BASIC RESEARCH www.jasn.org Mutations in NUP160 Are Implicated in Steroid-Resistant Nephrotic Syndrome Feng Zhao,1,2,3,4 Jun-yi Zhu ,2 Adam Richman,2 Yulong Fu,2 Wen Huang,2 Nan Chen,5 Xiaoxia Pan,5 Cuili Yi,1 Xiaohua Ding,1 Si Wang,1 Ping Wang,1 Xiaojing Nie,1,3,4 Jun Huang,1,3,4 Yonghui Yang,1,3,4 Zihua Yu ,1,3,4 and Zhe Han2,6 1Department of Pediatrics, Fuzhou Dongfang Hospital, Fujian, People’s Republic of China; 2Center for Genetic Medicine Research, Children’s National Health System, Washington, DC; 3Department of Pediatrics, Affiliated Dongfang Hospital, Xiamen University, Fujian, People’s Republic of China; 4Department of Pediatrics, Fuzhou Clinical Medical College, Fujian Medical University, Fujian, People’s Republic of China; 5Department of Nephrology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People’s Republic of China; and 6Department of Genomics and Precision Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC ABSTRACT Background Studies have identified mutations in .50 genes that can lead to monogenic steroid-resistant nephrotic syndrome (SRNS). The NUP160 gene, which encodes one of the protein components of the nuclear pore complex nucleoporin 160 kD (Nup160), is expressed in both human and mouse kidney cells. Knockdown of NUP160 impairs mouse podocytes in cell culture. Recently, siblings with SRNS and pro- teinuria in a nonconsanguineous family were found to carry compound-heterozygous mutations in NUP160. Methods We identified NUP160 mutations by whole-exome and Sanger sequencing of genomic DNA from a young girl with familial SRNS and FSGS who did not carry mutations in other genes known to be associated with SRNS. We performed in vivo functional validation studies on the NUP160 mutations using a Drosophila model. R11733 E803K Results We identified two compound-heterozygous NUP160 mutations, NUP160 and NUP160 . We showed that silencing of Drosophila NUP160 specifically in nephrocytes (fly renal cells) led to func- tional abnormalities, reduced cell size and nuclear volume, and disorganized nuclear membrane structure. These defects were completely rescued by expression of the wild-type human NUP160 gene in nephrocytes. R11733 By contrast, expression of the NUP160 mutant allele NUP160 completely failed to rescue nephrocyte E803K phenotypes, and mutant allele NUP160 rescued only nuclear pore complex and nuclear lamin localization defects. Conclusions Mutations in NUP160 are implicated in SRNS. Our findings indicate that NUP160 should be included in the SRNS diagnostic gene panel to identify additional patients with SRNS and homozygous or compound-heterozygous NUP160 mutations and further strengthen the evidence that NUP160 muta- tionscancauseSRNS. J Am Soc Nephrol 30: ccc–ccc, 2019. doi: https://doi.org/10.1681/ASN.2018080786 Received August 1, 2018. Accepted February 2, 2019. Research, Children's National Health System, 111 Michigan Ave NW, Washington DC 20010, or Dr. Zihua Yu, Department of F.Z. and J.-y.Z. contributed equally to this work. Pediatrics, Fuzhou Dongfang Hospital, 156 Xi Er Huan Bei Lu, Fuzhou, Fujian 350025, China. Email: zhan@childrensnational. Published online ahead of print. Publication date available at org or [email protected] www.jasn.org. Copyright © 2019 by the American Society of Nephrology Correspondence: Dr. Zhe Han, Center for Genetic Medicine J Am Soc Nephrol 30: ccc–ccc, 2019 ISSN : 1046-6673/3005-ccc 1 BASIC RESEARCH www.jasn.org Nephrotic syndrome is a renal disease caused by disruption of Significance Statement the glomerular filtration barrier, resulting in massive protein- uria, hypoalbuminemia, hyperlipidemia, and edema.1 With Mutations in .50 genes can lead to monogenic steroid-resistant ne- respect to responsiveness to standard steroid therapy, ne- phrotic syndrome (SRNS). The authors found that a young patient with phrotic syndrome is classified into steroid-sensitive nephrotic familial SRNS and FSGS carried novel compound-heterozygous mu- tations in NUP160; this gene encodes nucleoporin 160 kD, one of the syndrome and steroid-resistant nephrotic syndrome (SRNS). protein components of the nuclear pore complex. Using an in vivo renal SRNS can have either an immunologic or genetic etiology.2 cell assay on the basis of Drosophila nephrocytes (an experimental The contributions of genetic factors are increasingly empha- podocyte model previously used to validate candidate renal disease sized in the growing understanding of SRNS pathogenesis. To genes and specific patient-derived mutant alleles), they validated the NUP160 date, .50 monogenic genes have been identified that cause gene variants as factors implicated in kidney pathology. The findings indicate that NUP160 should be included in the SRNS di- 3 SRNS when mutated. These include genes encoding compo- agnostic gene panel to identify additional patients with SRNS carrying nents of the slit diaphragm, such as NPHS1, NPHS2,and homozygous or compound-heterozygous NUP160 mutations. CD2AP; genes encoding actin cytoskeleton proteins, such as ACTN4, INF2,andMYO1E; genes encoding actin-regulating NUP160 small GTPases of the Rho/Rac/Cdc42 family, including transgene but not by either patient-derived mutant in vivo NUP160 ARHGDIA, ARHGAP24,andKANK; and genes encoding allele, providing evidence to implicate these nucleoporins (Nups), including NUP93, NUP107,and mutations as pathogenic. NUP205. Mutations in NPHS1, NPHS2,andCD2AP disrupt the integrity of the slit diaphragm and lead to congenital nephrotic syndrome, early-onset autosomal recessive SRNS, METHODS and early-onset FSGS, respectively.4–6 Mutations in ACTN4 change the cytoskeletal dynamics of podocytes and lead Study Participants to adult-onset autosomal dominant FSGS.7 Mutations in Written informed consent from index family members and ARHGDIA alter podocyte migration capabilities and lead to control subjects was obtained under a protocol approved by early-onset SRNS.8 Mutations in NUP93 inhibit podocyte the institutional review boards of Shanghai Ruijin Hospital and proliferation, promote podocyte apoptosis, and lead to early Fuzhou Dongfang Hospital of China. DNA samples from 520 childhood-onset SRNS.9 Mutations in NUP107 cause hypo- healthy persons were used as controls. The nonconsanguineous plastic glomerular structures and abnormal podocyte foot Chinese index family included the proband, unaffected parents, processes and lead to early childhood-onset SRNS.10 Recently, and five siblings, two of whom died from SRNS in the 1990s Braun et al.11 described mutations in NUP107, NUP85, (Figure 1C, Supplemental Table 1). DNA samples of the index NUP133,andNUP160 in 13 families with SRNS. family were available from the proband (II6), a healthy sibling The NUP160 gene (mapping to chromosome 11p) encodes (II5), and both parents (I1 and I2). Nephrotic syndrome was Nup160, which is a component of the Nup107–160 complex diagnosed on the basis of urinary protein excretion .50 mg/kg required for early stages of nuclear pore complex (NPC) as- per day with hypoalbuminemia ,25 g/L. Steroid resistance was sembly.12,13 It is expressed in both human and mouse kidney defined as a failure of induction of complete remission after cells. Knockdown of the NUP160 gene damages mouse 4 weeks of standard therapy with prednisone (2 mg/kg per day podocytes cultured in vitro.14 Within a nonconsanguineous giveninthreedivideddoses;maximum60mg/d).ESRDwas 2 Chinese family two siblings, a brother with SRNS and a sister defined as a GFR,10 ml/min per 1.73 m or the necessity of E803K with proteinuria, were both found to carry NUP160 and any RRT. Tissue biopsies were evaluated by renal pathologists. R9103 NUP160 compound-heterozygous mutations.11 We excluded the possibility that the proband carried mutations We now describe two compound-heterozygous mutations in known genes associated with SRNS (Supplemental Table 2). R11733 E803K in NUP160, NUP160 and NUP160 , identified in a young girl with familial SRNS and FSGS. This patient did not Whole-Exome Sequencing carry mutations in genes previously associated with SRNS. Genomic DNAwas purified from blood samples collected from Furthermore, we functionally validated the NUP160 muta- the proband (II6), her surviving sister (II5), and her parents tions in vivo using a Drosophila model.15–21 The Drosophila (I1 and I2) using the DNeasy Blood & Tissue Kit (Qiagen) nephrocyte system has been previously proven to be very use- using standard procedures (Figure 1C). Whole-exome capture ful for validating candidate gene mutations for involvement in and sequencing were performed on a SureSelect platform monogenic SRNS and investigating molecular disease mech- (Agilent) with 3 mg of genomic DNA from each individual anisms underlying podocyte cellular pathologies.22–25 We first using SureSelect Exome Capture System V4 (5M). The resulting showed that nephrocyte-specific silencing of the conserved libraries were sequenced on a HiSeq 2000 Multiplexed Sequenc- endogenous fly Nup160 gene induced severe renal cell defects, ing platform (Illumina) according to the manufacturer’s instruc- thereby experimentally validating NUP160 as a novel candi- tions for paired end 100-bp reads. Reads were aligned to the date renal disease gene. We then showed complete rescue of human genome reference sequence (GRCh37/hg19; build of nephrocyte phenotypes by expression of a wild-type human the UCSC Genome Browser; http://genome.ucsc.edu) using 2 Journal of the American Society of Nephrology J Am Soc Nephrol 30: ccc–ccc,2019 www.jasn.org BASIC RESEARCH A ATG TGA 1 243 576108 9 11 12 13 14 15 16 17 1819 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 5,476 bp 196 bp B 803 1173 Nup160 1,436 aa pfam11715: nuleoporin Nup120/160 c.2407G>A(h) c.3517C>T(h) p.Glu803Lys p.Arg1173X low complexity region D RefSeq phosphorylation II6 (P) coiled coil C I1(F) I 12 I2(M) II 123456 II5(S) E p.Glu803Lys p.Arg1173X H. sapiens ESNL QHLLLSEV TDS D G ECTAAPTNR-QIIL E EL ED M. musculus ESNL QHLLLSEV TDS D G ECTAAPTNR - Q I E ILE L ED G.