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

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Somatic Mutations in Renal Cyst Epithelium in Autosomal Dominant Polycystic Kidney Disease BASIC RESEARCH www.jasn.org Somatic Mutations in Renal Cyst Epithelium in Autosomal Dominant Polycystic Kidney Disease Adrian Y. Tan,1,2 Tuo Zhang,2 Alber Michaeel,1 Jon Blumenfeld,3,4 Genyan Liu,1 Wanying Zhang,1 Zhengmao Zhang,1 Yi Zhu,1 Lior Rennert,5 Che Martin,1 Jenny Xiang,2 Steven P. Salvatore,1 Brian D. Robinson,1 Sandip Kapur,6 Stephanie Donahue,4 Warren O. Bobb,4 and Hanna Rennert1 1Departments of Pathology and Laboratory Medicine, 2Microbiology and Immunology, 3Medicine, and 6Surgery, Weill Cornell Medicine, New York, New York; 4The Rogosin Institute, NewYork-Presbyterian Hospital/Weill Cornell Medicine, New York, New York; and 5Department of Public Health Sciences, Clemson University, Clemson, South Carolina ABSTRACT Background Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy caused by mutations in PKD1 and PKD2 that is characterized by renal tubular epithelial cell proliferation and progressive CKD. Although the molecular mechanisms involved in cystogenesis are not established, concurrent inactivating constitutional and somatic mutations in ADPKD genes in cyst epithelium have been proposed as a cellular recessive mechanism. Methods We characterized, by whole-exome sequencing (WES) and long-range PCR techniques, the somatic mutations in PKD1 and PKD2 genes in renal epithelial cells from 83 kidney cysts obtained from nine patients with ADPKD, for whom a constitutional mutation in PKD1 or PKD2 was identified. Results Complete sequencing data by long-range PCR and WES was available for 63 and 65 cysts, re- spectively. Private somatic mutations of PKD1 or PKD2 were identified in all patients and in 90% of the cysts analyzed; 90% of these mutations were truncating, splice site, or in-frame variations predicted to be pathogenic mutations. No trans-heterozygous mutations of PKD1 or PKD2 genes were identified. Copy number changes of PKD1 ranging from 151 bp to 28 kb were observed in 12% of the cysts. WES also identified significant mutations in 53 non-PKD1/2 genes, including other ciliopathy genes and cancer-related genes. Conclusions These findings support a cellular recessive mechanism for cyst formation in ADPKD caused primarily by inactivating constitutional and somatic mutations of PKD1 or PKD2 in kidney cyst epithelium. The potential interactions of these genes with other ciliopathy- and cancer-related genes to influence ADPKD severity merits further evaluation. J Am Soc Nephrol 29: 2139–2156, 2018. doi: https://doi.org/10.1681/ASN.2017080878 Autosomal dominant polycystic kidney disease mechanism of cyst formation in ADPKD, disease (ADPKD) is clinically and genetically heteroge- severity has been attributed to the affected PKD neous, caused by mutations in PKD1 or PKD2, gene locus and the timing and type of somatic and is the most common inherited CKD.1 In 7%– 10% of individuals with ADPKD, the pathogenic variant of a PKD gene is not detected, suggesting Received August 15, 2017. Accepted June 5, 2018. other genetic causes including hypomorphic alleles, Published online ahead of print. Publication date available at 2 mosaicism, or rare mutations of other genes. In- www.jasn.org. activating, somatic mutations of PKD1/2 genes that Correspondence: Dr. Hanna Rennert, Department of Pathology occur in renal epithelial cells, together with a con- and Laboratory Medicine, Weill Cornell Medicine, 525 East 68 current, constitutional mutation of PKD1 or PKD2, Street, F544, New York, NY 10065. Email: [email protected]. cause clonal proliferation of tubular epithelium edu and cyst formation.3 In this cellular recessive Copyright © 2018 by the American Society of Nephrology J Am Soc Nephrol 29: 2139–2156, 2018 ISSN : 1046-6673/2908-2139 2139 BASIC RESEARCH www.jasn.org PKD gene mutations. Kidney cyst formation also occurs when Significance Statement there is a deletion of chromosomal regions of the correspond- ing PKD1 or PKD2 allele, termed loss of heterozygosity Autosomal dominant polycystic kidney disease (ADPKD) is a cili- (LOH).4–8 These mechanisms have also been identified in opathy caused by mutations in PKD1 and PKD2, characterized by malignant neoplasms, suggesting that ADPKD is a benign renal epithelial cell proliferation and progressive CKD. Among the genetic mechanisms proposed for cystogenesis are inactivating 9,10 neoplastic process. constitutional and somatic mutations in PKD1/2 genes, and PKD1/2 Recent studies, utilizing more technically advanced methods, gene dosage effects. This article describes the identification, by concluded that modestly reduced levels of PKD1/2 gene expres- genomic sequencing methods, of the high prevalence of inactivating sion (i.e., haploinsufficiency), rather than PKD1/2 gene inactiva- somatic mutations in PKD1/2 and non-PKD1/2 genes in epithelial tion, caused a spectrum of cystic renal phenotypes, ranging from cells obtained from kidney cysts in patients with ADPKD. The findings support a primary cellular recessive mechanism for cyst few, small cysts with relatively well compensated CKD, to mark- formation in ADPKD caused by mutations in these genes in the – edly enlarged kidneys with early onset ESRD.3,11 14 Although the renal epithelium. Potential interactions of PKD1/2 genes with prevalence of PKD gene haploinsufficiency in the ADPKD pop- other ciliopathy- and cancer-related genes that might influence ulation has not been established, it appears to be low.15 ADPKD severity is proposed. Primary cilia are microtubule-based, nonmotile projections of epithelial cells thatextendintothe lumenof renaltubules and other (357,999 exons, .20,000 genes) of renal cyst epithelia and 16 structures. Integral membrane proteins, including polycystin 1 peripheral blood lymphocytes (PBL) DNA was performed us- (PC1, TRPP1) and polycystin 2 (PC2, TRPP2), encoded by PKD1 ing the Agilent HaloPlex Target Enrichment and massive par- 17 and PKD2, respectively, localize to these cilia. Mammalian cil- allel sequencing (Illumina, San Diego, CA). Data analysis of iopathies are inherited disorders characterized by various pheno- the cyst and matched constitutional DNA was performed for types, including renal cyst formation. They are caused by simultaneous detection of single nucleotide variants (SNVs), mutations in genes that encode proteins associated with cilia indels, and copy number variations (CNVs) as previously de- fi 18 and traf cking pathways. The potential interactions of PKD1/ scribed.22,23 Schematics of WES data analysis pipeline is 2 genes with non-PKD1/2 genes, including other ciliopathy genes, shown is Supplemental Figure 2. The pathogenic potential fi have not been well de ned. of missense variants was evaluated using the computational Given the complex ADPKD phenotype, and multiple factors analysis tool Combined Annotation–Dependent Depletion affecting cyst development, we characterized the prevalence and (CADD; http://cadd.gs.washington.edu/). A CADD value of diversity of somatic PKD1/2 gene mutations arising in renal cyst $15 suggests the variant is likely pathogenic.24 Significantly epithelium and investigated whether somatic mutations in non- mutated WES variants were further filtered using a 30% ADPKD genes occur that might contribute to the pathogenesis of variant allele frequency (VAF) cutoff and a population allele kidney cysts. We report a comprehensive genomic analysis of so- frequency of 5% in The Exome Aggregation Consortium matic mutations, using whole-exome sequencing (WES) and (ExAC) database (http://exac.broadinstitute.org/). Sanger sequencing, in cyst epithelial cells that were isolated after The significance of mutated genes and pathways was ana- native nephrectomy in patients with ADPKD for whom consti- lyzed with the Mutational Significance In Cancer(MuSiC) suite fi tutional mutations in PKD1/2 were identi ed.Inthisstudy,in- of tools (http://gmt.genome.wustl.edu/packages/genome- activating somatic mutations in PKD1/2 genes occurred in 90% of music/index.html) using default settings, accounting for kidney cysts. We also characterized somatic variants in non- gene background mutation rates. Potential splice-site effects PKD1/2 genes (e.g., ciliopathy, cancer) from these cysts, including were evaluated using splicing prediction tools.20 Acustom fl those associated with other ciliopathies, which might in uence analysis pipeline analyzed constitutional mutations in 211 cili- cyst formation and disease severity in ADPKD. opathy and 565 cancer genes (http://cancer.sanger.ac.uk/census/; T. Zhang et al., unpublished data). Pathway analysis was performed by Reactome (https://reactome.org/). Statistical METHODS analysis to study the association between somatic variant and cyst size was performed using a linear mixed effects model Patients with ADPKD were enrolled in this study if they were with the R software environment (https://cran.r-project.org/ scheduled for a living donor kidney transplant, and removal of web/packages/nlme/index.html). Additional details are one or both native kidneys at the New York-Presbyterian Hos- provided in Supplemental Material. pital/Weill Cornell Medicine campus. Study eligibility was de- termined by the transplant surgeon (S.K.) before enrollment of each participant and informed consent was obtained (Supple- RESULTS mental Figure 1). The protocol was approved by the Weill Cornell Medicine Institutional Review Board. Cyst epithelial Clinical Cohort cells were isolated according to standard procedures.5,19 Nine unrelated patients with ADPKD were enrolled. All but PKD1/2 genetic analysis was performed by long-range PCR one had unilateral nephrectomy
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