Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease

Please cite this article in press as: Cornec-Le Gall et al., Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease, The American Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.03.013

ARTICLE Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease Emilie Cornec-Le Gall,1,4,5 Rory J. Olson,2 Whitney Besse,6 Christina M. Heyer,1 Vladimir G. Gainullin,1 Jessica M. Smith,1 Marie-Pierre Audre´zet,5 Katharina Hopp,7 Binu Porath,1 Beili Shi,8 Saurabh Baheti,3 Sarah R. Senum,1 Jennifer Arroyo,1 Charles D. Madsen,1 Claude Fe´rec,5 Dominique Joly,10 Franc¸ois Jouret,11 Oussamah Fikri-Benbrahim,12 Christophe Charasse,13 Jean-Marie Coulibaly,13 Alan S. Yu,14 Korosh Khalili,9 York Pei,8 Stefan Somlo,6 Yannick Le Meur,4 Vicente E. Torres,1 Genkyst Study Group, the HALT Progression of Polycystic Kidney Disease Group, the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease, and Peter C. Harris1,2,* Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by the progressive development of kidney cysts, often resulting in end-stage renal disease (ESRD). This disorder is genetically heterogeneous with
7% of families genetically unresolved. We performed whole-exome sequencing (WES) in two multiplex ADPKD-like pedigrees, and we analyzed a further 591 genetically unresolved, phenotypically similar families by targeted next-generation sequencing of 65 candidate genes. WES identified a DNAJB11 missense variant (p.Pro54Arg) in two family members presenting with non-enlarged polycystic kidneys and a frameshifting change (c.166_167insTT) in a second family with small renal and liver cysts. DNAJB11 is a co-factor of BiP, a key chaperone in the endoplasmic reticulum controlling folding, trafficking, and degradation of secreted and membrane proteins. Five additional multigenerational families carrying DNAJB11 mutations were identified by the targeted analysis. The clinical phenotype was consistent in the 23 affected members, with non-enlarged cystic kidneys that often evolved to kidney atrophy; 7 subjects reached ESRD from 59 to 89 years. The lack of kidney enlargement, histologically evident interstitial fibrosis in non-cystic parenchyma, and recurring episodes of gout (one family) suggested partial phenotypic overlap with autosomal-dominant tubulointerstitial diseases (ADTKD). Characterization of DNAJB11-null cells and kidney samples from affected individuals revealed a pathogenesis associated with maturation and trafficking defects involving the ADPKD protein, PC1, and ADTKD proteins, such as UMOD. DNAJB11-associated disease is a phenotypic hybrid of ADPKD and ADTKD, characterized by normal-sized cystic kidneys and progressive interstitial fibrosis resulting in late-onset ESRD.

Introduction Autosomal-dominant polycystic kidney disease (ADPKD) is the fourth leading cause of end-stage renal disease (ESRD) worldwide.1 It is characterized by the relentless development of renal cysts, causing kidney enlargement and ESRD in
50% of subjects by 60 years, although disease progression and prognosis is highly variable. Polycystic liver disease (PLD) is the most frequent extrarenal feature; other disease manifestations include a higher risk (53) of developing intracranial aneurysms (see GeneReviews in Web Resources).1 ADPKD is genetically heterogeneous, with pathogenic variants to PKD1 (MIM: 601313) and PKD2 (MIM: 173910) identified in
72%– 75% and
15%–18% of families, respectively.2–5 A third gene, GANAB (MIM: 104160), has been recently described in 12 families presenting with ADPKD or the related autosomal-dominant PLD (ADPLD).6–8 Genetic variability

strongly influences the severity of ADPKD: PKD1 truncating pathogenic variants are typically associated with earlier ESRD (median age
58 years) than PKD1 non-truncating (
67 years) and PKD2 pathogenic (
79 years) variants, while progression to ESRD has not been observed in GANAB (MIM: 600666).5,9 Presently, 7% to 10% of ADPKD-affected families remain genetically unresolved.4,10,11 PKD1 and PKD2 encode polycystin 1 (PC1) and 2 (PC2), respectively, both membrane glycoproteins functionally expressed at the primary cilium.12 PC1 is cleaved at a G protein-coupled receptor proteolytic site (GPS), and the level of mature, cleaved glycoforms of PC1 is associated with disease severity.13,14 GANAB encodes the a-subunit of glucosidase II (the ADPLD gene, PRKCSH [MIM: 177060], encodes the b subunit), a resident enzyme of the endoplasmic reticulum (ER) involved in asparagine (N)-linked glycosylation, a key quality control process

1

Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN 55905, USA; 2Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA; 3Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN 55905, USA; 4Department of Nephrology, University Hospital, European University of Brittany, Brest, Brittany 29200, France; 5Department of Molecular Genetics, National Institute of Health and Medical Sciences, INSERM U1078, Brest 29200, France; 6Section of Nephrology, Yale School of Medicine, New Haven, CT 06520, USA; 7Division of Renal Diseases and Hypertension, University of Colorado Denver Anschutz Medical Campus, Aurora, CO 80202, USA; 8Division of Nephrology, University Health Network, Toronto, ON M5G 2C4, Canada; 9Department of Medical Imaging, University Health Network, Toronto, ON M5G 2C4, Canada; 10Service of Nephrology, Necker Hospital, Paris 75231, France; 11Division of Nephrology, University of Lie`ge, Lie`ge 4000, Belgium; 12Service of Nephrology and Hemodialysis, Saintes 17108, France; 13Service of Nephrology, Yves Le Foll Hospital, Saint Brieuc 22000, France; 14Kidney Institute, University of Kansas Medical Center, Kansas City, KS 66160, USA *Correspondence: [email protected] https://doi.org/10.1016/j.ajhg.2018.03.013. Ó 2018 American Society of Human Genetics.

The American Journal of Human Genetics 102, 1–13, May 3, 2018 1

Please cite this article in press as: Cornec-Le Gall et al., Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease, The American Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.03.013

governing folding, maturation, and trafficking of membrane/secreted proteins.6,7 Additional ER proteins associated with translocation and folding of membrane/secreted proteins have been implicated in ADPLD (SEC63 [MIM: 608648], ALG8 [MIM: 608103], and SEC61B [MIM: 609214]), with PC1 particularly sensitive to perturbations in this pathway.6,9 A rarer group of dominant kidney diseases characterized by progressive tubulointerstitial fibrosis and progression to ESRD, with no or few small renal cysts, has recently been united under the term autosomal-dominant tubulointerstitial kidney diseases (ADTKD).15 ADTKD caused by UMOD pathogenic changes (ADTKD-UMOD [MIM: 191845]), the most frequent disorder of this group, accounts for close to 1% of ESRD prevalent cases.15–17 The UMOD protein, uromodulin (or Tamm-Horsfall protein), secreted in the thick ascendant limb of the loop of Henle (TAL), is the most abundant urinary protein. UMOD variants lead to protein misfolding and retention in the ER of TAL cells, likely resulting in progressive damage to these epithelial cells. Other genetic etiologies of ADTKD include pathogenic variants to MUC1 (MIM: 1583400), HNF1B (MIM: 189907), and more rarely REN (MIM: 179820) and SEC61A1 (MIM: 609213).9,15 In this study we employed global and focused sequencing of genetically unresolved ADPKD/ADPLD-affected families. We define the clinical and molecular features of a newly recognized autosomal-dominant disorder caused by pathogenic variants to DNAJB11, associating non-enlarged polycystic kidneys and chronic interstitial fibrosis, and resulting in renal insufficiency after the sixth decade.

Subjects and Methods Study Participants and Clinical Analysis A total of 722 subjects from 694 genetically unresolved ADPKD (n ¼ 502)- and ADPLD (n ¼ 192)-affected families were enrolled. The participants were recruited through different ADPKD cohorts: the French Genkyst study (n ¼ 123),5,11 the HALT-PKD clinical trial (n ¼ 58),4,18,19 the Toronto Genetic Epidemiology Study of Polycystic Kidney Disease (TGESP) (n ¼ 78),20,21 and the Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP) (n ¼ 4).22 The discovery cohort from Yale School of Medicine comprised 102 unrelated individuals with unresolved ADPLD.6 In addition, 285 pedigrees from the Mayo PKD Center and 44 from Brest University Hospital affected by ADPKD or ADPLD were included. The relevant Institutional Review Boards or ethics committees approved all studies, and participants gave informed consent. Clinical and imaging data were obtained by review of clinical records. Kidney function was calculated from clinical serum creatinine measurements with the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula.23 Blood samples for standard DNA isolation were collected from the probands and all available family members.

Whole-Exome Sequencing Analysis We performed whole-exome sequencing in two multiplex families (1 and 2), as previously described.6,7 Briefly, for family 1 (pedi-

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gree M624), total genomic DNA was quantified with a Qubit 2.0 fluorometer (dsDNA BR Assay Kit, Thermo Fisher Scientific). Subsequently, 1 mg of DNA was sheared by sonication (Covaris E210, 150–200 bp) and purified by AMPure XP beads (Agencourt). The shearing efficiency was checked on an Agilent Bioanalyzer 2100 (DNA1000 assay). Whole-exome capture was performed with the Agilent SureSelectXT Human All Exon V5UTRs Kit on an Agilent Bravo workstation. The enriched library was sequenced with 101 bp paired-end reads on an Illumina HiSeq 2000 in the Mayo Medical Genome Facility. Genome_GPS v.3.0.1 (Mayo Bioinformatics Core) was employed as a comprehensive secondary analysis pipeline for variant calling. FASTQ files were aligned to the hg19 reference genome (UCSC Genome Browser) with Novoalign (V2.08.01), and realignment and recalibration were performed with the Genome Analysis Toolkit (GATK) (3.3-0). Multi-sample variant calling was performed with the GATK (3.3-0) Haplotype Caller, and variants were filtered with Variant Quality Score Recalibration for both SNVs and indels. Variant mining was performed with Golden Helix SNP & Variation Suite v.8 (SVS). Exome sequencing and analysis was performed independently at Yale University in index case subject III.2 from family 2 (pedigree YU209), as previously described.6 Targeted exome capture from the proband employed the NimbleGen/Roche SeqCap EZ Exome v2 reagent, followed by 75-bp paired-end sequencing (Illumina HiSeq 2000). Sequence reads were aligned to the GRCh37/ hg19 human reference genome and realignment, recalibration, and variant calling were performed as described above. Family 1 and the proband from family 2 were independantly analyzed with the following filters: (1) read depth (R103) and genotype quality (R20), (2) selection for autosomal-dominant sample genotype pattern, (3) removal of Exome Aggregation Consortium (ExAC) browser variants with a minor allele frequency (MAF) > 0.1%, and (4) characterization of coding and non-coding SNVs within 14 bp of the splice site, and subsequent removal of SNPs predicted to be neutral by R2/6 dbNSFP tools (SIFT, PolyPhen-2 HVAR, MutationTaster, Mutation Assessor, FATHMM, and FATHMM MKL). The variants that remained after the SVS analysis are listed in Tables S1A and S1B.

Targeted Next-Generation Sequencing We developed a custom gene panel (SureSelect, Agilent) to capture the coding exons and
50 bp flanking intronic regions of 65 genes, including 14 genes known to be associated with polycystic kidney or liver diseases (Table S2). Similar to the wholeexome sequencing procedure, genomic DNA was quantified by Qubit and 500 ng of DNA was subsequently sheared by sonication. Libraries were prepared using the NEBNext Ultra DNA Preparation kit, and 24 samples were pooled before capture. The captured libraries were sequenced using the HiSeq4000 with 150 bp paired-end reads (Illumina). Because all the targeted regions cover less than 1 Mb, we indexed 48 samples per lane and still achieved high-depth coverage. Sequence alignment and variant calling was performed as described above for the whole-exome sequencing analysis. Variant mining was performed employing Golden Helix SNP and Variation Suite v.8 and copy number variations (CNVs) were detected with PatternCNV. Families were analyzed independently with the following filtering procedure: (1) variant-based read depth (R103) and quality (R20), (2) removal of Genome Aggregation Database (GnomAD) variants with a MAF of > 0.1%, and (3) characterization and removal of coding and non-coding SNVs

Please cite this article in press as: Cornec-Le Gall et al., Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease, The American Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.03.013

within 14 bp from the splice site and/or predicted to be neutral by R3/6 dbNSFP tools (tools selected as above).

Confirmation of the DNAJB11 Pathogenic Variants by Sanger Sequencing Primers to amplify exons 3, 4, 6, and 7 of DNAJB11 (GenBank: NM_016306.5) were designed with Primer-BLAST (NCBI) and 100 ng of genomic DNA used for PCR amplification (primers and PCR conditions are available upon request). Sanger sequencing was performed at Mayo Clinic Molecular Biology Core according to standard approaches. When samples were available, segregation analysis was performed by sequence analysis of the mutated exonic fragment.

Using CRISPR/Cas9 to Generate Targeted DNAJB11Mutated Cell Lines CRISPR/Cas9 targeting of DNAJB11 exon 3 in human primary renal cortical tubular epithelial (RCTE) cells generated clones with homozygous frameshifting changes. In short, a guide RNA (gRNA) predicted to have the lowest off-targeting effect was cloned into pX330 (SpCas9) and verified by sequencing (gRNA sequence: 50 -TGGATCATCAGGGTTCCGGT-30 ). DNAJB11 exon 3 was amplified by PCR (primer sequences are available on request) and cloned into pCAG-EGxxFP51 with restriction sites BamHI and EcoRI. The gRNA was then co-transfected with p-CAG-EG-DNAJB11(Ex3)-FP in renal cortical tubular epithelial (RCTE) cells and scored after 24 hr for EGFP-positive cells. pX330-gRNA was transfected into wild-type (WT) RCTE cells by electroporation, and the cells were allowed to recover for 36 hr prior to splitting and reseeding as single-cell suspensions in a 96-well plate. Cells were grown for 10 days for the establishment of single clone cell colonies, split in half, and re-seeded for screening. Screening was performed with genomic DNA extraction followed by amplification using DNAJB11 Exon 3 primers and a subsequent T7 mismatch assay. For this assay, PCR amplicons were denatured at 95 C for 2 min and cooled gradually to 25 C at a rate of 2 C per second. The reaction mixture was then subjected to T7 endonuclease (T7E1, New England BioLabs) for 20 min at 37 C and visualized in 2% agarose gels. Clones were additionally screened by western blotting (WB) for DNAJB11 and Sanger sequencing. A clone carrying a homozygous frameshifting deletion of 19 nucleotides in exon 3 of DNAJB11, c.164_182delACCGGAACCCTGATGATCC (p.Asp54fs), was selected for the functional characterization of DNAJB11 loss.

Western Blotting Studies, Glycosylation Analysis, and Immunoprecipitation For purification of crude membrane protein, cells were grown to confluence, washed with Dulbecco’s PBS (DPBS), scraped and re-constituted in LIS buffer (10 mM Tris-HCl [pH 7.4], 2.5 mM MgCl2, and 1 mM EDTA) plus protease inhibitors (Complete, Roche), and incubated on ice for 15 min. Cells were then homogenized by repeated passage through a 25.5G needle and centrifuged at 800 3 g for 5 min. The supernatant was collected and centrifuged at 20,000 3 g for 30 min. The resulting pellet was solubilized in IP buffer (20 mM HEPES [pH 7.5], 137 mM NaCl, 1% NP-40, 10% [wt/vol] glycerol, 2 mM EDTA, 2.5 mM MgCl2) supplemented with protease inhibitors (Roche) for 30 min on ice, followed by centrifugation at 12,000 3 g for 15 min. The supernatant was retained and normalized to total protein by BCA for subsequent analysis. All procedures employed pre-chilled

containers and were performed at 4 C to minimize protein degradation. For immunoprecipitation (IP), crude membrane protein (200 mg) was incubated overnight at 4 C in the presence of 2 mg of PC1 CT antibody and then incubated with 20 mL of packed washed A/G Agarose (Thermo) for 2 hr. The agarose was washed thrice in IP buffer and the protein was eluted with 40 mL 1 3 lithium dodecyl sulfate (LDS) plus 50 mM tris(2-carboxyethyl) phosphine (TCEP) for 15 min at 80 C. The eluate was split into three equal parts (untreated, EndoH, and PNGaseF) and subjected to deglycosylation analysis. Crude membrane protein and IP eluates were deglycosylated with EndoH and PNGaseF according to the manufacturer’s (New England BioLabs) instructions. 25 mg of input and 100% of the IP were loaded per SDS-PAGE lane.

Unfolded-Protein Response Assay To investigate the effect of loss of DNAJB11 on the unfolded protein response (UPR), we measured both its basal and induced activity in RCTE (WT) and three independent DNAJB11/ CRISPR clones. Cell lines were seeded with equal cell numbers and grown to confluence followed by treatment with 1 or 2 mg/mL Tunicamycin (Sigma-Aldrich) to induce the UPR or vehicle for 4 hr. Cells were then harvested, pelleted by centrifugation, and lysed in IP buffer containing protease inhibitors. Subsequent cell lysates were normalized to total protein by BCA and analyzed by western blotting against key downstream effectors for each UPR arm, i.e., XBP1s for IRE1a, ATF6a for ATF6, and CHOP for PERK.

Transfection, Confocal Microscopy, Immunofluorescence (IF), and Surface PC1 Labeling RCTE cells were split at a ratio of 1:2 the day before electroporation and transfected at
80% confluency. Electroporation of RCTE cells was performed with the Bio-Rad Gene Pulser with a square-wave protocol: 110V, 25 ms pulse, and 0.2 cm cuvettes (Bio-Rad) in electroporation buffer (20 mM HEPES, 135 mM KCl, 2 mM MgCl2, and 0.5% Ficoll 400 [pH 7.6]). TagGFP-PC2 and mCherry-PC1 constructs used in this study have been previously described.13 Transfected RCTE cells were grown on glass coverslips for 16 hr. For surface labeling of mCherry-PC1, transfected RCTE cells were cooled at 4 C for 30 min, washed once in ice-cold PBS, incubated with pre-chilled mCherry antibody 1:1,000 (BioVision) in 0.5% BSA in PBS for 30 min at 4 C. Cells were then fixed in 4% PFA (15 min at 4 C followed by 5 min at RT), and conjugated secondary antibody (AF647) was added to IF buffer for 45 min. Cells were washed once with DPBS, permeabilized with 0.1% Triton in DPBS (pH 7.5), washed again in PBS, and incubated in blocking buffer (10% normal goat serum, 1% BSA, and 0.1% Tween in PBS [pH 7.5]) for 30 min. After three PBS washes, primary antibodies (anti-mCherry and anti-GFP) were added to the IF buffer (1% BSA, PBS [pH 7.5], and 0.1% Tween) for 2 hr at room temperature or overnight at 4 C. After 33 PBS washes, conjugated secondary antibody (AlexaFluor594 and 488, respectively, Invitrogen) was added for 45 min. DAPI was added for 1 min to stain nuclei. Confocal microscopy was performed with a Zeiss Axiovert equipped with Apotome.

Histological Analysis Kidney samples were available in two affected subjects. Trichrome staining was carried out according to standard protocols. For immunohistochemical labeling, 2 mM paraffin sections of the healthy pole of a human tumor nephrectomy specimen and from two DNAJB11 nephrectomies (family 1, II.1, family 5,

The American Journal of Human Genetics 102, 1–13, May 3, 2018 3

Please cite this article in press as: Cornec-Le Gall et al., Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease, The American Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.03.013

II.2) were deparaffinized and rehydrated in EtOH. Antigen retrieval was obtained by steaming in sodium citrate buffer (pH 6.0). Nonspecific binding was blocked in PBS for 30 min at RT prior to incubation at 4 C overnight in 0.5% BSA with primary antibodies UMOD, MUC1, and BiP. Secondary antibodies (Alexa Fluor 488 donkey anti-rabbit IgG [1:1,000] and Alexa Fluor 594 donkey anti mouse IgG1 [1:1,000]) were applied for 30 min in 0.5% BSA followed by DAPI for 1 min. Slides were observed at 103 and 403 magnification (Zeiss AxioObserver, Carl Zeiss).

Primary Antibodies The following antibodies were used: PC1-NT (N-terminal, mouse monoclonal 7e12; 1 mg/mL for WB), PC1-CT (C-terminal, Everest Biotech, Cat.#EB08670; 2 mg for IP), EGFR (Cell Signaling, Cat.#2232; 1 mg/mL for WB), E-cadherin (BD, Cat.#610181; 1 mg/mL for WB), DNAJB11 (rabbit; Abcam Cat.#ab75107, 1 mg/mL for WB), mCherry (BioVision, Cat.#5993-100; 1/1,000 for surface labeling), UMOD (Santa Cruz; Cat #sc-271022; 1/100 for IF), BiP (Abcam; Cat #ab21685, 1/250 for IF), MUC1 (ThermoFisher; Cat #MA5-13168, 1/250 for IF), XBP1s (CST, Cat.#12782, 1 mg/mL for WB), IRE1a (CST, Cat.#65880, 1 mg/mL for WB), and CHOP (CST, Cat.#2895, 1 mg/mL for WB).

Results WES Suggested DNAJB11 as a Gene Mutated in ADPKD In family 1, subjects I.1 and II.1 were diagnosed at 79 years and 43 years with non-enlarged polycystic kidneys and a few millimeter-sized liver cysts (Figures 1A–1C; Table 1). In subject II.1, the diagnosis was concomitant with the identification of a metanephric adenoma during an evaluation for gastro-intestinal symptoms, which led to a partial right nephrectomy. At last follow-up (45 years), her kidney function was preserved and she was not hypertensive (Table 1). Subject I.1 started anti-hypertensive medication at 60 years and reached chronic kidney disease (CKD) stage 4 at 79 years. Whole-exome sequencing (WES), performed in both subjects as part of a previously described screen,7 identified DNAJB11 as the most promising candidate (Figures 2A and 2B; Table S1A). DNAJB11 has a 1,698 bp mRNA (GenBank: NP_016306.5) with a 1,077 bp coding segment, extending over 15,124 bp of genomic DNA (186,570,676– 186,585,800 nt) as 10 exons in chromosome region 3q27. DNAJB11 encodes a soluble glycoprotein (358 aa, MW ¼ 40.5 kDa; GenBank: NP_057390.1) of the ER lumen, one of the most abundant co-chaperones of binding immunoglobulin protein (BiP, also known as GRP78), a heat shock protein chaperone required for the proper folding and assembly of proteins in the ER.24,25 The DNAJB11 variant, c.161C>G, resulted in p.Pro54Arg, in the hallmark motif (His-Pro-Asp) of the highly conserved J domain of the protein, through which DNAJB11 interacts with BiP (Figures 2C and 2D). It was predicted to be deleterious by four variant prediction programs (Table S3) and absent in 133,070 exome or genome sequences of unrelated individuals sequenced as part of various disease-specific and population genetic studies compiled in the gnomAD database.26 The proband’s daughter, subject III.1, was sub-

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sequently found to harbor the p.Pro54Arg variant; at 19 years, no renal or hepatic cysts were detected by MRI and her kidney function was normal (Table 1). In a separate, previously described WES screen of 102 unrelated individuals,6 DNAJB11 was identified as the most likely candidate with the frameshifting change c.166_167insTT (p.Arg56Leufs*40) in subject III.2 from family 2 (Figures 2A and 2B; Table S1B). The pathogenic variant was subsequently identified in two other affected family members (Figure 1A). All affected individuals presented with non-enlarged polycystic kidneys, and three had multiple liver cysts (Figures 1D and S1A; Table 1). Subject III.2 was diagnosed with breast cancer at 38 years, but no likely pathogenic variants were identified in the breast cancer-associated genes BRCA1 and BRCA2. Her affected uncle, II.1, underwent parathyroid surgery for an adenoma (Table 1). Identification of Further ADPKD-Affected Families with DNAJB11 Pathogenic Variants Analysis of an additional 591 genetically unresolved families by targeted next generation sequencing (TNGS) of 65 known and candidate genes (Table S2) led to the identification of five additional families with DNAJB11 pathogenic variants. The familial and phenotypic characteristics of affected subjects are shown in Figures 1 and S1 and listed in Table 1, with details of the pathogenic variants in Figure 2. In family 3, subject III.2 (who was included in the HALTPKD clinical trial, Study B) was incidentally diagnosed with normal-sized polycystic kidneys and stage 3 CKD at 57 years (Figure S1C).19 She developed a multiform glioblastoma at 59 years and died shortly after diagnosis. Her mother (II.3) also had polycystic kidneys (Figure S1B) and started hemodialysis at 89 years, while her maternal aunt (II.1) reached ESRD at 75 years. A frameshifting deletion of a single nucleotide in DNAJB11 exon 6, c.479delC (p.Ala160Glufs*27) (Figure 2B), was identified in these subjects, her brother (III.4) and sister (III.5) with multiple small cysts (Figures 1E and S1D), and the son of III.2 (IV.2; 36 years) who had seven small bilateral kidney cysts (Figure S1E). The affected individuals in family 4 presented with normal-sized polycystic kidneys and no apparent liver cysts (Figures 1A, 1F, and 1G). Subject II.1 had severe renal insufficiency at 65 years and his mother (I.1) reached ESRD at 88 years. A missense change in DNAJB11 exon 4, c.230T>C (p.Leu77Pro), was identified in both affected subjects but was absent in the unaffected sister (II.2; Figure 2B). The substituted residue, in the J Domain of the protein, is invariant in orthologs and conserved in related proteins; it is not present in the gnomAD database and is predicted to be pathogenic by four in silico algorithms (Figures 2C and 2D; Table S3). The substitution c.616C>T, creating a nonsense change in exon 7, p.Arg206*, was identified in families 5, 6, and 7 (Figure 2B). In family 5, subjects I.1 and II.2 reached ESRD

Please cite this article in press as: Cornec-Le Gall et al., Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease, The American Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.03.013

Figure 1. Pedigrees of the Seven Families with DNAJB11-Associated Disorder, Abdominal Imaging, and Histological Characteristics (A) Seven pedigrees: black squares or circles indicate affected male or female subjects, respectively, presenting with bilateral renal cysts, liver cysts, renal failure, and/or genetically diagnosed, and gray symbols indicate case subjects where clinical information is unavailable. Clinical characteristics are detailed in Table 1. The DNAJB11 pathogenic variants identified in each family are indicated below each pedigree, ‘‘Mut’’ denotes that DNA sequencing proved the mutation, ‘‘WT’’ (wild-type) denotes the absence of the pathogenic variant, and ‘‘ND’’ (no data) indicates that no DNA sample was available. The DNAJB11-affected families originate from the US (families 1 [M624], 3 [M775], and 5 [M1231]), Canada (family 2 [YU209]), Belgium (family 4 [M1122]), and France (families 6 [PK20211] and 7 [PK4114]). Families came from various study cohorts: HALT-PKD (M775), CRISP (M1231), GeneQuest (NCT02112136, PK20211), TGESP (YU209), the French National Institute of Health and Medical Research Unit 1078, Brest, France (PK4114), and the Mayo PKD Center (M624, M1122). (B–J) Abdominal imaging of nine individuals from the seven families. Seven have magnetic resonance imaging (MRI; T2-weighted in B–E, G, J; T1-weighted in H) and two computed tomography (CT; non contrast enhanced in F; contrast enhanced in I). (K) Two representative kidney histology sections of family 5, II.2, who underwent a bilateral nephrectomy, stained with Masson’s trichrome (blue indicates fibrosis).

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6 Family 1d

2

3

d

Clinical Presentation and Pathogenic Variants in the 23 Affected Individuals from Seven DNAJB11-Affected Families

Pathogenic Variant c.161C>G (p.Pro54Arg)

c.166_167insTT (p.Arg56fs)

c.479delC (p.Ala160fs)

Subject

Sex

eGFRa (age) or ESRD (age)

I.1

F

28 (79)

II.1

F

III.1

Morphology of the Kidneys Context of Diagnosis (age)

Description of the Cysts

Kidney Length, (TKVml)c

Figure

Liver Cysts (number)

Other Significant Conditions (age)

HBP (age)b

Type

Age

renal insufficiency (78)

yes (60)

MRI

80

multiple bilateral small cysts (mostly < 1 cm, largest 2 cm)

R:9.2, L:10 (323)

1B

yes (3)

epilepsy (10)

96 (45)

incidental (43)

no (45)

MRI

44

multiple bilateral small cysts (largest 1.3 cm)

R:9.5, L:10 (335)

1C

yes (10)

metanephric adenoma, partial nephrectomy (43)

F

93 (19)

familial study (19)

no (19)

MRI

19

no renal cysts

R:9.7, L:10.0 (277)

NA

no

none

II.1

M

54 (66)

familial study (na)

yes (56)

MRI

53

multiple bilateral small cysts (largest 0.8 cm)

NA

NA

no

parathyroid adenoma, hypertrophic cardiomyopathy

II.2e

F

84 (76)

familial study (61)

yes (
50)

CT

70

small cortical cysts in the left kidney

R: 9.5, L: 9.6

S1A

yes (> 50)

Parkinson disease (60)

III.2

F

101 (45)

incidental (31)

no (45)

MRI

38

multiple bilateral small cysts

R:11.5, L:12

1D

yes (> 50)

breast cancer (36)

III.3

F

77 (56)

familial study (42)

yes (
50)

US

42

three parapelvic cysts on left kidney (largest 1 cm), no cysts detected on left kidney

R:9.7, L:10.6

NA

yes (> 20)

none

II.1e

F

ESRD (75)

renal insufficiency (
65)

NA

NA

NA

NA

NA

NA

NA

none

II.3

F

ESRD (89)

NA

NA

CTf

91

atrophic kidneys with multiple cysts

R:6; L:6

S1B

No

none

III.2

F

39 (59)

incidental (57)

yes (51)

US

57

multiple bilateral small cysts (largest 1.2 cm)

R:12, L:10

S1C

yes (na)

glioblastoma (59)

III.4

M

85 (66)

familial study (66)

no (66)

CT

62

multiple bilateral small cysts (largest 1.1 cm)

R:11, L:11 (483)

S1D

no

kidney stones (52)

III.5

F

82 (57)

familial study (57)

no (57)

MRI

57

multiple bilateral small cysts

R:11, L:11 (300)

1E

no

none

IV.2

M

97 (36)

familial study (35)

no (35)

MRI

36

seven bilateral small cysts (largest 0.3 cm)

R:10, L:9 (299)

S1E

yes (9)

none

(Continued on next page)

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The American Journal of Human Genetics 102, 1–13, May 3, 2018

Table 1.

Family 4

5

The American Journal of Human Genetics 102, 1–13, May 3, 2018 7

6

7

Continued

Pathogenic Variant c.230T>C (p.Leu77Pro)

c.616C>T (p.Arg206*)

c.616C>T (p.Arg206*)

c.616C>T (p.Arg206*)

Subject

Sex

eGFRa (age) or ESRD (age)

I.1

F

ESRD (88)

II.1

M

I.1e

Morphology of the Kidneys Context of Diagnosis (age)

Description of the Cysts

Kidney Length, (TKVml)c

Figure

Liver Cysts (number)

Other Significant Conditions (age)

HBP (age)b

Type

Age

renal insufficiency (78)

yes (79)

CT

90

multiple bilateral small cysts

R:9, L:10

1F

no

colorectal cancer (79)

25 (68)

incidental (65)

yes (48)

MRI

66

multiple bilateral small cysts

R:11, L:11 (280)

1G

no

type 2 diabetes (66)

F

ESRD (84)

autopsy (84)

NA

Autopsy

84

multiple bilateral small cysts (mostly < 1 cm, largest 1.5 cm)

R:9.5, L:10

NA

NA

none

II.2

F

ESRD (59)

incidental (56)

yes (57)

CT

57

multiple bilateral small cysts (largest 1 cm)

R:7.7, L:11.5

NA

no

gastrectomy for peptid ulcer disease (54), gout (50)

III.1

F

82 (56)

incidental (36)

no (57)

MRI

41

multiple bilateral small cysts (largest 0.8 cm)

R:9, L:10.5 (353)

1H

yes (> 10)

gout (39)

III.2

M

67 (55)

familial study (55)

yes (55)

MRI

55

four bilateral cysts (largest 0.9 cm)

R:10.4, L:10.7 (450)

NA

no

gout (40)

IV.1

F

92 (20)

familial study (20)

no (20)

NA

NA

NA

NA

NA

NA

epilepsy (8)

II.2

M

ESRD (74)

incidental (64)

yes (57)

CT

74

multiple bilateral small cysts (largest 2.7cm)

R:10, L:12 (371)

1I

no

type 2 diabetes (50), psoriatic arthritis (60)

II.3

F

ESRD (70)

renal insufficiency (60)

yes (56)

CT

71

multiple bilateral small cysts

R:10, L:9.3 (462)

S1F

no

ischemic cardiomyopathy (71), kidney stones (27)

II.2

F

100 (51)

flank pain (51)

no (52)

MRI

51

multiple small renal cysts (largest 2 cm), 3 angiomyolipomas (largest 5 cm)

R:13, L:13 (605)

1J

yes (> 20)

ischemic colitis (53), hearing loss (48)

Abbreviations: ACKD, acquired cystic kidney disease; CT, computed tomography; HBP, high blood pressure; MRI, magnetic resonance imaging; NA, not available; TKV, total kidney volume; US, ultrasound examination.aExpressed as mL/min/1.73 m2 on the basis of the last data available; obtained with the CKD-EPI formula b Age at HBP diagnosis or blood-pressure measurement c When available, obtained by stereological measurement; R, right kidney; L, left kidney d The pathogenic variant in this pedigree was first identified by whole-exome sequencing e A sample was unavailable, and so the DNAJB11 variant was not confirmed f Imaging performed 3 years after ESRD

Please cite this article in press as: Cornec-Le Gall et al., Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease, The American Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.03.013

Table 1.

Please cite this article in press as: Cornec-Le Gall et al., Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease, The American Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.03.013

Figure 2. Identification of DNAJB11 Pathogenic Variants in the Seven Pedigrees (A) Integrated Genomics Viewer (IGV, Broad Institute) visualization of the two DNAJB11 changes independently identified by WES (sequences from family 1, II.1 and family 2, III.2), with details of read depths and allele frequencies tabulated for each WES-identified pathogenic change. Targeted next-generation sequencing led to the identification of three additional pathogenic variants in five different pedigrees. (B) Sanger confirmations of the five identified DNAJB11 changes; WT sequences are shown for comparison. (C) Domain organization of DNAJB11, with the distribution of the pathogenic variants identified. DNAJB11 is a 358-amino-acid protein comprising a highly conserved J domain, with a characteristic His-Pro-Asp (HPD) motif through which it interacts with the chaperone BiP, a substrate binding domain, and a dimerization domain. The two identified missense changes occurred in the J-domain, one of them (p.Pro54Arg, identified in family 1) affecting the HPD motif. The other variants, all truncating, occurred in the J-domain or the substrate binding domain of the protein. (D) Multiple sequence alignments of DNAJB11 orthologous proteins, from human to rice (top), and of the J-domains of other human proteins from the ERdj family (bottom). The two amino acids affected by missense changes, proline at position 54 and leucine at position 77, are invariant in both cases.

at age 84 and 59, while subjects III.1 (a participant in the CRISP observational study; Figure 1H) and III.2 had preserved kidney function at 56 years and 55 years. Recurrent episodes of gout after 40 years were reported in three

8

The American Journal of Human Genetics 102, 1–13, May 3, 2018

affected family members (Table 1). A bilateral nephrectomy was performed prior to transplantation in II.2, and histological examination following Masson’s trichrome staining revealed significant and widespread

Please cite this article in press as: Cornec-Le Gall et al., Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease, The American Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.03.013

Figure 3. Loss of DNAJB11 Affects Maturation and Localization of Polycystin-1 (A) N-linked deglycosylation analysis of WT and DNAJB11/ RCTE membrane proteins either untreated (Un) or treated with Endoglycosidase H (þE; removing immature glycans) or PNGaseF (þP; removing all sugars). Immunoprecipitation was used to enrich the PC1 complex with a C-terminal PC1 (PC1-CT) antibody and immunodetected with the N-terminal PC1 (PC1-NT) antibody. Partial loss of mature PC1 (N-terminal PC1, resistant to EndoH, abbreviated NTR and indicated by a red arrow) was observed in DNAJB11deficient cells (see C for quantification), and increased levels of immature PC1 (including full-length [FL] and N-terminal EndoH sensitive [NTS] PC1). Similar analysis of E-cadherin and epidermal growth factor receptor (EGFR) did not show altered sensitivity to maturation in DNAJB11/ cells, although higher levels of E-cadherin were observed in wild-type as compared to DNAJB11/ cells. DNAJB11 loss was confirmed in null cells, and vinculin was employed as a loading control. (B) Schematic representation of the PC1 products in WT and DNAJB11/ cells presented in (A). (C) Decreased ratio of PC1 NTR compared to PC1 NTS in DNAJB11/ cells by more than 50%. The PC1 NTR/NTS bands density were measured in three different DNAJB11/ clones and three replicates of one clone, normalized to WT results, and presented here as the mean and standard deviation of all experiments. (D) Diagram of the WT-tagged PC1 and PC2 constructs used for the localization analysis presented in (E) with the site of PC1 GPS cleavage indicted with an arrow. (E) WT or DNAJB11/ cells were transfected with mCherry-PC1 and GFP-tagged-PC2 and examined for surface PC1 labeling (mCherry) in co-transfected cells. Surface PC1 expression was observed in 62.8% of the co-transfected WT cells, but only in 29.8% of the co-transfected DNAJB11/ (p < 0.001). This
50% decrease of surface PC1 expression in DNAJB11/ as compared to WT cells was confirmed on three different experiments. DNAJB11/ cells positive (middle) and negative (bottom) for surface PC1 are shown.

interstitial fibrosis in the non-cystic parenchyma (Figure 1K); no specific lesion was observed in the non-sclerotic glomeruli. In family 6, two affected siblings presented with atrophic polycystic kidneys (Figures 1I and S1F) and developed ESRD at 70 and 74 years. The family history was considered negative and the diagnosis of ‘‘chronic kidney disease of unknown etiology with acquired polycystic kidney disease’’ was initially considered for both until their familial connection was realized. In the only known affected subject of family 7, lower back pain prompted an abdominal MRI at 51 years, revealing multiple small bilateral cysts and three angiomyolipoma in the kidneys, plus mild PLD (Figure 1J). Kidney function and blood pressure were normal. Her past medical history was significant for severe sensorineural hearing loss of unknown etiology. A cerebral MRI performed at 51 years showed a frontal meningioma and

two arachnoid cysts. The subject did not present with any additional clinical or radiological feature suggestive of tuberous sclerosis complex (TSC), and no pathogenic variant was identified in TSC1 or TSC2. Characterization of the Effect of DNAJB11 Loss on PC1 Maturation DNAJB11 acts as a co-factor of the ER chaperone BiP, which regulates the folding and trafficking or degradation of membrane or secretory proteins. We therefore tested whether DNAJB11 loss was associated with impaired maturation of PC1.6,7 Analysis of the PC1 immunocaptured with a PC1-CT antibody in wild-type (WT) and DNAJB11/ cells and detected with a PC1-NT antibody showed that the mature, cleaved N-terminal PC1 (PC1NTR)13 was less abundant by >50% in the mutant cells (Figures 3A–3C). In contrast, full-length (FL; GPS uncleaved) and immature, N-terminal EndoH sensitive PC1

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Figure 4. Intracellular Distribution of UMOD, BiP, and MUC1 in Tubular Epithelial Cells Immunofluorescence labeling of uromodulin (UMOD; top), MUC1 (middle), and BiP (bottom) in kidney from an unaffected individual (control) and two DNAJB11-affected individuals (family 1, II.1 and family 5, II.2). The UMOD expression in the tubular ascending loop of Henle (TAL) of the unaffected individual is apical but appears to be retained intracellularly in the two DNAJB11-affected individuals (see insets a–c). Cellular distribution of BiP and MUC1 are not markedly different in the control and affected individuals, although in family 5, II.2 several TAL cells have apparent intracellular retention of MUC1 (yellow arrows) with increased intensity of BiP staining (red arrows; insets d and e).

(NTS-PC1) were elevated, which was confirmed on three different DNAJB11/ clones (Figure 3C), indicating that DNAJB11 plays a significant role in PC1 maturation. Analysis of the downstream effectors of the unfolded protein response (UPR) in WT and DNAJB11/ cells did not demonstrate constitutional or induced activation of the UPR (Figure S2). To determine the effect of DNAJB11 loss on PC1 localization by immunofluorescence, we co-transfected WT and DNAJB11/ cells with tagged PC1 and PC2 constructs (Figures 3D and 3E). In PC1-PC2 co-transfected cells, PC1 membrane expression in DNAJB11/ cells was less than half that seen in WT cells (results confirmed on three separate transfections), further indicating a role for DNAJB11 in PC1 trafficking.

10 The American Journal of Human Genetics 102, 1–13, May 3, 2018

Intracellular Retention of Uromodulin in TAL Epithelial Cells of DNAJB11-Affected Individuals The observation of late-onset renal insufficiency with renal atrophy, chronic interstitial fibrosis, and gout (family 5) suggested partial phenotypic overlap with ADTKD. Hypothesizing that DNAJB11 variants may affect folding and cellular trafficking of additional secretory proteins, such as uromodulin and MUC1, we examined the intracellular distribution of these two proteins in tubular epithelial cells on kidney tissue samples from two DNAJB11-affected subjects (family 1, II.1 and family 5, II.2). Strong intracellular staining for uromodulin was observed in both affected individuals’ thick ascending loop (TAL) epithelial cells, while it was strictly apical in the control kidney (Figure 4). In addition, intracellular retention of MUC1

Please cite this article in press as: Cornec-Le Gall et al., Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease, The American Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.03.013

was observed in some TAL cells of family 5, II.2, but not in the control subject or family 1, II.1 tissue (Figure 4).

Discussion We have identified a previously unrecognized form of cystic kidney disease due to monoallelic pathogenic variants to DNAJB11 by screening unresolved ADPKD- and ADPLD-affected families. This disease is characterized by the development of multiple small renal cysts and evolution to renal insufficiency, mostly after the sixth decade. DNAJB11-associated disease is phenotypically distinct from classical forms of ADPKD in several ways. Cyst size generally remains small, resulting in non-enlarged kidneys, but chronic interstitial fibrosis develops; hence, showing some characteristics of ADTKD. Liver cysts, but no liver fibrosis, was observed in some DNAJB11-affected individuals; their identification in subjects with clinical presentation and imaging consistent with ADTKD should therefore suggest the presence of a pathogenic variant in DNAJB11 rather than in other ADTKD-associated genes. Unlike ADPKD due to changes to PKD1 and PKD2, normal total kidney volume does not preclude the possibility of evolution to ESRD and should not be used as a prognostic marker in DNAJB11-affected individuals.27 Last, renal cysts can remain undetected in younger individuals, such as subject III.1 from family 1, hence ADPKD age-dependant diagnosis criteria based on MRI or ultrasound imaging cannot be used to exclude the diagnosis in young at-risk relatives.28,29 DNAJB11 accounts for
1.2% of the genetically unresolved ADPKD-affected families. However, some DNAJB11-affected individuals are probably not included in ADPKD cohorts, given the relatively advanced age at diagnosis, the absence of kidney enlargement and/or severe liver phenotype, and sometimes uncertain familial history, which may lead to the diagnosis of acquired cystic kidney disease.30 Determining the frequency in genetically unresolved ADTKD-affected cohorts will also be interesting. DNAJB11 plays a central role in the maintenance of the ER protein homeostasis (or proteostasis), a multi-step process including the glycosylation, folding, assembly, and trafficking of all secretory and membrane proteins, plus degradation of proteins failing the quality control processes.25 Protein folding is the most error-prone step in moving from gene expression to appropriately post-translationally modified and localized proteins, with disruption of ER proteostasis resulting in a wide range of human diseases.31,32 The association between proteostasis defects, causing loss of maturation and appropriate localization of PC1 and resulting in renal or liver cystogenesis, has been demonstrated or suggested in different forms of polycystic renal and/or liver diseases.6,7,33,34 Our data suggest that DNAJB11-related disease falls into this paradigm, with impaired maturation of PC1 causing cystogenesis. Interestingly, altered proteostasis caused by DNAJB11 pathogenic variants appears to also affect uromodulin, mimicking

ADTKD-UMOD cellular phenotypes, similar to that suggested for SEC61A1.34 DNAJB11 functions as a BiP co-chaperone, binding to misfolded proteins and delivering them to BiP for ATPdependent chaperoning. Evidence from murine models suggests that dysfunction of the chaperone BiP is associated with increased renal tubular dilatation and atrophy and interstitial fibrosis, similar to the phenotype detected here.35 Significant variants in two other genes encoding BiP cofactors, SIL1 and DNAJC3, have been described in autosomal-recessive multisystemic neurodegenerative disorders. Noteworthy, DNAJB11 is also secreted into the extracellular space, where it can bind misfolded proteins and attenuate proteotoxicity, notably in the brain.36 Idiopathic epilepsy was reported in two families. Although in our cellular studies we did not detect an activation of the UPR, such phenotypes could occur in DNAJB11-depleted disease organs. An oncogenic role of ER stress has been documented in a variety of studies,37 and activation of the UPR and increased expression of BiP were shown to be associated with shorter survival in glioblastoma.38 Of possible note, family 3, subject III.2 died from a glioblastoma; family 2, subject III.2 was diagnosed with breast cancer at 38 years; and family 1, subject II.1 developed a metanephric adenoma. However, proof of extra-cystic/ fibrotic manifestations of DNAJB11-related disease will need the characterization of more families. In aggregate, we propose that DNAJB11-related disease is an ER protein maturation disorder, leading to a hybrid phenotype that associates clinical features of ADPKD, resulting from PC1 maturation defects, and of ADTKD, resulting from the intra-cytoplasmic retention of tubular proteins such as uromodulin. The identification of this previously unrecognized disease expands our understanding of the etiology of renal fibrocystic disorders with implications for genetic counseling, prognostic assessment, and therapeutic management, while also extending the phenotypic range of chaperone-related diseases. Accession Numbers The accession numbers for the cDNA and protein sequence reported in this paper are GenBank: NM_016306.5 and NP_057390.1.

Supplemental Data Supplemental Data include two figures and three tables and can be found with this article online at https://doi.org/10.1016/j.ajhg. 2018.03.013.

Acknowledgments We thank the families and coordinators for involvement in the study, and Dr. Cornet (Verviers Hospital, Belgium) and Andrew Metzger and Marie Edwards (Mayo Clinic) for their assistance. The study was supported by NIDDK grant DK058816 (P.C.H.), the Mayo Translational PKD Center (DK090728; V.E.T.), an

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American Society of Nephrology (ASN) Foundation Kidney Research Fellowship (E.C.-L.G.), the Fulbright Association and the Foundation Monaham (E.C.-L.G.), a PKD Foundation fellowship (W.B.), an American Heart Association postdoctoral fellowship (B.P.), an ASN Ben J. Lipps Fellowship (K.H.), NIDDK Predoctoral Studentships (F31; R.J.O. and J.A.), Mayo Graduate School (R.J.O. and J.A.), the Zell Family Foundation, Robert M. and Billie Kelley Pirnie, and the Belgian Fonds National de la Recherche Scientifique (F.J.). The CRISP and HALT-PKD studies were supported by NIDDK cooperative agreements (DK056943, DK056956, DK056957, DK056961, DK062410, DK062408, DK062402, DK082230, DK062411, and DK062401), National Center for Research Resources General Clinical Research Centers, and National Center for Advancing Translational Sciences Clinical and Translational Science Awards. The Genkyst cohort was supported by National Plans for Clinical Research, Groupement Interregional de Recherche Clinique et d’Innovation (GIRCI Grand Ouest), and the French Society of Nephrology. The Yale Center for Mendelian Genomics (UM1 HG006504) is funded by the National Human Genome Research Institute. Funds were also provided by the National Heart, Lung, and Blood Institute under the Trans-Omics for Precision Medicine (TOPMed) program and by the Yale O’Brien Kidney Center (P30 DK079310). Acknowledgment to other HALT PKD, CRISP, and Genkyst investigators is given in the Supplemental Data.

3.

4.

5.

6.

7.

Declaration of Interests S.S. is a consultant for and a founder of Goldfinch Bio. Received: January 29, 2018 Accepted: March 8, 2018 Published: April 26, 2018

8.

Web Resources

9.

ADPKD Mutation Database, http://pkdb.mayo.edu Align GVGD, http://agvgd.iarc.fr ExAC Browser, http://exac.broadinstitute.org/ fathmm MKL, http://fathmm.biocompute.org.uk/fathmmMKL.htm GenBank, https://www.ncbi.nlm.nih.gov/genbank/ GeneReviews, Harris, P.C., and Torres, V.E. (2015). Polycystic kidney disease, autosomal dominant, https://www.ncbi.nlm. nih.gov/books/NBK1246/ gnomAD Browser, http://gnomad.broadinstitute.org/ MutationTaster, http://www.mutationtaster.org/ NCBI Nucleotide, https://www.ncbi.nlm.nih.gov/nuccore/ OMIM, http://www.omim.org/ PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/ Primer-BLAST, https://www.ncbi.nlm.nih.gov/tools/primer-blast/ RefSeq, https://www.ncbi.nlm.nih.gov/RefSeq SIFT, http://sift.bii.a-star.edu.sg/ UCSC Genome Browser, https://genome.ucsc.edu

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Please cite this article in press as: Cornec-Le Gall et al., Monoallelic Mutations to DNAJB11 Cause Atypical Autosomal-Dominant Polycystic Kidney Disease, The American Journal of Human Genetics (2018), https://doi.org/10.1016/j.ajhg.2018.03.013

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