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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.

INTRODUCTION

Genetically-heterogenous Steroid-Resistant Nephrotic Syndrome (SRNS) affects approximately 10% 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-[beta]2) 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/[m.sup.2]/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 [binia@pum.edu.pl]. Subsequently, PCR amplification products were purified using a GenElute PCR Clean-Up Kit (Sigma-Aldrich, Poznan, Poland). Sequencing of the products used BigDye[R] 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 intron 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 AmpFfSTR NGM PCR Amplification Kit and AmpFfSTR 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_105insG) 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.

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-[beta]2 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 haploin-sufficiency 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

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.

REFERENCES

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[3] McCarthy HJ, Bierzynska A, Wherlock M, Ognjanovic M, Kerecuk L, Hegde S. et al. Simultaneous sequencing of 24 genes associated with steroid-resistant nephrotic syndrome. Clin J Am Soc Nephrol 2013; 8(4/637-648

[4] Kestila M, Lenkkeri U, Mannikko M, Lamerdin J, McCready P, Putaala H. et al. Positionally cloned gene for a novel glomerular protein-nephrin-is mutated in congenital nephrotic syndrome. Mol Cell 1998; 1(4)375-582

[5] Hinkes BG, Mucha B, Vlangos CN, Gbadegesin R, Liu J, Hasselbacher K. 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 2007419(4):e907-e919.

[6] Lowik M, Levtchenko E, Westra D, Groenen P, Steenbergen E, Weening J. et al. Bigenic heterozygosity and the development of steroid-resistant focal segmental glomerulosclerosis. Nephrol Dial Transplant 2008; 23(10)3146-3151

[7] Rood IM, Deegens JK, Wetzels JF. Genetic causes of focal segmental glomerulosclerosis: implications for clinical practice. Nephrol Dial Transplant 2012; 27(3): 882-890

[8] Hasselbacher K, Wiggins RC, Matejas V, Hinkes BG, Mucha B, Hoskins BE. et al. Recessive missense mutations in LAMB2 expand the clinical spectrum of LAMB2-associated disorders. Kidney Int 2006;70(6):1008-1012

[9] Matejas V, Hinkes B, Alkandari F, Al-Gazali L, Annexstad E, Aytac MB. et al. Mutations in the human laminin beta2 (LAMB2) gene and the associated phenotypic spectrum. Hum Mutat 2010;31(9):992-1002

[10] Al-Hamed MH, Al-Sabban E, Al-Mojalli H, Al-Harbi N, Faqeih E, Al Shaya H. et al. A molecular genetic analysis of childhood nephrotic syndrome in a cohort of Saudi Arabian families. J Hum Genet 2013; 58(7)480-489

[11] Santln S, Garcla-Maset R, Ruiz P, Gimenez I, Zamora I, Pena A. et al. Nephrin mutations cause childhood- and adult-onset focal segmental glomerulosclerosis. Kidney Int. 2009; 76(12):1268-1276

[12] Koziell A, Grech V, Hussain S, Lee G, Lenkkeri U, Tryggvason K. 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(4): 379-388

[13] Hinkes B, Wiggins RC, Gbadegesin R, Vlangos CN, Seelow D, Nurnberg G. et al. Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible. Nat Genet 200638(12): 1397-1405

[14] Gbadegesin R, Hinkes BG, Hoskins BE, Vlangos CN, Heeringa SF, Liu J. et al. Mutations in PLCE1 are a major cause of isolated diffuse mesangial sclerosis (IDMS). Nephrol Dial Transplant 2008; 23(4): 1291-1297

[15] Schumacher V, Scharer K, Wuhl E, Altrogge H, Bonzel KE, Guschmann M. et al. Spectrum of early onset nephrotic syndrome associated with WT1 missense mutations. Kidney Int. 1998; 53(6):1594-1600.

[16] Mucha B, Ozaltin F, Hinkes BG, Hasselbacher K, Ruf RG, Schultheiss M. et al. Mutations in the Wilms' tumor 1 gene cause isolated steroid resistant nephrotic syndrome and occur in exons 8 and 9. Pediatr Res 200639(2): 325-331

[17] Ruf RG, Schultheiss M, Lichtenberger A, Karle SM, Zalewski I, Mucha B. et al. Prevalence of WT1 mutations in a large cohort of patients with steroid-resistant and steroid-sensitive nephrotic syndrome. Kidney Int 2004; 66(2): 364-570

[18] Benoit G, Machuca E, Antignac C. Hereditary nephrotic syndrome: a systematic approach for genetic testing and a review of associated podocyte gene mutations. Pediatr Nephrol 2010; 25(9):1621-1632

[19] Lipska BS, Balasz-Chmielewska I, Morzuch L, Wasielewski K, Vetter D, Borzecka H. et al. Mutational analysis in podocin-associated hereditary nephrotic syndrome in Polish patients: founder effect in the Kashubian population. J Appl Genet 2013; 54(3): 327-333

[20] Wasilewska A, Zoch-Zwierz W, Tenderenda E, Rybi-Szuminska A, Kolodziejczyk Z. WT1 mutation as a cause of progressive nephropathy in Frasier syndrome--case report. Pol Merkur Lekarski 2009; 26(156): 642-644

[21] Lipska BS, Iatropoulos P, Maranta R, Caridi G, Ozaltin F, Anarat A. et al. Genetic screening in adolescents with steroid-resistant nephrotic syndrome. Kidney Int 2013; 84(1): 206-213

[22] Denamur E, Bocquet N, Baudouin V, Da Silva F, Veitia R, Peuchmaur M. et al. WT1 splice-site mutations are rarely associated with primary steroid-resistant focal and segmental glomerulosclerosis. Kidney Int 2000; 57(5): 1868-1872

[23] Cho HY, Lee JH, Choi HJ, Lee BH, Ha IS, Choi Y et al. WT1 and NPHS2 mutations in Korean children with steroid-resistant nephrotic syndrome. Pediatr Nephrol 2008; 23(1): 63-70

[24] Klamt B, Koziell A, Poulat F, Wieacker P, Scambler P, Berta P. et al. Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms. Hum Mol Genet 1998; 7(4): 709-714

[25] Aucella F, Bisceglia L, De Bonis P, Gigante M, Caridi G, Barbano G. et al. WT1 mutations in nephrotic syndrome revisited High prevalence in young girls, associations and renal phenotypes. Pediatr Nephrol 2006; 21(10):1393-1398

[26] Fujita S, Sugimoto K, Miyazawa T, Yanagida H, Tabata N, Okada M. et al. A female infant with Frasier syndrome showing splice site mutation in Wilms' tumor gene (WT1) intron 9. Clin Nephrol 2010; 73(6): 487-491

[27] Lipska BS, Ranchin B, Iatropoulos P, Gellermann J, Melk A, Ozaltin F et al. Genotype-phenotype associations in WT1 glomerulopathy. Kidney Int 2014: doi:10.1038/ki.2013.519

Agnieszka Binczak-Kuleta (1), Jacek Rubik (2), Mieczysiaw Litwin (2), Malgorzata Ryder (1), Klaudyna Lewandowska (1), Olga Taryma-Lesniak (1), Jeremy S. Clark (1) *, Ryszard Grenda (2), Andrzej Ciechanowicz (1)

(1) Department of Clinical and Molecular Biochemistry, Pomeranian Medical University, al. Powstancow Wlkp. 72, 70-111 Szczecin, Poland. (2) Department of Nephrology, Kidney Transplantation and Hypertension, Children's Memorial Health Institute, Al. Dzieci Polskich 20, 04730 Warsaw, Poland.

* Corresponding author: Jeremy Simon Clark,

Department of Clinical & Molecular Biochemistry, Pomeranian Medical University, Powstancow Wlkp. 72, 70-111 Szczecin, Poland Phone: +48 91 466 1490 Fax: +48 91 466 1492

e-mail: jeremyclarkmel@gmail.com; binia@sci.pum.edu.pl

Submitted: 7 February 2014/Accepted: 1 March 2014
TABLE 1. Background characteristics and genetic variants
in children with steroid-resistant FSGS (patients listed
in order of age of NS onset).

Patient   Age of NS   Time to    Gender
          onset       ESRD       phenotype
          (months)    (months)

P1        1           160        female
P2        3           3          male
P3        5           39         male
P4        5           59         female
P5        8           135        female
P6        8           n.a.       female
P7        9           63         male
P8        14          28         male
P9        16          46         male
P10       17          11         male
P11       19          70         female
P12       20          64         female
P13       20          64         female
P14       21          102        female
P15       26          n.a.       female
P16       27          13         female
P17       36          0          female
P18       41          6          male
P19       45          21         male
P20       47          n.a.       female
P21       50          55         male
P22       51          8          male
P23       61          n.a.       female
P24       72          36         female
P25       72          66         male
P26       76          0          male
P27       78          44         female
P28       80          n.a.       male
P29       95          66         female
P30       99          0          male
P31       103         26         female
P32       126         18         male
P33       132         38         female

Patient   NPHS2 variants *        WT1 variants *
          [allele1];[allele2]     [allele1];[allele2]

P1        [c.413G>A];[c.413G>A]
P2
P3        [c.413G>A];[c.413G>A]
P4        [c.868G>A];[c.868G>A]
P5        [c.413G>A];[c.413G>A]
P6                                [c.1432+4C>T];[=]
P7        [c.104dup];[=]
P8        [c.686G>A];[=]
P9
P10
P11
P12
P13
P14
P15
P16
P17
P18
P19
P20       [c.686G>A];[=]          [c.1432+5G>A];[=]
P21
P22
P23
P24
P25
P26
P27
P28
P29
P30       [c.413G>A];[c.413G>A]
P31
P32
P33

ESRD = End Stage Renal Disease:

n.a. = not available;

[=] = reference sequence;

* Blank spaces indicate that reference sequences
were detected for the genes analysed.
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Author:Binczak-Kuleta, Agnieszka; Rubik, Jacek; Litwin, Mieczyslaw; Ryder, Malgorzata; Lewandowska, Klaudyn
Publication:Bosnian Journal of Basic Medical Sciences
Article Type:Report
Geographic Code:4EXBO
Date:May 1, 2014
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