NPHS2 p.V290M mutation in late-onset steroid-resistant nephrotic syndrome

For Peer Review

Draft Manuscript for Review

NPHS2 p.V290M mutation in late-onset steroid-resistant

nephrotic syndrome

Journal: Pediatric Nephrology

Manuscript ID: pedneph-12-07-0412.R1

Manuscript Type: Original Article

Date Submitted by the Author: 19-Oct-2012

Complete List of Authors: Kerti, Andrea; Semmelweis University, Ist Department of Pediatrics Csohány, Rózsa; Semmelweis University, Ist Department of Pediatrics Szabó, Attila; Semmelweis University, Ist Department of Pediatrics Árkossy, Ottó; FMC Szépvölgyi Dialysis Centre, Szent János Hospital, Nephrology Outpatient Dept Sallay, Péter; Semmelweis University, Ist Department of Pediatrics Moriniére, Vincent; Hôpital Necker, INSERM U983 Vega-Warner, Virginia; University of Michigan, Departments of Pediatrics and Human Genetics Nyírı, Gábor; Semmelweis University, Clinical and Research Center for Molecular Neurology Lakatos, Orsolya; University of Pécs, Department of Pediatrics Szabó, Tamás; University of Debrecen, Medical and Health Science Center, Pediatric Institute Lipska, Beata S.; Medical University of Gdansk, Department of Biology and Genetics; University of Heidelberg, Division of Pediatric Nephrology, Center for Pediatrics and Adolescent Medicine Schaefer, Franz; University of Heidelberg, Division of Pediatric Nephrology, Center for Pediatrics and Adolescent Medicine Antignac, Corinne; Hôpital Necker, INSERM U983 Reusz, George; Semmelweis University, Ist Department of Pediatrics Tulassay, Tivadar; Semmelweis University, Joint Research Laboratory of the Hungarian Academy of Sciences and the Semmelweis University Tory, Kalman; Semmelweis University, Ist Department of Pediatrics

Keywords: genetics, nephrotic syndrome, NPHS2, podocin, mutation screening, algorithm

Pediatric Nephrology Editorial Office, phone 214-648-3438, email: [email protected]

Pediatric Nephrology

For Peer Review

1

NPHS2 p.V290M mutation in late-onset steroid-resistant nephrotic syndrome

Andrea Kerti1, Rózsa Csohány1, Attila Szabó1, Ottó Árkossy2, Péter Sallay1, Vincent

Moriniére3, Virginia Vega-Warner4, Gábor Nyírı5, Orsolya Lakatos6, Tamás Szabó7,

Beata S. Lipska8,9, Franz Schaefer9, Corinne Antignac3, George Reusz1, Tivadar

Tulassay10, Kálmán Tory1

1 Ist Department of Pediatrics, Semmelweis University, Budapest, Hungary

2 FMC Szépvölgyi Dialysis Centre, Szent János Hospital, Nephrology Outpatient Dept, Budapest, Hungary

3 INSERM U983, Hôpital Necker, Paris, France

4 Departments of Pediatrics and Human Genetics, University of Michigan, Ann Arbor, Michigan, USA

5 Clinical and Research Center for Molecular Neurology, Semmelweis University

6 Department of Pediatrics, University of Pécs, Hungary

7 University of Debrecen, Medical and Health Science Center, Pediatric Institute, Debrecen, Hungary

8 Department of Biology and Genetics, Medical University of Gdansk, Poland

9 Division of Pediatric Nephrology, Center for Pediatrics and Adolescent Medicine, University of Heidelberg, Germany

10 Joint Research Laboratory of the Hungarian Academy of Sciences and the Semmelweis University, 1st Department of Pediatrics

Corresponding author:

Kálmán Tory

[email protected]

Cell: +36-20-8258166; Fax: +36-1-3138212

Ist Department of Pediatrics, Semmelweis University, Budapest

Bókay J. u. 53., Budapest, 1083 Hungary

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Abstract

Background. The most frequently mutated gene of steroid-resistant nephrotic

syndrome (SRNS) is NPHS2. Current guidelines propose the sequencing of all

NPHS2 exons only in childhood-onset SRNS.

Methods. A cohort of 38 Hungarian patients with childhood-onset nephrotic-range

proteinuria was screened for NPHS2 mutations. The frequency of the p.V290M

mutation in late-onset SRNS was examined in the French and PodoNet cohorts.

Results. Seven of 38 Hungarian patients carried NPHS2 mutations on both alleles.

Two of them – diagnosed with proteinuria through school-screening programs at the

age of 9.7 and 14 years – have not developed nephrotic syndrome in childhood. The

first, an 18-year-old man, homozygous for p.V290M, has never developed edema.

The second, a 31-year-old woman – compound heterozygous for p.V290M and

p.R138Q – was first detected to develop hypoalbuminemia (<30g/l) and edema at the

age of 24.3 and 27.5 years, respectively. Both patients have a normal GFR. The

mutation p.V290M was carried by 3/38 patients in the Hungarian cohort, by 2/95

patients with late-onset SRNS in the PodoNet cohort and 0/83 patients in the French

cohort.

Conclusions. We propose that not only the p.R229Q variant, but also the p.V290M

mutation should be screened in Central and Eastern European patients with a late-

onset SRNS.

Keywords: genetics, nephrotic syndrome, NPHS2, podocin, mutation screening,

algorithm

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Introduction

Nephrotic syndrome is the consequence of the glomerular filtration barrier’s damage

and refers the clinical picture of heavy proteinuria, hypalbuminemia, edema and

hyperlipidemia. The majority of patients with nephrotic syndrome respond to steroid

therapy and have a good clinical prognosis. In contrast, approximately 10% of

children and 40% of adults are steroid-resistant and progress to ESRD [1-3].

Based on its etiology, two forms of steroid-resistant nephrotic syndrome (SRNS) can

be distinguished, an immune and a genetic form. As the treatment in the two forms is

different, their differentiation is clinically important. This is primarily based on the

identification of the causative mutation in one of the ten genes identified thus far in

isolated SRNS [4-14]. Out of them, NPHS2, encoding podocin, is the most frequently

mutated gene in all but the neonatal age group [13, 15, 16].

NPHS2 mutations were first described in children presenting with SRNS before the

age of 6 years who progressed to ESRD during the first decade of life. Histologically,

minimal change disease (MCD) were found on early biopsy samples and focal

segmental glomerulosclerosis (FSGS) at later stages. [13]

Based on large cohort studies, among patients carrying NPHS2 mutations on both

alleles, the onset of SRNS depends significantly on the type of the mutation [17, 18].

An onset above the age of 10 years is exceptional and have been reported

exclusively in patients carrying a missense or a splice site mutation [17-21].

Nevertheless, even these patients have developed nephrotic syndrome till the age of

16 years, and progressed to ESRD till the age of 26, the latest [17, 22].

However, the clinical phenotype caused by NPHS2 can be even less severe due to

the variant p.R229Q [23]. This variant when found in trans-association with one

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pathogenic NPHS2 mutation, was reported in patients developing SRNS as late as

39 years of age and ESRD at the age of 43.5 years [22].

Based on these observations, sequencing of all NPHS2 exons has been proposed

only in children presenting with SRNS before the age of 14 years. In patients with a

late-onset SRNS, only the p.R229Q variant is proposed to be screened at the first

step, followed by sequencing of the other exons in case of its presence [4, 16].

Here we present a late-onset, slowly progressing SRNS in two unrelated patients

carrying the p.V290M NPHS2 mutation. Mutation-screening in these patients – based

on the current guidelines – would have failed. We propose that this missense

mutation should also be screened in Central and Eastern European patients with

late-onset SRNS, where its allele frequency is not negligible.

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Methods

Out of a cohort of 47 Hungarian unrelated patients with nephrotic-range proteinuria

(>40mg/m2/h) diagnosed before 14 years of age and unresponsive to steroid-

treatment, 7 patients were excluded for syndromic SRNS, 1 patient for a histology of

diffuse mesangial sclerosis and 1 for recurrence after renal transplantation. Thus, a

total of 38 patients with nephrotic-range proteinuria with FSGS (n=30) or MCD (n=8)

on histology have been selected for NPHS2 mutation-screening. Thirty-six of 38

patients developed nephrotic syndrome either at diagnosis (n=32) or later, but before

the age of 14 years (n=4). Eight patients were later reported to achieve remission

after cyclosporine therapy. None of the families was known to be consanguineous.

One case was familial with an affected sibling pair.

Genomic DNA was isolated from peripheral blood by standard methods, after

obtaining informed consent from the patients or their parents. The 8 exons and the

adjacent intronic junctions of NPHS2 have been directly sequenced by one of three

laboratories: (1) Ist Department of Pediatrics, Semmelweis University, Budapest,

Hungary (2) INSERM U983, Necker Hospital, Paris, France (3) University of

Michigan, Ann Arbor, Michigan, USA.

Screening of the p.V290M mutation in large SRNS cohorts

The frequency of the mutation c.868G>A (p.V290M) was examined in late-onset

SRNS in two large cohorts: (1) in 83 patients from the French cohort originating

mainly from Europe and North Africa with an onset of SRNS between the ages of 14

and 70 years (average: 29 years) and (2) in 95 patients from the PodoNet cohort

originating mainly from Italy (n=42), Turkey (n=27), Germany (n=9), and Poland (n=9)

with an onset between the ages of 14 and 40 years (average 19 years). Screening of

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the French cohort was performed by the direct sequencing of exon 7 in (1) Ist

Department of Pediatrics, Semmelweis University, Budapest, Hungary (2) INSERM

U983, Necker Hospital, Paris, France. The results from PodoNet consortium were

obtained through direct sequencing of the entire NPHS2 coding sequence performed

in the laboratories of the project partners.

Primers are available upon request. For sequence analysis, the software Sequencher

(Gene Codes, Ann Arbor, MI) was used. Segregation was confirmed by direct

sequencing of parental samples when available. All experiments were in accordance

with the Hungarian ethical committee recommendations and with the Declaration of

Helsinki Principles.

Haplotype analysis

Family members of patients carrying p.V290M mutation were genotyped using

markers D1S3760, D1S215, D1S3759 and D1S2751 neighboring the NPHS2 gene

locus. Genotyping was performed by PCR using fluorescent dye-labeled forward

primers and subsequent capillary electrophoresis using an 3500 Genetic Analyzer

(ABI), the results were analysed using GeneMapper software (Applied Biosystems).

Haplotype frequencies were compared using Fisher's exact test.

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Results

NPHS2 mutations

Seven of 38 unrelated patients (18%) were found to carry homozygous or compound

heterozygous NPHS2 mutations (Table 1). One of them, a nonsense mutation

(c.790G>T, p.E264*) was novel. While 5 patients carried a truncating or the similarly

severe p.R138Q mutation [24] on both alleles, 2 patients carried the p.V290M

mutation either in homozygous state (VM86) or in association to the p.R138Q

mutation (VM163) (Figure 1). In 3 patients (VM70, VM71 and VM84) only one allele

was found to be mutated carrying either a truncating, the p.V290M or the p.R138Q

mutation, respectively (Table 1).

Four patients carried the p.R229Q variant in heterozygous state. However, as no

pathogenic mutation was found on the second NPHS2 allele, p.R229Q was not

considered to be pathogenic in these patients.

Clinical phenotype of patients with NPHS2 mutations

The clinical presentation of the 5 patients with truncating or the p.R138Q mutation on

both alleles corresponded to the classical NPHS2-related phenotype [4] (Table 1). All

of them presented with nephrotic syndrome between the ages of 3 months and 4

years, and 4 of them progressed to ESRD between the ages of 2.5 and 9.5 years.

None of them responded to cyclosporin therapy and no recurrence was seen after

transplantation. While the phenotype of 2 patients (VM70, VM71) with a single

heterozygous mutation was similar (Table 1), patient VM84 achieved complete

remission (proteinuria<4mg/m2/hour) 8 weeks after the start of cyclosporine therapy.

Her proteinuria has been remaining in the normal range for 1,5 years, with no

medication in the last 6 months.

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In contrast to patients with truncating or p.R138Q mutations, the two adults carrying

the p.V290M mutation followed a markedly milder clinical course (Table 1). Patient

VM86 carrying the homozygous p.V290M mutation was diagnosed with proteinuria at

the age of 9.7 years through a school screening program. His proteinuria has been

persisted with an average value of 34mg/m2/hour (range: 3-104, n=119

measurements) (Figure 2). However, till now, the age of 18 years, he has never

developed edema and his serum albumin concentration dropped below 30g/l only

once (Figure 2). At the age of 16 years, a renal biopsy was performed which showed

FSGS. Currently, his GFR is estimated to be 108ml/min/1,73m2 by the MDRD

formula.

The second patient (VM163), being compound heterozygous for p.V290M and

p.R138Q, was diagnosed with proteinuria just before her 14th birthday through a

school screening program as well. Her proteinuria has been persisted with an

important variation (average: 35mg/m2/hour [range: 1-228, n=91]) (Figure 2). A renal

biopsy was performed at the age of 17.6 years which showed MCD. Her serum

albumin level first dropped below 30g/l at the age of 24.3 years, but had not

developed edema till the age of 27.5 years (Figure 2). She became pregnant at the

age of 28.3 years. At the 34th gestational week severe edema developed which

necessitated the termination of the pregnancy by cesarean section. The following

days, the edema resolved quickly. Currently, at the age of 31 years, her renal

function is still normal with an estimated (MDRD) GFR of 84 ml/min/1,73m2.

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Allele frequency of p.V290M in late-onset SRNS in the French and the PodoNet

cohorts

As the c.868G>A (p.V290M) mutation was associated with a late-onset SRNS in two

patients and its allele frequency was important (4/76 [5%] alleles) in the Hungarian

cohort, we examined its allele frequency in late-onset SRNS in two large European

cohorts. While none of the 83 patients in the French cohort, 2 out of 95 patients in the

PodoNet cohort was found to carry the p.V290M mutation, either in compound

heterozygous state in association to a splice site mutation (c.451+3A>T, intron 3) in a

German patient or as a single heterozygous mutation in a Turkish patient giving an

allele frequency of 1,1% in the PodoNet cohort.

Haplotype analysis of the p.V290M allele

To investigate whether a founder effect can explain the observed geographical

differences, the 12 parental alleles of the 3 patients carrying the p.V290M were

genotyped. Indeed, a common V290M haplotype was found with allele numbers

arbitrarily designated 8-3-1 based on (CA)n repeat number of 174 bp, 193 bp, and

180 bp at markers D1S3760, D1S215 and D1S3759 (Figure 3). The 8 non-V290M

alleles were all different from each other, and only one of them was haploidentical

with the V290M alleles within the conserved region of 530kb (Figure 3). Thus, the

marked difference in the frequency of this haplotype (4/4 vs. 1/8, p=0.01), supports

the notion that the p.V290M allele derives from a common ancestor.

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Discussion

Identification of a mutation in a patient with SRNS has direct clinical consequences, it

makes unnecessary further immunosuppressive treatment and unlikely the

recurrence of nephrotic syndrome in a kidney graft [25]. In the present study, the

finding of a single heterozygous NPHS2 mutation in a patient who achieved complete

remission after cyclosporine therapy does not contradict to this, instead it rather

emphasizes that the identification of an NPHS2 mutation carrier by chance is not

impossible in the immune form of SRNS. Therefore, despite of the general

expectation that the second mutation goes undetected; patients with one

heterozygous mutation should be treated as potential carriers in whom NPHS2 is not

pathogenic. Thus, considering only patients with mutations on both alleles, the

mutation rate of NPHS2 in isolated FSGS and MCD in the presented cohort (7/38

patients) corresponds to previous studies [17-19, 26].

Current guidelines propose the routine sequencing of NPHS2 only in childhood-onset

SRNS [4] or in patients with an onset before the age of 13 years [16]. This approach

is based on the observation that even an onset above the age of 10 years is rare and

has only been reported in patients with splice or missense mutations as p.V180M,

p.R238S and p.V290M [17-21]. Weber et al. found the p.V180M and the R238S

mutations in seven patients with a median age of onset of 11±1 years [18]. In a large

survey, among patients with two NPHS2 mutations, an onset above 10 years of age

was only found in five patients carrying either p.V180M or p.V290M [17]. The latest

onset associated with these mutations was 16.6 and 14.3 years, respectively.

Nevertheless, the mutations p.R238S and p.V180M – encoded by the same exon 5

as the p.R229Q variant – cannot be missed in adolescent-onset SRNS if the

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proposed screening of p.R229Q is performed by direct sequencing of the whole

exon.

In contrast, the mutation p.V290M – reported to lead to SRNS between the ages of 5

months and 14.7 years in 10 patients [17, 19, 27-30] – is encoded by exon 7. The

proposed sequencing of all NPHS2 exons till the age of onset of 14 years, could

identify this mutation in all but one case. In this study, we report two patients carrying

p.V290M mutation with an unexpectedly late onset and mild clinical course. The 18-

year-old boy has never had edema and his serum albumin level is still in the normal

range. The 31-year-old woman developed first edema at the age of 27.5 years, and

currently has a normal renal function. As patients with two NPHS2 mutations have

not been reported to progress to ESRD later than 26 years of age [17-20, 22], this

clinical course is unexpectedly mild and comparable to that of patients carrying

p.R229Q in trans-association to a mutation. Along the same line, 2 patients with

p.V290M were reported to progress to ESRD, at the ages of 20 and 24 years [29,

30]. In the current study, both patients carrying p.V290M had been diagnosed with

proteinuria by school screening programs. However, based on the age at the onset of

clinical symptoms and the current guidelines, only the p.R229Q variant should have

been screened, and thus the mutation screening would have failed.

The clinical significance of this observation depends on the geographic region. While

the allele frequency of the p.V290M mutation was found to be 1/13.000 in the general

American population [31], it has never been reported in SRNS patients from Western

Europe or North Africa [13, 18, 22]; but was found in 11 of 124 patients with two

NPHS2 mutations from Central and Eastern European cohorts, including the present

one [17, 19, 25, 27, 28], suggesting a founder effect. Accordingly, in the present

study, none of the French or Italian patients with adolescent- and adult-onset SRNS,

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but 2 of 46 patients from Germany, Poland or Turkey carried this mutation. The

founder effect of the p.V290M mutation was further supported by the finding that the

four V290M alleles found in the present study were haploidentical in an at least

0.5Mb region.

Since the original descriptions, the spectrum of SRNS phenotypes linked to a single

gene has been continuously expanding. Mutations of NPHS1 were found in

childhood- and adult-onset SRNS [32, 33], as such as NPHS2 mutations in neonatal-

onset SRNS [18]. It has been pointed out that most of this phenotypic variability is

secondary to the mutations’ different severity and effect on the encoded protein’s

function [17]. As the size of the cohorts and the clinical experience are growing, the

mutation screening can be expected to become more specific based on subtle

phenotype-differences. Along this line, while the mutation screening of NPHS2 in

adolescent and adult-onset SRNS was focused on p.R229Q [4, 16], recently,

sequencing of the entire coding region of NPHS2 in patients with an onset of SRNS

below 20 years of age has been recently proposed by the PodoNet cohort [34].

In conclusion, based on the presented observations, we are going to screen patients

with an onset of SRNS below 30 years not only for the p.R229Q variant but also for

the p.V290M mutation. We propose that this missense mutation should be screened

in young adults with SRNS from Central and Eastern Europe.

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Acknowledgement

This work was supported by Pfizer, TÁMOP-4.2.1/B-09/1/KMR-2010-0001, Zoltán

Magyary and János Bólyai fellowship (KT). The authors thank Mária Bernáth for

expert technical assistance.

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17. Hinkes B, Vlangos C, Heeringa S, Mucha B, Gbadegesin R, Liu J, Hasselbacher K, Ozaltin F,

Hildebrandt F (2008) Specific podocin mutations correlate with age of onset in steroid-

resistant nephrotic syndrome. J Am Soc Nephrol 19:365-371.

18. Weber S, Gribouval O, Esquivel EL, Moriniere V, Tete MJ, Legendre C, Niaudet P, Antignac C

(2004) NPHS2 mutation analysis shows genetic heterogeneity of steroid-resistant nephrotic

syndrome and low post-transplant recurrence. Kidney Int 66:571-579.

19. Berdeli A, Mir S, Yavascan O, Serdaroglu E, Bak M, Aksu N, Oner A, Anarat A, Donmez O, Yildiz

N, Sever L, Tabel Y, Dusunsel R, Sonmez F, Cakar N (2007) NPHS2 (podicin) mutations in

Turkish children with idiopathic nephrotic syndrome. Pediatr Nephrol 22:2031-2040.

20. Bower M, Salomon R, Allanson J, Antignac C, Benedicenti F, Benetti E, Binenbaum G, Jensen

UB, Cochat P, DeCramer S, Dixon J, Drouin R, Falk MJ, Feret H, Gise R, Hunter A, Johnson K,

Kumar R, Lavocat MP, Martin L, Moriniere V, Mowat D, Murer L, Nguyen HT, Peretz-Amit G,

Pierce E, Place E, Rodig N, Salerno A, Sastry S, Sato T, Sayer JA, Schaafsma GC, Shoemaker L,

Stockton DW, Tan WH, Tenconi R, Vanhille P, Vats A, Wang X, Warman B, Weleber RG, White

SM, Wilson-Brackett C, Zand DJ, Eccles M, Schimmenti LA, Heidet L (2012) Update of PAX2

mutations in renal coloboma syndrome and establishment of a locus-specific database. Hum

Mutat 33:457-466.

21. Hildebrandt F, Heeringa SF (2009) Specific podocin mutations determine age of onset of

nephrotic syndrome all the way into adult life. Kidney Int 75:669-671.

22. Machuca E, Hummel A, Nevo F, Dantal J, Martinez F, Al-Sabban E, Baudouin V, Abel L,

Grunfeld JP, Antignac C (2009) Clinical and epidemiological assessment of steroid-resistant

nephrotic syndrome associated with the NPHS2 R229Q variant. Kidney Int 75:727-735.

23. Tsukaguchi H, Sudhakar A, Le TC, Nguyen T, Yao J, Schwimmer JA, Schachter AD, Poch E,

Abreu PF, Appel GB, Pereira AB, Kalluri R, Pollak MR (2002) NPHS2 mutations in late-onset

focal segmental glomerulosclerosis: R229Q is a common disease-associated allele. J Clin

Invest 110:1659-1666.

24. Huber TB, Simons M, Hartleben B, Sernetz L, Schmidts M, Gundlach E, Saleem MA, Walz G,

Benzing T (2003) Molecular basis of the functional podocin-nephrin complex: mutations in

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the NPHS2 gene disrupt nephrin targeting to lipid raft microdomains. Hum Mol Genet

12:3397-3405.

25. Buscher AK, Kranz B, Buscher R, Hildebrandt F, Dworniczak B, Pennekamp P, Kuwertz-Broking

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27. Karle SM, Uetz B, Ronner V, Glaeser L, Hildebrandt F, Fuchshuber A (2002) Novel mutations

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Ruf EM, Mucha B, Bagga A, Neuhaus T, Fuchshuber A, Bakkaloglu A, Hildebrandt F (2004)

Patients with mutations in NPHS2 (podocin) do not respond to standard steroid treatment of

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improvement in a patient with steroid-resistant nephrotic syndrome due to podocin (NPHS2)

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31. Exome Variant Server NGESPE Seattle, WA (URL: http://evs.gs.washington.edu/EVS/).

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Barquero E, Fernandez-Llama P, Ballarin J, Ars E, Torra R (2009) Nephrin mutations cause

childhood- and adult-onset focal segmental glomerulosclerosis. Kidney Int 76:1268-1276.

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nephrotic syndrome (SRNS): finding in the PodoNet cohort 26:1577.

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Legends to figures

Figure 1

Sequencing chromatogram of the c.868 G>A (p.V290M) mutation in heterozygous

(patient VM163) and homozygous state (patient VM86)

Figure 2

Proteinuria and serum albumin level of patients VM86 and VM163 carrying p.V290M

mutation

Figure 3

Parental haplotypes of patients carrying the p.V290M mutation (P: paternal, M:

maternal alleles). The common parts of the four V290M alleles are highlighted (gray).

Only one of the 8 non-V290M alleles is haploidentical with them (outlined by a

dashed line). All the non-V290M alleles are different from each other.

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Table 1. Genetic and clinical characteristics of patients with NPHS2 mutations

Ind Gender Nucleotide change Amino acid

change Histology

Age at dg of

proteinuria (years)

Age at onset of

edema (years)

Response to

cyclosporin

Age at ESRD

(years)

Recurrence after

Tx

Patients carrying truncating or p.R138Q mutations on both alleles

VM1 F c.413G>A het (m)

c.948delT het (p)

p.R138Q

p.A317Lfs*31 FSGS 4 4 resistent N (7.5) no Tx

VM83 M c.413G>A het (p)

c.790G>T het (m)

p.R138Q

p.E264* FSGS 4 4 resistent ESRD (9.5) no recurrence

VM161 M c.413G>A hom p.R138Q FSGS 0.3 0.3 resistent ESRD (6) no recurrence

VM162 F c.413G>A het

c.419delG het

p.R138Q

p.G140Dfs*41 FSGS 1.5 1.5 resistent ESRD (6) no recurrence

VM164 F c.467dupT het

c.948delT het

p.L156Ffs*11

p.A317Lfs*31 FSGS 3 3 resistent ESRD (9) no recurrence

Patients carrying p.V290M mutation in homozygous or compound heterozygous state

VM86 M c.868G>A hom (m, p) p.V290M FSGS 9 not yet (18) resistent N (18) no Tx

VM163 F c.413G>A het (m)

c.868G>A het (p)

p.R138Q

p.V290M MCD 14 27.5 resistent N (31) no Tx

Patients with a single heterozygous mutation

VM70 M c.948delT het p.A317Lfs*31 FSGS 0.7 0.7 resistent ESRD (2.5) no recurrence

VM71 F c.868G>A het p.V290M FSGS 3 6 not treated ESRD (8) no recurrence

VM84 F c.413G>A het p.R138Q FSGS 5 5 complete

remission N (6.5) no Tx

dg: diagnosis, FSGS: focal segmental glomerulosclerosis, het: heterozygous, hom: homozygous, m: maternal allele, MCD: minimal change disease,

N: normal renal function, p: paternal allele, Tx: renal transplantation

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Sequencing chromatogram of the c.868 G>A (p.V290M) mutation in heterozygous (patient VM163) and homozygous state (patient VM86)

23x27mm (300 x 300 DPI)

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Proteinuria and serum albumin level of patients VM86 and VM163 carrying p.V290M mutation 82x123mm (300 x 300 DPI)

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Parental haplotypes of patients carrying the p.V290M mutation (P: paternal, M: maternal alleles). The common parts of the four V290M alleles are highlighted (gray). Only one of the 8 non-V290M alleles is haploidentical with them (outlined by a dashed line). All the non-V290M alleles are different from each

other.

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NPHS2 p.V290M mutation in late-onset steroid-resistant nephrotic syndrome

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