“It’s In Your Genes”

Approximately 10% of children presenting with nephrotic syndrome demonstrate resistance to steroid therapy. Up to two thirds of these patients do not respond to intensified immunosuppression with calcineurin inhibitors or B cell–depleting therapy (1). In many of these “multidrug resistant” cases, causative genetic abnormalities are identified. The number of monogenic disease entities causing the clinical phenotype of steroid-resistant nephrotic syndrome has rapidly increased in recent years. Currently, more than 40 “podocytopathies” related to genes that are expressed exclusively or predominantly by podocytes have been characterized. Large studies using next-generation sequencing gene panel screening have found genetic causes in up to 30% of patients with this condition (2).

Although next-generation sequencing gene panels currently are the most widely used technology to screen for disease groups involving several or even multiple causative genes, they are rapidly outdated as new disease genes continue to be discovered. In view of the continued drop in sequencing cost and advancing bioinformatic capacities, many laboratories turn to whole-exome sequencing as an even more efficient, unbiased approach to genetic diagnostics (3). The ethical and legal dilemmas that can result from incidental findings in genes unrelated to the disease of interest can be circumvented by filtering the exome information for a limited number of genes associated with the wider disease group of interest, and extending the analysis only in case of negative findings in the first-line analysis.

In a study published in this issue of CJASN, Landini et al. (4) applied this approach in 64 young patients diagnosed with steroid-resistant nephrotic syndrome. The exome results were filtered in silico for 298 genes related to CKD, including but not limited to the podocyte-specific genes primarily associated with steroid-resistant nephrotic syndrome. As expected, 19 patients (30%) carried disease-causing variants in podocytopathy genes typically associated with steroid-resistant nephrotic syndrome and FSGS. Remarkably, in another 18 patients clinically diagnosed with, and treated for steroid-resistant nephrotic syndrome, pathogenic mutations were found in genes usually associated with other phenotypes, including Alport syndrome (n=6), Dent disease (n=3), renal-coloboma syndrome (n=2), Fabry disease, cystinosis, and five other rare monogenic syndromic disorders. Post hoc reverse phenotyping by thorough multidisciplinary assessment revealed the presence of distinct extrarenal features typical of the genetic diagnoses in all patients and/or first-degree relatives.

These findings confirm several recent reports in which mutations in the collagenopathy genes (5), CLCN5 (6), and PAX2 (7) were found to cause “phenocopies” of steroid-resistant nephrotic syndrome. In these conditions, proteinuria is due to pathomechanisms that are not directly related to podocyte function (i.e., structural abnormalities of the glomerular basement membrane in Alport syndrome and defective tubular endocytosis of filtered albumin in Dent disease) or altered nephron and/or podocyte development, as hypothesized for Pax2-associated disease. Although Landini et al. are not the first to describe unexpected genetic causes of steroid-resistant nephrotic syndrome, the fact that screening of a very wide range of nephropathy genes gives a detection rate twice as high as that achieved by studying only podocytopathy genes stricto sensu impressively illustrates the power of the whole-exome sequencing approach.

“Reverse phenotyping,” i.e., the alignment of unexpected genetic findings with the clinical phenotype by post hoc clinical examination, is about to become a common task for nephrologists as comprehensive genetic diagnostics are gaining ground, and it is not limited to proteinuric glomerulopathies (8). Patients with Alport syndrome have also been misdiagnosed as having membranoproliferative GN, and CLCN5 mutations (defining Dent disease) have been found also in patients presenting with a Bartter-like phenotype. A frequently mistaken clinical diagnosis is that of nephronophthisis: among 79 consanguineous patients with familial childhood-onset CKD in whom nephronophthisis was suspected on the basis of ultrasound criteria, genetic analysis revealed a tubulopathy in eight patients, Alport syndrome in four patients, congenital anomalies of the kidneys and urinary tract in three patients, and autosomal recessive polycystic kidney disease in two patients.

In syndromic disorders involving the kidneys, the need to revise the initial diagnosis on the basis of genetic findings may be a result of incomplete disease penetrance, but more commonly results from the failure to detect subtle extrarenal disease features by clinical examination. Such features may consist of abnormalities of the optical nerve or retina, hearing, or bone morphology, which often escape detection by routine diagnostic work-up. Weber et al. (9) identified mutations in four genes associated with mild syndromic disease with dominant transmission (PAX2, HNF1B, EYA1, and SALL1) in 17 out of 99 children with CKD caused by apparently isolated kidney malformations. In many children, subtle extrarenal disease features were recognized after the genetic diagnosis was made, and in a few families disease features and even advanced CKD were detected in a parent only after the diagnosis was made through the genetic study in the affected child.

In view of the high risk of missing correct diagnoses on clinical grounds, there is an ongoing discussion in the pediatric and clinical geneticist community as to whether an “exome-first” approach should be adopted in patients with a potential genetic condition. The study by Landini et al. is an excellent illustration of this debate. On the one hand one might argue that in many cases the correct diagnosis could have been suspected from a standardized, comprehensive clinical examination and the family history, which could have been followed by targeted screening of the most likely genes. On the other hand, if a noninvasive, increasingly rapid and cheap diagnostic measure can provide an unequivocal disease classification, why not go for it right away and complete the phenotyping in a cost-effective manner after the genetic diagnosis is established? In this context, it is important to note that genetic screening does not always yield unambiguous results. Indeed, Landini et al. identified variants of unknown significance in podocytopathy genes in another 16% of their cohort. In such cases, careful clinical phenotyping can support the interpretation of the pathogenicity of a variant in a given gene.

The clinical benefit of establishing a genetic disease cause in steroid-resistant nephrotic syndrome is obvious. An early diagnosis obtained soon after disease onset can save resources spent for unnecessary, invasive, and sometimes misleading diagnostic interventions (including kidney biopsy), and avoid ineffective therapies fraught with side effects. Many of the patients diagnosed with genetic disease in the study by Landini et al. had received frustraneous immunosuppressive therapies before screening, which could have been avoided if the diagnosis had been sought beforehand. Establishing a genetic diagnosis early on in the course of disease also allows for early detection and management of extrarenal anomalies in syndromic disorders, potentially minimizing the effects of such comorbidities. Moreover, the prognostic value of a genetic diagnosis is usually superior to that of clinical, biochemical, and histopathologic findings. Although long-term kidney survival is poor for most genetic entities, the patients are not at risk of disease recurrence after transplantation, contrasting with a high recurrence risk in patients with nongenetic forms of steroid-resistant nephrotic syndrome. Finally, an exciting perspective in the management of podocytopathies is the emergence of effective causative therapies specific to individual genetic disorders, as first demonstrated by coenzyme Q10 supplementation in mitochondriopathies affecting the podocytes (10). Further gene-specific molecular therapies are on the horizon.

Another ongoing debate relates to the preferred technical approach to diagnostic gene screening. One might argue whether the high detection rate observed by Landini et al. necessarily required whole-exome sequencing with bioinformatic filtering for nephropathy genes. The latest generation podocytopathy gene panels include the collagenopathy genes and most of the syndromic genes that were not listed as “podocytopathy” genes in the study, so these would have also been detected with the gene panel approach. However, the option to expand the search filter as needed to identify unusual or novel disease-associated genes, as well as the steadily decreasing cost of genetic testing and the diminishing cost difference between exome and targeted gene panels, tend to tip the balance toward exome sequencing.

The study by Landini et al. illustrates the large genetic heterogeneity of steroid-resistant nephrotic syndrome and provides further support for early comprehensive genetic screening of this condition. Even in the era of genomics, precise clinical phenotyping remains essential to support the interpretation of the genetic findings.

Disclosures

Dr. Schaefer is a member of the European Reference Network for Rare Kidney Diseases and receives funding from the European Union within the framework of the Third Health Programme (ERN-2016 - Framework Partnership Agreement 2017–2021).