anti-Cytokeratin 8, Cy-3 dUTP (reference) was incorporated using the Klenow and anti-SIX2 at 4°C overnight. They were then incubated fragment. The array was hybridized with labeled DNAs in with biotin-conjugated rabbit anti-rabbit Ig (Dako, Kyoto, the presence of Cot-1 DNA (Life Technologies) and blocking Japan) for 30 minutes at room temperature, followed by agents (Agilent Technologies) for 24 hours at 65°C, washed, the addition of peroxidase-conjugated streptavidin (Nichirei, and scanned on a scanner (Agilent Technologies). Data were Tokyo, Japan) for 5 minutes. Peroxidase activity was visualized extracted using Agilent Feature Extraction software version by diaminobenzidine. The sections were counterstained with 10 with the default settings for aCGH analysis. Statistically Mayer hematoxylin (MUTO, Tokyo, Japan), dehydrated, and significant aberrations were determined using the ADM-II then mounted with Malinol (MUTO). algorithm in Agilent Genomic Workbench software version 6.5 (Agilent Technologies). Genomic locations were refer- Generation of Cbwd1-Deficient Mice enced according to Genome Reference, GRCh37/hg19. We generated two lines of Cbwd1-deficient mice as follows. Cbwd1 gRNA vectors were constructed with pT7-sgRNA and Whole-Genome Sequencing pT7-hCas9 plasmids (gifts from Dr. M. Ikawa, Osaka Univer- DNA libraries for whole-genome sequencing were construc- sity).24 After digestion of pT7-hCas9 with EcoRI, hCas9 ted using TrueSeq Nano DNA Library Kit (Illumina), and mRNA was synthesized using an in vitro RNA transcription sequenced using 150-bp paired-end reads on a NovaSeq6000 kit (mMESSAGE mMACHINE T7 Ultra kit; Ambion, Austin, sequencer (Illumina). The average fold coverage was .30 (to- TX), in accordance with the manufacturer’s instructions. A tal number of bases ranging from 103.9 to 126.8 Gbp). pair of oligonucleotides targeting Cbwd1 was annealed and After quality-based read trimming, sequence reads were inserted into the BbsI site of the pT7-sgRNA vector. The aligned to the human reference genome GRCh37/hg19 using sequences of the gRNAs were as follows: 59-GGCGGAAGAA- BWA-MEM,18 and then processed by various structural vari- GAGTACGCGG-39 and 59-ACAATTGTCACCGGGTACTT- ation calling tools: BreakDancer,19 BreakSeq2,20 Pindel,21 39. Both are located in exon 1 of Cbwd1 to generate CNVnator,22 and their integration method MetaSV.23 Cbwd1-deficient mice. After the digestion of pT7-sgRNA with XbaI, gRNAs were synthesized using the MEGAshortscript Read Depth–Based Copy Number Analyses using WES kit (Ambion). We used C57BL/6N female mice (purchased Data from Clea-Japan Inc., Tokyo, Japan) to obtain C57BL/6N The read depth of coverage for each exon in the RefSeq Genes eggs, and performed in vitro fertilization with these eggs. In was calculated and normalized using CalculateHsMetrics in brief, Cas9 mRNA and gRNA were introduced into fertilized Picard Tools (https://broadinstitute.github.io/picard/). Copy eggs by electroporation (Genome Editor electroporator; BEX number states around the CBWD1 locus were assessed by Co. Ltd., Tokyo, Japan), in accordance with previously reported comparing the normalized coverage values excluding large protocols,25 after which we transferred the eggs to the oviducts outliers in the present family members. EXCAVATOR2 of pseudopregnant females on the day when a vaginal plug was (https://github.com/matheuscburger/Excavator2) was also found. Founder mice harboring a mutant Cbwd1 allele were used to confirm the exact copy number of the presumed hemi- crossed with wild-type mice to obtain Cbwd1 heterozygous zygous members. The window size was set to 1000 bases, and mice. After segregating the Cbwd1 mutant alleles, homozygous analysis was performed in the paired mode using II.3 as a mice were used for additional analysis. normal control.

Immunohistochemistry RESULTS CBWD1 protein expression was examined in embryonic mouse kidneys (embryonic days 11.5–16.5), in neonatal Case Report mouse kidneys and in a kidney from an adult human donor. A kindred with CAKUT was referred to our institution for Approval for research on human subjects was obtained from ESKD treatment (Figure 1A). The family consisted of non- the Central Ethics Board of Tokyo Women’s Medical Univer- consanguineous parents (I.1 and I.2), two male siblings (II.1 sity. The antibodies against the following were used: and II.3), and one female sibling (II.2). Both the eldest male CBWD1 (catalog number GTX120748; GeneTex, Irvine, CA), sibling and the female sibling presented with unilateral renal Cytokeratin 8 (catalog number ab53280; Abcam, Cambridge, agenesis and contralateral renal hypoplasia (Figure 1B). Al- MA), and SIX2 (catalog number 11562–1-AP; Proteintech, though CAKUT can appear as part of a systemic condition Chicago, IL). Tissue sections were deparaffinized with xylene with extrarenal manifestations, the current cases were not and rehydrated through serially gradated ethanol solutions accompanied by any other birth defects, such as coloboma, and Tris-buffered saline. Endogenous peroxidase was blocked skeletal/digital, or genital anomalies or other clinical findings, with 0.3% H2O2 in methanol for 30 minutes, followed by including diabetes, hypocalcemia, hypomagnesemia, or

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A B 1 2 R L I

1 23 II

C Chr9: 163253 -164850 Chr9: 177574 -179736

N I.1 I.2 II.1 II.3 II.2 C N I.1 I.2 II.1 II.3 II.2 C M

D Chr9 deletion: g. 178351_182100 Distance from pter 168000 170000 172000 174000 176000 178000 180000 182000 184000 186000 188000

Coverage

Alignment

RefGene CBWD1 E 1

0.5 II.3 I.2 0 I.1 II.1 II.2

CBWD1 CDS5 CBWD1 CDS3 CBWD1 CDS2 CBWD1 CDS1C9orf66 CDS1

Figure 1. CBWD1 is identified as a candidate gene for CAKUT. (A) Pedigree of the family containing some members affected by CAKUT. Squares represent males, circles represent females, and shading indicates members with CAKUT. Carriers of genetic anomalies are indicated by a dot within the square or circle. (B) An arrow depicts hypoplasia of the right kidney on computed tomography images in Patient II.1. (C) Agarose gel electrophoresis demonstrating genomic amplification of the coding region of CBWD1 exon 1. Validating PCRs using genomic DNAs from examined subjects indicated that a genomic segment between 177574 and 179736, which includes exon 1 of CBWD1 and harbors an initiating codon, was homozygously deleted in diseased siblings (II.1 and II.2) but not in the parents and the healthy sibling (II.3). Parents are I.1 (father) and I.2 (mother). A genomic segment between 163253 and 164850 at 9p was unaffected. (D) Whole-genome sequencing reads of an affected sibling (II.2) showing the 3.7 kb homozygous deletion (Chr9:178351_182100). A deletion at the CBWD1 locus involving the entirety of coding exon 1 was identified. (E) The relative normalized coverage of CBWD1 coding sequence (CDS) 1 of both parents (I.1 and I.2)

142 JASN JASN 31: 139–147, 2020 www.jasn.org BASIC RESEARCH hyperuricemia. The other family members had no health of the deleted regions, we carried out additional whole- problems, including in their kidneys. The renal function of genome sequencing for the affected sibling (II.2) and the parents the eldest son (II.1) gradually deteriorated such that at 6 years (I.1 and I.2), which uncovered a homozygous deletion spanning of age, he started chronic peritoneal dialysis, and at 10 years of from Chr9:178351 to Chr9:182100 involving the entire coding age, he received a cadaveric kidney transplant. The female exon 1 of CBWD1 in the affected sibling (Figure 1D). sibling (II.2) also demonstrated ESKD and received a living- To analyze whether the parents had heterozygous deletion related preemptive kidney transplant from her grandmother or not, we calculated normalized coverage values of coding at 11 years of age. sequences around CBWD1 using CollectHsMetrics (Picard), and found that the patients’ coverage was zero. Although Identification of CBWD1 as a Candidate Gene for the younger healthy brother’s coverage was 0.39, the parents’ CAKUT coverage was 0.27 (69%) and 0.23 (59%), which were within Under the hypothesis that a gene harboring deleterious ho- the range fitting heterozygosity (Figure 1E, Supplemental mozygous or compound heterozygous mutations in autoso- Figure 1). We also analyzed the copy number profiles of the mal recessive fashion may have caused CAKUT in this family, region containing a potential deletion in using we performed WES using peripheral-blood DNA from all EXCAVATOR2.30 Heterozygous exon 1 deletion in the CBWD1 family members to identify candidate single-nucleotide gene was also validated in the parents’ sample (Supplemental variants and insertions and/or deletions. We did not find Figure 2). any single-nucleotide variants or insertions and/or deletions that existed only in the affected siblings including all 39 cur- Expression Patterns of Cbwd1 rently established causing genes for CAKUT (Supplemental To examine the significance of CBWD1 in the development of Table 2).5 However, we identified a deletion in the af- the genitourinary system, we first investigated the localization fected siblings in the region of chromosome 9p24.3. The de- of CBWD1 by immunohistochemistry. Antibodies against letion seemed to span between at least Chr9:177574 and CBWD1, SIX2, and Cytokeratin 8 were used for immunohis- Chr9:179736 on GRCh37/hg19, which involved exon 1 of tochemical staining of mouse embryonic kidneys. SIX2 and CBWD1, harboring an initiating codon. We designed primers Cytokeratin 8 are known as markers of mesenchyme cells and to validate the deleted region and confirmed that this region the ureteric bud in the developing kidney, respectively.31,32 We was indeed homozygously deleted in the affected siblings but found that Cbwd1 was present in kidneys at E13.5 but not at not in the parents and the healthy sibling (Figure 1C). Search- E11.5 or E12.5 (Figure 2, A and B, Supplemental Figure 3). ing of the Database of Genomic Variants (http://dgv.tcag.ca/ Cbwd1 was primarily observed in the ureteric bud epithe- dgv/app/home?ref=GRCh37/hg19) showed that a heterozy- lial cells labeled with anti-Cytokeratin 8 in serial sections gous deletion of this region was found in four out of 2538 (Figure 2B). At E16.5, the staining became more intense in individuals (0.16%) (accession number: gssvL128711).26 In the ureteric bud (Figure 2C). In an adult human kidney spec- this population, there were 2504 individuals from the 1000 imen, CBWD1 was not observed (Supplemental Figure 4). Genomes Project, including 104 Japanese individuals. The de- letion was not observed in this Japanese population. Because Kidney Defects in Cbwd1-Deficient Mice copy number variants (CNVs) might cause the disease,27,28 To uncover the role of CBWD1 in the development of we also performed array-CGH analyses, which resulted in the urinary tract, we generated Cbwd1-deficient mice the detection of no obvious CNVs in the affected siblings (C57BL/6N-Cbwd1em1) by CRISPR-Cas9 gene targeting and (data not shown). This result suggested that any CNVs that examined their phenotypes. Homozygous mice were born at might cause the disease could be less than detectable range or Mendelian frequency and did not suffer premature mortality out of probing regions for the array-CGH we used. (Supplemental Table 3). We determined the edited sequence We attempted to determine breakpoints of the deletion by (Supplemental Figure 5) and confirmed the loss of Cbwd1 in performing long-range PCR and primer walking; however, we the nuclei of the ureteric bud cells (Figure 3, A and B). To could not determine them because of the presence of highly characterize the CAKUT phenotypes of Cbwd1-deficient 2 2 repetitive sequences in this region. CBWD1 is located at mice, we examined the morphology of 34 Cbwd1 / mice, 2 9p24.3:121038–179075 (GRCh37/hg19). This region is 54 Cbwd1+/ mice, and 42 wild-type mice. Overall, 29% of 2 2 2 known to be segmentally quadruplicated and rearranged Cbwd1 / mice and 4% of Cbwd1+/ mice had grossly iden- with transposition and translocation, which results in highly tifiable CAKUT, whereas no abnormal phenotype was found in homologous sequences in two closely located segments at any of the wild-type mice (Figure 3C). CAKUT found in the 9q21.11 and one segment at 2q13.29 For precise determination Cbwd1-deficient mice were hydronephrosis, hydroureters, and

was 0.69 and 0.59, compared with the coverage of the younger healthy brother (II.3). The coverage of the patients (II.1 and II.2) was zero. C, a nonrelated control individual; M, DNA Molecular Weight Marker VIII (Roche); N, negative control amplifying without template DNA.

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Cytokeratin 8 Six2 HE Cbwd1 (ureteric bud) (mesenchyme) A E12.5

embryonic kidney

100m

B E13.5

embryonic kidney

100m

C E16.5

 renal pelvis 100 m

Figure 2. Cbwd1 is expressed in the ureteric bud cells. (A–C) Serial sections of developing mouse kidneys at E12.5 (A), E13.5 (B), and E16.5 (C). The first section shows hematoxylin and eosin (HE) staining and the other sections show immunoperoxidase labeling for Cbwd1 (second section), SIX2 (third section), and Cytokeratin 8 (fourth section). Cbwd1 was detected in the nuclei of ureteric bud cells at E13.5 and at E16.5 (arrow). duplicated ureters (Figure 3D). These phenotypes were con- Cbwd1-deficient mice showed duplicated ureters. Although sidered to be associated with failed expression of Cbwd1 in these phenotypic differences may be attributable to species dif- the ureteric bud during the development of the urinary tract. ferences,33 both phenotypes could be interpreted as primary 2 2 2 Cbwd1+/ and Cbwd1 / mice survived to adulthood, for defects at the level of ureter budding. For example, the nephric which prospective dissection also identified CAKUT, including duct–specific inactivation of Gata3, a transcription factor ex- duplicated ureters and hydronephrosis (Figure 3, E and F). Torule pressed in the ureteric bud, also results in a spectrum of urinary out off-target effects of CRISPR-Cas9, we also analyzed an inde- tract malformations including kidney agenesis and duplex sys- pendent line of Cbwd1-deficient mice (C57BL/6N-Cbwd1em2) tems and hydroureter.34 This kind of phenotypic variation is and confirmed similar results (Supplemental Figures 6–8, Sup- frequently observed in genetic studies of CAKUT.14 Many estab- plemental Table 4). lished CAKUT-causing genes encode transcription factors. Be- cause Cbwd1 was also localized in the nuclei of the ureteric bud cells (Figure 3A), it may influence the transcription network in DISCUSSION the developing kidney. However, the molecular mechanisms of CBWD1-mediated kidney development remain to be resolved. We identified that a deletion in CBWD1 is involved in CAKUT The deletion detected in this study involved exon 1 of etiology, as supported by several lines of evidence. First, CBWD1 harboring the initiating codon. Deletion of the first we found a homozygous deletion in CBWD1 in patients coding exon usually results in a gross deletion of the affected with autosomal recessive CAKUT. Second, we showed that transcripts. If there are multiple transcriptional variants, loss Cbwd1 was expressed in the kidney during development. may be avoided depending on the variant using alterna- Third, and most significantly, Cbwd1-deficient mice showed tive first exons. By applying the public database GTEx CAKUT phenotypes. Portal (https://gtexportal.org/home/documentationPage), we The function of CBWD1 was not known until now. Our found that two types of transcriptional variants are mainly ex- results show that CBWD1 is associated with the development pressed in human tissues: [ENST00000356521.8] and of the ureteric bud into the urinary tract. Some phenotypic [ENST00000382447.8]. Because the initiating codons of both differences were observed between the patients and the mouse variants are in the deleted region detected in this study, it can model. Specifically, the patients with homozygous deletion be said that the expression of the CBWD1 gene is almost lost in involving CBWD1 showed renal agenesis; however, some the patients’ tissues.

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A WT B Cbwd1-/- C (%) 29 (P0) (P0) 30

20

10 4 0 0 WT +/- -/- (n=42) (n=54) (n=34) D E F Cbwd1+/- Cbwd1+/- Cbwd1-/- (P0) (adult) (4week)

adrenal gland duplex collecting system

kidney

ureter

duplex bladder collecting system hydronephrosis

Figure 3. Cbwd1 mutants (C57BL/6N-Cbwd1em1) show kidney and urinary tract defects. (A and B) Cbwd1 localization in the renal 2 2 pelvis of neonatal (P0) mice. (A) Cbwd1 is localized in nuclei of the epithelium in wild-type mice. (B) Nuclear staining is lost in Cbwd1 / mice. (C) Percentages of newborn mice with CAKUT. (D) Representative images of gross anatomy (left) and light microscopic histology 2 of CAKUT found in a Cbwd1+/ pup at P0 (right). The left kidney presented with hydronephrosis, hydroureter, and duplicated ureter 2 (arrow). (E) Hydronephrosis, hydroureter, and duplicated ureter observed in a Cbwd1+/ adult mouse. (F) Hydronephrosis found in a 2 2 4-week-old Cbwd1 / mouse.

CBWD1 is located at 9p24.3:121038–179075 (GRCh37/ patients. Dr. Ohmuraya and Dr. Araki generated knockout mice. hg19), which maps near the 9p telomere. The deletion 9p Dr. Akagawa, Dr. Yamamoto, and Dr. Furukawa performed genetic syndrome is clinically characterized by dysmorphic facial fea- analyses. Dr. Kanda, Dr. Horita, and Dr. Yoshida performed mouse tures, hypotonia, and mental retardation. Although abnormal analyses and immunohistochemical experiments. Dr. Kanda wrote external genitals are frequently seen (15 out of 36 cases),35 the original draft of the manuscript. Dr. Kanda, Dr. Ohmuraya, renal malformation is one of the rare features (one out of Dr. Akagawa, Dr. Harita, Dr. Oka, Dr. Furukawa, and Dr. Hattori 13 cases).36 The patients in our study presented with CAKUT contributed to manuscript review and editing. Dr. Kanda, Dr. Oka, without other clinical manifestations and the parents were and Dr. Hattori were involved in funding acquisition. All authors healthy. It is thought that these phenotypic differences be- approved the final version of the manuscript. tween reported patients with the deletion 9p syndrome and We gratefully acknowledge the hospital staff for their help the family in our study are attributable to cytogenetic dif- and support. We also thank Mitsuhiro Amemiya and Akira Saito ferences. The deletion 9p syndrome is caused by constitu- (StaGen Co. Ltd., Tokyo, Japan) for help with processing of the tional monosomy of part of the short arm of chromosome 9. next-generation sequencing data. We are also grateful to GenoStaff In addition, the deletion is large, varying from 800 kb to Co. for their support with the immunohistochemistry. Finally, we 12.4 Mb.37 In contrast, the deletion observed in our patients thank Jeremy Allen, from Edanz Group (www.edanzediting.com/ was homozygous and 3.7 kb in size. ac) for editing a draft of this manuscript. In conclusion, our results implies a role for CBWD1 in CAKUT etiology. Cbwd1 is localized in the ureteric bud and DISCLOSURES promotes its development into the kidney collecting system. None.

ACKNOWLEDGMENTS FUNDING

Dr. Kanda and Dr. Hattori designed the study. Dr. Kanda, Dr. Kaneko, The study was supported by the Ministry of Education, Culture, Sports, Dr. Sugawara, Dr. Ishizuka, Dr. Miura, and Dr. Hattori treated the Science and Technology, Japan (Grant-in-Aid for Scientific Research

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[KAKENHI] 25870536, 15K21385, and 17K09689); by the Japanese Associa- 8. McMahon AP: Development of the mammalian kidney. Curr Top Dev tion of Dialysis Physicians, Japan (grant 2014-13); and by research funds from Biol 117: 31–64, 2016 the Kawano Masanori Memorial Public Interest Incorporated Foundation for 9. Costantini F: Genetic controls and cellular behaviors in branching Promotion of Pediatrics, the Mother and Child Health Foundation, and the morphogenesis of the renal collecting system. Wiley Interdiscip Rev Takeda Science Foundation, Japan. Dev Biol 1: 693–713, 2012 10. Sanna-Cherchi S, Sampogna RV, Papeta N, Burgess KE, Nees SN, Perry BJ, et al.: Mutations in DSTYK and dominant urinary tract malformations. SUPPLEMENTAL MATERIAL NEnglJMed369: 621–629, 2013 11. Vivante A, Kohl S, Hwang DY, Dworschak GC, Hildebrandt F: Single- This article contains the following supplemental material gene causes of congenital anomalies of the kidney and urinary tract (CAKUT) in . Pediatr Nephrol 29: 695–704, 2014 online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ 12. Weber S, Moriniere V, Knüppel T, Charbit M, Dusek J, Ghiggeri GM, ASN.2019040398/-/DCSupplemental. et al.: Prevalence of mutations in renal developmental genes in children Supplemental Table 1. Sequence performance data of the whole- with renal hypodysplasia: Results of the ESCAPE study. JAmSoc exome sequence using SOLiD 5500 system. Nephrol 17: 2864–2870, 2006 Supplemental Table 2. The coverage of the WES for 39 currently 13. Hwang DY, Dworschak GC, Kohl S, Saisawat P, Vivante A, Hilger AC, et al.: Mutations in 12 known dominant disease-causing genes clarify established monogenic causing genes for CAKUT. many congenital anomalies of the kidney and urinary tract. Kidney Int Supplemental Table 3. Genotype analysis of 4-week-old mice 85: 1429–1433, 2014 (C57BL/6N-Cbwd1em1). 14. van der Ven AT, Vivante A, Hildebrandt F: Novel insights into the Supplemental Table 4. Genotype analysis of 4-week-old mice pathogenesis of monogenic congenital anomalies of the kidney and – (C57BL/6N-Cbwd1em2). urinary tract. JAmSocNephrol29: 36 50, 2018 15. van der Ven AT, Connaughton DM, Ityel H, Mann N, Nakayama M, Supplemental Figure 1. Normalized coverage of the coding se- Chen J, et al.: Whole-exome sequencing identifies causative mutations quence around CBWD1. in families with congenital anomalies of the kidney and urinary tract. Supplemental Figure 2. The copy number profiles of the parents in JAmSocNephrol29: 2348–2361, 2018 the region containing a potential deletion in chromosome 9 from 16. Furukawa T, Kuboki Y, Tanji E, Yoshida S, Hatori T, Yamamoto M, et al.: EXCAVATOR2. Whole-exome sequencing uncovers frequent GNAS mutations in in- traductal papillary mucinous neoplasms of the pancreas. Sci Rep 1: Supplemental Figure 3. Cbwd1 was not observed in E11.5 mouse 161, 2011 kidneys. 17. 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