A Single-Gene Cause in 29.5% of Cases of Steroid-Resistant Nephrotic Syndrome

CKDs take one of the highest tolls on human health. They insidiously lead to ESRD, necessitating dialysis or kidney transplantation for survival. More than 20 million individuals in the United States have CKD, and the treatment cost exceeds $29 billion Medicare expenses annually. The prevalence of CKD has been rising continuously during the last 20 years.1 Nephrotic syndrome (NS) is a CKD defined by significant proteinuria (>40 mg/m2 per hour) with resulting hypoalbuminemia, which in turn causes edema. In young adults and children, NS is classified by its response to a standardized steroid therapy as steroid-sensitive NS (SSNS) versus steroid-resistant NS (SRNS). SRNS constitutes the second most frequent cause of ESRD in the first two decades of life (North American Pediatric Renal Trials and Collaborative Studies, 2008). For most patients, no curative treatment is available. The most frequent renal histologic feature of SRNS is FSGS. Moreover, in patients with FSGS the risk of recurrence after kidney transplantation is estimated to be 11%–50%, an event that again leads to terminal renal failure.2–4 The risk for recurrence of FSGS in patients with inherited forms of SRNS is lower than that in patients with nonhereditary FSGS.5,6 SRNS is one of the most intractable diseases in nephrology. Its etiology and pathogenesis have been a conundrum for decades. However, discovery of >27 recessive or dominant genes that, if mutated, cause SRNS has recently provided fundamental insights into mechanisms of this disease.7 Identification of these single-gene causes of SRNS has revealed that the glomerular podocyte and the glomerular slit membrane that it maintains are the primary sites at which the pathogenesis of SRNS unfolds.8 The power of identification of such single-gene causes of SRNS lies in the fact that recessive mutations almost always convey full penetrance and thereby represent the cause of SRNS (i.e., the etiology) rather than conveying only an increased risk for the disease. Thus, if strict genetic criteria are being followed for the decision in which mutations are considered causative, identification of the causative mutation allows for (1) unequivocal molecular genetic diagnostics, (2) establishment of genotype-phenotype correlations, (3) transfer of mutations into genetic animal models with detailed study of their detrimental effects, (4) etiologic stratification of participants for therapeutic studies by specific causative gene and mutation, and (5) discovery of specific mutations that may be amenable to treatment. We have previously shown that 85% of SRNS cases with onset by 3 months of age and 66% with onset by 1 year of age can be explained by recessive mutations in one of four genes only (NPHS1, NPHS2, LAMB2, or WT1).9 However, few data are available on the fraction of monogenic causes of SRNS manifesting later in life, no data are available on large international cohorts,5,10–13 and the generation of data on single-gene causes of SRNS in large cohorts has been impractical until recently. Because no large cohorts have been studied for the known 27 SRNS-causing genes regarding the frequency of mutations that are considered causative (as defined by strict genetic criteria), we developed a strategy of high-throughput, barcoded exon sequencing using the Fluidigm platform with consecutive next-generation sequencing.14–16 Using this approach we have established a high-throughput, low-cost strategy for exon sequencing of all 27 known SRNS-causing genes for proof of principle in a small cohort of 96 individuals.16 To generate data on the percentage of single-gene causes of SRNS in more individuals, we applied this technique to a large cohort of 2016 individuals (1783 families) with SRNS. We found that a high fraction of SRNS manifesting before 25 years of age is caused by single-gene mutations and is inversely correlated to age of onset. We observed that specific genotype-phenotype correlations exist for PLCE1 mutations and that certain founder mutations prevail in specific geographic regions. Our data demonstrate the heuristic power of mutation analysis in SRNS and will guide the expectations regarding the frequency and nature of causative mutations throughout selected regions of the world. Our high-throughput sequencing strategy will accelerate the generation of causation-based genotype data for large cohorts of therapeutic studies.