Rodney D. Gilbert

Simultaneous sequencing of 24 genes associated with steroid-resistant nephrotic syndrome

BACKGROUND AND OBJECTIVES Up to 95% of children presenting with steroid-resistant nephrotic syndrome in early life will have a pathogenic single-gene mutation in 1 of 24 genes currently associated with this disease. Others may be affected by polymorphic variants. There is currently no accepted diagnostic algorithm for clinical genetic testing. The hypothesis was that the increasing reliability of next generation sequencing allows comprehensive one-step genetic investigation of this group and similar patient groups. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This study used next generation sequencing to screen 446 genes, including the 24 genes known to be associated with hereditary steroid-resistant nephrotic syndrome. The first 36 pediatric patients collected through a national United Kingdom Renal Registry were chosen with comprehensive phenotypic detail. Significant variants detected by next generation sequencing were confirmed by conventional Sanger sequencing. RESULTS Analysis revealed known and novel disease-associated variations in expected genes such as NPHS1, NPHS2, and PLCe1 in 19% of patients. Phenotypically unexpected mutations were also detected in COQ2 and COL4A4 in two patients with isolated nephropathy and associated sensorineural deafness, respectively. The presence of an additional heterozygous polymorphism in WT1 in a patient with NPHS1 mutation was associated with earlier-onset disease, supporting modification of phenotype through genetic epistasis. CONCLUSIONS This study shows that next generation sequencing analysis of pediatric steroid-resistant nephrotic syndrome patients is accurate and revealing. This analysis should be considered part of the routine genetic workup of diseases such as childhood steroid-resistant nephrotic syndrome, where the chance of genetic mutation is high but requires sequencing of multiple genes.

Rituximab therapy for steroid-dependent minimal change nephrotic syndrome

We present a patient with steroid-sensitive but high-dose steroid-dependent nephrotic syndrome who was treated with rituximab. For 9 months following therapy she had undetectable CD19 cells in the peripheral circulation. She remained in remission during this period even though therapy was reduced to low-dose, alternate day prednisolone only. After 9 months, CD19 cells were once again detectable. Shortly after CD19 cells became detectable again she relapsed. We conclude that B-lymphocytes play a central role in the pathogenesis of idiopathic minimal change nephrotic syndrome (MCNS) and that rituximab may have a useful role in the management of steroid-dependent patients.

Mutations in PIK3R1 Cause SHORT Syndrome

SHORT syndrome is a rare, multisystem disease characterized by short stature, anterior-chamber eye anomalies, characteristic facial features, lipodystrophy, hernias, hyperextensibility, and delayed dentition. As part of the FORGE (Finding of Rare Disease Genes) Canada Consortium, we studied individuals with clinical features of SHORT syndrome to identify the genetic etiology of this rare disease. Whole-exome sequencing in a family trio of an affected child and unaffected parents identified a de novo frameshift insertion, c.1906_1907insC (p.Asn636Thrfs*18), in exon 14 of PIK3R1. Heterozygous mutations in exon 14 of PIK3R1 were subsequently identified by Sanger sequencing in three additional affected individuals and two affected family members. One of these mutations, c.1945C>T (p.Arg649Trp), was confirmed to be a de novo mutation in one affected individual and was also identified and shown to segregate with the phenotype in an unrelated family. The other mutation, a de novo truncating mutation (c.1971T>G [p.Tyr657*]), was identified in another affected individual. PIK3R1 is involved in the phosphatidylinositol 3 kinase (PI3K) signaling cascade and, as such, plays an important role in cell growth, proliferation, and survival. Functional studies on lymphoblastoid cells with the PIK3R1 c.1906_1907insC mutation showed decreased phosphorylation of the downstream S6 target of the PI3K-AKT-mTOR pathway. Our findings show that PIK3R1 mutations are the major cause of SHORT syndrome and suggest that the molecular mechanism of disease might involve downregulation of the PI3K-AKT-mTOR pathway.

Mosaicism of the UDP-Galactose Transporter SLC35A2 Causes a Congenital Disorder of Glycosylation

Biochemical analysis and whole-exome sequencing identified mutations in the Golgi-localized UDP-galactose transporter SLC35A2 that define an undiagnosed X-linked congenital disorder of glycosylation (CDG) in three unrelated families. Each mutation reduced UDP-galactose transport, leading to galactose-deficient glycoproteins. Two affected males were somatic mosaics, suggesting that a wild-type SLC35A2 allele may be required for survival. In infancy, the commonly used biomarker transferrin showed abnormal glycosylation, but its appearance became normal later in childhood, without any corresponding clinical improvement. This may indicate selection against cells carrying the mutant allele. To detect other individuals with such mutations, we suggest transferrin testing in infancy. Here, we report somatic mosaicism in CDG, and our work stresses the importance of combining both genetic and biochemical diagnoses.

Genomic and clinical profiling of a national Nephrotic Syndrome cohort advocates a precision medicine approach to disease management

Steroid Resistant Nephrotic Syndrome (SRNS) in children and young adults has differing etiologies with monogenic disease accounting for 2.9-30% in selected series. Using whole exome sequencing we sought to stratify a national population of children with SRNS into monogenic and non-monogenic forms, and further define those groups by detailed phenotypic analysis. Pediatric patients with SRNS were identified via a national United Kingdom Renal Registry. Whole exome sequencing was performed on 187 patients, of which 12% have a positive family history with a focus on the 53 genes currently known to be associated with nephrotic syndrome. Genetic findings were correlated with individual case disease characteristics. Disease causing variants were detected in 26.2% of patients. Most often this occurred in the three most common SRNS-associated genes: NPHS1, NPHS2, and WT1 but also in 14 other genes. The genotype did not always correlate with expected phenotype since mutations in OCRL, COL4A3, and DGKE associated with specific syndromes were detected in patients with isolated renal disease. Analysis by primary/presumed compared with secondary steroid resistance found 30.8% monogenic disease in primary compared with none in secondary SRNS permitting further mechanistic stratification. Genetic SRNS progressed faster to end stage renal failure, with no documented disease recurrence post-transplantation within this cohort. Primary steroid resistance in which no gene mutation was identified had a 47.8% risk of recurrence. In this unbiased pediatric population, whole exome sequencing allowed screening of all current candidate genes. Thus, deep phenotyping combined with whole exome sequencing is an effective tool for early identification of SRNS etiology, yielding an evidence-based algorithm for clinical management.

Mutations in phospholipase C epsilon 1 are not sufficient to cause diffuse mesangial sclerosis.

Diffuse mesangial sclerosis occurs as an isolated abnormality or as a part of a syndrome. Recently, mutations in phospholipase C epsilon 1 (PLCE1) were found to cause a nonsyndromic, autosomal recessive form of this disease. Here we describe three children from one consanguineous kindred of Pakistani origin with diffuse mesangial sclerosis who presented with congenital or infantile nephrotic syndrome. Homozygous mutations in PLCE1 (also known as KIAA1516, PLCE, or NPHS3) were identified following genome-wide mapping of single-nucleotide polymorphisms. All affected children were homozygous for a four-basepair deletion in exon 3, which created a premature translational stop codon. Analysis of the asymptomatic father of two of the children revealed that he was also homozygous for the same mutation. We conclude this nonpenetrance may be due to compensatory mutations at a second locus and that mutation within PLCE1 is not always sufficient to cause diffuse mesangial sclerosis.

Eculizumab therapy for atypical haemolytic uraemic syndrome due to a gain-of-function mutation of complement factor B

BackgroundAtypical haemolytic uraemic syndrome (aHUS) is caused by dysregulated complement activation. A humanised anti-C5 monoclonal antibody has recently become available for treatment of this conditionCase-Diagnosis/TreatmentWe present the first description of an infant with an activating mutation of complement factor B successfully treated with eculizumab. On standard doses she had evidence of ongoing C5 cleavage despite a good clinical response.ConclusionsEculizumab is effective therapy for aHUS associated with factor B mutations, but recommended doses may not be adequate for all patients.

Does dysregulated complement activation contribute to haemolytic uraemic syndrome secondary to Streptococcus pneumoniae

We describe two patients with haemolytic uraemic syndrome (HUS) associated with invasive Streptococcus pneumoniae infection. Both patients had transiently reduced serum concentrations of complement C3. One had reduced expression of CD46 and never recovered renal function. No constitutive defect in regulation of the alternative pathway of complement activation was demonstrated in the second patient but there was an apparent improvement in her condition after administration of eculizumab. The most widely accepted mechanism for pneumococcal HUS is endothelial cell damage by pre-formed antibodies against the Thomsen-Friedenreich antigen. This explanation does not bear rigorous scrutiny. We postulate that transiently dysregulated complement activation may play a role in the pathogenesis of pneumococcal disease. We further postulate that the mechanism could be enhanced binding of factor H to the neuraminidase-altered surface of endothelial cells or reduced binding of factor H to the endothelial cell surface mediated by competitive binding of factor H by pneumococcal surface protein C (pspC).

Cisplatin-induced haemolytic uraemic syndrome associated with a novel intronic mutation of CD46 treated with eculizumab

A 2-year-old patient with a neuroblastoma developed haemolytic uraemic syndrome (HUS) following treatment with cisplatin and carboplatin. Following treatment with eculizumab, there was a substantial improvement in renal function with the recovery of the platelet count and the cessation of haemolysis. Subsequent investigations showed a novel, heterozygous CD46 splice site mutation with reduced peripheral blood neutrophil CD46 expression. Withdrawal of eculizumab was followed by the recurrence of disease activity, which resolved with re-introduction of therapy. Abnormal regulation of complement may be associated with other cases of cisplatin-induced HUS and treatment with eculizumab may be appropriate for other affected individuals.

Microscopic polyarteritis with renal and cerebral involvement.

Abstract We report a 15-year-old girl who presented with renal failure requiring dialysis and a diagnosis of p-ANCA-associated vasculitis. She subsequently developed cerebral vasculitis during treatment with oral cyclophosphamide and prednisolone. Treatment with intravenous cyclophosphamide and plasma exchange produced a complete recovery.