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NPHS2 mutations in late-onset focal segmental glomerulosclerosis: R229Q is a common disease-associated allele.

ABSTRACT: Mutations in NPHS2, encoding podocin, have been identified in childhood onset focal and segmental glomerulosclerosis (FSGS). The role of NPHS2 in adult disease is less well defined. We studied 30 families with FSGS and apparent autosomal recessive inheritance and 91 individuals with primary FSGS. We screened family members for NPHS2 mutations. NPHS2 mutations appeared to be responsible for disease in nine of these families. In six families, the affected individuals were compound heterozygotes for a nonconservative R229Q amino acid substitution. This R229Q variant has an allele frequency of 3.6% in a control population. In these families, R229Q was the only mutation identified on one of the two disease-associated NPHS2 alleles. We used in vitro-translated podocin and purified nephrin to investigate the effect of R229Q on their interaction and found decreased nephrin binding to the R229Q podocin. These data suggest that this common polymorphism contributes to the development of FSGS. Chromosomes bearing the R229Q mutation share a common haplotype defining an approximately 0.2-Mb region. R229Q appears to enhance susceptibility to FSGS in association with a second mutant NPHS2 allele. Identification of R229Q mutations may be of clinical importance, as NPHS2-associated disease appears to define a subgroup of FSGS patients unresponsive to corticosteroids.

SUBMITTER: Tsukaguchi H

PROVIDER: S-EPMC151634 | biostudies-literature | 2002 Dec

REPOSITORIES: biostudies-literature

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NPHS2 mutations in late-onset focal segmental glomerulosclerosis: R229Q is a common disease-associated allele.

Tsukaguchi Hiroyasu H Sudhakar Akulapalli A Le Tu Cam TC Nguyen Trang T Yao Jun J Schwimmer Joshua A JA Schachter Asher D AD Poch Esteban E Abreu Patricia F PF Appel Gerald B GB Pereira Aparecido B AB Kalluri Raghu R Pollak Martin R MR

The Journal of clinical investigation 20021201 11

Mutations in NPHS2, encoding podocin, have been identified in childhood onset focal and segmental glomerulosclerosis (FSGS). The role of NPHS2 in adult disease is less well defined. We studied 30 families with FSGS and apparent autosomal recessive inheritance and 91 individuals with primary FSGS. We screened family members for NPHS2 mutations. NPHS2 mutations appeared to be responsible for disease in nine of these families. In six families, the affected individuals were compound heterozygotes fo ...[more]

PMID: 12464671

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Project description:BACKGROUND:Focal segmental glomerulosclerosis is a kidney disease that is manifested as the nephrotic syndrome. It is often resistant to glucocorticoid therapy and progresses to end-stage renal disease in 50 to 70% of patients. Genetic studies have shown that familial focal segmental glomerulosclerosis is a disease of the podocytes, which are major components of the glomerular filtration barrier. However, the molecular cause in over half the cases of primary focal segmental glomerulosclerosis is unknown, and effective treatments have been elusive. METHODS:We performed whole-genome linkage analysis followed by high-throughput sequencing of the positive-linkage area in a family with autosomal recessive focal segmental glomerulosclerosis (index family) and sequenced a newly discovered gene in 52 unrelated patients with focal segmental glomerulosclerosis. Immunohistochemical studies were performed on human kidney-biopsy specimens and cultured podocytes. Expression studies in vitro were performed to characterize the functional consequences of the mutations identified. RESULTS:We identified two mutations (A159P and Y695X) in MYO1E, which encodes a nonmuscle class I myosin, myosin 1E (Myo1E). The mutations in MYO1E segregated with focal segmental glomerulosclerosis in two independent pedigrees (the index family and Family 2). Patients were homozygous for the mutations and did not have a response to glucocorticoid therapy. Electron microscopy showed thickening and disorganization of the glomerular basement membrane. Normal expression of Myo1E was documented in control human kidney-biopsy specimens in vivo and in glomerular podocytes in vitro. Transfection studies revealed abnormal subcellular localization and function of the A159P-Myo1E mutant. The Y695X mutation causes loss of calmodulin binding and of the tail domains of Myo1E. CONCLUSIONS:MYO1E mutations are associated with childhood-onset, glucocorticoid-resistant focal segmental glomerulosclerosis. Our data provide evidence of a role of Myo1E in podocyte function and the consequent integrity of the glomerular filtration barrier.

Project description:Focal segmental glomerulosclerosis (FSGS) is a leading cause of kidney disease worldwide. The presumed etiology of primary FSGS is a plasma factor with responsiveness to immunosuppressive therapy and a risk of recurrence after kidney transplant-important disease characteristics. In contrast, adaptive FSGS is associated with excessive nephron workload due to increased body size, reduced nephron capacity, or single glomerular hyperfiltration associated with certain diseases. Additional etiologies are now recognized as drivers of FSGS: high-penetrance genetic FSGS due to mutations in one of nearly 40 genes, virus-associated FSGS, and medication-associated FSGS. Emerging data support the identification of a sixth category: APOL1 risk allele-associated FSGS in individuals with sub-Saharan ancestry. The classification of a particular patient with FSGS relies on integration of findings from clinical history, laboratory testing, kidney biopsy, and in some patients, genetic testing. The kidney biopsy can be helpful, with clues provided by features on light microscopy (e.g, glomerular size, histologic variant of FSGS, microcystic tubular changes, and tubular hypertrophy), immunofluorescence (e.g, to rule out other primary glomerulopathies), and electron microscopy (e.g., extent of podocyte foot process effacement, podocyte microvillous transformation, and tubuloreticular inclusions). A complete assessment of renal histology is important for establishing the parenchymal setting of segmental glomerulosclerosis, distinguishing FSGS associated with one of many other glomerular diseases from the clinical-pathologic syndrome of FSGS. Genetic testing is beneficial in particular clinical settings. Identifying the etiology of FSGS guides selection of therapy and provides prognostic insight. Much progress has been made in our understanding of FSGS, but important outstanding issues remain, including the identity of the plasma factor believed to be responsible for primary FSGS, the value of routine implementation of genetic testing, and the identification of more effective and less toxic therapeutic interventions for FSGS.

Project description:Natural mutations such as lysine 255 to glutamic acid (K to E), threonine 259 to isoleucine (T to I) and serine 262 to proline (S to P) that occur within the actin binding domain of alpha-actinin-4 (ACTN4) cause an autosomal dominant form of focal segmental glomerulosclerosis (FSGS) in affected humans. This appears due to elevated actin binding propensity in podocytes resulting in a 'frozen' cytoskeleton. What is challenging is how this cellular behavior would be compatible with other cell functions that rely on cytoskeleton plasticity. Our previous finding revealed that wild type ACTN4 can be phosphorylated at tyrosine 4 and 31 upon stimulation by epidermal growth factor (EGF) to reduce the binding to actin cytoskeleton. We queried whether the elevated actin binding activity of FSGS mutants can be downregulated by EGF-mediated phosphorylation, to discern a mechanism by which the actin-cytoskeleton can be released in FSGS. In this manuscript, we first constructed variants with Y4/31E to mimic the phosphorylation at tyrosines 4 and 31 based on earlier modeling simulations that predicted that this would bury the actin binding domains and lead to a decrease in actin binding activity. We found that Y4/31E significantly reduced the actin binding activity of K255E, T259I and S262P, dramatically preventing them from aggregating in, and inhibiting motility of, podocytes, fibroblasts and melanoma cells. A putative kinase target site at Y265 in the actin binding domain was also generated as a phosphomimetic ACTN4 Y265E that demonstrated even greater binding to actin filaments than K255E and the other FSGS mutants. That the tyrosine kinase regulation of FSGS mutation binding to actin filaments can occur in cells was shown by phosphorylation on Y4 and Y31 of the K225E after extended exposure of cells to EGF, with a decrease in ACTN4 aggregates in fibroblasts. These findings will provide evidence for targeting the N-termini of FSGS ACTN4 mutants to downregulate their actin binding activities for ameliorating the glomerulosclerotic phenotype of patients.

Dataset Information

NPHS2 mutations in late-onset focal segmental glomerulosclerosis: R229Q is a common disease-associated allele.

Publications

NPHS2 mutations in late-onset focal segmental glomerulosclerosis: R229Q is a common disease-associated allele.

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