Nephrin mutations cause childhood- and adult-onset focal segmental glomerulosclerosis
2009; Elsevier BV; Volume: 76; Issue: 12 Linguagem: Inglês
10.1038/ki.2009.381
ISSN1523-1755
AutoresSheila Santín, Rafael García‐Maset, Patricia Ruíz, Isabel Giménez, I. Zamora, Antonia Peña, A. Madrid, Juan Antonio Camacho, Gloria Fraga, Ana Sánchez‐Moreno, María Ángeles Cobo, Carmen Bernis, Alberto Ortíz, Augusto Luque de Pablos, Guillem Pintos‐Morell, Maria Luisa Justa, Emilia Hidalgo-Barquero, Patricia Fernández‐Llama, José Ballarín, Elisabet Ars, Roser Torrá, on behalf of the FSGS Spanish Study Group,
Tópico(s)Tuberous Sclerosis Complex Research
ResumoMutations in the NPHS1 gene cause congenital nephrotic syndrome of the Finnish type presenting before the first 3 months of life. Recently, NPHS1 mutations have also been identified in childhood-onset steroid-resistant nephrotic syndrome and milder courses of disease, but their role in adults with focal segmental glomerulosclerosis remains unknown. Here we developed an in silico scoring matrix to evaluate the pathogenicity of amino-acid substitutions using the biophysical and biochemical difference between wild-type and mutant amino acid, the evolutionary conservation of the amino-acid residue in orthologs, and defined domains, with the addition of contextual information. Mutation analysis was performed in 97 patients from 89 unrelated families, of which 52 presented with steroid-resistant nephrotic syndrome after 18 years of age. Compound heterozygous or homozygous NPHS1 mutations were identified in five familial and seven sporadic cases, including one patient 27 years old at onset of the disease. Substitutions were classified as 'severe' or 'mild' using this in silico approach. Our results suggest an earlier onset of the disease in patients with two 'severe' mutations compared to patients with at least one 'mild' mutation. The finding of mutations in a patient with adult-onset focal segmental glomerulosclerosis indicates that NPHS1 analysis could be considered in patients with later onset of the disease. Mutations in the NPHS1 gene cause congenital nephrotic syndrome of the Finnish type presenting before the first 3 months of life. Recently, NPHS1 mutations have also been identified in childhood-onset steroid-resistant nephrotic syndrome and milder courses of disease, but their role in adults with focal segmental glomerulosclerosis remains unknown. Here we developed an in silico scoring matrix to evaluate the pathogenicity of amino-acid substitutions using the biophysical and biochemical difference between wild-type and mutant amino acid, the evolutionary conservation of the amino-acid residue in orthologs, and defined domains, with the addition of contextual information. Mutation analysis was performed in 97 patients from 89 unrelated families, of which 52 presented with steroid-resistant nephrotic syndrome after 18 years of age. Compound heterozygous or homozygous NPHS1 mutations were identified in five familial and seven sporadic cases, including one patient 27 years old at onset of the disease. Substitutions were classified as 'severe' or 'mild' using this in silico approach. Our results suggest an earlier onset of the disease in patients with two 'severe' mutations compared to patients with at least one 'mild' mutation. The finding of mutations in a patient with adult-onset focal segmental glomerulosclerosis indicates that NPHS1 analysis could be considered in patients with later onset of the disease. Idiopathic nephrotic syndrome (NS) represents a heterogeneous group of glomerular disorders occurring mainly in children. It is generally divided into steroid sensitive (SSNS) and steroid resistant (SRNS), depending on the patient's response to steroid therapy. Over the past decade, mutations in genes encoding podocyte proteins have been identified in several forms of hereditary NS.1.Jeanpierre C. Denamur E. Henry I. et al.Identification of constitutional WT1 mutations, in patients with isolated diffuse mesangial sclerosis, and analysis of genotype/phenotype correlations by use of a computerized mutation database.Am J Hum Genet. 1998; 62: 824-833Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 2.Boute N. Gribouval O. Roselli S. et al.NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome.Nat Genet. 2000; 24: 349-354Crossref PubMed Scopus (1134) Google Scholar, 3.Kaplan J.M. Kim S.H. North K.N. et al.Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis.Nat Genet. 2000; 24: 251-256Crossref PubMed Scopus (989) Google Scholar, 4.Kim J.M. Wu H. Green G. et al.CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility.Science. 2003; 300: 1298-1300Crossref PubMed Scopus (393) Google Scholar, 5.Winn M.P. Conlon P.J. Lynn K.L. et al.A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis.Science. 2005; 308: 1801-1804Crossref PubMed Scopus (815) Google Scholar, 6.Hinkes B. Wiggins R.C. Gbadegesin R. et al.Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible.Nat Genet. 2006; 38: 1397-1405Crossref PubMed Scopus (406) Google Scholar Mutations in the NPHS1 gene are responsible for congenital NS of the Finnish type (CNF), which is an autosomal recessive disorder characterized by massive proteinuria often starting in utero.7.Hvenainen E.K. Hallman N. Hjelt L. Nephrotic syndrome in newborn and young infants.Ann Paediatr Fenn. 1956; 2: 227-241PubMed Google Scholar Kidney biopsy shows irregular microcystic dilatation of proximal tubules8.Patrakka J. Kestila M. Wartiovaara J. et al.Congenital nephrotic syndrome (NPHS1): features resulting from different mutations in Finnish patients.Kidney Int. 2000; 58: 972-980Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar and the disease used to lead to death in the neonatal period, but nowadays it can be treated by dialysis and nutritional support, followed by renal transplantation in early childhood.9.Holmberg C. Antikainen M. Ronnholm K. et al.Management of congenital nephrotic syndrome of the Finnish type.Pediatr Nephrol. 1995; 9: 87-93Crossref PubMed Scopus (160) Google Scholar The human NPHS1 gene is located at the long arm of chromosome 19, 19q13.1, and contains 29 exons.10.Kestila M. Lenkkeri U. Mannikko M. et al.Positionally cloned gene for a novel glomerular protein—nephrin—is mutated in congenital nephrotic syndrome.Mol Cell. 1998; 1: 575-582Abstract Full Text Full Text PDF PubMed Scopus (1480) Google Scholar The protein product, termed 'nephrin,' is a putative member of the immunoglobulin-like superfamily.11.Khoshnoodi J. Sigmundsson K. Ofverstedt L.G. et al.Nephrin promotes cell–cell adhesion through homophilic interactions.Am J Pathol. 2003; 163: 2337-2346Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar,12.Patari-Sampo A. Ihalmo P. Holthofer H. Molecular basis of the glomerular filtration: nephrin and the emerging protein complex at the podocyte slit diaphragm.Ann Med. 2006; 38: 483-492Crossref PubMed Scopus (57) Google Scholar Mutations in the NPHS2 gene were initially described in early onset SRNS.2.Boute N. Gribouval O. Roselli S. et al.NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome.Nat Genet. 2000; 24: 349-354Crossref PubMed Scopus (1134) Google Scholar However, some cases with late onset disease have been described by Tsukaguchi et al.13.Tsukaguchi H. Sudhakar A. Le T.C. et al.NPHS2 mutations in late-onset focal segmental glomerulosclerosis: R229Q is a common disease-associated allele.J Clin Invest. 2002; 110: 1659-1666Crossref PubMed Scopus (208) Google Scholar and Machuca et al.14.Machuca E. Hummel A. Nevo F. et al.Clinical and epidemiological assessment of steroid-resistant nephrotic syndrome associated with the NPHS2 R229Q variant.Kidney Int. 2009; 75: 727-735Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Variants in both NPHS1 and NPHS2 genes have been reported to occur together in a few number of families with congenital focal segmental glomerulosclerosis (FSGS)15.Koziell A. Grech V. Hussain S. et al.Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration.Hum Mol Genet. 2002; 11: 379-388Crossref PubMed Scopus (219) Google Scholar or CNF,16.Schultheiss M. Ruf R.G. Mucha B.E. et al.No evidence for genotype/phenotype correlation in NPHS1 and NPHS2 mutations.Pediatr Nephrol. 2004; 19: 1340-1348Crossref PubMed Scopus (51) Google Scholar suggesting the presence of 'oligogenicity' in this disorder. At present, more than 90 mutations in NPHS1 have been identified. These mutations are scattered along the NPHS1 gene, most of them being private mutations.15.Koziell A. Grech V. Hussain S. et al.Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration.Hum Mol Genet. 2002; 11: 379-388Crossref PubMed Scopus (219) Google Scholar, 16.Schultheiss M. Ruf R.G. Mucha B.E. et al.No evidence for genotype/phenotype correlation in NPHS1 and NPHS2 mutations.Pediatr Nephrol. 2004; 19: 1340-1348Crossref PubMed Scopus (51) Google Scholar, 17.Lenkkeri U. Mannikko M. McCready P. et al.Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations.Am J Hum Genet. 1999; 64: 51-61Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 18.Beltcheva O. Martin P. Lenkkeri U. et al.Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome.Hum Mutat. 2001; 17: 368-373Crossref PubMed Scopus (141) Google Scholar, 19.Lahdenkari A.T. Kestila M. Holmberg C. et al.Nephrin gene (NPHS1) in patients with minimal change nephrotic syndrome (MCNS).Kidney Int. 2004; 65: 1856-1863Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 20.Heeringa S.F. Vlangos C.N. Chernin G. et al.Thirteen novel NPHS1 mutations in a large cohort of children with congenital nephrotic syndrome.Nephrol Dial Transplant. 2008; 23: 3527-3533Crossref PubMed Scopus (55) Google Scholar The high percentage of NPHS1 missense mutations represents a diagnostic challenge as in some cases it is difficult to differentiate between disease-causing variant and a neutral one. In silico approaches have recently been developed for the evaluation of amino-acid substitutions in several human disease genes.21.Abkevich V. Zharkikh A. Deffenbaugh A.M. et al.Analysis of missense variation in human BRCA1 in the context of interspecific sequence variation.J Med Genet. 2004; 41: 492-507Crossref PubMed Scopus (128) Google Scholar, 22.Tavtigian S.V. Deffenbaugh A.M. Yin L. et al.Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral.J Med Genet. 2006; 43: 295-305Crossref PubMed Scopus (476) Google Scholar, 23.Rossetti S. Consugar M.B. Chapman A.B. et al.Comprehensive molecular diagnostics in autosomal dominant polycystic kidney disease.J Am Soc Nephrol. 2007; 18: 2143-2160Crossref PubMed Scopus (298) Google Scholar, 24.Barnetson R.A. Cartwright N. van V.A. et al.Classification of ambiguous mutations in DNA mismatch repair genes identified in a population-based study of colorectal cancer.Hum Mutat. 2008; 29: 367-374Crossref PubMed Scopus (68) Google Scholar These approaches take into consideration a combination of a multi-sequence alignment (MSA) of orthologous proteins and a measure of the chemical difference between the amino acids observed at the mutation point. These factors were used as in silico predictors in a scoring matrix for the evaluation of missense substitutions in the NPHS1 gene. Classically, NPHS1 mutations have been observed in children presenting with NS within days after birth and before 3 months of life.25.Hinkes B.G. Mucha B. Vlangos C.N. et al.Nephrotic syndrome in the first year of life: two thirds of cases are caused by mutations in 4 genes (NPHS1, NPHS2, WT1, and LAMB2).Pediatrics. 2007; 119: e907-e919Crossref PubMed Scopus (323) Google Scholar Recently, Philippe et al.26.Philippe A. Nevo F. Esquivel E.L. et al.Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome.J Am Soc Nephrol. 2008; 19: 1871-1878Crossref PubMed Scopus (105) Google Scholar identified NPHS1 mutations in children with later onset SRNS (between 5 months and 8 years). However, the role of NPHS1 in adults with FSGS remains unknown. Our aim was to study if NPHS1 mutations could be responsible not only for congenital-onset and childhood-onset but also for adult-onset FSGS. NPHS1 mutation screening was performed in 97 patients from 89 families by direct DNA sequencing. Homozygous or compound heterozygous NPHS1 substitutions were identified in 12 cases, of whom 1 was familial with two affected siblings, 4 were only children of consanguineous parents, and 7 were sporadic cases (Table 1). Two of these twelve cases carried additional variants in the NPHS2 gene. In one CNF case (patient 19) we found the p.R229Q NPHS2 variant in heterozygous state with two NPHS1 mutations. Moreover, two compound heterozygous NPHS2 variants (p.P20L and p.E237Q) in conjunction with a homozygous NPHS1 mutation were detected in one patient with FSGS congenital NS (patient 41; Table 2).Table 1Overview of genotypic and phenotypic dataFamilial SRNSSporadic SRNSCongenital onsetEarly childhood onsetLate childhood onsetAdolescent onsetAdult onsetNo. of patients (families) studied21 (13)76 (76)11 (10)21 (16)8 (6)5 (5)52 (52)No. of patients (families) with two NPHS1 mutations6 (5)7 (7)8 (8)3 (2)1 (1)1 (1)Mutation detection rate38.5%10%80%13%17%2%No. of patients (families) with one NPHS1 mutation2 (1)2 (2)2 (1)2 (2)No. of patients (families) with one NPHS1 variant of unknown effect2 (2)2 (2)Age at onset in patients with 2 'severe' NPHS1 mutations1.1±1.2 months; n=8 patientsAge at onset in patients with 1 'mild'/1 'severe' or 2 'mild' NPHS1 mutations100±130 months; n=5 patientsSRNS, steroid-resistant nephrotic syndrome.When 2 members of a single family presented an age at onset of the disease that fell between two different categories, we included both in the category of the patient presenting the earlier age at onset of NS. Open table in a new tab Table 2Clinical data of patients with SRNS and NPHS1 substitutionsPatientGenderAge of onset NS (months)Renal biopsyTherapyEvolutionTx/RecurrenceNPHS1 mutation 1 (MG)aNPHS1 mutations, defined in this table as substitutions detected in the NPHS1 gene; MG, mutation group (classification of NPHS1 substitutions according to Table 3).NPHS1 mutation 2 (MG)aNPHS1 mutations, defined in this table as substitutions detected in the NPHS1 gene; MG, mutation group (classification of NPHS1 substitutions according to Table 3).NPHS2 variantsPatients with CNFbThese patients presented with 2 NPHS1 mutations in homozygous or heterozygous state. 18M0.6CNF—ESRD at 4 yearsYes/Noc.1701C>Ap.C567X (A)c.2417C>Gp.A806D (B)Not identified 19F0.2CNF—ESRD at 1 yearYes/2 TxcThese patients presented with chronic rejection nephropathy.c.1701C>Ap.C567X (A)c.3343G>Tp.E1115X (A)c.686G>Ap.R229Q 20M0.2CNF—ESRD at 1 yearYes/2 TxcThese patients presented with chronic rejection nephropathy.c.1701C>Ap.C567X (A)c.1868G>Tp.C623F (B)Not identified 38dOnly child of consanguineous parents.F1Not performed—ESRD at 1 yearNoc.2540_43delp.T847fsX903 (A)c.2540_43delp.T847fsX903 (A)Not identified 256dOnly child of consanguineous parents.M1Not performed—ESRD at 3 monthsNoc.1379G>Ap.R460Q (I)eThis mutation has previously been described in patients with a severe phenotype.25c.1379G>Ap.R460Q (I)eThis mutation has previously been described in patients with a severe phenotype.25Not identifiedPatients with congenital FSGSbThese patients presented with 2 NPHS1 mutations in homozygous or heterozygous state. 40dOnly child of consanguineous parents.M3FSGSCsESRD at 2 yearsYes/2 TxcThese patients presented with chronic rejection nephropathy.c.2143G>Cp.G715R (B)c.2143G>Cp.G715R (B)Not identified 41dOnly child of consanguineous parents.M3FSGSCsNormal Cr at 3 yearsNoc.1538T>Cp.L513P (B)c.1538T>Cp.L513P (B)c.59C>Tp.P20 L+c.709G>Cp.E237Q 177F0DMS—CKD stage IV at 2 yearsNoc.139delGp.A47fsX127 (A)c.3478C>Tp.R1160X (A)Not identifiedPatients with childhood FSGSbThese patients presented with 2 NPHS1 mutations in homozygous or heterozygous state. 79-1fSiblings with the same parents.F72FSGSCs, CsA, MMFNormal Cr at 15 yearsNoc.1099 C>Tp.R367C (B)c.361G>Ap.E121K (I)Not identified 79-2fSiblings with the same parents.M12FSGS*Cs, CsANormal Cr at 7 yearsNoc.1099 C>Tp.R367C (B)c.361G>Ap.E121K (I)Not identified 182F84FSGSCs, CsA, CPNormal Cr at 11 yearsNoc.1379G>Ap.R460Q (I)eThis mutation has previously been described in patients with a severe phenotype.25c.2928G>Tp.R976S (B)gThis mutation has previously been described in patients with a mild phenotype.26Not identified 198F8FSGSCs, CsANormal Cr at 2 yearsNoc.791C>Gp.P264R (C)c.2026C>Tp.P676S (I)Not identifiedPatient with adulthood FSGSbThese patients presented with 2 NPHS1 mutations in homozygous or heterozygous state. 140F324FSGS*Cs, CP, TacrohThis patient responded partially to CP treatment and she is now treated with angiotensin-converting enzyme inhibitor and Tacro.Normal Cr at 29 yearsNoc.2479C>Ap.R827X (A)c.2928G>Tp.R976S(B)gThis mutation has previously been described in patients with a mild phenotype.26/ c.2971G>Cp.V991 L (NV)iThis patient has 3 heterozygous missense substitutions, but the p.V991L was considered a neutral variant.Not identifiedPatients with one NPHS1 mutation 21-1fSiblings with the same parents.F24FSGSCs, CsA, MMFNormal Cr at 8 yearsNoc.3250_3251insGp.V1084fsX1095 (A)Not identifiedc.59C>Tp.P20 L 21-2fSiblings with the same parents.M24FSGSCs, CsA, MMFNormal Cr at 6 yearsNoc.3250_3251insGp.V1084fsX1095 (A)Not identifiedc.59C>Tp.P20 L 85M115FSGSCs, CsA, CPESRD at 12 yearsYes/Yesc.3250_3251insGp.V1084fsX1095 (A)Not identifiedNot identified 88F132FSGS*Cs, CsA, CPESRD at 14 yearsYes/Yesc.1610C>Tp.T537 M (B)c.1223G>Ap.R408Q (P)Not identifiedPatients with one NPHS1 variant of unknown effect 122M324FSGSCs, CsAESRD at 28 yearsYes/Yesc.563A>Tp.N188I (I)Not identifiedNot identified 189F348FSGSCs, CsAESRD at 34 yearsYes/NocThese patients presented with chronic rejection nephropathy.c.791C>Gp.P264R (C)Not identifiedNot identifiedCKD, chronic kidney disease; CP, cyclophosphamide; Cr, creatinine; Cs, corticosteroids; CsA, cyclosporin A; DMS, diffuse mesangial sclerosis; ESRD, end-stage renal disease; F, female; FSGS, focal segmental glomerulosclerosis; FSGS*, mesangioproliferative lesions with FSGS; M, male; MMF, mophetil micophenolate; Tacro, tacrolimus; Tx, kidney transplantation.a NPHS1 mutations, defined in this table as substitutions detected in the NPHS1 gene; MG, mutation group (classification of NPHS1 substitutions according to Table 3).b These patients presented with 2 NPHS1 mutations in homozygous or heterozygous state.c These patients presented with chronic rejection nephropathy.d Only child of consanguineous parents.e This mutation has previously been described in patients with a severe phenotype.25.Hinkes B.G. Mucha B. Vlangos C.N. et al.Nephrotic syndrome in the first year of life: two thirds of cases are caused by mutations in 4 genes (NPHS1, NPHS2, WT1, and LAMB2).Pediatrics. 2007; 119: e907-e919Crossref PubMed Scopus (323) Google Scholarf Siblings with the same parents.g This mutation has previously been described in patients with a mild phenotype.26.Philippe A. Nevo F. Esquivel E.L. et al.Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome.J Am Soc Nephrol. 2008; 19: 1871-1878Crossref PubMed Scopus (105) Google Scholarh This patient responded partially to CP treatment and she is now treated with angiotensin-converting enzyme inhibitor and Tacro.i This patient has 3 heterozygous missense substitutions, but the p.V991L was considered a neutral variant. Open table in a new tab SRNS, steroid-resistant nephrotic syndrome. When 2 members of a single family presented an age at onset of the disease that fell between two different categories, we included both in the category of the patient presenting the earlier age at onset of NS. CKD, chronic kidney disease; CP, cyclophosphamide; Cr, creatinine; Cs, corticosteroids; CsA, cyclosporin A; DMS, diffuse mesangial sclerosis; ESRD, end-stage renal disease; F, female; FSGS, focal segmental glomerulosclerosis; FSGS*, mesangioproliferative lesions with FSGS; M, male; MMF, mophetil micophenolate; Tacro, tacrolimus; Tx, kidney transplantation. In addition, only a single pathogenic NPHS1 mutation was identified in two patients with sporadic SRNS, one of them (patient 88) carrying also the p.R408Q NPHS1 neutral variant in heterozygosity (Table 2). Moreover, we identified two siblings with only one NPHS1 mutation and the p.P20L variant in the NPHS2 gene (family 21). NPHS1 variants of unknown significance (p.N188I and p.P264R) were heterozygous in two cases (Table 2) and four patients carried a single new highly neutral NPHS1 variant (p.Q259E in one case, p.L392P in three cases). Finally, the p.R408Q neutral variant was found in seven cases without any other variant in compound heterozygosity. On the other hand, causative mutations in either NPHS2 or WT1 gene were found in eight cases (unpublished results), bearing no NPHS1 variants. These patients were also screened for TRPC6, ACTN4, and CD2AP and no mutations were found. The detection rate of NPHS1 mutations for familial cases was 38% (5 of 13) and 10% (7 of 76) for sporadic cases. Patients with only one NPHS1 mutation were not included to calculate this mutation detection rates (Table 1). If we discard patients clinically diagnosed as CNF, mutations were identified in 27% (3 of 11) of the familial cases and 6% (4 of 73) of the sporadic cases studied in the present paper. The frequency of NPHS1 mutations in adults was 2% (1 of 52) but seven times higher in children (14%, 3 of 22). Of the 25 NPHS1 substitutions hereby detected, 72% (18 of 25) were missense. A large majority of these missense changes were within the immunoglobulin motifs of the extracellular domain. As shown in Table 3, nine variants were novel (36%), consisting of seven missense, one nonsense, and one frameshift mutation. Although most mutations in this gene are private, we identified one mutation (p.C567X) in three out of five nonrelated patients with CNF (60%). Moreover, the p.R408Q neutral variant has an allele frequency of 4.5% (8 of 178 alleles) and the p.L392P neutral variant of 2% in our population with SRNS. Four more variants were present in at least more than one patient: p.P264R (exon 7), p.R460Q (exon 11), p.R976S (exon 22), and p.V1084fsX1095 (exon 24). No other hot spot was found in the NPHS1 gene in the present study.Table 3Classification of NPHS1 substitutionsNPHS1 substitutionsExonPrevious descriptionGDaGD (Grantham distance); score of chemical difference between the normal and mutated residue (high score, greater difference).GVbGV (Grantham variation); score of chemical difference between 14 orthologs (ranging from orangutan to fruit fly) (0=completed conserved).GD/GV matrix scorecGD/GV matrix score; lower matrix scores corresponded to low GD and high GV (conservative change and strong variation within the MSA), whereas higher matrix scores corresponded to high GD and low GV (nonconservative change and strong conservation within the multi-sequence alignment).GDevdGDev (Grantham deviation); score of chemical difference between the mutated residue and the range of variation between orthologs (GD similar to GDev, higher difference).Defined domain (degree of conservation)eDomain-containing residue: Ig-like C2 type domains (1–8), fibronectin type II (FTIII), Cter (1160–1241): binding to podocin. C, conserved (80–50%); domains; HC, highly conserved (>80%); NHC, not highly conserved (49–30%); No, not defined.Splicing predictionfNot predicted by Splice Site Prediction Neural Network. Score of the acceptor site (AS).Control chromosomesDescribed in SNP databasePolyphen predictiongPolyphen assessment; ratio Polyphen >2 (probably damaging), ratio Polyphen >1 (possibly damaging), ratio Polyphen 11 → mutation group (MG)=B; VS=5–10 → MG=C; VS=0–4 → MG=I; VS <-1 → MG=NV; A, pathogenic; B, highly likely pathogenic; C, likely pathogenic; I, unknown pathogenicity; NV, neutral variant.Positive controls Protein is retained in endoplasmic reticulum: 'severe mutation'26.Philippe A. Nevo F. Esquivel E.L. et al.Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome.J Am Soc Nephrol. 2008; 19: 1871-1878Crossref PubMed Scopus (105) Google Scholar R367C915.Koziell A. Grech V. Hussain S. et al.Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration.Hum Mol Genet. 2002; 11: 379-388Crossref PubMed Scopus (219) Google Scholar, 17.Lenkkeri U. Mannikko M. McCready P. et al.Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations.Am J Hum Genet. 1999; 64: 51-61Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 18.Beltcheva O. Martin P. Lenkkeri U. et al.Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome.Hum Mutat. 2001; 17: 368-373Crossref PubMed Scopus (141) Google Scholar18085+4127 (0)Ig 4 (HC) (+4)Not predicted (0)0/6017.Lenkkeri U. Mannikko M. McCready P. et al.Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations.Am J Hum Genet. 1999; 64: 51-61Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar (+2)No (+1)2.16 (probably damaging) (+2)No (+2)15B C623F1415.Koziell A. Grech V. Hussain S. et al.Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration.Hum Mol Genet. 2002; 11: 379-388Crossref PubMed Scopus (219) Google Scholar, 16.Schultheiss M. Ruf R.G. Mucha B.E. et al.No evidence for genotype/phenotype correlation in NPHS1 and NPHS2 mutations.Pediatr Nephrol. 2004; 19: 1340-1348Crossref PubMed Scopus (51) Google Scholar, 17.Lenkkeri U. Mannikko M. McCready P. et al.Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations.Am J Hum Genet. 1999; 64: 51-61Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 18.Beltcheva O. Martin P. Lenkkeri U. et al.Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome.Hum Mutat. 2001; 17: 368-373Crossref PubMed Scopus (141) Google Scholar, 25.Hinkes B.G. Mucha B. Vlangos C.N. et al.Nephrotic syndrome in the first year of life: two thirds of cases are caused by mutations in 4 genes (NPHS1, NPHS2, WT1, and LAMB2).Pediatrics. 2007; 119: e907-e919Crossref PubMed Scopus (323) Google Scholar2050+8205 (+2)Ig 6 NHC) (+2)Not predicted (0)0/6017.Lenkkeri U. Mannikko M. McCready P. et al.Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations.Am J Hum Genet. 1999; 64: 51-61Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar (+2)No (+1)3.30 (probably damaging) (+2)No (+2)19B A806D1817.Lenkkeri U. Mannikko M. McCready P. et al.Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations.Am J Hum Genet. 1999; 64: 51-61Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar,18.Beltcheva O. Martin P. Lenkkeri U. et al.Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome.Hum Mutat. 2001; 17: 368-373Crossref PubMed Scopus (141) Google Scholar12664+3124 (+2)Ig 7 (NHC) (+2)Not predicted (0)0/6017.Lenkkeri U. Mannikko M. McCready P. et al.Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations.Am J Hum Genet. 1999; 64: 51-61Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar (+2)No (+1)1.85 (possibly damaging) (+1)No (+2)13B L832P*Substitutions not detected in our cohort of patients.1826.Philippe A. Nevo F. Esquivel E.L. et al.Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome.J Am Soc Nephrol. 2008; 19: 1871-1878Crossref PubMed Scopus (105) Google Scholar980+698 (+2)Ig 7 (NHC) (+2)Not predicted (0)0/18226.Philippe A. Nevo F. Esquivel E.L. et al.Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome.J Am Soc Nephrol. 2008; 19: 1871-1878Crossref PubMed Scopus (105) Google Scholar (+2)No (+1)2.20 (probably damaging) (+2)No (+2)17B Protein traffics normally in the cell: 'mild mutation'26.Philippe A. Nevo F. Esquivel E.L. et al.Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome.J Am Soc Nephrol. 2008; 19: 1871-1878Crossref PubMed Scopus (105) Google Scholar L96V*Substitutions not detected in our cohort of patients.326.Philippe A. Nevo F. Esquivel E.L. et al.Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome.J Am Soc Nephrol. 2008; 19: 1871-1878Crossref PubMed Scopus (105) Google Scholar320+232 (+2)Ig 1 (NHC) (+2)Not predicted (0)0/18826.Philippe A. Nevo F. Esquivel E.L. et al.Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome.J Am Soc Nephrol. 2008; 19: 1871-1878Crossref PubMed Scopus (105) Google Scholar (+2)No (+1)1.34 (possibly benign) (-1)No (+2)10C A107T*Substitutions not detected in our cohort of patients.326.Philippe A. Nevo F. Esquivel E.L. et al.Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome.J Am Soc Nephrol. 2008; 19: 1871-1878Crossref PubMed Scopus (105) Google Scholar5860-230 (+1)Ig 1 (NHC) (+2)Not predicted (0)0/18826.Philippe A. Nevo F. Esquivel E.L. et al.Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome.J Am Soc Nephrol. 2008; 19: 1871-1878Crossref PubMed Scopus (105) Google Scholar (+2)No (+1)1.50 (possibly benign) (-1)No (+2)5C R460Q1115.Koziell A. Grech V. Hussain S. et
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