Article Reverse Phenotyping after Whole-Exome Sequencing in Steroid-Resistant Nephrotic Syndrome Samuela Landini,1,2,3 Benedetta Mazzinghi ,4 Francesca Becherucci ,4 Marco Allinovi,2,3 Aldesia Provenzano ,1,3 Viviana Palazzo,1 Fiammetta Ravaglia,4 Rosangela Artuso ,1 Emanuele Bosi ,2 Stefano Stagi ,5 Giulia Sansavini,4 Francesco Guzzi ,2,3,4 Luigi Cirillo ,4 Augusto Vaglio ,2,3,4 Luisa Murer ,6 Licia Peruzzi ,7 Andrea Pasini ,8 Marco Materassi,4 Rosa Maria Roperto,4 Hans-Joachim Anders ,9 Mario Rotondi ,10 Sabrina Rita Giglio ,1,2,3 and Paola Romagnani 2,3,4 Due to the number of contributing authors, Abstract the affiliations are Background and objectives Nephrotic syndrome is a typical presentation of genetic podocytopathies but listed at the end of occasionally other genetic nephropathies can present as clinically indistinguishable phenocopies. We hypoth- this article. esized that extended genetic testing followed by reverse phenotyping would increase the diagnostic rate for these Correspondence: patients. Prof. Paola Romagnani or Prof. Design, setting, participants, & measurements All patients diagnosed with nephrotic syndrome and referred to our Sabrina Giglio, center between 2000 and 2018 were assessed in this retrospective study. When indicated, whole-exome sequencing Department of fi Clinical and and in silico ltering of 298 genes related to CKD were combined with subsequent reverse phenotyping in patients Experimental and families. Pathogenic variants were defined according to current guidelines of the American College of Medical Biomedical Sciences Genetics. “Mario Serio”, University of Results A total of 111 patients (64 steroid-resistant and 47 steroid-sensitive) were included in the study. Not a Florence, Viale Pieraccini 6, 50139, single pathogenic variant was detected in the steroid-sensitive group. Overall, 30% (19 out of 64) of steroid- Florence, Italy. resistant patients had pathogenic variants in podocytopathy genes, whereas a substantial number of variants E-mail:paola. were identified in other genes, not commonly associated with isolated nephrotic syndrome. Reverse romagnani@unifi.it or phenotyping, on the basis of a personalized diagnostic workflow, permitted to identify previously unrecognized sabrina.giglio@ meyer.it clinical signs of an unexpected underlying genetic nephropathy in a further 28% (18 out of 64) of patients. These patients showed similar multidrug resistance, but different long-term outcome, when compared with genetic podocytopathies. Conclusions Reverse phenotyping increased the diagnostic accuracy in patients referred with the diagnosis of steroid-resistant nephrotic syndrome. CJASN 15: 89–100, 2020. doi: https://doi.org/10.2215/CJN.06060519 Introduction nephropathies outside the podocytopathy spectrum Isolated nephrotic syndrome is classified, according (e.g., Alport syndrome, Dent disease, or Fabry to the response to steroids, as steroid-sensitive or disease) are usually recognized upon standard diag- steroid-resistant nephrotic syndrome (1,2). Although nostic work-up. However, steroid-resistant nephrotic steroid-sensitive nephrotic syndrome usually has a syndrome can rarely be the only evident clinical sign, favorable prognosis, steroid-resistant nephrotic at disease onset or even later (14–18). When this syndrome can progress to ESKD (3–7). Indeed, happens, these genetic nephropathies are often mis- despite some patients’ response to immunosuppres- classified and treated as isolated steroid-resistant sion in terms of proteinuria reduction, many are nephrotic syndrome when genetic testing for com- multidrug resistant (6). Genetic testing using ex- monly reported disease-causing genes (i.e., podocyt- tended panels of podocytopathy genes has become a opathy genes) proves to be negative. These conditions valuable diagnostic tool to identify monogenic are referred to as phenocopies of monogenic podocyto- podocytopathies, which account for about 30% of pathies (19–25). A phenocopy is defined as “a pheno- patients affected by steroid-resistant nephrotic syn- typic trait or disease that resembles the trait expressed drome (5,8–12). In addition, a recent report sugges- by a particular genotype, but in an individual who is ted that steroid-sensitive nephrotic syndrome may not a carrier of that genotype” (26). Recently, by also occasionally be of genetic origin (13). All other applying whole-exome sequencing, Warejko et al.(27) cases of isolated nephrotic syndrome are usually reported the diagnosis of a phenocopy in 5% of patients assumed to be of nongenetic cause. Genetic with steroid-resistant nephrotic syndrome. We www.cjasn.org Vol 15 January, 2020 Copyright © 2020 by the American Society of Nephrology 89 90 CJASN 252 patients diagnosed with nephrotic syndrome and referred to our center between January 2000 and December 2018 47 patients presenting with: - clinical, laboratory or biopsy signs of an immune-mediated disease, n=17 - macroscopic hematuria, predominant tubular proteinuria, n=2 - syndromic nephrotic syndrome and/or extra-renal involvement, n=4 - known family history of nephrotic syndrome, n=10 - parents DNA untraceable, n=14 205 evaluated patients 141 steroid-sensitive nephrotic syndrome 94 patients affected by unfrequently relapsing and not steroid-dependent nephrotic syndrome 64 47 steroid-resistant steroid-sensitive nephrotic syndrome nephrotic syndrome 111 patients sent to genetic analysis Figure 1. | Flowchart for the selection of 111 patients included in the study. hypothesized that establishing standardized criteria to define nephrologists and from medical records. Inclusion criteria podocytopathy versus phenocopy genes, and adding re-eval- were onset of symptoms before 30 years of age and a uation of patients and their family (i.e., “reverse phenotyp- clinical diagnosis of nephrotic syndrome (e.g.,nephrotic ing”) after genetic testing, could correctly segregate the range proteinuria, hypoalbuminemia, edema) or nephrotic identified genetic variants to previously unrecognized range proteinuria with kidney histology of FSGS, minimal clinical symptoms and increase the sensitivity of the genetic change disease, or diffuse mesangial sclerosis. Exclusion analysis. criteria were (1) evidence of clinical, laboratory, or kidney biopsy signs of an immune-mediated disease; (2)macro- scopic hematuria or predominantly tubular proteinuria Materials and Methods (low-molecular-weight proteins .50% according to urine Patients protein electrophoresis) (28); (3) syndromic nephrotic syn- All consecutive patients diagnosed with nephrotic syn- drome and/or presence of extrarenal signs or symptoms drome and referred to the Nephrology Unit of the Meyer (e.g., sensorineural hearing loss, ocular abnormalities); (4) Children’s University Hospital of Florence from January known family history of nephrotic syndrome; and (5) 2000 to December 2018 were assessed for inclusion in this untraceable parents’ DNA (Figure 1). The remaining pa- retrospective study (Figure 1, Supplemental Table 1). tients were then subclassified in steroid-resistant or steroid- Participants were followed from the day of referral until sensitive nephrotic syndrome. The majority of patients 31 December 2018, with no loss to follow-up. Demographic, with steroid-resistant nephrotic syndrome were diagnosed clinical, and laboratory data were retrospectively collected in another nephrology center and subsequently referred to from direct interview of patients, families, and referring our hospital, therefore detailed laboratory information at CJASN 15: 89–100, January, 2020 Reverse Phenotyping in Nephrotic Syndrome, Landini et al. 91 disease onset was not always available. Those affected count approach as detailed in Supplemental Appendix 1. by congenital nephrotic syndrome, or with a histologic Synonymous variants and intronic variants that were not diagnosis of diffuse mesangial sclerosis, or advanced kidney located within splice site regions were excluded. Vari- failure at diagnosis were considered comparable with ants were confirmed in the patients’ and families’ DNA patients who were steroid-resistant, although not treated by Sanger sequencing. with steroids. Among patients who were steroid-sensitive, only frequently relapsing or steroid-dependent patients Definition of Podocytopathy and Phenocopy Genes were selected for genetic testing and inclusion in the study. We systematically defined podocytopathy versus phe- First-degree relatives were either included before the study nocopy genes on the basis of Online Mendelian Inheri- fi or asked to participate after the identi cation of potentially tance in Man (OMIM; https://www.omim.org). causative gene variants in the patient. The local Ethics Accordingly, genes identified in OMIM as causing “ne- ’ Committee of the Meyer Children s University Hospital of phrotic syndrome” or “FSGS” were considered “podo- Florence approved the study. The study was conducted cytopathy genes.” In contrast, genes identified in OMIM according to the Declaration of Helsinki. A clinical geneticist to cause a syndromic disorder, with nephrotic syndrome counseled all patients and their families regarding the beingonlyoneamongmanyotherclinicalsignsoreven whole-exome sequencing procedure, and all participants not mentioned at all, were considered “phenocopy genes.” or their legal guardians gave written informed consent. Recently published literature indicating a causative path- ogenic role for a gene in nephrotic syndrome was also Sequencing and Bioinformatic Analysis considered to stratify cases as podocytopathy versus pheno-