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ALG9 Mutation Carriers Develop Kidney and Liver Cysts

Whitney Besse ,1 Alex R. Chang,2 Jonathan Z. Luo ,3 William J. Triffo,4 Bryn S. Moore,3 Ashima Gulati,1 Dustin N. Hartzel ,5 Shrikant Mane,6 Regeneron Genetics Center, Vicente E. Torres,7 Stefan Somlo,1,6 and Tooraj Mirshahi5

Departments of 1Internal Medicine (Nephrology) and 6Genetics, Yale University School of Medicine, New Haven, Connecticut; Departments of 2Nephrology, 3Molecular and Functional Genomics, 4Radiology, and 5Biomedical and Translational Informatics, Geisinger Clinic, Danville, Pennsylvania; and 7Division of Nephrology and Hypertension, Mayo Clinic, Rochester, Minnesota

ABSTRACT Background Mutations in PKD1 or PKD2 cause typical autosomal dominant polycystic kidney disease (ADPKD), the most common monogenic kidney disease. Dominantly inherited polycystic kidney and liver diseases on the ADPKD spectrum are also caused by mutations in at least six other genes required for protein biogenesis in the endoplasmic reticulum, the loss of which results in defective production of the PKD1 gene product, the membrane protein polycystin-1 (PC1). Methods We used whole-exome sequencing in a cohort of 122 patients with genetically unresolved clinical diagnosis of ADPKD or polycystic liver disease to identify a candidate gene, ALG9,andin vitro cell-based assays of PC1 protein maturation to functionally validate it. For further validation, we identified carriers of ALG9 loss-of-function mutations and noncarrier matched controls in a large exome-sequenced popula- tion-based cohort and evaluated the occurrence of polycystic phenotypes in both groups. Results Two patients in the clinically defined cohort had rare loss-of-function variants in ALG9,which encodes a protein required for addition of specific mannose molecules to the assembling N-glycan pre- cursors in the endoplasmic reticulum lumen. In vitro assays showed that inactivation of Alg9 results in impaired maturation and defective glycosylation of PC1. Seven of the eight (88%) cases selected from the population-based cohort based on ALG9 mutation carrier state who had abdominal imaging after age 50; seven (88%) had at least four kidney cysts, compared with none in matched controls without ALG9 mutations. Conclusions ALG9 is a novel disease gene in the genetically heterogeneous ADPKD spectrum. This study supports the utility of phenotype characterization in genetically-defined cohorts to validate novel disease genes, and provide much-needed genotype-phenotype correlations.

JASN 30: 2091–2102, 2019. doi: https://doi.org/10.1681/ASN.2019030298

Individuals with autosomal dominant polycystic Received March 26, 2019. Accepted June 26, 2019. kidney disease (ADPKD) develop fluid-filled cysts that originate from renal tubules and enlarge over Published online ahead of print. Publication date available at time, eventually leading to renal failure in at least www.jasn.org. half of affected individuals by the sixth decade. In Correspondence: Dr.StefanSomlo,SectionofNephrology, addition to kidney cysts, patients with ADPKD Yale University School of Medicine, P.O. Box 208029, 333 Cedar Street, New Haven, CT 06520-8029, or Dr. Tooraj Mirshahi, frequently develop liver cysts originating from Department of Molecular and Functional Genomics, Geisinger thebileducts.1 ADPKD most commonly results Clinic, 100 North Academy Avenue, Danville, PA 17822-2621. from mutations in PKD1 (77%) or PKD2 (15%), E-mail: [email protected] or [email protected] but approximately 8% of cases have no mutation Copyright © 2019 by the American Society of Nephrology

JASN 30: 2091–2102, 2019 ISSN : 1046-6673/3011-2091 2091 BASIC RESEARCH www.jasn.org detected (NMD) in either gene (ADPKD-NMD).2 Several Significance Statement genes which encode proteins in the endoplasmic reticulum (ER) that function in the biogenesis and quality control of Dominantly inherited polycystic kidney and liver phenotypes occur integral membrane proteins have been implicated as disease when epithelial cells in these organs have reduction of polycystin-1 genes for dominant polycystic kidney and liver phenotypes functional dosage. In a cohort of genetically unresolved polycystic liver and kidney disease, the authors identified heterozygous loss clinically characterized as isolated polycystic liver disease of function mutations in ALG9. ALG9 encodes an endoplasmic re- – (PCLD) or ADPKD-NMD.2 8 PCLD, also known as autoso- ticulum enzyme that builds N-glycans, and the authors show that mal dominant polycystic liver disease, presents with liver Alg9 inactivation results in impaired polycystin-1 maturation. Using findings that are histologically and radiographically indis- a novel ‘genotype-first’ approach to ascertain individuals based tinguishable from those in ADPKD, but with few or absent strictly on their ALG9 genotype from a large cohort of exome- sequenced individuals, the authors find that 7/8 (88%) of ALG9 9 kidney cysts. Although the ADPKD-PCLD diseases are in- mutation carriers over age 50 have multiple kidney cysts. Together, herited as dominant traits, accrual of somatic mutations these findings identify ALG9 as a novel human polycystic kidney resulting in biallelic loss of the respective disease genes in and liver disease gene and support the utility of a genotype- renal tubular and biliary epithelial cells is required for cyst driven approach to candidate disease gene validation and genotype- initiation and accounts for the focal nature of the resulting phenotype correlation. polycystic phenotypes.10–14 Although there are a large num- ber of client proteins that may be affected by loss of these PKHD1, and DNAJB11) have been excluded. Extraction of ge- ER-associated gene products, studies have shown that it is nomic DNA, WES, variant calling, and analysis for this cohort specifically the impaired post-translational maturation of have been described previously.7 New samples were captured by the PKD1 gene product polycystin-1 (PC1) that produces the xGen Exome Research Panel reagent from Integrated DNA the polycystic liver and kidney phenotypes.15 The factors Technologies and sequenced on the Illumina HiSeq4000 plat- that determine the relative occurrence of liver cysts and form. Variants were filtered to consider only rare heterozygous kidney cysts are not well understood but the ESKD that likely deleterious variants (stop gain, frameshift, canonical occurs in ADPKD due to PKD1 and PKD2 has not been splice, missense with Combined Annotation Dependent Deple- attributed to mutations in any of the disease genes in the tion [CADD]22 score .20) of high sequencing quality. Rare PCLD–ADPKD-NMD spectrum.2,6–8,15 variants were considered to be those with minor allele frequency In this study, we identified ALG9 as a novel candidate gene (MAF) of ,0.001 in the gnomAD Browser.23 False positive in our unsolved cases for the PCLD–ADPKD-NMD pheno- variant calls as defined by PLDiff/depth ,724 were excluded typic spectrum. ALG9 encodes a-1,2-mannosyltransferase, from consideration in all analyses. As no single gene was en- which catalyzes the addition of the seventh and ninth mannose riched with variants among cohort cases in statistical analyses at molecules to a growing N-glycan precursor in the ER lu- a genome-wide level, we ranked all genes represented by at least men.16,17 Autosomal recessive loss of ALG9 results in a severe one loss-of-function variant based on the number of individuals congenital disorder of glycosylation (CDG) with a multiorgan carrying potentially deleterious variants in the gene. ALG9 was phenotype that includes kidney cysts.16,18,19 We validated then selected from among top candidates by review of available ALG9 as a disease gene by in vitro functional bioassays showing literature and resources, including data on protein function and both quantitative and qualitative defects in PC1 maturation localization, and mouse or human phenotypes. ALG9 variants after inactivation of Alg9.AmongstALG9 loss-of-function investigated as part of this study are deposited in ClinVar mutation carriers identified in a large population-based co- (SCV000914195, SCV000914194, SCV000930618-SCV000930621). hort that had undergone whole exome sequencing (WES), we identified significant enrichment of kidney cysts. In Vitro Gene Knockout Theparentalcelllinenotedas“control” in this study is a mouse renal tubule epithelial cell line which contains a three- METHODS copy Pkd1F/H bacterial artificial chromosome (BAC) with amino-terminal (N-terminal) FLAGx3 and carboxy-terminal PCLD–ADPKD-NMD Cohort and Exome Analysis (C-terminal) HAx3 epitope tags.15 The cell line has stable This study is approved by the Institutional Review Board at expression of Cas9 introduced by lentiviral transduction using Yale University. The studied cohort consists of 122 unrelated lentiCas9-Blast (Addgene 52962). Guide-RNA sequence target- cases of PCLD or ADPKD-NMD enrolled at Yale or received ing the reverse strand of Alg9 exon 6 (59-TCCCGTCATGGCGAT- de-identified from collaborators, as well as selected mild– CAGCG-39) was introduced transiently using electroporation of a moderate severity ADPKD-NMD cases from the Consortium modified pGL3-U6-sgRNA-PGK-puromycin plasmid (Addgene of Radiologic Imaging Study of PKD20 and HALT21 clinical 51133) lacking the puromycin cassette to direct the Cas9 protein cohorts obtained from the National Institute of Diabetes and to make a double-stranded cut at this site. Transfected cells were Digestive and Kidney Diseases Central Repository. Cases clonally diluted after 72 hours and grown until sufficient quantity solved by pathogenic variants in established disease genes to passage. Cell line clones were screened by PCR and Sanger (PKD1, PKD2, PRKCSH, SEC6, GANAB, ALG8, SEC61B, sequencing for frameshift mutations (Supplemental Figure 1).

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Immunoblotting and Glycosylation Analysis and variant calling in this cohort have been previously de- Immunoblotting was performed on whole cell lysate prepared scribed. Briefly, NimbleGen (SeqCap VCRome) probes were using RIPA buffer as reported previously.7 For quantitative used for exome capture of the first approximately 61,000 comparisons, cell lysate concentration was measured by Lowry samples and a modified version of Integrated DNA Technolo- protein assay using standard reagents. For Ponceau staining, gies xGen probes was used for the remaining approximately membrane was washed in Tris-buffered saline/Tween 20 (TBST), 31,000 samples.30,31 Variants were annotated using the En- submerged in Ponceau S Solution (Sigma) for 10 minutes, rinsed sembl Variant Effect Predictor with Ensembl 90 definitions.32 briefly in water to remove background staining, and dried on PLdiff/depth and gnomAD were used as described above to tissue. Membrane was rehydrated in 100% methanol before filter out common and false positive variants. Carriers of proceeding with blocking in 5% nonfat milk in TBST. The ALG9 loss-of-function variants were identified from this co- following primary antibodies were used at 1:3000 dilution in hort and confirmed with Sanger sequencing. We accessed 5% milk in TBST unless otherwise noted: anti-hemagglutinin pedigree reconstruction data assembled from exome-based (anti-HA) (3F10; Roche), anti–PC1-LRR (7e12, dilution identity-by-descent analysis to determine the relatedness 1:1000; Santa Cruz Biotechnology), chicken anti-green fluo- between individuals to identify any first or second degree rescent protein (anti-GFP) antibody (GFP-1020; Aves Labs), relatives for the ALG9 carriers in the cohort.31 and rabbit anti-Hsp90 antibody (Cell Signaling). For glycosyl- ation studies, cell lysate obtained in RIPA buffer was treated MyCode Cohort Case versus Control Study with Endoglycosidase H (EndoH) per nondenaturing protocol The study of the MyCode cohort was approved by the Insti- (NEB) for 3 hours at 37°C. tutional Review Board at Geisinger. Patients carrying the ALG9 mutation who were eligible for matching were defined as those Missense Variant Analysis with abdominal imaging studies available for digital review In silico analysis of missense variants was performed using with a definablenativekidneyeGFR—i.e., not on dialysis prediction algorithms including CADD,22 MetaSVM and or with kidney transplant. One imaging exam was selected MetaLR,25 REVEL,26 and FATHMM-MKL.27 Biochemical val- for each case by a predefined algorithm: magnetic resonance idation studies were conducted on stable cell lines generated by imaging (MRI) . computed tomography (CT) . ultrasound, lentiviral transduction of C-terminal GFP fusion with human and exams with contrast . those without. Clinical informa- ALG9 cDNA (ENST00000616540.4; 618 amino acid transcript) tion for carriers of ALG9 mutations was obtained from clinical 2 2 into the Alg9 / cell line. Human ALG9 cDNA sequence was databases and used to select random matched controls in a 2:1 amplified from total RNA of human embryonic kidney 293 T ratio (see power calculation below) based on six parameters cells using a two-step PCR strategy. The 59 untranslated region (see Results). A radiologist with expertise in abdominal imag- to the middle of the transcript was amplified separately from ing (W.J.T.), blinded to patient genotypes and clinical infor- overlapping region spanning from the middle of the transcript mation, provided interpretation of kidney and liver findings to the 39 untranslated region, and these products were then in the selected imaging studies using clinical radiologic diag- used as a template for a second PCR reaction, using primers nostic criteria. Peripelvic cysts, although infrequent (data not designed to add restriction sites to each end. This product was shown), were excluded from the analysis given their lymphatic cloned into Zero Blunt TOPO vector using a cloning kit from origin and inability to reliably delineate discrete cysts from the ThermoFisher, and the correct clone was confirmed by Sanger collecting system. For CT scans, a cyst was defined as a homo- sequencing. This ALG9 sequence in the TOPO vector was mod- geneous mass of water attenuation (210 to 20 HU) with an ified using a site-directed mutagenesis protocol.28 Briefly, Pfu imperceptible wall and no enhancement after administration of polymerase (Promega) amplified a modified plasmid primed contrast or, in the case of hemorrhagic or proteinaceous cysts, by complimentary forward and reverse primers containing the as a homogeneous, nonenhancing lesion with precontrast density sequence edit (Supplemental Methods). The resultant product .20 HU. Size thresholds were set based on the heterogeneity of was transformed into DH5a competent cells after digestion of the CT data, discussed below (see Results). For MRI, a cyst was parental template plasmid with DpnI (NEB). A single modified defined as a lesion distinct from the surrounding parenchyma clone for each missense variant was confirmed by sequencing, with either homogenous internal T2-weighted signal intensity cut by restriction digest, ligated into pLVX-IRES-Puro (Clontech), consistent with simple fluid, or a lesion with T1 and T2 signal and again each was confirmed with Sanger sequencing. GFP characteristics consistent with a hemorrhagic or proteinaceous expression and localization for each cell line was examined by cyst. In cases where postcontrast imaging was available, the le- immunoblotting and fluorescence microscopy, respectively. sions showed no enhancement. For ultrasound imaging, a cyst was defined as an anechoic structure with a distinct posterior MyCode Cohort Exome Analysis wall and posterior enhancement. Whole-exome sequencing and variant calling were performed before this study on 92,455 MyCode Community Health Statistical Analyses Initiative participants29 as part of the Geisinger-Regeneron A two-tailed test was used to compare means of biologic DiscovEHR collaboration. Methods for whole-exome capture repeats on Western blots. Sample size calculations for case

JASN 30: 2091–2102, 2019 Alg9 Mutations Cause Kidney Cysts 2093 BASIC RESEARCH www.jasn.org versus control analysis were performed using Power Analysis used CRISPR/Cas9 to introduce a frameshift in exon 6 of Sample Size (PASS) with the goal of achieving .90% power in Alg9 to inactivate it in a mouse kidney tubular epithelial cell detecting a difference if cases and controls had an 80% and very line. The isogenic unmodified parental cell expressing Cas9 conservatively selected 20% incidence of kidney cysts, respec- was used as control. The parental cell line expresses three copies tively. Case versus control comparisons were performed using of a BAC transgene, Pkd1F/H-BAC, containing the full genomic the Fisher exact test. sequence of mouse Pkd1 modified with addition of N-terminal FLAGx3 and C-terminal HAx3 epitope tags.7,15 Sanger sequenc- ing of the genomic region of Alg9 targeted by the guide RNA in 2 2 RESULTS a clonally selected candidate Alg9 / cell line confirmed the presence of a homozygous frameshifting mutation (Supplemental 2 2 ALG9 Is a Candidate Gene for PCLD–ADPKD-NMD Figure 1). Alg9 / cells had no apparent morphologic changes We performed WES on 122 unrelated patients with ADPKD- on light microscopy compared with the parental cell line. 2 2 NMD or PCLD without pathogenic mutations in previously Alg9 / cells showed a significant decrease in the steady-state established nor proposed disease genes. We identified one case level of PC1-CTF to approximately 70% of the control intensity, with a heterozygous loss-of-function mutation in exon 6 of quantified in four biologic samples per genotype by anti-HA 2 ALG9, p.W227X, and five other cases with rare (MAF,1310 3) (Figure 1A, left panel; Supplemental Figure 2). A total of 57 heterozygous missense variants of unknown consequence in of the 60 predicted N-glycosylation sites on PC1 are in the ALG9 (Table 1). Each of the five missense variants was predic- N-terminal fragment. After exit from the ER, enzymes in the ted to be in the top 0.3% most likely deleterious of all potential Golgi modify the N-glycans conferring resistance to EndoH genome single nucleotide variants (CADD-phred $25) but had which is associated with slower migration on protein gel elec- differing predictions with other in silico algorithms (Table 1).33 trophoresis. This allows experimental distinction of the imma- In vitro inactivation of the previously reported PCLD–ADPKD- ture EndoH-sensitive PC1 N-terminal fragment (PC1-NTS) NMD disease genes that encode ER proteins have shown signif- that has not traversed the Golgi from the EndoH-resistant frag- icant quantitative reduction and qualitative alteration of mature ment (PC1-NTR) that reaches the cell surface. Immunoblot PC1 protein.6–8,15 In vitro biallelic inactivation models the cel- analysis with antibody directed to the PC1 N-terminal resolves lular recessive genotype resulting from somatic second hits in three fragments: PC1-FL, PC1-NTR, and PC1-NTS (Figure 1A, 2 2 cyst-forming epithelium. We therefore investigated the effects right panel). Alg9 / cells show quantitatively decreased PC1- of inactivation of ALG9 on PC1 in a cell culture system. The NTR, indicating reduced mature, cell surface–expressed PC1. full-length PC1 protein (PC1-FL) is normally cleaved into an In addition to quantitative differences, PC1-FL and PC1-NTS 2 2 N-terminal fragment and an 11-transmembrane-spanning from Alg9 / cells have faster migration in denaturing gel elec- C-terminal fragment (PC1-CTF) before leaving the ER and trophoresis compared with control cells (Figure 1A, Supple- trafficking to functional destinations on the cell surface. We mental Figure 2). This may also be true of PC1-NTR, but the

Table 1. ALG9 heterozygous mutation carriers in 122 genetically unresolved patients with PCLD–ADPKD-NMD Amino Nucleotide MAF FATHMM- Age/ Kidney Liver Identifier Acid CADDb MetaSVMb MetaLRb REVELc Changea gnomAD MKLb Gender Cystsd Cysts Change Cases with pathogenic ALG9 mutations G8261813e c.681G.A p.W227X Novel N/A N/A N/A N/A D 60/M Innumerable N/A 2 YU202f c.1109G.A p.R370K 4310 6 33 D D 0.912 D 56/M 7:11 Innumerable Cases not explained by ALG9: benign missense variants only 2 YU22g c.694G.C p.A232P 3310 4 25 D T 0.395 D 67/F N/A Multiple 2 YU36h c.839C.T p.A280V 7310 6 31 T T 0.324 D 66/F — Multiple 2 453976i c.944A.G p.N315S 6310 5 25 T T 0.574 D 42/F Multiple Innumerable 2 YU394 c.1550G.T p.R517L 4310 6 27 T T 0.462 D 63/F 1:0 Multiple Yale case cohort comprised of PCLD and mild-moderate ADPKD-NMD cases from Consortium of Radiologic Imaging Study of PKD (CRISP)/HALT Progression of Polycystic Kidney Disease (HALT PKD) cohorts. N/A, not applicable; D, deleterious; T, tolerated. aENST00000616540.4 (CCDS73380). bVariant prediction (see Methods). cREVEL scores range from zero to one, with one representing all simulations suggest pathogenicity. dCyst counts (right:left) or description from available imaging. eHALT identifier. fMayo Clinic Family T57. gMayo Clinic Family T39. hMayo Clinic Family T17. iCRISP identifier.

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-/- -/- -/- -/- A Alg9 Alg9 B -/- -/- C Alg9 Alg9

ControlAlg9 ControlAlg9 ––++EndoH Control- ALG9 Control- ALG9 ALG9p.R370KControl- p.Y287CALG9 PC1-FL

PC1-FL = } PC1-FL PC1-NTR 250 = } PC1-FL * PC1-NTS *PC1-NTS 75 Hsp90 250 250

150 PC1-CTF 150 PC1-CTF

100 75 Hsp90 100 Hsp90 75

Alg9-/-

D -/- E Alg9 Controlp.A280Vp.A232Pp.Y287Cp.N315Sp.R370Kp.R517L

= } PC1-FL higher exposure

= } PC1-FL

250

150 PC1-CTF

100 Hsp90 75

75 GFP

Figure 1. Alg9 loss causes abnormal biogenesis of PC1. (A) Immunoblots of cell lysate with anti-HA (left panel) and anti-LRR PC1–N-terminal antibody (7e12) (right panel)showquantitativedecreaseinPC1–C-terminal fragment (PC1-CTF) and the mature EndoH-resistant fraction of PC1–N-terminal fragment (PC1-NTR), as well as faster migration of PC1 full-length (PC1-FL) and the 2 2 immature EndoH-sensitive PC1–N-terminal fragment (PC1-NTS) in Alg9 / cells compared with controls. Re-expression of human 2 2 ALG9-GFP in Alg9 / cells rescues both the quantitative and migration differences (third lane). (B) Cell lysate was treated with EndoH 2 2 and blotted with anti-HA. The migration difference of PC1-FL between control and Alg9 / cell lysates is eliminated after EndoH treatment, showing that there is altered glycosylation of PC1 in the absence of Alg9. (C and D) Anti-HA immunoblots of cell lysate from 2 2 Alg9 / cells with or without stable re-expression of human ALG9-GFP with either wild-type (ALG9) or the indicated missense variants. The known pathogenic missense mutation, p.Y287C, and the experimental missense variant, p.R370K, do not rescue the PC1 2 2 phenotype in Alg9 / cells. (D) The other experimental missense variants rescue the PC1 phenotypes. This bioassay identifies p.R370K as a deleterious missense mutation and p.A232P, p.A280V, pN315S, and p.R517L as benign variants. (E) Imaging for YU202 with ALG9-p.R370K. Serial CT scan sections show multiple small kidney cysts (arrows) many of which are likely hemorrhagic/ proteinaceous (left panel) and innumerable liver cysts (right panel).

2 2 overlapping migration of PC1-FL and PC1-NTR from Alg9 / migration between genotypes (Figure 1B), confirming 2 2 cells prevents definitive conclusion. The genotype-dependent that the faster migration of PC1-FL from Alg9 / cells is differences in migration of both cleaved and uncleaved PC1 due to altered N-glycosylation. Finally, both the altered pro- suggest that, in the absence of Alg9, there is altered glyco- tein expression levels and altered glycosylation of PC1 in 2 2 sylation of PC1. Enzymatic removal of N-glycans, which on Alg9 / cells showed expression indistinguishable PC1-FL are all EndoH sensitive, results in identical from control cells when human ALG9-GFP is re-expressed

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2 2 in Alg9 / cells (Figure 1A). This confirms that the effects compound heterozygous ALG9 loss-of-function mutations. on PC1 maturation are a specific consequence of loss of Alg9 Principle component analysis and relatedness analysis of and provides strong support for the conclusion that ALG9 exome data indicated that all 21 of these patients were of is a human polycystic disease gene. European descent and no close familial relations were present between them. Of the 21 patients, 14 (67%) had at least one Assessment of ALG9 Missense Variants Found in abdominal imaging report available for evaluation. The ALG9 Patients with PCLD–ADPKD-NMD variants (Table 2) in these 14 individuals were confirmed by We next used this system to evaluate the functional conse- Sanger sequencing. None carried loss-of-function mutations quences of the ALG9 missense variants identified in our pa- in established cystic disease genes, and missense variants in tients (Table 1). We expected that re-expression of benign these genes were benign by in silico prediction (Supplemental human ALG9 missense variants would rescue PC1 properties Table 1). Clinical radiology reports described kidney cysts 2 2 in Alg9 / cells, whereas deleterious missense variants would (n=7) or hypodensities suggestive of cysts (n=4) in these 14 fail to do so. We modified the human wild-type ALG9-GFP patients, and liver cysts in only one. To characterize and quan- cDNA sequence to introduce each of the five missense muta- tify these findings and determine the specificity of these tions (Table 1). In addition, we produced a clone with a known phenotypes to the ALG9 genotype in light of the sporadic oc- deleterious missense variant ALG9-p.Y287C16 as a positive currence of kidney and liver cysts in some individuals with 2 2 control. Each cDNA was introduced into the Alg9 / cell increasing age, we designed a case versus control analysis in line by lentiviral transduction and stably expressing cell lines which a radiologist blinded to case-control status evaluated and were selected. Expression of each clone was verified by GFP quantitated cyst burden. 2 2 epifluorescence in cell culture (data not shown). Alg9 / cells expressing the positive control ALG9-p.Y287C, as well as the Case versus Matched Control Analysis line expressing the patient-derived variant ALG9-p.R370K, We included 11 of the 14 ALG9 cases for the case-control study. showed quantitatively reduced PC1-CTF and slower migrat- The excluded patients include two for which only a scanned 2 2 ing PC1-FL indistinguishable from that seen in the Alg9 / radiology report but no digital imaging was available for re- 2 2 (Figure 1C). The Alg9 / cells expressing ALG9-p.A232P, evaluation (MC6, MC13; Table 2). The third patient had ALG9-p.A280V, ALG9-p.N315S, or ALG9-p.R517L each reached ESKD before first available imaging (MC11; Table 2). showed complete rescue of PC1 quantitative and qualitative We selected two random controls for each of the 11 remaining maturation defects (Figure 1D). The data indicate that, cases from exome-sequenced individuals lacking any coding 2 among the rare missense mutations found in the patients, region variants in PKD1 or PKD2 (MAF,2310 3) or other 2 only ALG9-p.R370K resulted in ALG9 loss of function (Table 1). established PCLD gene (MAF,1310 2); they were matched for The demonstration of significant impairment of PC1 matura- six parameters: age at time of imaging, type of imaging study tion resulting from ALG9 exon 6 truncation or ALG9-p.R370K, including use of contrast, year of imaging, CKD stage at time of and the occurrence of these variants in patients with the imaging, gender, and ethnicity. We defined four or more cysts ADPKD-PCLD phenotype support these as the causative vari- in patients aged 50 years or more as a threshold for positive ants for these two patients in our cohort (Table 1). YU202, with evidence of cystic disease. This criterion was informed by Rule pathogenic missense variant ALG9-p.R370K, is a 56-year-old et al.34 who report one, two, or three cysts of $5mmseenin male with clinically diagnosed PCLD (Figure 1E). His father 26%, 9.8%, and 4.3%, respectively, in healthy organ donors also had PCLD, but no genetic diagnosis. G8261813, with aged 50 or more. Four or more cysts only occurred 1.2% of ALG9-p.W227X, was diagnosed with ADPKD-NMD as an en- those healthy individuals.34 We assessed kidney and liver lesions rolled participant in the HALT PKD study (imaging not avail- in a blinded fashion using prespecified radiographic criteria able). These clinical data indicate that ALG9-related disease can (see Methods) for all 33 individuals (11 cases, 22 matched span the spectrum from PCLD to ADPKD. controls; Supplemental Table 2; Table 2). Although matched between cases and controls, the 33 imaging studies included Genotype-First Approach To Characterize ALG9 different imaging modalities with variable technical parame- Mutation Carriers ters inherent in a cohort spanning multiple decades. In ag- To evaluate these findings further, we determined the burden gregate, the CT exams, which were the majority of the data, of polycystic kidney and liver phenotypes in cases selected dictated that a size threshold of $8 mm allowed for a definitive based solely on the presence of ALG9 loss-of-function muta- application of the specified criteria for a cyst, and a minimum tions from a large population-based cohort in which WES size threshold of $4 mm allowed for lesions to be detectable data are linked to electronic health record data.30 Among the across all exams. As such, lesions $8 mm were classified as 92,455 exomes queried, we identified 21 carriers of rare either “cyst” or “indeterminate mass,” and lesions ,8mmbut 2 (MAF,1310 3) heterozygous ALG9 loss-of-function mu- $4 mm were reported as “too small to characterize” (TSTC) per tations (nonsense, frameshift, canonical splice, or start loss) common nomenclature. To facilitate direct comparison to the meeting sequence quality control thresholds (see Methods). CT data, the same TSTC thresholds were applied to the MRI There were no instances of individuals with homozygous or data. In contrast to CTand MRI, the concept of TSTC does not

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Table 2. Alg9 loss-of-function mutations carriers with abdominal imaging among 92,455 patients in a health system Nucleotide Identifier/Gender Amino Acid Change MAF gnomAD Imaging Type (age)b Kidney Cystsc TSTCc Nephrolithiasisd eGFR (age)e Changea Cases in case-control analysis Kidney cysts MC2/F c.427C.T p.R143X Novel MRI (65) 10 18 Y 60 (65) MC3/M c.1295C.A p.S432X Novel US (73) Innumerable — + 26 (75) MC5/F c.1018+1G.A Splice variant Novel CT+ (86) 39f 51 Yg 38 (96) MC7/M c.1472delA p.N491IfsTer33 Novel CT+ (69) 6 (9) 22 Y 74 (73) MC9/F c.506dupT p.S170EfsTer24 Novel US (66) 5 — + 90 (66) MC10/M c.883_885delCCT_insGTAAA p.P295VfsTer13 Novel CT+ (68) 9 21 Y 52 (75) MC12/F c.511C.T p.R171X 8.13E206 CT+ (61) 4 (9) 20 — 46 (63) One or fewer cysts MC1/M c.1018+1G.A Splice variant Novel CT+ (42) — 1 — 50 (51) MC4/F c.1088_1091delTCCA p.I363SfsTer52 4.06E206 CT+ (82) —— — 47 (85) MC8/F c.3G.A Start loss Novel CT+ (37) — 1 — 72 (44) MC14/F c.3G.A Start loss Novel MRI+ (37) 1 3 + 90 (44) Cases not matched for case-control analysis MC6/M c.1018+1G.A Splice variant Novel CT+ (76) Yh — 47 (78) MC11/F c.1363C.T p.R455X 4.06E206 CT (70) Yh,i — ESKD (66) MC13/F c.566-1G.A Splice variant 1.22E205 CT+ (49) Yh — 97 (50) US, ultrasound. aENST00000616540.4 (CCDS73380). b Alg9 +, with contrast. cKidney cysts (.8 mm) and lesions TSTC (4–8 mm) as described in Methods and Results. When additional imaging allowed for recharacterization of indeterminate masses as cysts, cyst count inclusive of these is uain as inyCysts Kidney Cause Mutations noted in parentheses after count from originally selected image. d Noted during blinded analysis. Y, yes; +, nephrolithiasis noted on additional CT scan if available. www.jasn.org eMost recent outpatient eGFR. fAmong cases with liver imaging, this was the only case with liver cysts noted. gPathology of bilateral nephrolithiasis demonstrated uric acid composition. hBased on clinical radiology report. iNative kidneys by CT 2/4 yr after transplant/ESKD. AI RESEARCH BASIC 2097 BASIC RESEARCH www.jasn.org

A B C Right Left

MC3 MC5 MC10 c.1295C>A; p.R432X c.1018+1G>A; splice variant c.883_885delCCT_insGTAA; p.P295VfsTer13

Figure 2. Cystic kidney phenotypes in carriers of ALG9 heterozygous loss-of-function variants are of varying severity. (A) Ultrasound long-axis views from anteroposterior sweep, and (B and C) axial CT scan sections. Quantification of lesions in all cases is presented in Table 2. apply to the ultrasound data, as such findings would not be The severity of the cystic phenotype in cases is highly vari- reliably detected by that modality. able between individuals. Cyst numbers ranged from zero to Among the 11 ALG9 mutation carriers (cases) analyzed in the innumerable kidney cysts (Figure 2, Table 2). In contrast, no case-control analysis, seven (64%) had four or more kidney cysts controls had more than two cysts. Cysts were bilateral in all (Figure 2, Table 2). None of the 22 controls met this threshold seven affected cases, and were mentioned in the clinical (P,0.001; Tables 2 and 3; see Methods). The four ALG9 cases with less than four kidney cysts included the three youngest Table 3. Case versus matched control analysis patients, aged 37, 37, and 42, each of whom also had at least P one TSTC in addition to the observed cysts. The fourth case was Cyst count Cases Controls Value an 82-year-old female with no cysts. Considering only the cases Case-control cohort n=11 n=22 $ , and controls $50 years of age, seven of eight cases (88%) versus Cyst count 4700.001 none of 16 controls had four or more kidney cysts (P,0.001; Cysts plus TSTC combined 8 4 0.0055 count $4 Table 3). The background occurrence of a single cyst or TSTC Bilateral cystsa 7 2 0.0021 was relatively common in controls (ten of 22; 45%), but signif- Subset of age over 50 n=8 n=16 icantly less than in cases (ten of 11; 91%; P=0.02). The case Cyst count $470,0.001 versus control cyst count comparison for the selected threshold Cysts plus TSTC combined 7 4 0.0087 of four or more kidney cysts remained robustly significant count $4 2 (P,0.01) even when criteria were modified to include TSTCs Bilateral cystsa 7 2 7.5310 4 or any number of bilateral cysts (Table 3). aBilateral cysts: at least one cyst in each kidney.

2098 JASN JASN 30: 2091–2102, 2019 www.jasn.org BASIC RESEARCH radiology reports for all seven cases. Five of the six cases with DISCUSSION a finite number of cysts had 18 or more lesions of 4–8mm diameter, suspected to be early cysts but noted as TSTCs Our study is the first to complement gene discovery in clinically (Figure 2, Table 2). The largest kidney cysts in cases ranged ascertained polycystic kidney and liver disease with phenotypic from 1.2 to 4.8 cm. For two cases with four and six cysts characterization in genetically defined at-risk patients from respectively, specific additional kidney lesions counted as population-based exome sequencing efforts. We show the sig- indeterminate masses could be categorically defined to be nificant benefits of combining these complementary strategies. cysts upon review of additional imaging studies (Table 2). Recent implication and analyses of disease genes for ADPKD The liver was not imaged in the two ALG9 carriers who only and PCLD demonstrate that these clinically defined human had renal ultrasound available; only one of the nine cases phenotypes are found in individuals with heterozygous muta- with liver imaging (MC5) had liver cysts. MC5’sonlypar- tions in genes required for sufficient PC1 functional dosage. As tially imaged liver contained two cysts, up to 1.1 cm in size, such, the implication and study of novel disease genes for this and four TSTCs. genetically heterogeneous phenotype offers concrete entry Therewas limitedavailabilityof familial data in thisstudy.At points for novel biologic investigations into PC1 function, an thetimeofYU202’s enrollment, he reported that his father had entity of central importance to the design of targeted therapies multiple liver cysts at age 83, and mother was living and well. for ADPKD and PCLD–ADPKD-NMD. These phenotypes ex- For the cases in the MyCode cohort, we used the available ist both mechanistically and phenotypically on more of a con- pedigree reconstruction from the 92,455 individuals to sup- tinuum than was realized before these studies.6–8,15 This study plement the absence of relevant family history data in clinical adds the novel benefit of being able to detail findings in muta- charts. Only MC1 and MC3 have relatives in the cohort, a tion carriers of a disease gene independent from the influence mother and a brother, respectively, and each are noncarriers of the clinical polycystic cohort from which it was discovered. of the respective ALG9 mutations. MC3 (Figure 2A, Table 2) is The population-based ascertainment of ALG9 mutation the most significantly affected case in this cohort, and notably carriers in the MyCode cohort provides several insights. Firstly, the brother of this individual did not share the ALG9 mutation because human variation assures that no additional gene var- and did not have kidney cysts on a CTscan performed at age 70. iants would be over-represented in the carriers of ten novel or The blinded review incidentally noted nephrolithiasis in ultra-rare ALG9 variants, it provides the strength of a random- four ALG9 mutation carrier cases (two CT scan, one ultra- ized study investigating the effect of the ALG9 variant alone, not sound, and one MRI) on the single imaging study used in requiring a co-occurring variant. We found that the penetrance the study. This was only noted in one control (ultrasound of four or more definitive kidney cysts in individuals over age exam). As MRI and ultrasound are inferior for identifying 50 with ALG9 mutations is 88% (seven of eight). The size and nephrolithiasis, and nephrolithiasis can be transient on imag- number of cysts found in ALG9 carriers is generally milder than ing, we performed additional chart review to consider other typical ADPKD, showing large variation in expressivity. ALG9 available CT scans for the cases and controls. CT scan clinical carriers show a kidney-predominant phenotype, differing from reports confirmed the nephrolithiasis found in the four cases, hypothesis based on four of the six PCLD–ADPKD-NMD and reported nephrolithiasis in an additional three cases for a disease genes encoding proteins in the ER which have been total of seven of 11 (64%) cases with digital imaging (Table 2). described in patients exclusively with PCLD. This raises the Six of these seven were among the cases with multiple kidney possibility that similar genotype-first analyses for other cysts and the seventh had one kidney cyst and three TSTCs. PCLD genes related to ER biogenesis may establish whether The most recent CT scan clinical reports for the controls did the stronger correlation with PCLD is a reflection of the disease not describe any additional cases of nephrolithiasis, however, mechanism or related to the criteria for ascertainment used in we did not review every available scan for each control as this previous discovery cohorts.3–5,7 The generally mild phenotype study was not designed to make a case versus control compar- in human ALG9 disease is likely determined by two factors: the ison for this finding. Of the remaining ten ALG9 mutation relative frequency of somatic second hit mutations inactivating carriers who lacked digital imaging data, there was no notation the normal copy of ALG9, and the level of damage to the PC1 of nephrolithiasis by International Classification of Diseases functional dose that results from loss of ALG9.7 code, an insensitive measure, nor on the imaging report for ALG9 joins two other genes, ALG8 and PMM2, implicated MC6, MC11, or M13. in kidney and liver cyst phenotypes whose recessive loss results In aggregate, this genotype-first analysis shows a signifi- in rare CDG.7,35 Kidney cystic disease attributed to PMM2 cantly greater incidence of multiple kidney cysts in ALG9 loss- mutations is described as part of a hyperinsulinemic hypogly- of-function carriers compared with individuals without this cemia and polycystic kidney disease syndrome attributed to genotype. Given the expanding availability of exome- recessively inherited biallelic nontruncating mutations, at least sequenced cohorts tied to the live electronic health record, one of which is a unique promotor variant proposed to result in this genotype-first approach will be a valuable adjunct in val- decreased expression of PMM2 specifically in the pancreas, idating the pathogenicity of candidate genes for rare Mendelian kidney, and liver, avoiding the typical neurologic symptoms disease traits. of PMM2-CDG.35 Due to a germline recessive genotype, the

JASN 30: 2091–2102, 2019 Alg9 Mutations Cause Kidney Cysts 2099 BASIC RESEARCH www.jasn.org kidney cystic phenotype in hyperinsulinemic hypoglycemia in patients with ADPKD by CTscan imaging.37 Nephrolithiasis and polycystic kidney disease does not require accrual of prevalence in the United States is reported at approximately somatic second hit mutations, is diagnosed at a younger 9% when self-reported by patients,38,39 and similarly when age, and affects the organ diffusely. Recessive loss of the 1220 veterans were screened with ultrasound,40 but compara- a-1,2-mannosyltransferase encoded by ALG9 causes the ble CTscan studies have not been done. The available data thus rare ALG9-CDG. Unique from other CDGs, kidney cysts suggest that nephrolithiasis in ALG9 carriers is of similar are present in all five of five cases in which kidney examination prevalence to that in typical ADPKD, which is considered was included among the 12 reported cases of ALG9-CDG. The enriched, but that comparisons cannot be drawn to the general pathogenic ALG9 missense variant p.R370K described in this population. report, as well as the variants that cause ALG9-CDG (p.Y287C, Our novel use of the genotype-first approach to disease gene p.N527K, p.E530K; ENST00000616540.4; CCDS73380), are all validation and phenotype characterization was able to uncover predicted to be on the ER luminal side of the ALG9 ER trans- a kidney-predominant cystic phenotype which would not have membrane protein.16,19,36 ALG9 functions to add specific man- been possible from our phenotypically defined cohort of nose molecules to the assembling N-glycan precursors in the predominantly PCLD. It also allows for quantification ER lumen.17 The presence of kidney cysts in ALG9-CDG at of additional imaging findings such as nephrolithiasis. birth, in contrast to the age-dependent and incomplete pene- This approach required the initial implication of ALG9 in trance in patients heterozygous for ALG9, is supportive of the phenotype-based cohorts. Although our data describe polycys- hypothesis that a cellular recessive mechanism established for tic kidneys and suggest that a liver-specific (PCLD) phenotype ADPKD and PCLD is required to initiate cysts due to ALG9 may occur in fewer than one in 14 ALG9 mutation carriers, one mutations. As the proteins encoded by ALG9, ALG8,and of our two clinically ascertained patients had PCLD, thus some PMM2 are enzymes with nonredundant roles in the assem- patients may manifest this way. As is the case for GANAB and bly of N-glycans, our findings suggest a requirement of DNAJB11, and potentially other genes on the PCLD–ADPKD- N-glycosylation in renal tubular and biliary epithelial NMD spectrum, ALG9 mutations will likely explain only a homeostasis. very small fraction of cases with clinically diagnosed ALG9 is now the third ER protein–encoding polycystic dis- ADPKD. Nonetheless, ALG9 may be considered when mu- ease gene, after GANAB and DNAJB11, described to explain tations in PKD1 or PKD2 are not found in mild–moderate either kidney-predominant polycystic phenotypes or casesofADPKD-NMD,aswellasforPCLDandphenotypes PCLD.6,8 We also reported a young female with a mutation spanning these clinical distinctions. We suggest the genotype- in ALG8 who had eight bilateral kidney cysts without liver based ascertainment approach be applied to other diseases with cysts.7 DNAJB11 is the only of these genes with reported cases extensive genetic heterogeneity to help validate candidate genes of ESKD, but this was proposed to result from tubulointersti- and better inform genotype-phenotype correlations and tial fibrosis rather than polycystic kidney disease.8 In this prognostic advice in the era of precision medicine. study, one ALG9 mutation carrier reached ESKD at age 66 with atrophic native kidneys containing bilateral renal cysts on imaging 4 years after onset of ESKD. Lack of records from ACKNOWLEDGMENTS earlier time points precludes drawing any conclusions in this case. In the two ALG9 cases with the largest number of kidney cysts (MC3, MC5), the eGFRs were 26 and 38, and the kidney We thank Dr. F. Perry Wilson and Dr. Brandon K. Fornwalt for advice size was mildly enlarged or normal at 14/15 and 10/12 cm, on study design. respectively. The patient we report from the HALT ADPKD Dr. Besse performed experiments and cowrote the manuscript. cohort, for whom imaging and organ size measurements were Dr. Chang, Mr. Luo, Dr. Triffo, Ms. Moore, Dr. Gulati, and Mr. Hartzel not available, had an eGFR of approximately 30 ml/min at analyzed data. Dr. Mane provided exome sequencing. Regeneron age 60. It remains possible that ALG9 falls in the group of Genetics Center (RGC) provided access to exome sequencing data. ER-associated polycystic kidney disease genes that may on Dr. Torres contributed patient samples and data. Dr. Besse, Dr. Chang, rare occasions be associated with CKD, although additional Mr. Luo, Dr. Triffo, Dr. Somlo, and Dr. Mirshahi designed the study. study will be required to validate this clinical association. Dr. Somlo and Dr. Mirshahi cowrote the manuscript. The finding of nephrolithiasis in the clinical imaging reports The list of contributors from RGC and Yale Center for Mendelian of seven of 11 ALG9 carrier cases is of interest but should be Genomics (CMG) is provided in the Supplemental Acknowledgments. interpreted with caution. Clinical indication for CT scanning was not noted in our analyses, therefore indication bias en- DISCLOSURES riching nephrolithiasis in the patients who had imaging is possible. Nonetheless, considering the unbiased denominator Dr. Torres reports grants and honoraria from Otsuka Pharmaceuticals, of 21 ALG9 mutation carriers, we can report a lifetime prev- grantsfrom Palladio Biosciences, Mironid, SanofiGenzyme, AcceleronPharma alence of at least 33% for nephrolithiasis in ALG9 loss-of- Inc., Regulus Therapeutics, and Blueprint Medicines, all outside the submitted function mutation carriers. This is comparable to the 28% found work. Dr. Somlo reports personal fees from Goldfinch Bio, outside the

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submitted work. Dr. Mirshahi reports grants from National Institutes of Polycystic Kidney Disease: Monoallelic mutations to DNAJB11 cause Health (NIH) National Institute of General Medical Sciences and grants atypical autosomal-dominant polycystic kidney disease. Am J Hum from NIH National Human Genome Research Institute, during the conduct of Genet 102: 832–844, 2018 the study. 9. Qian Q, Li A, King BF, Kamath PS, Lager DJ, Huston J 3rd, et al.: Clinical profile of autosomal dominant polycystic liver disease. Hepatology 37: 164–171, 2003 FUNDING 10. Qian F, Watnick TJ, Onuchic LF, Germino GG: The molecular basis of focal cyst formation in human autosomal dominant polycystic kidney diseasetypeI.Cell 87: 979–987, 1996 This work was supported by PKD Foundation Research Grant and Fellow- 11. Wu G, D’Agati V, Cai Y, Markowitz G, Park JH, Reynolds DM, et al.: ships to Dr. Besse (217G18a and 190F15a) and Dr. Gulati (207F17a); NIH Somatic inactivation of Pkd2 results in polycystic kidney disease. grants DK100592 and DK051041 to Dr. Somlo, DK090728 to Dr. Torres, Cell 93: 177–188, 1998 GM111913 to Dr. Mirshahi, and DK106515-01 to Dr. Chang; the George 12. Watnick TJ, Torres VE, Gandolph MA, Qian F, Onuchic LF, Klinger KW, ’ M. O Brien Kidney Center at Yale (P30 DK079310); the Mayo Clinic Robert et al.: Somatic mutation in individual liver cysts supports a two-hit M. and Billie Kelley Pirnie Translational PKD Center; and the Yale CMG (NIH model of cystogenesis in autosomal dominant polycystic kidney dis- M#UM1HG006504-05). ease. Mol Cell 2: 247–251, 1998 13. Janssen MJ, Salomon J, Cnossen WR, Bergmann C, Pfundt R, Drenth JP: Somatic loss of polycystic disease genes contributes to the forma- SUPPLEMENTAL MATERIAL tion of isolated and polycystic liver cysts. Gut 64: 688–690, 2015 14. Tan AY, Zhang T, Michaeel A, Blumenfeld J, Liu G, Zhang W, et al.: Somatic mutations in renal cyst epithelium in autosomal dominant This article contains the following supplemental material online at polycystic kidney disease. J Am Soc Nephrol 29: 2139–2156, 2018 http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019030298/-/ 15. Fedeles SV, Tian X, Gallagher AR, Mitobe M, Nishio S, Lee SH, et al.: A DCSupplemental. genetic interaction network of fi ve genes for human polycystic kidney Supplemental Table 1. Additional coding region genetic variants and liver diseases defines polycystin-1 as the central determinant of with MAF ,1% by whole exome sequencing in published ADPKD- cyst formation. Nat Genet 43: 639–647, 2011 PCLD genes in ALG9 loss-of-function carriers. 16. Davis K, Webster D, Smith C, Jackson S, Sinasac D, Seargeant L, et al.: ALG9-CDG: New clinical case and review of the literature. Mol Genet Supplemental Table 2. Matched Control Phenotype Data. Metab Rep 13: 55–63, 2017 2/2 Supplemental Figure 1. Sanger sequencing of Alg9 cell line. 17. Freeze HH: Genetic defects in the human glycome. Nat Rev Genet 7: Supplemental Figure 2. Quantification of relative PC1 protein 537–551, 2006 2 2 expression level in Alg9 / cells. 18. Tham E, Eklund EA, Hammarsjö A, Bengtson P, Geiberger S, Lagerstedt- Supplemental Methods. Robinson K, et al.: A novel phenotype in N-glycosylation disorders: Gillessen-Kaesbach-Nishimura skeletal dysplasia due to pathogenic Supplemental Acknowledgments. variants in ALG9. Eur J Hum Genet 24: 198–207, 2016 19. Arora V, Shah N, Khatter S, Puri RD, Kumar R, Gupta P, et al.: ALG9 associated gillessen-kaesbach–nishimura syndrome (GIKANIS): An un- REFERENCES common aetiology of enlarged foetal kidneys. J Foetal Med 5: 237–239, 2018 1. Torres VE, Harris PC, Pirson Y: Autosomal dominant polycystic kidney 20. Chapman AB, Guay-Woodford LM, Grantham JJ, Torres VE, Bae KT, disease. Lancet 369: 1287–1301, 2007 Baumgarten DA, et al.; Consortium for Radiologic Imaging Studies of 2. Cornec-Le Gall E, Torres VE, Harris PC: Genetic complexity of autoso- Polycystic Kidney Disease cohort: Renal structure in early autosomal- mal dominant polycystic kidney and liver diseases. J Am Soc Nephrol dominant polycystic kidney disease (ADPKD): The Consortium for 29: 13–23, 2018 Radiologic Imaging Studies of Polycystic Kidney Disease (CRISP) – 3. Li A, Davila S, Furu L, Qian Q, Tian X, Kamath PS, et al.: Mutations in cohort. Kidney Int 64: 1035 1045, 2003 PRKCSH cause isolated autosomal dominant polycystic liver disease. 21. Torres VE, Abebe KZ, Chapman AB, Schrier RW, Braun WE, Am J Hum Genet 72: 691–703, 2003 Steinman TI, et al.; HALT-PKD Trial Investigators: Angiotensin block- 4. Drenth JP, te Morsche RH, Smink R, Bonifacino JS, Jansen JB: Germline ade in late autosomal dominant polycystic kidney disease. N Engl J Med – mutations in PRKCSH are associated with autosomal dominant poly- 371: 2267 2276, 2014 ’ cystic liver disease. Nat Genet 33: 345–347, 2003 22. Kircher M, Witten DM, Jain P, O Roak BJ, Cooper GM, Shendure J: A 5. Davila S, Furu L, Gharavi AG, Tian X, Onoe T, Qian Q, et al.: Mutations in general framework for estimating the relative pathogenicity of human SEC63 cause autosomal dominant polycystic liver disease. Nat Genet genetic variants. Nat Genet 46: 310–315, 2014 36: 575–577, 2004 23. Lek M, Karczewski KJ, Minikel EV, Samocha KE, Banks E, Fennell T, 6. Porath B, Gainullin VG, Cornec-Le Gall E, Dillinger EK, Heyer CM, et al.; Exome Aggregation Consortium: Analysis of protein-coding Hopp K, et al.; Genkyst Study Group; HALT Progression of Polycystic genetic variation in 60,706 humans. Nature 536: 285–291, 2016 Kidney Disease Group; Consortium for Radiologic Imaging Studies of 24. Zook JM, Chapman B, Wang J, Mittelman D, Hofmann O, Hide W, et al.: Polycystic Kidney Disease: Mutations in GANAB, encoding the gluco- Integrating human sequence data sets provides a resource of benchmark sidase IIa subunit, cause autosomal-dominant polycystic kidney and SNP and indel genotype calls. Nat Biotechnol 32: 246–251, 2014 liver disease. Am J Hum Genet 98: 1193–1207, 2016 25. Dong C, Wei P, Jian X, Gibbs R, Boerwinkle E, Wang K, et al.: 7. Besse W, Dong K, Choi J, Punia S, Fedeles SV, Choi M, et al.: Isolated Comparison and integration of deleteriousness prediction methods for polycystic liver disease genes define effectors of polycystin-1 function. nonsynonymous SNVs in whole exome sequencing studies. Hum Mol JClinInvest127: 3558, 2017 Genet 24: 2125–2137, 2015 8. Cornec-Le Gall E, Olson RJ, Besse W, Heyer CM, Gainullin VG, Smith 26. Ioannidis NM, Rothstein JH, Pejaver V, Middha S, McDonnell SK, Baheti S, JM, et al.; Genkyst Study Group; HALT Progression of Polycystic et al.: REVEL: An ensemble method for predicting the pathogenicity of Kidney Disease Group; Consortium for Radiologic Imaging Studies of rare missense variants. Am J Hum Genet 99: 877–885, 2016

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27. Shihab HA, Rogers MF, Gough J, Mort M, Cooper DN, Day IN, et al.: An contrast-enhanced computed tomography of potential kidney integrative approach to predicting the functional effects of non-coding donors. AmJKidneyDis59: 611–618, 2012 and coding sequence variation. Bioinformatics 31: 1536–1543, 2015 35. Cabezas OR, Flanagan SE, Stanescu H, García-Martínez E, Caswell R, 28. Maniatis T, Fritsch EF, Sambrook J: Molecular cloning: A laboratory Lango-Allen H, et al.: Polycystic kidney disease with hyperinsulinemic manual, New York, Cold Spring Harbor Laboratory, 1982 hypoglycemia caused by a promoter mutation in phosphomannomu- – 29. Carey DJ, Fetterolf SN, Davis FD, Faucett WA, Kirchner HL, Mirshahi U, tase 2. JAmSocNephrol28: 2529 2539, 2017 et al.: The Geisinger MyCode community health initiative: An electronic 36. The UniProt Consortium: UniProt: The universal protein knowledge- – health record-linked biobank for precision medicine research. Genet base. Nucleic Acids Res 45[D1]: D158 D169, 2017 37. Nishiura JL, Neves RF, Eloi SR, Cintra SM, Ajzen SA, Heilberg IP: med 18: 906–913, 2016 Evaluation of nephrolithiasis in autosomal dominant polycystic kidney 30. Dewey FE, Murray MF, Overton JD, Habegger L, Leader JB, Fetterolf SN, disease patients. Clin J Am Soc Nephrol 4: 838–844, 2009 et al.: Distribution and clinical impact of functional variants in 50,726 whole- 38. Pfau A, Knauf F: Update on nephrolithiasis: Core curriculum 2016. exome sequences from the DiscovEHR study. Science 354: aaf6814, 2016 Am J Kidney Dis 68: 973–985, 2016 31. Staples J, Maxwell EK, Gosalia N, Gonzaga-Jauregui C, Snyder C, 39. Scales CD Jr, Smith AC, Hanley JM, Saigal CS; Urologic Diseases in fi Hawes A, et al.: Pro ling and leveraging relatedness in a precision med- America Project: Prevalence of kidney stones in the United States. Eur – icine cohort of 92,455 exomes. Am J Hum Genet 102: 874 889, 2018 Urol 62: 160–165, 2012 32. McLaren W, Gil L, Hunt SE, Riat HS, Ritchie GR, Thormann A, et al.: The 40. Bansal AD, Hui J, Goldfarb DS: Asymptomatic nephrolithiasis detected ensembl variant effect predictor. Genome Biol 17: 122, 2016 by ultrasound. Clin J Am Soc Nephrol 4: 680–684, 2009 33. Ghosh R, Oak N, Plon SE: Evaluation of in silico algorithms for use with ACMG/AMP clinical variant interpretation guidelines. Genome Biol 18: 225, 2017 34. Rule AD, Sasiwimonphan K, Lieske JC, Keddis MT, Torres VE, Vrtiska See related editorial, “New Ways of Finding New Genes for Old Diseases,” on TJ: Characteristics of renal cystic and solid lesions based on pages 2037–2039.

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