Retrospective mutational analysis of NPHS1, NPHS2, WT1 and LAMB2 in children with steroid-resistant focal segmental glomerulosclerosis – a single-centre experience

  • Agnieszka Bińczak-Kuleta Department of Clinical and Molecular Biochemistry, Pomeranian Medical University
  • Mieczysław Litwin Department of Nephrology, Kidney Transplantation and Hypertension, Children`s Memorial Health Institute
  • Małgorzata Ryder Department of Clinical and Molecular Biochemistry, Pomeranian Medical University
  • Klaudyna Lewandowska Department of Clinical and Molecular Biochemistry, Pomeranian Medical University
  • Olga Taryma-Leśniak Department of Clinical and Molecular Biochemistry, Pomeranian Medical University
  • Jeremy S. Clark Department of Clinical and Molecular Biochemistry, Pomeranian Medical University
  • Ryszard Grenda Department of Nephrology, Kidney Transplantation and Hypertension, Children`s Memorial Health Institute
  • Andrzej Ciechanowicz Department of Clinical and Molecular Biochemistry, Pomeranian Medical University
Keywords: gene variant, NPHS2, WT1, children, steroid-resistant nephrotic syndrome, FSGS

Abstract

The aim of our study was to examine NPHS1, NPHS2, WT1 and LAMB2 mutations, previously reported in two thirds of patients with nephrotic syndrome with onset before the age of one year old. Genomic DNA samples from Polish children (n=33) with Steroid-ResistantNephrotic Syndrome (SRNS) due to focal segmental glomerulosclerosis (FSGS), manifesting before the age of 13 years old, underwent retrospective analysis of NPHS1, NPHS2, WT1 (exons 8, 9 and adjacent exon/intron boundaries) and LAMB2. No pathogenic NPHS1 or LAMB2 mutations were found in our FSGS cohort. SRNS-causing mutations of NPHS2 and WT1 were detected in 7 of 33 patients (21%), including those with nephrotic syndrome manifesting before one year old: five of seven patients. Four patients had homozygous c.413G>A (p.Arg138Gln) NPHS2 mutations; one subject was homozygous for c.868G>A (p.Val290Met) NPHS2. A phenotypic female had C>T transition at position +4 of the WT1 intron 9 (c.1432+4C>T) splice-donor site, and another phenotypic female was heterozygous for G>A transition at position +5 (c.1432+5G>A). Genotyping revealed a female genotypic gender (46, XX) for the first subject and male (46, XY) for the latter. In addition, one patient was heterozygous for c.104dup (p.Arg36Profs*34) NPHS2; two patients carried a c.686G>A (p.Arg229Gln) NPHS2 non-neutral variant. Results indicate possible clustering of causative NPHS2 mutations in FSGS-proven SRNS with onset before age one year old, and provide additional evidence that patients with childhood steroid-resistant nephrotic syndrome due to focal segmentalglomerulosclerosis should first undergo analysis of NPHS2 coding sequence and WT1 exons 8 and 9 and surrounding exon/intron boundary sequences, followed by gender genotyping.

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Retrospective mutational analysis of NPHS1, NPHS2, WT1 and LAMB2 in children with steroid-resistant focal segmental glomerulosclerosis – a single-centre experience
Published
2015-05-20
How to Cite
1.
Bińczak-Kuleta A, Litwin M, Ryder M, Lewandowska K, Taryma-Leśniak O, S. Clark J, Grenda R, Ciechanowicz A. Retrospective mutational analysis of NPHS1, NPHS2, WT1 and LAMB2 in children with steroid-resistant focal segmental glomerulosclerosis – a single-centre experience. Bosn J of Basic Med Sci [Internet]. 2015May20 [cited 2021Oct.18];14(2):89-3. Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/2270
Section
Molecular Biology

INTRODUCTION

Genetically-heterogenous Steroid-Resistant Nephrotic Syndrome (SRNS) affects approximately io% of children with idiopathic nephrotic syndrome, progresses toward End-Stage Renal Disease (ESRD), and typically shows Focal Segmental GlomeruloSclerosis (FSGS) in renal histology [1, 2]. To the best of our knowledge, mutations of at least 24 genes have been associated with hereditary SRNS [3]. In 1998, Kestila et al. [4] discovered that the NPHS1 gene, encoding the podocytic protein nephrin, is mutated in the Finnish type of congenital nephrotic syndrome (CNS). This finding was the first proof of concept for the heredity of childhood nephrotic syndrome. In 2007, Hinkes et al. [5] found that mutations in NPHS1, NPHS2 (encoding podocin), WT1 (exons 8 and 9) and LAMB2 (encoding laminin-β2) are causes of two thirds of cases of nephrotic syndrome with onset in the first year of life. Mutational analysis of seven podocyte genes (NPHS1, NPHS2, WT1, CD2AP, ACTN4, TRPC6 and PLCE1) in 19 non-familial childhood-onset, steroid resistant, biopsy-proven FSGS patients revealed variants of NPHS1, NPHS2, WT1 and CD2AP that could be the cause of the disease in four subjects (21%). In addition, these results have suggested the role of combinations of genetic variants (bigenic heterozygosity) in the pathogenesis of steroid-resistant FSGS [6]. Santin et al. [2] proved the clinical utility of a genetic testing algorithm, for SRNS occurring before adolescence, based on an analysis of NPHS1, NPHS2, WT1 and PLCE1 genes. Recently, Rood et al. [7] suggested that there might be no need for PLCE1 analysis, for steroid-resistant FSGS occurring before adolescence, in this genetic testing algorithm. On the other hand, Hasselbacher et al. [8] and Matejas et al. [9] indicated that analysis of the LAMB2 gene, which is mutated in Pierson syndrome (OMIM #609049), could be included in the diagnostics of early onset nephrotic syndrome with absence of extrarenal abnormalities. Therefore, to further address this issue we decided to analyse retrospectively NPHS1, NPHS2, WT1 (exons 8 and 9 and the adjacent exon/intron boundaries) and LAMB2 genes in DNA samples from 33 patients with steroid-resistant focal segmental glomerulosclerosis manifesting before adolescence.

MATERIALS AND METHODS

Samples

Genomic DNA samples (n=33) for analysis were chosen from the DNA Repository for Childhood Nephrotic Syndrome (NS) at the Department of Clinical and Molecular Biochemistry, Pomeranian Medical University, Szczecin, Poland. The DNA samples were selected from children of Central European descent (Polish origin) who met the following criteria for inclusion: age of NS onset before adolescence (before the age of 13 years), biopsy-proven FSGS, primary steroid resistance of nephrotic syndrome defined as the persistence of heavy proteinuria (> 50 mg/kg/day) after eight weeks of prednisone treatment (60 mg/m2/day) and no significant extrarenal manifestations. The study was approved by the ethics committees of the Pomeranian Medical University, Szczecin, Poland and the Children’s Memorial Health Institute, Warsaw, Poland, and conforms to the ethical standards laid down in the 1964 Declaration of Helsinki; parents gave informed consent.

Procedures

Genomic DNA from peripheral blood leucocytes was extracted using a commercially available DNA isolation kit (QIAamp Blood DNA Mini Kit, QIAGEN, Hilden, Germany). Amplification of NPHS1 (NCBI Reference Sequence NG_013356), NPHS2 (NCBI Reference Sequence NG_007535), WT1 (NCBI Reference Sequence NG_009272) and LAMB2 (NCBI Reference Sequence NG_008094) were performed by PCR. Primer data are available on request []. Subsequently, PCR amplification products were purified using a GenElute PCR Clean-Up Kit (Sigma-Aldrich, Poznan, Poland). Sequencing of the products used BigDye® Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems, Life Technologies Polska, Warsaw, Poland). Electrophoresis and analysis were performed using an ABI PRISM 3100-Avant machine (Data Collection Software v2.0, Sequencing Analysis Software v5.1; Applied Biosystems).

RESULTS

The NPHS1, NPHS2, WT1 and LAMB2 genes were analyzed in genomic DNA samples of 33 Polish children with primary SRNS due to FSGS. The study cohort (15 phenotypic males and 18 phenotypic females) consisted of seven children with SRNS with onset in the first year of life, 15 subjects with early childhood-onset SRNS (13 to 60 months) and 11 subjects with late childhood-onset SRNS (61 to 132 months) (Table 1). The median age of NS onset was 36 months (range: 1 to 132 months); for males: 45 months (range: 3 to 126 months); for females: 27 months (range: 1 to 132 months). No pathogenic NPHS1 or LAMB2 variants were found in our FSGS cohort. The SRNS-causing mutations of NPHS2 and WT1 were detected in 7 of 33 patients (21%), and in those with nephrotic syndrome manifesting within the first year of life: five out of seven patients. Five patients had homozygous missense NPHS2 mutations; four of these with c.413G>A (p.Arg138Gln) mutations, and the fifth with a c.868G>A (p.Val290Met) mutation. DNA sequencing revealed a C>T transition at position +4 of the splice-donor site in WT1 in-tron 9 (c.1432+4C>T, previous nomenclature: IVS9ds+4C>T) from a phenotypic female (subject P20) and a G>A transition at position +5 of the splice-donor site in WT1 intron 9 (c.1432+5G>A, previous nomenclature: IVS9ds+5G>A) from another phenotypic female (subject P6) (Table 1). No parental DNA samples were available for analysis. Genotyping using the AmpFℓSTR NGM PCR Amplification Kit and AmpFℓSTR Yfiler PCR Amplification Kit revealed a female origin (46, XX) for the DNA sample from subject P20 and a male origin (46, XY) for the DNA sample from subject P6. In addition, the P7 subject was heterozygous for c.104dup (p.Arg36Profs*34) (previous nomenclature: c.104_1051nsG) without any additional pathogenic NPHS2 variant and two patients (P8 and P20 subjects) were heterozygous for the c.686G>A (p.Arg229Gln) NPHS2 non-neutral polymorphism.

TABLE 1: Background characteristics and genetic variants in children with steroid-resistant FSGS (patients listed in order of age of NS onset).

DISCUSSION

Results of our study revealed that one fifth of cases of steroid-resistant FSGS which occurred before adolescence were caused by mutations in either the NPHS2 or WT1 gene. Neither NPHS1 nor LAMB2 mutations were found in our FSGS cohort. It has also not escaped of our notice that 5 out of 7 from the NPHS2 or WT1 disease-causing mutations were detected in a small subgroup of children with SRNS (n=7) manifesting in the first year of life. Recent results, from similarly-designed studies with SRNS children which focused on searching for mutations in panels of genes, were published by Al-Hamed et al. [10] and McCarthy et al. [3]. Al-Hamed et al. detected disease-causing NPHS1 and NPHS2 variants in 12% (6 of 49) and 22% (11 of 49) of Saudi Arabian families with childhood nephrotic syndrome, respectively. The NPHS1 and NPHS2 mutations were exclusively detected in children of consanguineous parents [10]. McCarthy et al. sequenced 24 hereditary SRNS-associated genes, including the NPHS1, NPHS2, WT1 and LAMB2 genes, in 36 SRNS patients from the UK Renal Registry. These patients were ethnically-heterogeneous and between 1 month and 16 years of age at onset of disease. Molecular analysis revealed causative NPHS1 and NPHS2 mutations in 14% (5 of 36) and 8% (3 of 36) of the patients, respectively [3]. The NPHS1 mutations identified by Al-Hamed et al. [10] and McCarthy et al. [3] in congenital nephrotic syndrome have also been detected in early childhood-onset SRNS [10] and late childhood-onset SRNS [3]. In addition, the NPHS1 mutations were also identified in adult-onset SRNS [2], including SRNS due to FSGS [11]. Therefore, the lack of NPHS1 mutations in our group is somewhat surprising. However, the homogeneity of ethnic origin (of Central European/ Polish descent) and renal histology (FSGS), as well as the absence of children from consanguineous marriages, should be taken into consideration in relation to this lack of NPHS1 mutation. In addition, Hinkes et al. reported that NPHS1 mutations in patients of Central European descent were solely found in nephrotic syndrome with congenital onset (0–3 months)[5], which would only apply to two patients in our cohort (Table 1). Note also that, in one of these two patients, the homozygous NPHS2 mutation found confirms previous reports that CNS may be caused by mutations in genes other than NPHS1 [12-17]. Benoit et al. [18] has even recommended that patients with nephrotic syndrome occurring later than the congenital period (and with FSGS in renal histology) should first undergo screening for NPHS2 mutations followed by NPHS1 analysis. Lipska et al. reported recessive pathogenic NPHS2 mutations in 20 of 141 (14%) Polish children with steroid-resistant nephrotic syndrome [19]. The detection rate of NPHS2 mutations was very similar to that revealed in our study (15%). If from the study of Lipska et al. [19] the c.1032delT NPHS2 variant is excluded, as it is found solely in Kashubian patients who were not enrolled in our study, then the c.413G>A (p.Arg138Gln) NPHS2 mutation was the most frequently detected genetic variant both by us and by Lipska et al. [19], and is the most common in populations of European descent. Lipska et al. [19] also reported that NPHS2 mutations were to be found in the highest prevalence (26 %, 9/35) in patients with SRNS diagnosed within the first year of life, and it is of some interest that in our study 5 out of 7 patients with FSGS-proven SRNS caused by NPHS2 mutations were found with age of onset within the first year of life (Table 1). Both McCarthy et al. [3] and Al-Hamed et al. [10] found no LAMB2 or WT1 mutations in their studied cohorts.

The lack of LAMB2 mutations in our FSGS cohort, in patients from the UK Renal Registry [3] and in Saudi Arabian SRNS children [10] fully justifies removal of laminin-β2 genetic analysis from the algorithm introduced by Santin et al. [2] for molecular genetic diagnostics of non-syndromic childhood-onset primary steroid-resistant nephrotic syndrome. Despite a suggestion to consider PLCE1 testing only in children with diffuse mesangial sclerosis [7], Al-Hamed et al. [10] showed the clinical utility of PLCE1 analysis in childhood-onset steroid resistant FSGS, and this could be further considered (but was not analysed in our study). As well as NPHS1, NPHS2 and PLCE1 sequencing, Santin et al. also proposed the analysis of WT1 exons 8 and 9 in molecular genetic diagnostics of childhood onset SRNS [2]. To the best of our knowledge, Wasilewska et al. in 2009 [20] identified the first Polish patient with Frasier syndrome caused by a WT1 splice-site mutation (c.1432+sG>A), and Lipska et al. (in 2013) [21], in a SRNS cohort from the international PodoNet registry (including 22 patients from Poland), found one Polish patient with classical Frasier syndrome caused by a de novo c.1432+sG>A WT1 mutation. The prevalence of WT1 splice-site mutations with sporadic steroid-resistant FSGS in our study (6%) was similar to results published previously by other authors [6, 17, 21-23]. Both c.1432+5G>A or c.1432+4C>T mutations affect WT1 alternative splicing, leading to an imbalance between two WT1 isoforms with or without (+KTS or −KTS, respectively) three amino acids lysine (K)-threonine (T)-serine (S) between zinc fingers 3 and 4 [24]. According to Klamt et al. [24] the pathology of Frasier syndrome suggests that gonadal development may be especially sensitive to under-representation of the WT1 +KTS isoforms. Patients with c.1432+5G>A or c.1432+4C>T WT1 and FSGS present either an XY disorder of sex differentiation or no gonadal dysgenesis in XX subjects [6, 15-17, 20-23, 25, 26], and we can assume that this also applied to the WT1 splice-site mutations identified in our FSGS cohort. Note, however, that although Lipska et al. [27] recently reported WT1 mutations in 6% (46/746) of sporadic SRNS cases in the PodoNet registry, only one third of these (15 out of 17 with FSGS) were due to intronic KTS splice-site variants. The exonic WT1 mutations were associated with an early age of SRNS onset: mean 1.1 years, whereas KTS intronic splice site variants were not associated: mean age of onset 4.5 years. Recently, Lipska et al. [27] reported NPHS2 mutations in 6% (11 of 179; [21]) and WT1 mutations in 6% (46 of 746;) of patients with adolescent-onset sporadic SRNS from the international PodoNet registry, and recommended routine screening of WT1, in addition to NPHS2, in children with isolated, sporadic SRNS because of the high prevalence of “truly or apparently isolated SRNS”. Lowik et al. indicated the cost-effectiveness and the clinical value of genetic screening based on sequencing of NPHS2 and WT1 exons 8 and 9, with the adjacent exon/intron boundaries, also for childhood-onset steroid-resistant FSGS [6]. In addition, although the series of FSGS patients in their study was rather small (n=19), the results strongly suggest that both recessive homozygous CD2AP mutations or combined haploinsufficiency in NPHS2 and CD2AP might cause primary steroid-resistant FSGS in children [6]. Therefore, it seems to reasonable to consider the screening of PLCE1, as discussed earlier, and CD2AP as second-choice targets in a genetic testing approach for the diagnosis of primary steroid-resistant FSGS occurring before adolescence [2, 6, 10].

CONCLUSION

In conclusion, our results indicate possible clustering of causative NPHS2 mutations in FSGS-proven SRNS with onset in the first year of life, and provide additional evidence that children with steroid-resistant nephrotic syndrome due to focal segmental glomerulosclerosis, in whom NS occurs before the age of 13 years, should first undergo analysis of the NPHS2 coding sequence and the WT1 gene, especially focused on exons 8 and 9 and the surrounding exon/intron boundary DNA sequences, followed by, in cases with WT1 KTS intronic splice site variants, genotyping for gender.

DECLARATION OF INTEREST

The authors declare no conflict of interest.

Acknowledgements

ACKNOWLEDGEMENTS

This study was supported by Research Grant No. N402 420 438 from the Polish Ministry of Science and Higher Education. We are grateful to Dr. Andrzej Ossowski for the gender genotyping of WT1 patients and to the anonymous native speaker of English for help with preparation of the manuscript.

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