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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2020  |  Volume : 31  |  Issue : 1  |  Page : 144-149
NPHS2 gene mutations in azerbaijani children with steroid-resistant nephrotic syndrome

1 Department of Pediatrics, Pediatric Nephrology Division, Azerbaijan State Medical University, Baku, Azerbaijan
2 Department of Pediatric Molecular Medicine Laboratory, Ege University Medical Faculty, Izmir, Turkey

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Date of Submission19-Sep-2018
Date of Decision02-Nov-2018
Date of Acceptance04-Nov-2018
Date of Web Publication3-Mar-2020


Nephrotic syndrome (NS) is characterized by proteinuria in children. Steroid- resistant NS (SRNS) is defined by resistance to standard steroid therapy, and it continues to be one of the most common causes of chronic renal failure. Molecular studies have revealed specialized molecules in different regions of the podocytes that play a role in proteinuria. Mutations in NPHS2 that encode for podocin constitute a frequent cause of SRNS worldwide. This study aimed to screen for podocin mutations in Azerbaijani patients with SRNS. Our study included 21 pediatric patients with SRNS aged between 0 and 18 years and the same number of healthy control groups. Mutational analysis of the NPHS2 gene was performed using direct sequencing methods. Disease-causing mutations in the NPHS2 gene were detected in eight patients (38%). Thirteen patients (62%) had NPHS2 mutations without causing the disease. Two patients had p.Val290Met homozygous mutation; two had p.Arg229Gln homozygous mutations; and one each had p.Pro20Leu homozygote, p.Leu169Pro homozygote, p.Arg138Gln homozygote, and p.Arg168His homozygous mutations. When we correlated the NPHS2 mutation status with disease progression, there was a statistically significant increase in serum creatinine, proteinuria, and serum albumin values in patients with NPHS2 gene mutations compared to the group without mutation (P <0.05). Our study concludes that mutations of the NPHS2 gene (38%) are heterogeneous in Azerbaijani SRNS patients. Based on our results, we support a model in which ethnicity plays an important role in certain NPHS2 mutations. NPHS2 mutation analysis may help to better predict the course of the disease, remove unnecessary long-term immunosuppressive therapy, and develop specific treatment.

How to cite this article:
Baylarov R, Senol O, Atan M, Berdeli A. NPHS2 gene mutations in azerbaijani children with steroid-resistant nephrotic syndrome. Saudi J Kidney Dis Transpl 2020;31:144-9

How to cite this URL:
Baylarov R, Senol O, Atan M, Berdeli A. NPHS2 gene mutations in azerbaijani children with steroid-resistant nephrotic syndrome. Saudi J Kidney Dis Transpl [serial online] 2020 [cited 2020 Jun 2];31:144-9. Available from: http://www.sjkdt.org/text.asp?2020/31/1/144/279934

   Introduction Top

Nephrotic syndrome (NS) is an uncommon childhood disease that is characterized by edema, severe proteinuria, hypoalbuminemia, and hyperlipidemia. NS is not common in the 1st year of life, and there is a heterogeneous group of diseases. If the NS starts between 0 and 3 months of age, it is called congenital NS; when the disease starts between 3 and 12 months of age, it is called infantile NS. NS is clinically divided according to the response of patients to steroid treatment as follows: steroidsensitive NS (SSNS) and steroid-resistant NS (SRNS). Seventy-five percent of patients with SRNS exhibit focal and segmental glomerulo- sclerosis histology in renal biopsies, and a significant number of these patients develop end-stage renal disease (ESRD).[1],[2],[3],[4] Nephrin (NPHS1), α-actinin 4 (ACT4), CD2AP, TRPC6, and podocin (NPHS2) are podocyte proteins that play important roles in glome- rular slit diaphragm homeostasis. If the genes that encode these proteins mutate, it is known that a different NS table has emerged.[5],[6],[7],[8] Podocin is a raft-associated component of the glomerular foot-process membrane where it is localized at the insertion of the slit-diaphragm. The NPHS2 gene (OMIM No. 604766) locates at chromosome 1q25-q31. This gene was first mapped by linkage analysis in families with an autosomal recessive SRNS.[9] Boute et al[6] reported NPHS2 mutation in SRNS for the first time. Caridi et al,[9] Ruf et al,[10] Weber et al,[11] and Berdeli et al[12] in Turkey studied NPHS2 gene mutation in both familial and sporadic SRNS in detail. These studies are the most comprehensive studies. The ethnic distribution of this disease is defined by the identification of the NPHS2 mutation in patients from different countries.[13],[14],[15],[16],[17] However, there are no studies on NPHS2 mutation in patients with SRNS from Azerbaijan in literature.

In this study, it was aimed to determine the distribution of podocin gene (NPHS2) mutation in Azerbaijani patients with SRNS and to show genotype-phenotype relation by comparing this mutation distribution with other ethnic groups.

   Materials and Methods Top

Patient group

Twenty-one children with a diagnosis of SRNS aged between 0 and 18 years who were admitted to the Department of Pediatric Nephrology of Azerbaijan State Medical University Hospital were included in the study. The same number of healthy controls that included Azerbaijani children were also included in order to detect DNA variants in the NPHS2 gene. NS was defined by the presence of edema, severe proteinuria (>40 mg/m2/h or protein/creatinine ratio >2.0 mg/mg), hypo- albuminemia (<2.5 g/dL), and hyperlipidemia. Remission was defined as urine protein excretion below 4 mg/m2/h or protein/creatinine ratio at 0.2 mg/mg over the following three days. Steroid resistance was accepted as nonremission despite treatment. For genetic analysis, a consent form was taken from each patient’s and control’s parents, and the ethical committee approval was taken before the study.

Podocin (NPHS2) mutation analysis

Genetic analysis of the study group was carried out at the Molecular Medicine Laboratory, Ege University Medical Faculty Children’s Hospital. We collected 2 mL of peripheral blood from the patients and controls in ethylenediaminetetraacetic acid tubes. Genomic DNA was obtained by taking 200 μL of this sample. Synthetic oligo-nucleotide primers for 8 exons encoding the NPHS2 gene were designed according to the NCBI Reference Sequence (NG_007535.1) GenBank sequence. The cDNA of 1853 bp mRNA (NM_ 014625.3) was read according to the NCBI sequence. How the read sequences are reflected in the protein structure is interpreted according to the protein sequence (NP_055440.1) consisting of 383 amino acids. Mutation analysis was performed by Sanger DNA sequencing in all the eight exons of the NPHS2 gene.

Polymerase chain reaction amplification

A volume of 25 μL polymerase chain reaction (PCR) reaction mixture comprised the following: 100 ng of genomic DNA, 2.5 μL of 10x PCR buffer solution (Invitrogen Enhancer Buffer, Thermo Fisher Scientific, USA), 2.5 Mm MgCl2, 200 mM each of the four dNTPs (Promega, Madison, USA), 5 pmol forward and reverse primers each, and 1.0 U platinum Taq (Polymerase Co, Paisley, UK). PCR amplification was carried out using a gradient- based PCR program (PE Applied Biosystems, Foster City, CA, USA). DNA was run on ethidium bromide 2% agarose gel electropho- resis to confirm the PCR amplification product. For DNA sequence analysis, positive PCR products were subjected to enzymatic PCR purification.

Purification of polymerase chain reaction products

The PCR products obtained were checked in agarose gel electrophoresis, and positive PCR fragments were purified using Exo-SAP enzyme mixture (Affymetrix Inc., Central Expressway, Santa Clara, CA 95051, USA).

DNA sequence analysis

Purified PCR products were obtained from BigDye® Terminator v3.1 (Applied Biosystems, USA) for fluorescence labeling of nucleotides prior to DNA sequencing using the kit (PE Applied Biosystems, Foster City, CA, USA). The second round of PCR products obtained after cycle-sequencing PCR was purified from residual fluorescent stain using BigDyeXT kit (PE Applied Biosystems). The re-purified PCR products were loaded into the ABI 3130XL Genetic Analyzer automated DNA sequencing system (PE Applied Biosystems, Foster City, CA, USA), and the nucleotide sequences were read. Evaluation was performed with the SEQSCAPE 2.0 (850 Lincoln Centre Drive Foster City, CA 94404, USA) computer program.

Statistical analysis

Statistical analyzes were performed using Statistical Package for Social Sciences (SPSS) version 16.0 (SPSS Inc., Chicago, IL., USA).

   Results Top

Molecular findings

We detected mutations in the NPHS2 gene in eight (38%) of the 21 patients. Two of the patients had p.Val290Met homozygote mutation, two patients had p.Arg229Gln homozygote mutations, one patient had p.Pro20Leu homozygote mutation, one patient had p.Leu169Pro homozygote mutation, one patient had p.Arg138Gln homozygote mutation and one patient had p.Arg168His homozygote mutation. We detected p.Gly34Gly/p.Ala318Ala polymorphism in NPHS2 gene in 10 patients and p.Gly34Gly polymorphism in NPHS2 gene were detected in three patients [Table 1]. Since gene polymorphisms do not affect the function of the encoded protein, the genotype- phenotype association of polymorphisms in the NPHS2 gene has not been evaluated in this study.
Table 1: Mutation distribution, patient number, and mutation status of patient group.

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NPHS2 gene mutation and disease association

When we compared the NPHS2 mutation status with disease progression, there was a significant increase in serum creatinine, protei- nuria and serum albumin values in patients with NPHS2 gene mutations compared to the group without mutation (P <0.05). The p.Arg168His and p.Val290Met mutations in the NPHS2 gene, which were detected in our study, were classified as pathogenic mutations according to the NCBI ClinVar database. Also, according to PolyPhen-2 (Polymorphism Phenotyping v2) database, the scores of these mutations are 1.00 and 0.99, respectively. p.Arg229Gln and p.Arg138Gln mutations were classified as variants of uncertain (or unknown) significance (VUS) in the NCBI ClinVar database. The scores of these genes in the PolyPhen-2 database were 0.90 and 1.00, respectively. In the NCBI ClinVar database, p.Pro20Leu is benign and the p.Leu169Pro mutation is not specified in the database. In PolyPhen-2 database, these genes’ scores were 0.098 and 0.99, respectively. The mean protei- nuria value in individuals with these mutations was 198 ± 114, the mean serum albumin level was 1.7 ± 0.4, and the mean creatinine level was 0.77 ± 0.5. In individuals who did not have these mutations, these values were as follows: the mean proteinuria value was 168 ± 120, the mean serum albumin value was 0.7 ± 0.19, and the mean creatinine level was 0.51 ± 0.16. When we compared the two groups, the mean proteinuria, albumin, and creatinine levels in patients with NPHS2 mutations were statistically significantly higher than the values in patients without mutations (P <0.05) [Table 2].
Table 2: The comparison of clinical evaluation of patients (mean proteinuria, albumin, and creatinine levels) with their mutation.

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   Discussion Top

Podocin is a protein that is usually expressed as a primer in glomerular podocyte cells and has a role in both structural and functional changes of the glomerular filtration barrier. Mutations in the NPHS2 gene that encode this protein have been associated with both familial and sporadic SRNS.[18] The detection of the NPHS2 mutation in children with SRNS has a critical importance because homozygous or compound heterozygous mutation in this gene form creates steroid resistance and forms severe SRNS, characterized by rapid deterioration to ESRD. Therefore, the finding of mutation in NS with steroid resistance has clinical significance.[19] A clinical assessment with the finding of mutation helps avoiding useless or more immunosuppressive treatment in patients and recurrence of NS may occur in renal grafts. Different NPHS2 mutations have been identified in Turkish, Italian, French, German, and Israeli-Arab children.[13],[14],[15],[16],[17] Podocin mutation studies are fewer in Israel-Jewish, Chinese, and Japanese children. There are no studies in Azerbaijani children that demonstrate the association of NS and NPHS2 gene mutation. Ruf et al studied 190 patients with SRNS from 165 different families and 124 patients with SSNS from 120 different families. In 43 of 165 families with SRNS (26%), homozygous or compound heterozygous mutations in NPHS2 were observed. Caridi et al found NPHS2 mutation in 19% of sporadic NS patients when they screened for NPHS2 gene in 671 patients with sporadic NS and in 205 familial cases with recessive inheritance.[9] Weber et al performed NPHS2 mutation analysis in 338 patients from 272 families with SRNS; 81 families with AR SRNS, 172 patients with sporadic SRNS, and 19 patients with diffuse mesangial sclerosis. They found that NPHS2 mutations were detected in 42% of familial SRNS and 10% of sporadic cases.[11] Berdeli et al in the study of Turkish children with NS identified new mutations in the NPHS2 gene and identified other mutations in their own other studies. In their study, they had 41 patients (13.8%) who had familial NS and 254 patients (86.2%) who had sporadic NS; the podocin mutation in the familial SRNS was 29.2% and the podocin mutation in sporadic SRNS was 24%. They reported that the mutation in the NPHS2 gene was associated with the disease.[12] Chernin et al observed that the knowledge of the NPHS2 mutation rate in different populations of SRNS patients was the source of choice for the appropriate genetic evaluation scheme. They also reported that homozygous or compound heterozygous mutations in the NPHS2 gene were absent in African-American children with SRNS in their study which included 18 patients.[22] Tsukaguchi et al reported NPHS2 variants in 23% of familial SRNS and 2% of sporadic cases.[23] Lipska et al detected NPHS2 mutations in 14% of 20 Polish SRNS patients.[24] In 2009, Otukesh et al had identified mutations in exon 5 and exon 7 in 20 Iranian children with SRNS.[25] Abid et al[13] in Pakistan identified low-frequency mutation in the patients and similarly Vasudevan et al detected mutation in 25 Indian children; only 4% of pathologic NPHS2 mutations were detected.[26]

In our study, the mutations which were detected are heterogeneously distributed as in the literature and are related to the development of the disease. The most common p.Gly34Gly and p.Ala318Ala polymorphisms mentioned in the literature were found in 13 patients in our study. Thep.Arg168His and p.Val290Met mutations in NPHS2 gene, which were described as pathogens in the literature and in the NCBI ClinVar database and PolyPhen-2 database; p.Arg138Gln and p.Arg229Gln mutations, which were designated as VUS according to NCBI ClinVar database; p.Pro20Leu, which was implied as benign in the NCBI ClinVar database; and p.Leu169Pro, which is not in the NCBI ClinVar database but its PolyPhen-2 score is 0.99, were detected in eight patients. The prognosis of disease in patients with the NPHS2 mutation is worse than in patients who do not have the mutation when compared to the clinical data of the patients. As the patient group is small, a more comprehensive study is needed on the same population. Nevertheless, these data show us how to treat this disease and how to treat it in Azerbaijani children with SRNS.

In patients with SRNS, identification of NPHS2 mutations is important not only for therapeutic reasons but also for genetic evaluation. However, there is no clear genotype/ phenotype correlation in NPHS2-mutated children with SRNS. Therefore, further studies with more patients are needed to investigate genotype/phenotype associations for homo- zygous or compound heterozygous mutations in NPHS2 and to respond to other immuno- suppressive treatments such as CsA, tacro- limus, cyclophosphamide, methylprednisolone, pulse therapy, or mycophenolate mofetil. For the importance of single heterozygous sequence variants, functional studies should be performed.

   Conclusion Top

Our study concludes that the mutations detected in the NPHS2 gene are heterogeneously seen in Azerbaijani children with SRNS. Mutations in the NPHS2 gene should be investigated for each child with SRNS in terms of disease progression and treatment. We recommend that further studies are needed to find the certain frequency of NPHS2 mutations in the Azerbaijan population and such an analysis may help to better predict the course of the disease, remove long-standing unnecessary immunosuppressive therapies, and develop specific therapeutic interventions against the mutations.

Conflict of interest: None declared.

   References Top

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Correspondence Address:
Ozgur Senol
Department of Pediatric Molecular Medicine Laboratory, Ege University Medical Faculty, Izmir
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DOI: 10.4103/1319-2442.279934

PMID: 32129207

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