Laura Massella

Broadening the Spectrum of Diseases Related to Podocin Mutations

A total of 179 children with sporadic nephrotic syndrome were screened for podocin mutations: 120 with steroid resistance, and 59 with steroid dependence/frequent relapses. Fourteen steroid-resistant patients presented homozygous mutations that were associated with early onset of proteinuria and variable renal lesions, including one case with mesangial C3 deposition. Single mutations of podocin were found in four steroid-resistant and in four steroid-dependent; five patients had the same mutation (P20L). Among these, two had steroid/cyclosporin resistance, two had steroid dependence, and one responded to cyclosporin. The common variant R229Q of podocin, recently associated with late-onset focal segmental glomerulosclerosis, had an overall allelic frequency of 4.2% versus 2.5% in controls. To further define the implication of R229Q, a familial case was characterized with two nephrotic siblings presenting the association of the R229Q with A297V mutation that were inherited from healthy mother and father, respectively. Immunohistochemistry with anti-podocin antibodies revealed markedly decreased expression of the protein in their kidneys. All carriers of heterozygous coding podocin mutation or R229Q were screened for nephrin mutation that was found in heterozygosity associated with R229Q in one patient. Finally, podocin loss of heterozygosity was excluded in one heterozygous child by characterizing cDNA from dissected glomeruli. These data outline the clinical features of sporadic nephrotic syndrome due to podocin mutations (homozygous and heterozygous) in a representative population with broad phenotype, including patients with good response to drugs. The pathogenetic implication of single podocin defects per se in proteinuria must be further investigated in view of the possibility that detection of a second mutation could have been missed. A suggested alternative is the involvement of other gene(s) or factor(s).

Recurrence of focal segmental glomerulosclerosis after renal transplantation in patients with mutations of podocin.

BACKGROUND Posttransplant recurrence of focal segmental glomerulosclerosis (FSGS) occurs in a relevant proportion of FSGS patients and represents an important clinical emergency. It is taken as a proof of the existence of circulating permeability plasma factor(s) that are also putative effectors of original proteinuria in these patients. Familial forms of FSGS do not recur, but the discovery of numerous patients with sporadic FSGS and mutations of podocin (NPHS2, that is actually an inherited disease) who received a renal graft require a re-evaluation of the problem. METHODS To evaluate the incidence of posttransplant recurrence of FSGS in patients with NPHS2, the authors screened for podocin mutations in 53 patients with the clinical and pathologic stigmata of FSGS who had renal failure and who had undergone renal transplantation.Results. Twelve children were found to carry a homozygous (n9) or a heterozygous (n4) mutation of podocin and were classified, according to current criteria, as patients with inherited FSGS. In 5 patients of this group (38%), proteinuria recurred after renal graft and in 2, renal biopsy results showed recurrence of FSGS. Prerecurrence serum of 3 patients of this cohort was tested for antipodocin antibodies with indirect immuno-Western utilizing human podocyte extracts and were found negative. The rate of FSGS recurrence was comparable in non-NPHS2-FSGS children (12 of 27) and adults (3 of 13). Also clinical outcome of recurrence and response to plasmapheresis and immunosuppressors were comparable, suggesting a common mechanism. CONCLUSION These data show a high rate of FSGS recurrence in patients with NPHS2 mutations that is comparable with idiopathic FSGS and describe the successful therapeutic approach. Recurrence of an apparently inherited disease should stimulate a critical review of the mechanisms of recurrence and of original proteinuria in these cases.

Rituximab in Steroid-Dependent or Frequently Relapsing Idiopathic Nephrotic Syndrome

The outcome of steroid-dependent or frequently relapsing nephrotic syndrome of minimal change disease (MCD), mesangial proliferative GN (MesGN), or FSGS may be poor and with major treatment toxicity. This academic, multicenter, off-on trial (ClinicalTrials.gov #NCT00981838) primarily evaluated the effects of rituximab therapy followed by immunosuppression withdrawal on disease recurrence in 10 children and 20 adults with MCD/MesGN (n=22) or FSGS who had suffered ≥2 recurrences over the previous year and were in steroid-induced remission for ≥1 month. Participants received one dose (n=28) or two doses of rituximab (375 mg/m(2) intravenously). At 1 year, all patients were in remission: 18 were treatment-free and 15 never relapsed. Compared with the year before rituximab treatment, total relapses decreased from 88 to 22 and the per-patient median number of relapses decreased from 2.5 (interquartile range [IQR], 2-4) to 0.5 (IQR, 0-1; P<0.001) during 1 year of follow-up. Reduction was significant across subgroups (children, adults, MCD/MesGN, and FSGS; P<0.01). After rituximab, the per-patient steroid maintenance median dose decreased from 0.27 mg/kg (IQR, 0.19-0.60) to 0 mg/kg (IQR, 0-0.23) (P<0.001), and the median cumulative dose to achieve relapse remission decreased from 19.5 mg/kg (IQR, 13.0-29.2) to 0.5 mg/kg (IQR, 0-9.4) (P<0.001). Furthermore, the mean estimated GFR increased from 111.3±25.7 to 121.8±29.2 ml/min per 1.73 m(2) (P=0.01), with the largest increases in children and in FSGS subgroups. The mean height z score slope stabilized in children (P<0.01). Treatment was well tolerated. Rituximab effectively and safely prevented recurrences and reduced the need for immunosuppression in steroid-dependent or frequently relapsing nephrotic syndrome, and halted disease-associated growth deficit in children.

Rituximab in Children with Resistant Idiopathic Nephrotic Syndrome

Idiopathic nephrotic syndrome resistant to standard treatments remains a therapeutic dilemma in pediatric nephrology. To test whether the anti-CD20 monoclonal antibody rituximab may benefit these patients, we conducted an open-label, randomized, controlled trial in 31 children with idiopathic nephrotic syndrome unresponsive to the combination of calcineurin inhibitors and prednisone. All children continued prednisone and calcineurin inhibitors at the doses prescribed before enrollment, and one treatment group received two doses of rituximab (375 mg/m(2) intravenously) as add-on therapy. The mean age was 8 years (range, 2-16 years). Rituximab did not reduce proteinuria at 3 months (change, -12% [95% confidence interval, -73% to 110%]; P=0.77 in analysis of covariance model adjusted for baseline proteinuria). Additional adjustment for previous remission and interaction terms (treatment by baseline proteinuria and treatment by previous remission) did not change the results. In conclusion, these data do not support the addition of rituximab to prednisone and calcineurin inhibitors in children with resistant idiopathic nephrotic syndrome.

Advances in Alport syndrome diagnosis using next-generation sequencing

Alport syndrome (ATS) is a hereditary nephropathy often associated with sensorineural hypoacusis and ocular abnormalities. Mutations in the COL4A5 gene cause X-linked ATS. Mutations in COL4A4 and COL4A3 genes have been reported in both autosomal recessive and autosomal dominant ATS. The conventional mutation screening, performed by DHPLC and/or Sanger sequencing, is time-consuming and has relatively high costs because of the absence of hot spots and to the high number of exons per gene: 51 (COL4A5), 48 (COL4A4) and 52 (COL4A3). Several months are usually necessary to complete the diagnosis, especially in cases with less informative pedigrees. To overcome these limitations, we designed a next-generation sequencing (NGS) protocol enabling simultaneous detection of all possible variants in the three genes. We used a method coupling selective amplification to the 454 Roche DNA sequencing platform (Genome Sequencer junior). The application of this technology allowed us to identify the second mutation in two ATS patients (p.Ser1147Phe in COL4A3 and p.Arg1682Trp in COL4A4) and to reconsider the diagnosis of ATS in a third patient. This study, therefore, illustrates the successful application of NGS to mutation screening of Mendelian disorders with locus heterogeneity.

TRPC6 Mutations in Children with Steroid-Resistant Nephrotic Syndrome and Atypical Phenotype

BACKGROUND AND OBJECTIVES Mutations in the TRPC6 gene have been recently identified as the cause of late-onset autosomal-dominant focal segmental glomerulosclerosis (FSGS). To extend the screening, we analyzed TRPC6 in 33 Italian children with sporadic early-onset SRNS and three Italian families with adult-onset FSGS. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS TRPC6 mutation analysis was performed through PCR and sequencing. The effects of the detected amino acid substitutions were analyzed by bioinformatics tools and functional in vitro studies. The expression levels of TRPC6 and nephrin proteins were evaluated by confocal microscopy. RESULTS Three heterozygous missense mutations (c.374A>G_p.N125S, c.653A>T_p.H218L, c.2684G>T_p.R895L) were identified. The first new mutation, p.H218L, was found in a 18-year-old boy who presented a severe form of FSGS at the age of 8 years. The second, p.R895L, a new de novo mutation, was identified in a girl with collapsing glomerulosclerosis at the age of 2 years. The former mutation, p.N125S, was found in two siblings with early-onset steroid-resistant nephrotic syndrome (SRNS) at the ages of 4 and 14 years. Renal immunofluorescence revealed upregulated expression of TRPC6 and loss of nephrin in glomeruli. The intracellular calcium concentrations were significantly higher in the cells expressing all mutant TRPC6 channels compared with cells expressing wild-type TRPC6. CONCLUSIONS Our findings suggest that TRPC6 variants can also be detected in children with early-onset and sporadic SRNS (4 of 33 patients). Moreover, in one patient a new de novo TRPC6 mutation was associated with a rare severe form of childhood collapsing glomerulosclerosis with rapid progression to uremia.

Minimal Change Disease

Minimal change disease (MCD) is a major cause of idiopathic nephrotic syndrome (NS), characterized by intense proteinuria leading to edema and intravascular volume depletion. In adults, it accounts for approximately 15% of patients with idiopathic NS, reaching a much higher percentage at younger ages, up to 70%-90% in children >1 year of age. In the pediatric setting, a renal biopsy is usually not performed if presentation is typical and the patient responds to therapy with oral prednisone at conventional doses. Therefore, in this setting steroid-sensitive NS can be considered synonymous with MCD. The pathologic hallmark of disease is absence of visible alterations by light microscopy and effacement of foot processes by electron microscopy. Although the cause is unknown and it is likely that different subgroups of disease recognize a different pathogenesis, immunologic dysregulation and modifications of the podocyte are thought to synergize in altering the integrity of the glomerular basement membrane and therefore determining proteinuria. The mainstay of therapy is prednisone, but steroid-sensitive forms frequently relapse and this leads to a percentage of patients requiring second-line steroid-sparing immunosuppression. The outcome is variable, but forms of MCD that respond to steroids usually do not lead to chronic renal damage, whereas forms that are unresponsive to steroids may subsequently reveal themselves as FSGS. However, in a substantial number of patients the disease is recurrent and requires long-term immunosuppression, with significant morbidity because of side effects. Recent therapeutic advances, such as the use of anti-CD20 antibodies, have provided long-term remission off-therapy and suggest new hypotheses for disease pathogenesis.

Risk Factors for Cyclosporin A Nephrotoxicity in Children with Steroid-Dependant Nephrotic Syndrome

BACKGROUND AND OBJECTIVES Cyclosporin A (CsA) is a well-established treatment for steroid-dependent nephrotic syndrome (SDNS) that may, however, cause chronic ischemic renal lesions. The objective of the study was to assess the prevalence of CsA nephrotoxicity (CsAN) in protocol biopsies of children with SDNS. DESIGN, SETTINGS, PARTICIPANTS, & MEASUREMENTS From 1990 through 2008, we performed 71 renal biopsies in 53 patients with SDNS. The mean CsA C2 levels were 466 +/- 134 ng/ml, and the mean duration of treatment was 4.7 +/- 2.0 yr before biopsy (range 2.9 to 12.7 yr). RESULTS CsAN was observed in 22 (31%) of 71 renal biopsies. Of these, 11 corresponded to isolated vascular or tubular lesions, and 11 corresponded to combined vascular and tubular lesions. The majority of CsAN lesions were mild (17 of 22). In no cases were lesions graded as severe. By regression analysis, CsAN was positively associated with the use of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs) and with hyperuricemia and negatively associated with minimal-change lesions. By multivariate analysis, only association with the use of ACEIs or ARBs retained significance. Stratification of the population according to CsA C2 levels showed increased risk for CsAN for C2 levels >600 ng/ml. CONCLUSIONS Mild to moderate CsAN occurs in approximately one third of patients who have SDNS and are treated with CsA for >3 yr. Our data suggest that patients who require high dosages of CsA or treatment for hypertension, in particular when ACEIs/ARBs are used, are at higher risk for CsAN.

Reduced elastogenesis: a clue to the arteriosclerosis and emphysematous changes in Schimke immuno-osseous dysplasia?

BackgroundArteriosclerosis and emphysema develop in individuals with Schimke immuno-osseous dysplasia (SIOD), a multisystem disorder caused by biallelic mutations in SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1). However, the mechanism by which the vascular and pulmonary disease arises in SIOD remains unknown.MethodsWe reviewed the records of 65 patients with SMARCAL1 mutations. Molecular and immunohistochemical analyses were conducted on autopsy tissue from 4 SIOD patients.ResultsThirty-two of 63 patients had signs of arteriosclerosis and 3 of 51 had signs of emphysema. The arteriosclerosis was characterized by intimal and medial hyperplasia, smooth muscle cell hyperplasia and fragmented and disorganized elastin fibers, and the pulmonary disease was characterized by panlobular enlargement of air spaces. Consistent with a cell autonomous disorder, SMARCAL1 was expressed in arterial and lung tissue, and both the aorta and lung of SIOD patients had reduced expression of elastin and alterations in the expression of regulators of elastin gene expression.ConclusionsThis first comprehensive study of the vascular and pulmonary complications of SIOD shows that these commonly cause morbidity and mortality and might arise from impaired elastogenesis. Additionally, the effect of SMARCAL1 deficiency on elastin expression provides a model for understanding other features of SIOD.

Dental Abnormalities in Schimke Immuno-osseous Dysplasia

Described for the first time in 1971, Schimke immuno-osseous dysplasia (SIOD) is an autosomal-recessive multisystem disorder that is caused by bi-allelic mutations of SMARCAL1, which encodes a DNA annealing helicase. To define better the dental anomalies of SIOD, we reviewed the records from SIOD patients with identified bi-allelic SMARCAL1 mutations, and we found that 66.0% had microdontia, hypodontia, or malformed deciduous and permanent molars. Immunohistochemical analyses showed expression of SMARCAL1 in all developing teeth, raising the possibility that the malformations are cell-autonomous consequences of SMARCAL1 deficiency. We also found that stimulation of cultured skin fibroblasts from SIOD patients with the tooth morphogens WNT3A, BMP4, and TGFβ1 identified altered transcriptional responses, raising the hypothesis that the dental malformations arise in part from altered responses to developmental morphogens. To the best of our knowledge, this is the first systematic study of the dental anomalies associated with SIOD.