RCAN1) Gene Confers Enhanced Calcineurin Activity and May Cause FSGS
Total Page：16
File Type：pdf, Size：1020Kb
BASIC RESEARCH www.jasn.org A Rare Autosomal Dominant Variant in Regulator of Calcineurin Type 1 (RCAN1) Gene Confers Enhanced Calcineurin Activity and May Cause FSGS Brandon M. Lane ,1 Susan Murray,2 Katherine Benson,3 Agnieszka Bierzynska,4 Megan Chryst-Stangl,1 Liming Wang ,5 Guanghong Wu,1 Gianpiero Cavalleri ,3 Brendan Doyle,6 Neil Fennelly,6 Anthony Dorman,6 Shane Conlon,2 Virginia Vega-Warner,7 Damian Fermin ,7 Poornima Vijayan ,8 Mohammad Azfar Qureshi,8 Shirlee Shril,9 Moumita Barua,8 Friedhelm Hildebrandt,9 Martin Pollak,10 David Howell,11 Matthew G. Sampson,9,12 Moin Saleem ,4 Peter J. Conlon ,2,13 Robert Spurney,5 and Rasheed Gbadegesin 1,5 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Podocyte dysfunction is the main pathologic mechanism driving the development of FSGS and other morphologic types of steroid-resistant nephrotic syndrome (SRNS). Despite significant prog- ress, the genetic causes of most cases of SRNS have yet to be identified. Methods Whole-genome sequencing was performed on 320 individuals from 201 families with familial and sporadic NS/FSGS with no pathogenic mutations in any known NS/FSGS genes. Results Two variants in the gene encoding regulator of calcineurin type 1 (RCAN1) segregate with disease in two families with autosomal dominant FSGS/SRNS. In vitro,lossofRCAN1 reduced human podocyte viability due to increased calcineurin activity. Cells expressing mutant RCAN1 displayed increased calci- neurin activity and NFAT activation that resulted in increased susceptibility to apoptosis compared with wild-type RCAN1. Treatment with GSK-3 inhibitors ameliorated this elevated calcineurin activity, suggest- ing the mutation alters the balance of RCAN1 regulation by GSK-3b, resulting in dysregulated calcineurin activity and apoptosis. Conclusions These data suggest mutations in RCAN1 can cause autosomal dominant FSGS. Despite the widespread use of calcineurin inhibitors in the treatment of NS, genetic mutations in a direct regulator of calcineurin have not been implicated in the etiology of NS/FSGS before this report. The findings highlight the therapeutic potential of targeting RCAN1 regulatory molecules, such as GSK-3b, in the treatment of FSGS. JASN 32: ccc–ccc, 2021. doi: https://doi.org/10.1681/ASN.2020081234 Glomerular diseases, including diabetic nephropathy, aretheprimaryknowncauseofCKDintheUnited States and the rest of the world.1,2 Most glomerular Received August 27, 2020. Accepted February 25, 2021. diseases are due to primary dysfunction or secondary Published online ahead of print. Publication date available at injury to the podocyte, the visceral epithelial cell of the www.jasn.org. trilayer glomerular filtration barrier. Primary podocyte Correspondence: Dr. Rasheed Gbadegesin, Division of Ne- dysfunction, referred to as podocytopathy, typically phrology, Duke Molecular Physiology Institute, Carmichael manifests as steroid-resistant nephrotic syndrome building, RM 51-104, 300 N Duke St., Durham, NC 27701-2047. (SRNS) with morphologic changes of FSGS or minimal Email: [email protected] change disease apparent on kidney biopsy specimens.3,4 Copyright © 2021 by the American Society of Nephrology JASN 32: ccc–ccc, 2021 ISSN : 1046-6673/3207-ccc 1 BASIC RESEARCH www.jasn.org It is estimated that 5%–30% of all podocytopathies are due Significance Statement to mutation in single genes, especially in children and young adults.5–7 More than 60 genes have been identified as causes of Whole-genome sequencing of 320 individuals with nephrotic syn- monogenic SRNS; however, these genes are responsible for drome (NS) of unclear genetic etiology and data from several only 20% of all genetic SRNS, suggesting there are other, un- independent patient cohorts provided insight into the genetic ar- chitecture of the condition. The strategy identified a disease- fi 6,8–11 fi identi ed, single-gene causes of SRNS. Identi cation of causing autosomal dominant mutation in regulator of calcineurin these causal genes has the potential to improve our under- type 1 (RCAN1) that increased cellular calcineurin (CN) activity, standing of disease pathogenesis, the identification of disease NFAT (NF of activated T cells) activation, and susceptibility to ap- biomarkers, the identification of new therapeutic agents, and optosis of podocytes in vitro. Inhibition of an RCAN regulator, GSK- b RCAN1 the repurposing of existing agents to treat nephrotic 3 , rescued the increased CN activation. Mutations in are a novel cause of NS and reveal a potential target for developing syndrome (NS). personalized therapy. To identify new, single-gene causes of SRNS, we carried out whole-genome sequencing (WGS) on 320 individuals from 201 families with familial and sporadic NS, and reviewed METHODS whole-exome sequencing data from patients with NS of un- clear genetic etiology. We identified two segregating, hetero- WGS fi zygous mutations in the regulator of calcineurin (CN) type 1 WGS was performed at GENEWIZ (South Plain eld, NJ). fl (RCAN1) in two large Northern European families. There are Brie y, genomic DNA samples were assessed for purity, quan- three genes in the RCAN family RCAN1–3, all of which encode tity, and quality by using the NanoDrop 2000 Spectrophotom- proteins capable of interacting with CN and inhibiting CN- eter (Thermo Fisher), Qubit 2.0 Fluorometer, Qubit dsDNA dependent signaling pathways.12–22 Therefore, we screened HS Assay Kit (Thermo Fisher), and agarose gel electrophore- families with hereditary and sporadic NS in other independent sis. Library construction was then performed using Illumina’s cohorts for rare variants in RCAN1–3 genes. We identified TruSeq DNA PCR-Free library preparation kit following the four possible disease-causing variants: three in RCAN2,and manufacturer’s protocol. Genomic DNA was fragmented by one in RCAN3. acoustic shearing with a Covaris S220 instrument. Sheared The RCAN family of proteins form a complex with the DNA was then end repaired and A-tailed, followed by adaptor catalytic subunit of CN, and regulate both CN phosphatase ligation. Final libraries were analyzed on the Agilent TapeSta- activity and its ability to bind key substrates like NF of acti- tion, for library sizing, and quantified using the Qubit dsDNA vated T cells (NFAT).12–14,16–18,20,23–27 Unregulated CN acti- HS Assay Kit along with the KAPA Library Quantification Kit vation is central to the pathogenesis of multiple glomerular for quantitative PCR. DNA libraries were sequenced using disease processes, and CN inhibitors (CNIs) are often used to Illumina platforms to generate $120 Gb of raw data per sam- treat glomerular diseases.28–36 The rationale for treating ac- ple, with a 23150-bp, paired-end sequencing configuration. quired forms of NS with CNIs has historically been that the immune system was thought to play a significant role in ac- Variant Calling and Annotation quired forms of NS, such as minimal change disease DNA-sequencing data were processed using fastp1 to trim low- and FSGS.28,29 However, CN has nonimmunologic actions quality bases and Illumina sequencing adapters from the 39 end that are important in the pathogenesis of kidney diseases. of reads.39 Reads were then aligned to the GRCh37 version of the For example, CN causes cytoskeletal instability by dephosphor- human genome with the BWA2 algorithm.40 PCR duplicates ylating synaptopodin and promoting its degradation.28–31,33 were flagged using the PICARD Tools3 software suite.41 Align- Moreover, podocyte loss plays a key role in pathogenesis ment processing and variant calling were performed using the of FSGS, and CN promotes a decrease in the number of GATK4 toolkit following the Broad Institute’s Best Practices glomerular podocytes by both genetic and nongenetic Workflow.42,43 Functional consequences and genotype prove- mechanisms.28–31,34,37,38 nances of variants were annotated using Ensembl Variant In this study, we discovered that a disease-causing RCAN1 Predictor.44 After annotation, variants meeting the following variant in individuals with FSGS had a reduced ability to in- criteria were selected for further analysis: having a “pass” status hibit activated CN compared with wild-type (WT) RCAN1. after GATK’s Variant Quality Score Recalibration, found to re- The increase in CN activation induced by the RCAN1 variant side in a coding region, and had an allele frequency of ,5% in at was inhibited by treatment with antagonists of glycogen syn- least one population of the Genome Aggregation Database (gno- thase kinase 3 (GSK-3). In addition, cells expressing this mAD).45 Second-level filtering to identify disease-causing vari- RCAN1 variant were more sensitive to apoptotic stimuli, ants is as shown in Supplemental Figure 1. Variants of interest which could be rescued by CNI treatment. Collectively, our were confirmed by Sanger sequencing. findings suggest mutations in RCAN1 are a novel genetic cause of NS, and use of CNIs and GSK antagonists may represent RCAN1 Knockdown Podocytes targeted or personalized therapy for individuals with NS/FSGS Multiple, conditionally immortalized, human podocyte lines due to RCAN1 mutations. (courtesy of Dr. Jeffrey Kopp) with reduced RCAN1 expression 2 JASN JASN 32: ccc–ccc,2021 www.jasn.org BASIC RESEARCH were created using lentiviral transduction of short hairpin RNA total phosphatase levels. EGTA-supplemented buffer was (shRNA) against RCAN1 (TRCN0000019848; Millipore added to additional wells for each sample to measure the Sigma). Lentiviral control lines were created using shRNA non-CN phosphatase activity. All sample wells received water with no known target (SHC016V; Millipore Sigma). Podocyte and CN substrate (RII phosphopeptide), except for back- lentiviral transduction was performed as described previ- ground wells in which water was substituted for substrate. A ously.46 RCAN1 KD was confirmed through immunoblotting positive control (CN enzyme supplied by the kit) was used to (LS-C162511; LifeSpan Biosciences). ensure assay effectiveness. The plate was incubated at 30°C for 10 minutes, lysate was added to all sample wells, and then it Immunoprecipitation Studies was incubated at 30°C for 30 minutes.