CG/CA genotypes represent novel markers in the NPHS2 gene region associated with nephrotic syndrome

Nephrotic syndrome (NS) is considered as a primary disease of the kidney that represents a heterogeneous group of glomerular disorders occurring mainly in children. It is generally divided into steroid-sensitive and steroid-resistant forms, depending upon the patient’s response to steroid therapy. Among the genes involved, the NPHS2 gene has been reported as the causative gene in steroid resistant form of nephrotic syndrome. In the present study, heterozygosity rate, allelic frequency and linkage of rs2274625 and rs3829795 markers were investigated in the NPHS2 gene region. To determine the SNP alleles, tetra-primer ARMS PCR was used. After genotyping rs2274625 and rs3829795 polymorphic markers in 120 unrelated individuals and nine trios families, the data were analysed using various computer programs such as UCSC Genome Browser, dbSNP and SNPper. Based on the statistical analysis of the results, for rs2274625 marker, allele frequency for C and T alleles was 97% and 3%, respectively. For rs3829795 marker allele frequency for G and A alleles was 55% and 45%, respectively. The values of heterozygosity index for the examined markers were 5% for rs2274625 and 45/8% for rs3829795. Consequently, two informative haplotypes, CG/CA, were identified in the NPHS2 gene region through combination of these two markers. These haplotypes can serve as appropriate tools for the identification of heterozygous carriers and linkage analysis of nephrotic syndrome disease in the Iranian families with an affected child.

Mutation analysis of NPHS2 in Chinese Han adult-onset focal segmental glomerulosclerosis / 上海交通大学学报（医学版）

Objective·To screen NPHS2 mutations in adult focal segmental glomerulosclerosis(FSGS)patients based on a large Chinese FSGS cohort. Methods · All patients were biopsy determined FSGS by the Department of Nephrology at Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine. FSGS secondary to systemic disease and other hereditary kidney disease were excluded. After extraction of genomic DNA of peripheral blood,NPHS2 was screened by directly sequencing the exon/intron junction or high-throughput sequencing,including whole exon sequencing and Panel sequencing, and then verified by Sanger sequencing. One hundred healthy controls were enrolled to validate candidate mutations. Results · Two hundred and four FSGS patients were enrolled,including 52 familial(25.5%) and 152 sporadic patients(74.5%),of which steroid-resistant FSGS patients accounted for 30.3%(46/152).By sequencing NPHS2 in all patients of the cohort(Sanger sequencing in 61 patients and high-throughput sequencing in 143 patients), 2 novel conserved mutations were identified, one homozygous mutation in sporadic steroid-resistant FSGS, p.N199I and one heterozygous mutation in familial FSGS, p.L321fx346. Both of them were not detected in 100 healthy controls. These two variants were predicted to be damaging by Polyphen,SIFT and Mutation Taster.Totally,the mutation rate of NPHS2 in the FSGS cohort was 1%. Conclusion·Since the overall frequency of NPHS2 mutations is considerably low in Chinese adult-onset FSGS,NPHS2 is not the main disease-causing gene of this group of people.

Identificación de variantes del gen NPHS2 en niños con síndrome nefrótico corticorresistente / NPHS2 Mutation analysis study in children with steroid-resistant nephrotic syndrome

Resumen: La podocina es una proteína localizada en el diafragma de filtración glomerular donde participa en la regulación de la filtración glomerular. Las mutaciones del gen NPHS2, que codifica a la podocina, son la principal causa de síndrome nefrótico corticorresistente (SNCR) autosómico recesivo en niños. Objetivos: Identificar mutaciones de NPHS2 en niños chilenos con SNCR, y establecer la prevalencia de las variantes más frecuentes en un grupo de adultos sanos. Pacientes y método: Análisis mutacional de NPHS2 en 34 niños chilenos con SNCR. Una vez identificadas las dos variantes de NPHS2 de mayor frecuencia, se realizó un screening de estas mutaciones en 223 adultos sanos. El análisis mutacional se realizó por secuenciación directa de los ocho exones codificantes amplificados por reacción de polimerasa en cadena. La secuenciación del DNA se realizó mediante método fluorométrico y las secuencias fueron evaluadas con el software SeqPilot. La asociación entre la presencia de variantes de NPHS2 y SNCR se calculó comparando las frecuencias alélicas entre los pacientes con SNCR y los voluntarios sanos utilizando prueba exacta de Fisher. Se consideró significativo p < 0,05. Resultados: Se detectaron mutaciones patogénicas de NPHS2 en siete de los 34 pacientes (21%) estudiados, de los cuales seis resultaron heterocigotos para p.R229Q y p.A284 V. En voluntarios sanos la prevalencia de p.R229Q fue de 2,46%. Conclusiones: Este estudio muestra que p.R229Q y p.A284 V son las variantes de NPHS2 más frecuentes en niños chilenos con SNCR. Por primera vez se describe esta asociación en niños chilenos, en base a la cual es posible proponer una estrategia de screening para estudio genético en pacientes con SNCR y sus familias. Se propone una estrategia de búsqueda de p.R229Q y p.A284 V en forma paralela o secuencial en estos pacientes.

Abstract: Podocin is a protein located in the glomerular slit diaphragm where it takes part in the regulation of glomerular filtration. Mutations of the NPHS2 gene that codes podocin are the main cause of autosomal recessive steroid resistant nephrotic syndrome (SRNS). Objectives: To identify the NPHS2 mutations in Chilean children with SRNS, and to determine the prevalence of the most common variants in a group of healthy adults. Patients and methods: Mutation analysis of NPHS2 in 34 Chilean children with SRNS. Once the two most common variants of NPHS2 were identified, screening for these mutations was performed on 233 healthy adults. The mutation analysis was performed by the direct sequencing of the eight coding exons by polymerase chain reaction amplification. The DNA sequencing was performed using a fluorometric method, and then evaluated with SeqPilot™ software. The relationship between the presence of NPHS2 variants and SRNS was calculated by comparing the allele frequency between patients with SRNS and those of the healthy volunteers using the exact Fisher test. A P < .05 was considered significant. Results: Pathogenic NPHS2 mutations were detected in 7 (21%) of the 34 patients studied, of which 6 were heterozygotes for p.R229Q and p.A284 V. The presence of p.R229Q was 2.46% in the healthy volunteers. Conclusions: This study shows that p.R229Q and p.A284 V are the most frequent variants in Chilean children with SRNS. It is the first time that this relationship has been reported in Chilean children. Based on this, a screening strategy is proposed for the genetic study in patients with SRNS and their families. A parallel or sequential search strategy for p.R229Q and p.A284 V in these patients is proposed.

The clinical features of hereditary nephrotic syndrome caused by NPHS 2 mutation in two pediatric patients / 临床儿科杂志

Objective To explore the clinical features of steroid resistant nephrotic syndrome caused by NPHS2 gene mutation. Methods The clinical data of two pediatric patients with steroid resistant nephrotic syndrome were retrospectively analyzed. The pertinent literatures were reviewed. Results Both patients were male with onset age at 2 and 3 years old. The clinical features were heavy proteinuria, hypoalbuminemia, and hypercholesterolemia, which met the diagnostic criteria of nephrotic syndrome. Renal pathology found one patient with focal segmental glomerulosclerosis, and other with minimal-change. Both of them suffered from recurrent inguinal hernioplasty and one was accompanied with hypoplasia of left testis. Gene detection verified a NPHS2 gene mutation. Both of them were hormone resistant at the beginning of onset and later hormone combined with different kinds of immunosuppressive therapy was still ineffective. Both of them entered the end-stage of renal disease 3 years after onset. Conclusions For male pediatric patients with steroid resistant nephrotic syndrome, combined with non-renal manifestations such as multiple hernia or testicular abnormalities, the possibility of the hereditary nephrotic syndrome caused by NPHS2 mutations should be considered.

Familial steroid-sensitive idiopathic nephrotic syndrome: seven cases from three families in China

OBJECTIVES: Familial steroid-sensitive idiopathic nephrotic syndrome is rare, and only approximately 3% of patients have affected siblings. METHODS: Herein, we report seven cases of patients with steroid-sensitive idiopathic nephrotic syndrome from three Chinese families. Mutational screening of the Nphs2 gene was performed in all the patients. RESULTS: All seven of the familial steroid-sensitive idiopathic nephrotic syndrome cases in our sample exhibited minimal change disease, and one case also presented with mesangial proliferative glomerulonephritis, according to the renal pathology. No significant was associations were found between Nphs2 gene mutations and the onset of proteinuria and nephrotic syndrome in these familial cases. CONCLUSIONS: The presence of minimal change disease is important, but it is not an unusual finding in patients with familial steroid-sensitive idiopathic nephrotic syndrome, which appears to be clinically benign and genetically distinct from other types of nephrosis. .

NPHS2 and CD2AP gene mutation in children with primary steroid-resistant nephrotic syndrome in Guangdong province / 中华实用儿科临床杂志

Objective To study NPHS2 and CD2AP gene mutation with primary steroid-resistant nephroticsyndrome (SRNS) children in Guangdong province,and to investigate the relationship between NPHS2,CD2AP genemutation and SRNS,so as to provide a theoretical basis for the diagnosis and treatment of SRNS in children.Methods Twenty-six SRNS children and 20 cases of the healthy children as controls were chosen randomly in Guangdong province.Genomic DNA was isolated from peripheral blood leucocytes of these patients and the healthy children.Mutational analysis was performed in 8 exons of NPHS2 gene and 18 exons of CD2AP gene after sequencing directly.The results were compared with United States National Center for Biotechnology Information (NCBI) gene database,the detected gene mutation.Results The variation analysis revealed 3 polymorphisms (288C ＞ T,954T ＞ C,1038A ＞ G) in 14 cases out of 26 patients and 4 cases of the healthy children studied,which had been reported before,but there was no significant difference in the genotypic and allelic frequencies of these polymorphisms between the patients and the controls (all P ＞ 0.05).One CD2AP heterozygous mutation (1917 + 20 C ＞ G) was detected in intron in 2 cases of SRNS children.Conclusions NPHS2 gene variation may not be the main mechanism of SRNS in Guangdong province.CD2APgene mutation may increase the possibility of SRNS and focal segmental glomerulosclerosis in children.CD2AP mutation in intron may involve in the pathogenesis of SRNS.

Síndromes nefróticos congénitos y hereditarios / Congenital and heritable nephrotic syndromes

En los últimos años se han identificado muchos síndromes nefróticos familiares y esporádicos que no responden a los tratamientos habituales (esteroides e inmunosupresores), evolucionan con relativa rapidez a la insuficiencia renal crónica y se producen por mutaciones genéticas. La mayoría de los síndromes nefróticos que se trasmiten genéticamente y que pueden ser congénitos, presentarse en el primer año de la vida, o en el niño mayor, son atribuidos a mutaciones en los genes NPHS1, NPHS2, WT1 y LAMB2. Otros síndromes nefróticos producidos por mutaciones genéticas pueden no manifestarse hasta la adultez. El objetivo fundamental de esta revisión fue llamar la atención sobre los síndromes nefróticos producidos por mutaciones genéticas en los que no sólo no se obtienen resultados con los tratamientos inmunosupresores, si no en los que dichos tratamientos pueden ser perjudiciales para el paciente

In past years many familial and sporadic nephrotic syndromes refractory to usual treatments (steroids and immunosuppressives), evolve quickly to a chronic renal failure produced by genetic mutations. Most of nephrotic syndromes genetically transmitted and that may be congenital, present in the first year of life or in the older child, are attributable to NPHS1, NPHS2, WT1 and KLAMB2 gen mutations. Other nephrotic syndromes produced by genetic mutations may not appear until adulthood. The main objective of present review was to alert on the nephrotic syndromes produced by genetic mutations without response to immunosuppressive treatments, but on those in which such treatment may be dangerous for patient

El síndrome nefrótico y el diagnóstico genético en pediatría / Inheritance genetics of nephrotic syndrome

Steroid-Resistant Nephrotic Syndrome (SRNS) is found in approximately 20 percent of patients with Nephrotic Syndrome (NS). Podocyte-gen mutations are diagnosed in a half of these children. Nephrin (NPHS1), podocin (NPHS2) and Wilms tumor suppressor gene (WT1) are the most frequently founded mutations. These patients usually progress to End Stage Renal Disease (ESRD). Objective: Current concepts in genetic diagnostic of NS in pediatrics are presented. A local experience is analyzed. In Chilean pediatric patients with SRNS, a mutational analysis of the NPHSl and NPHS2 gene was carried out by direct sequencing of the coding regions following polymerase chain reaction (PCR) amplification of genomic leukocyte DNA with flanking intronic primers. For WTl (exon 8 and exon 9), PCR of these fragments were done. Thirty-three patients were included, 17 males, 11,1 +/- 6.8 years. 54 percent of them developed ESRD, 12 patients were transplanted at the time of the analysis, 5 were under dialysis therapy, and 16 children correspond to ESRD Stage 3 and 4. Genetic analysis showed a gen mutation in 9 patients, NPHSl in 3 and NPHS2 in 6 of them. All genetic NS patients were cyclosporine-resistant. Post transplant relapse of NS was lower in genetic patients (p < 0.05). Conclusion: SRNS in children should be always evaluated from a genetic approach in order to avoid long-term immunosuppression, and to anticipate a clinical evolution after kidney transplantation.

En pediatría, el 20 por ciento de los pacientes portadores de Síndrome Nefrótico Idiopático son corticoresistentes (SNCR). Aproximadamente la mitad de ellos corresponden a mutaciones de genes que codifican proteínas del podocito. Las mutaciones más frecuentes corresponden al gen de la nefrina (NPHS1), la podocina (NPHS2) y del gen supresor del tumos de Wilms (WT1). Estas formas hereditarias no responden a tratamientos inmu-nosupresores y pueden progresar a enfermedad renal terminal (ERT). Objetivo: Revisar el estado actual del diagnóstico genético en Síndrome Nefrótico en niños, y presentar esta experiencia nacional de esta patología. Para el estudio de pacientes pediátricos chilenos, se realizó análisis de mutación del gen NPH2 por secuen-ciación directa de la regiones codificantes por PCR para la amplificación del DNA genómico leucocitario con partidores de acompañamiento intrónico. Para nefrina se procedió a extraer el DNA genómico, y se realizó la búsqueda de mutaciones de NPHSl por secuenciación directa de los 29 exones codificantes y las uniones intrónicas adyacentes, mientras que para el estudio de WTl se practicó el análisis mutacional de los exones 8 y 9, realizado por secuenciación directa del producto amplificado de WT1-PCR. Se han estudiado 33 pacientes provenientes de 29 familias, 17 varones, edad 11,1 +6,8 años. Dieciocho pacientes (54 por ciento) evolucionaron a ERT. Doce pacientes estaban trasplantados, 5 en diálisis, y 16 estaban en etapas 3-4 de enfermedad renal crónica. El estudio genético identificó mutaciones en 9 pacientes (27 por ciento), 3 correspondieron a NPHS1, 6 a NPHS2. Ningún caso con mutación respondió a tratamiento de Ciclosporina A (CsA), y las recaídas posttrasplante fueron significativamente menores en el grupo con mutación (+). Conclusión: Las mutaciones en estos genes deben ser estudiadas en cada niño con SNCR con el fin de evitar tratamientos prolongados e inefectivos, y anticipar la evolución después del trasplante renal.

The correlation between NPHS2 polymorphism and IgA nephropathy in northern Chinese patients / 中华内科杂志

Objective To examine the polymorphism in NPHS2 gene of IgA nephropathy in northern Chinese patients and to investigate the possible association of the NPHS2 polymorphism with the development of IgA nephropathy, as well as its clinical and histologic manifestations.Methods The polymorphism of NPHS2 was analyzed by direct DNA sequencing in 32 northern Chinese patients with IgA nephropathy (16 with heavy proteinuria and 16 with isolated hematuria).According to preliminary results, a total of 537 IgA nephropathy patients were genotyped for the NPHS2 C357T polymorphism by PCR combined with restriction fragment length polymorphism (PCR-RFLP). We collected clinical and histologic manifestations for gene analysis in patients with IgA nephropathy, such as age, sex, urine protein excretion and so on.ResultsEight NPHS2 polymorphisms (-931A＞T, -601C ＞T, 19G＞T, 171A＞G, 357C ＞ T, IVS3-21C ＞ T, 1023C ＞ T and 1107A ＞ G) were identified.The preliminary results of gene sequencing showed that the frequency of 357T allele in nephrotic syndrome group was obviously lower than isolated hematuria group (0.038 vs 0.125, P ＜0.05).In 537 IgA nephropathy patients with clinical and histologic data, the average urinary protein excretion in the patients with the 357CT/TT genotype was less (P =0.023).The incidence of urinary protein of more than 3.5 g/d was significantly lower in patients with T allele and TT/CT genotype, respectively (P =0.017 and 0.011).The logistic regression analysis indicated that, even after adjusting for the effect of hypertension and age of patients, the CT/II genotype of NPHS2 C357T was an independent protective factor for the urinary protein excretion more than 3.5 g/d(P =0.012,OR = 0.485, 95％ CI 0.275-0.84).ConclusionsEight NPHS2 polymorphisms were identified in northern Chinese IgA nephropathy patients. The frequencies of NPHS2 T allele and TT/CT genotype were the protective factors for urinary protein, especially with that of more than 3.5 g/d.

Mutations of podocyte molecules in patients with late onset familial focal segmental glomerular sclerosis / 中华肾脏病杂志

Objective To investigate the mutations of pedocyte molecules in patients with late onset familial focal segmental glomerular sclerosis (FSGS). Methods Thirty-one pedigrees of late onset familial FSGS in Department of Nephrology, Shanghai Ruijin Hospital from Sep 1997 to Oct 2007 were enrolled in this study. The diagnosis standard of familial FSGS was as follows:(1) the age of presentation was more than 12 years old. (2) in one pedigree, two or more individuals were proven as FSGS by renal biopsy, or at least one was proven to be FSGS by renal biopsy, the others presented renal insufficiency or pmteinuria without precise causes. One hundred unrelated healthy people were screened as control group. Genomic DNA extracted from peripheral blood cells were amplified by PCR and then sequenced for mutations of NPHS2, ACTN4 and TRPC6. Results A novel missense heterozygotic mutation L316P of ACTN4 was identified inone pedigree. The mean onset age of the affected members of this pedigree was (38.7±7.4) years old and their kidney injury progress was slow. Proteinuria of the proband's brother was not improved by immunosuppressor. All 3 affected members of this family had such heterozygotic mutation. A novel missense heterozygotic mutation Q889K of TRPC6 was found in another pedigree. The mean onset age of the affected members in this pedigree was (38.0±4.2) years old. Three members presenting renal disease in this family all had such heterozygotic mutation but with different clinical manifestations. A quiescent mutation G467G of TRPC6 was also identified. Above variants were not found in healthy controls. No NPHS2 mutation was found to cause familial FSGS in these pedigrees. Conclusions A novel mutation L316P of ACTN4 and a new mutation Q889K of TRPC6 are identified in Chinese patients of late onset familial FSGS. No NPHS2 mutation is found to induce FSGS in these pedigrees.

Mutations in NPHS2 Gene in A Child with Steroid-Dependent Nephrotic Syndrome / 实用儿科临床杂志

T were also detected in the controls.Conclusion Nephrotic syndrome may be associated with NPHS2 identified in children.

A familial steroid-responsive nephrotic syndrome report / 中华肾脏病杂志

Objective To report a familial steroid responsive idiopathic nephrotic syndrome,describe the clinic and pathologic features of the family and screen the potential mutations of NPHS2 in the kindred. Methods Clinopathological data of the family members were collected(including patients) . The reports on familial steroid responsive idiopathic nephrotic syndrome were reviewed. The characteristics were compared between them. NPHS2 mutation was screened through DHPLC for the patients. Resutts There were two brothers with steroid responsive nephrotic syndrome in this family,and the renal pathology was minimal change disease. One of them had massive proteinuria and hypertension,whose renal biopsy showed mild mesangial proliferative nephritis. In this family,any mutation in NPHS2 gene was not found as it was reported in other populations. Conclusions The present study is the first report of familial steroid responsive idiopathic nephrotic syndrome in Chinese population. No relationship between the disease and NPHS2 gene mutation in this family is found.